Summer Studentship reports 2012

Biochemical Society Summer Studentship 2012

Investigating the Mechanisms of Intestinal Heam Transport

Student: Bethan Tempest Supervisor: Dr Yemisi Latunde-Dada

Background and Aims

Haem iron accounts for only 5-10% of dietary iron intake, yet a relatively high bioavailability (20-30%) means haem is still the major contributor to body iron status. (1)As such, understanding the mechanisms of intestinal haem iron absorption is a major goal in iron metabolism research. Earlier work suggests apical haem uptake to be a saturable, energy dependent process indicating the presence of a haem transporter(2)In 2005 the elusive haem transporter was identified and isolated and known as HCP-1 (haem carrier protein-1). However subsequent research shows that this protein also serves as a folate/proton symporter with a higher affinity for folate than haem at low pH.(3)Thus the quest to understand the mechanisms of haem absorption continues. This project aimed to investigate molecules that could possible absorb haem from the diet and deliver it to the circulation.

Recent evidence from microarray data has revealed the up-regulation of peptide transporters in iron-deficient mouse duodenal samples(unpublished data)(4)In this project it was hypothesised that these peptide transporters may putatively act as haem transporters and that amino acids, such as histidine, or proteins such as albumin form ligands with haem to generate a complex for absorption. Seven haem binding peptides were identified from microarrays and the literature and were studied for haem absorption capacity.(5)

Description of Work Most experiments involved determination of haem uptake via ferritin assay, and protein quantification. Cells and Culture Conditions:

• Duodenal HuTu cells obtained from the American Type Culture Collection. • Cells were cultured in DMEM medium supplemented with 10% foetal calf serum, 100kU/L of penicillin, and

100 mg/L streptomycin and maintained at 37oC in a humidified incubator containing 95% air/5% CO2.

siRNA depletions: • HuTu cells were seeded into 12-well-plates to give about 50% confluency 24 h after plating. • Cells transfected with 10 nM of either siCONTROL non-targeting, non-homologous siRNA (4390843) or siRNA-

targeting putative haem binding peptides and Ribocellin reagent as per manufacturer’s protocol. • Transfected cells were assayed after 72 h for haem uptake.

Haem treatment:

• Ten micro molar haem arginate was applied to the cells and uptake was for 1 h. • The uptake medium was then removed and the cells washed with 0.1% BSA to remove unbound haem. • Cells were incubated in serum free medium overnight for ferritin formation. • Cells washed with Phosphate-Buffered Saline and lysed with Mammalian Protein Extraction Reagent (MPER®,

Thermo Fisher Scientific, Cramlington, UK). • Cell extract was then centrifuged ( 5 min, 16,000g)

Ferritin Assay: using Alpha Diagnostic Int Human Ferritin Elisa Kit according to manufacturers protocol

• 20μl aliquot’s of supernatant were incubated with 200μl of Ab-enzyme conjugate on assay plate for 30 min. • Wells washed 300 ul of wash buffer x5 • 150 μl of TMB substrate was added per well and mixed for 15 min before addition of stop solution (50μl) • Absorbance read at 450nm in a spectrophotometer

Protein Assay: By using BIO RAD DC protein assay according to manufacturers instruction

• 70 μl of supernatant was added to 96 well plate with 30 μl of working reagent, and 100 μl of reagent B per well and incubated for 15min

• Absorbance measured at 750nm in spectrophotometer.

This protocol was also used to investigate novel nanoiron compounds presumed to be absorbed by the same mechanism as haem iron. Results Seven putative haem transporters were investigated. Silencing of P2, P5 and P6 genes produced a statistically significant reduction in haem uptake measured as ferritin concentration. (Fig.1) Silencing of P1 gene produced a modest reduction in haem uptake, though no other statistically significant results were obtained.

Iron utilization from novel nano iron compounds was also investigated. Utilization as ferritin from Fe-A and Fe-B compounds was not inhibited by a ferrous iron chelator (ferrozine). Absorption of control inorganic ions Fe-C and Fe-D was reduced significantly in the presence of ferritin. (Fig.2)

Future Directions These preliminary findings will in lead to further investigation of the P2, P5 and P6 as putative haem binding peptides. Cloning and characterisation of the proteins will be conducted. In addition the novel nano iron compounds will be characterised and kinetics of transport investigated. Together these investigations will increase understanding of haem transport.

Departures from proposal It was not possible to run qPCR and Western Blot analysis for all experiments so no data is presented however I still had the opportunity to practice these assays as part of other projects being conducted in the lab.

Value of Studentship Student: The studentship has been an incredibly enjoyable and rewarding experience. The techniques I have been exposed to include ferritin and protein assaying, qPCR and western blotting. I have improved upon my basic laboratory skills and learnt to use new techniques and equipment which will place me at a significant advantage in future studies. The experience has influenced me to reconsider my future career ambitions. I now hope to seek a research or PhD position after completing my undergraduate studies.

Lab: Bethan’s contributions to studies on the putative haem binding peptides constitute a part of on-going research in the lab. She was hard-working, conscientious and also did some data processing and analysis in the lab. The data generated from the novel iron compounds will be a useful part of a manuscript in preparation. Her summer studentship has been very productive and rewarding.

References: (1) Sharp PA. Intestinal iron absorption: regulation by dietary & systemic factors. Int J Vitam Nutr Res 2010 Oct;80(4-5):231-242. (2) Worthington MT, Cohn SM, Miller SK, Luo RQ, Berg CL. Characterization of a human plasma membrane heme transporter in intestinal and hepatocyte cell lines. Am J Physiol Gastrointest Liver Physiol 2001 Jun;280(6):G1172-7. (3) Qiu A, Jansen M, Sakaris A, Min SH, Chattopadhyay S, Tsai E, et al. Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption. Cell 2006 Dec 1;127(5):917-928. (4) Shayeghi M, Latunde-Dada GO, Oakhill JS, Laftah AH, Takeuchi K, Halliday N, et al. Identification of an intestinal heme transporter. Cell 2005 Sep 9;122(5):789-801. (5) Kuhl T, Sahoo N, Nikolajski M, Schlott B, Heinemann SH, Imhof D. Determination of hemin-binding characteristics of proteins by a combinatorial peptide library approach. Chembiochem 2011 Dec 16;12(18):2846-2855.

Peptides silencing and haem uptake in HuTu cells

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Figure 1: Haem absorption (measured as ferritin concentration) in HuTu cells after gene silencing

Figure 2: Summary graph of assays of iron uptake from novel nano iron compounds (Fe-A_Fe-D)

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Biochemical Society Summer Studentship 2012

Biochemical Society Studentship Report 2012 Alvaro Martinez Guimerá [email protected] Imperial College London Background: VASE isoform of NCAM has been identified as a novel CNS myelin- and reactive astrocyte-expressed inhibitor of axon regeneration. Neutralising it has potential use in therapies to treat stroke, spinal injuries or neurodegenerative diseases, and RNAi approaches have been successfully used for the downregulation of human VASE in NIH3T3 fibroblasts without affecting non-VASE NCAM levels. These promising early results suggest the use of RNAi as a potential therapy for axon regeneration through VASE-targeting, but to demonstrate the therapeutic potential of this approach, in vivo studies in a suitable animal model will be required, to this end, the downregulation efficiency and specificity of a rat RNAi to the rat isoform of VASE NCAM must be evaluated. Aims: The aim of this 8-week project is to investigate if a rat sequence RNAi that targets 30bp of the rodent VASE NCAM transcript can be used as a molecular biology tool to selectively downregulate this protein.

Project Description and Results: -Characterizing existing cell lines: The first step to the project was to establish the clonality of two existing cell lines, both being transgenic and G418-resistant rat NIH3T3 fibroblasts, one expressing VASE NCAM (hereafter referred to as ‘VASE’) and the other expressing non-VASE NCAM (hereafter referred to as N24). Results showed significant loss of transgene expression (Figure 1). -G418 Titration: Since a 100% clonal VASE cell line would be needed for future work, this cell line was selected using a range of G418 concentrations. Results from this titration would be used for further experiments involving G418 selection. Secondary immunocytochemistry was used again to confirm the 100% clonality of the selected VASE cells (Figure 2) where differential transgenic expression between cells could already be appreciated. -Anti-VASE Antibody Characterisation: VASE expression on cell lines was examined initially using an N16 antibody specific for NCAM, but successful characterisation of the specificity, and optimisation of the staining protocol of a new anti-VASE antibody that was specific for the 10 amino acid insertion that differentiates VASE NCAM from non-VASE NCAM was used for later analysis of VASE expression. -Plasmid Purification: The next step was to isolate and assess the purity and concentration of the ‘rat VASE’-containing plasmid from a bug stock using a standard Qiagen- Maxi purification kit. NCAM-/VASE- NIH3T3 fibroblast cells (3T3s) were then transfected with the plasmid using FuGENETM reagent and after two days incubation, selection was started. In addition, four GFP-containing plasmids consisting of two different siRNA target sequences for rat VASE and two different siRNA sequences for the human isoform of VASE were purified and the concentration and purity of the plasmids established in the same manner as performed for the ‘rat VASE’-containing plasmid. -siRNA Transfection: The low number of rat VASE+ 3T3 cells left after selection and their slow growth following the end of selection indicated the transfection of these cells with the purified siRNA-containing plasmids wouldn’t be possible in the time available. Therefore, the 100% clonal VASE cell line (which had been left selecting on 4mg/ml G418 to reduce loss of transgene expression) was transfected with each siRNA isolated, since if the rat VASE siRNA is able to successfully downregulate the human isoform of VASE, it is very likely it will downregulate the rat isoform of VASE. Due to the high background fluorescence obtained in the secondary immunocytochemistry procedure used to examine VASE expression, only one of the rat siRNA sequences suggested VASE downregulation had occurred in a relatively clear manner (Figure 3). Departures from Original Proposal: Due to the lack of time primarily caused by the slow growth of selected rat VASE+ 3T3 cells, there wasn’t enough time to prove these cells were VASE+ through immunocytochemistry and so, as a contingency plan, ‘human VASE’-expressing 3T3s were transfected with the rat RNAi. Also, the downregulation selectivity of the rat siRNA couldn’t be assessed by transfection of N24 cells for the same reason. A separate but relevant experiment was also performed based on previous observations where live staining was used to examine the fate of the primary antibody, both NCAM-specific (N16) and VASE-specific, after binding to their protein targets. It was observed that the fate of these two antibodies was different since the VASE-antibody seemed to be quickly endocytosed and degraded upon binding to VASE NCAM, as was observed in experiments involving live staining at different primary antibody incubation times under conditions of permeabilisation and no permeabilisation. It was quite remarkable to note that the same polypeptide responds so differently to two antibodies that recognise different sites.

mailto:[email protected]

Figures:

Future Directions: The results obtained suggest it is very likely that the rat VASE siRNA sequence downregulates the rat isoform of the VASE NCAM protein and so future in vivo models in rodents could be used to test a combination of VASE- targeting RNAi and growth factors as a therapy to promote axon regeneration in neurodegenerative diseases. Furthermore, the finding that binding of the VASE-specific antibody is likely to promote the intake and degradation of the VASE NCAM expressed on cells suggests this antibody could be an interesting therapy to be tested. Value of the Studentship: The two most noteworthy outcomes I’ve had from my internship are firstly, the confidence I have gained regarding lab work and secondly finding out what it’s like to work in a lab. Whilst at the beginning I felt insecure about performing experiments with the fear of wasting lab resources such as expensive antibodies and was only comfortable when working under supervision, I became more independent throughout the internship and by the end became confident enough to design my own experiments and discuss them with my supervisors. Whilst before the internship I was unsure of wanting to pursue a career in research I am now quite keen on taking a PhD in the near future, now that I know how massively different working in a real lab is to the practicals I have performed so far at university. I have also learned the value of organisation and patience in a lab and this, as well as the whole internship experience will help me with my final year project. Value to the lab: Welcoming Alvaro to the lab over the summer has been a thoroughly positive experience. In addition to his contribution to our research, which has moved a key project along significantly, Alvaro’s contribution to lab life has been an unexpected bonus. He came to the lab with a determined yet flexible outlook and a willingness to learn, and sufficiently mastered cell culture, fluorescence immunocytochemistry, plasmid preparations and cell transfections to generate a new clonal cell line and show that a species-targeted RNAi tool was effective, the aims of the project. By the end of the project, Alvaro was designing well thought-out experiments and helped investigate an exciting observation that indicates we have a new tool for neutralising an inhibitor of axon regeneration in the CNS.

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Investigation into the modulatory effects of Zinc (Zn), Manganese (Mn) and

Inositol Phosphoglycans on the activity of the enzyme Glycogen Synthase

Kinase 3β (GSK3β)

Andras Solt

Aims 1. To establish a working assay to measure the activity of GSK3β

2. To investigate the concentration dependent effects of Zinc on GSK3β activity

3. To investigate the concentration dependent effects of Manganese on GSK3β activity

4. To investigate the effects of IPGs on GSK3β activity

Departures from the initial project

No departures were observed

Work carried out

GSK3β activity assay

The initial assay was an adaptation of a 32

P transfer assay as developed by Ishiguro et al. Tau was

chosen as the GSK3β phosphorylation substrate. Tau-412 protein (Sigma, Product No: T0326) was incubated

with the enzyme (Sigma-Aldrich, Recombinant Rabbit, N terminal His tagged, Product No: G 1663) at a 250ng

μl-1

concentration. The reaction was MES buffered at pH 6.5 (100mM). ATP and 32

PγATP was supplied at 4mM

concentration with Magnesium supplied as MgAcetate (0.5mM). For inhibitory Calcium chelation EGTA was

included in the reaction mix at a concentration of 1mM. To prevent oxidative damage of the proteins, 2-

Mercaptoethanol was added at a final concentration of 2mM. Enzyme was available at a stock concentration

of 200U per 100μL. Reaction concentration was at 1/5th

stock. The reaction was allowed to proceed for an

hour at 37°C. The reaction mixture was blotted on phosphocellulose paper and left over phosphorimager film

overnight to develop. The film was scanned using a laser imager the following day.

The method did not yield consistent results, even under increased enzyme and substrate concentrations.

Hence instead of Tau, a substrate peptide was chosen instead, as applied by numerous sources (Ryves

et al, Harwood et al).

The peptide analogue GSM (Millipore, Catalogue number #12-553) of 1mg mL-1

was diluted to a reaction

concentration of 5ng/20μL. The reaction was buffered at pH 7.5 by 50mM TRIS. The following components

were also included; 1X Protease Inhibitor (Boehringer tablet), 2-Mercaptoethanol (2mM), *ATP (25μM),

MgAcetate (10mM). GSK3β was added at 1/5th

stock concentration. 5μL of the total reaction mixture was

allocated to the metal ions (Zn and Mn) to be investigated.

This method was consistent and repeatable.

Effects of Zinc and Manganese

The investigation was extended to

changing substrate peptide concentrations

(0.25μg/μL,0.5g/μL, 0.75μg/μL, 1.0μg/μL)

IPG Assay

Conclusions and future developments IPGs elicit a more modulated response compared to mimetic metal ion ratios and concentrations

Substrate concentration plays no effect in the control of GSK3β by Manganese, while it does in case of

Zinc.

IC50 values for Zinc were elusive and results were not always consistent, the assay must be optimised

to gain consistent results.

Kinetic analysis of the Zn and Mn assays is needed.

Brain IPG analysis has not been done.

Assessment of gains from project

The Thomas lab has gained a working platform onto which future investigations of GSK3β can be built.

Collaboration has been established between working groups via the harnessing of expertise of Patricia

MacLean and Sirilaksana Kunjara.

I have gained notable skills in terms of assay development, experimental design, safe practices of working with

radioisotopes in biological research. I would like to thank the Biochemical society and the relevant research

groups for their kind support and efforts that have made this project a reality.

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One unit of IPG is the amount needed to cause 50% inhibition of Pyruvate Dehydrogenase. Metal ion

concentrations and ratios found in IPGs (McLean et al) were used to analyse the effect of IPGs on

GSK3β activity as opposed to the metal ions alone. Metal concentrations were only available for liver

IPGs. Mn and Zn in 2:1 (6μM:3μM) and 1.5:1 (22.5μM:15μM) ratios were used to stimulate IPGs.

Andrew Foster - Biochemical Society Studentship Report

Supervisor – Dr. Peter Roach, University of Southampton Introduction and aims: The aim of this project was to investigate the role played by the intracellular protein RelA in mediating the bacterial stringent response. When placed under stress, bacteria modify cellular processes to aid survival, including transcription levels and targets. Central to this adaptation is the small signalling molecule guanosine tetraphosphate ((p)ppGpp), which has been shown to regulate RNA polymerase activity both directly and indirectly 1. Cellular levels of the (p)ppGpp nucleoside are closely controlled by a set of enzymes with ppGpp synthetase and/or hydrolase activity. The protein SpoT displays both synthetase activity to generate the alarmone from ATP and GTP, and hydrolase activity to break down the nucleotide, yielding pyrophosphate and either GDP or GTP. In comparison RelA shows just a synthetase activity, and upon amino acid starvation catalyses (p)ppGpp synthesis. This process is thought to rely upon the binding of RelA and ribosomes, as well as the presence of deacylated t-RNA and the ribosomal protein L112. The aim of this project is to study the interactions of RelA, specifically looking at the binding of RelA and ribosomes and its effect on (p)ppGpp synthesis.

By utilising fluorescence polarization, fluorescently tagged RelA will be used for binding studies with ribosomes, with the effect other cellular factors have on this relationship also open for investigation. Fluorescence polarization is based on the principal that, following excitation, light is emitted in different planes relative to the amount of rotation of the fluorescent molecule during excitation. Binding of fluorescent RelA to ribosomes should result in a decrease in molecular rotation due to the increased size of the combined molecules, hence the emitted light should be more polarized than unbound RelA3. DsRed, a red fluorescent protein with emission at 580nm, will be attached to the N-terminus of the RelA gene. Following purification of DsRed/RelA and ribosomes, fluorescence polarization will be used to determine a binding constant of tagged RelA.

Description of work carried out/results: Construction of Fusion Expression Plasmid: The RelA gene, encoded in a pET vector, and a DsRed encoding plasmid were separately transformed into XL10-Gold competent cells. The RelA plasmid was digested by SacI-HF and ZhoI restriction enzymes to isolate the RelA gene of 2001bp. The DsRed gene was opened via a double digestion with SacI-HF and XhoI to remove the MthK gene from the

5311bp DsRed containing backbone (fig. 1). Ligation of the two fragments resulted in the construction of a 7312bp plasmid encoding for N-tagged Dsred/RelA, with a C-terminal His-tag on RelA. Following transformation (XL10-Gold) and growth on ampicillin agar plates, analytical digestion with ScaI-HF confirmed the successful ligation with the presence of two bands at the expected sizes

pRJW/4444/8812AA7QQP_1239028_RelA7310 bp

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Figure 1 - DsRed/RelA Ligation

of 5159bp and 2151bp on agarose gel. The plasmid was then successfully transformed into BL10-Rosetta cells and grown on Amp/Chl agar plates.

Expression and purification: BL21-rosetta cells were grown in 2YT media before inducing protein expression with IPTG. Previous work by the group on RelA had optimised growth and purification for the wild-type protein, and so this was adapted for DsRed/RelA. After overnight induction, the cells were harvested to yield bright red cell paste. Purification was investigated utilising the terminal His-tagged protein with a Ni-affinity column. A useful feature of the DsRed fluorescence is the ability to easily follow its path during the Ni-Column. After loading the cell lysate in with 10mM imidazole as a loading buffer, the red protein was eluted with a gradient to 500mM imidazole, easily followed by eye. (fig. 2)

To increase purity, a further purification step was utilised. The size exclusion properties of the column separated the large DsRed/RelA from smaller protein impurities and salts, as visualised on SDS-PAGE gel (fig. 3). Concerns over the reported RelA instability to repeated freeze-thaw cycles dictated the need for all purification steps to be carried out over the course of a maximum of two days.

By the end of the studentship, DsRed/RelA had been successfully constructed and purified, finding an optimised expression and purification protocol for the fusion protein based on that of RelA. The fluorescence was confirmed by emission at 588nm following excitation, and ribosome preparation had begun. However, full ribosome purification was not achieved in time to run fluorescence polarization experiments.

Future directions:

As seen in the SDS-PAGE gel in figure 3, the DsRed/RelA protein presents as a double band. Further research will be needed to ascertain the identity of the bands, with one possible cause being hydrolysis of the protein by proteases. Isolation of the bands followed by electrospray ionisation mass spectrometry should help identification. Once ribosomes have been prepared, fluorescence polarization studies can be run. By addition of ribosomes to a fixed concentration of DsRed/RelA, a curve of polarization against ribosome

Figure 2 - DsRed/RelA binding to blue Ni-Column

Figure 3 - SDS-PAGE gel showing DsRed/RelA protein at 103kDa in lanes 6-9

concentration can be plotted which should allow calculation of a binding constant for DsRed/RelA. By then adding wild-type RelA to a fixed concentration of ribosomes with saturated binding of DsRed/RelA, a displacement curve can be found, resulting in an IC50 for RelA. This can then be used in conjunction with the binding constant for the displaced DsRed/RelA, and after suitable analysis, a binding constant for wild-type RelA can be found. The effect of other factors such as uncharged t-RNA and the ribosome associated protein L11, both shown to affect RelA mediated (p)ppGpp synthesis, can then be seen in the same way.

Value of the studentship:

The 2 month studentship for me was not only beneficial to my development, but was also an extremely enjoyable opportunity. As a chemistry student, the opportunity to experience a more biology orientated laboratory gave me a good insight into the work involved. I not only reinforced my current understanding of biological chemistry, I also learnt many new practical techniques that would otherwise not be available to me. The idea of working towards a goal that is not only interesting but also potentially beneficial to the field of medicine is extremely rewarding. Following these 2 months, the idea of further postgraduate work in a biological lab is more attractive and I have gained a greater insight into the work of postgraduate research.

References:

1. Z. D. Dalebroux and M. S. Swanson, Nat Rev Micro, 2012, 10, 203-212. 2. R. Payoe and R. P. Fahlman, Biochemistry, 2011, 50, 3075-3083. 3. K. Yan, E. Hunt, J. Berge, E. May, R. A. Copeland and R. R. Gontarek, Antimicrobial Agents and

Chemotherapy, 2005, 49, 3367-3372.

The type 3 ryanodine receptor as a target for breast cancer therapy

Ashling Lavery

Supervised by Dr. John Mackrill, Dept. of Physiology, University College Cork

Aims

The aims of this project were to investigate the effects of dantrolene on MDA-MB-231 breast cancer cell (note – in the original proposal, MCF-7 breast cancer cells were to be used) proliferation and calcium (Ca2+) signalling, to determine the potential of this inhibitor of ryanodine receptor (RyR; Ca2+ channel) gating (which is upregulated in some breast cancers) as an agent for breast cancer therapy. In addition to the original proposal, the effects of another RyR inhibitor, tetracaine, were also studied.

Methods

Cells were cultured in suspension in Dulbeco’s modified Eagle’s medium with 10% v/v bovine serum and penicillin/streptomycin (DMEM).

Proliferation assay The crystal violet staining assay, as described by Senaratne et al., 2000 [1], was used to determine the effects of dantrolene, tetracaine or vehicle on cell density.

Calcium Imaging Using calcium imaging, as described by Hammoud et al., 2012 [2], a record of changes in the fura-2 ratio (340/380 nm) due to one of 8 dantrolene concentrations, and subsequently 500 µM 4-chloro-m-cresol (CmC; RyR agonist), was made.

Statistical Analysis The statistical analysis of the data was performed using one-way analyses of variance (ANOVA) with a Tukey post test.

Results and Conclusions

Figure 1: Cell density over time for cells that are blank (untreated), treated with vehicle (0.1% v/v ethanol) or treated with tetracaine (50 μM).

The crystal violet assays performed on blank, vehicle-treated and tetracaine-treated cells at specific time points showed a significant inhibition of cell growth due to tetracaine treatment. However, it is possible that inhibition occurs before the first assayed time point, day 6. This indicates that future studies should assay earlier time points, beginning with a day 0 time point, to confirm the minimum incubation time needed for the drug to effectively inhibit growth.

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Figure 2: Cell density over time for cells that are blank (untreated), treated with vehicle (0.1% v/v DMSO) or treated with dantrolene (50 μM) The decrease in cell viability due to dantrolene seen in figure 2 is not statistically significant. When compared to the tetracaine experiments, it appears that dantrolene

has little effect on growth, suggesting that tetracaine affects targets other than RyR.

Figure 3: Cell density when treated with various concentrations of dantrolene for 7 days.

Statistical analysis of adjacent increments indicates significant variance between 100 and 50 μM, 20 and 10 μM, and 10 and 5 μM. This suggests that a maximal effect occurs with a concentration of 100 μM and the main effects are at the higher concentrations. Concentration dependency of the inhibition does not appear to be strong.

Figure 4: Percent fall in mean fura-2 ratio due to various concentrations of dantrolene.

Statistical analysis indicates that the fall in mean fura-2 ratio due to dantrolene is only significantly different between 100 and 50 μM.

Neither sets of data from the two concentration response experiments ([dantrolene] versus cell density and [dantrolene] versus fall in fura-2 ratio) fit to the sigmoidal relationship expected; they do not reach a trough or plateau within the expected range of dantrolene concentrations. This implies that the effects of the highest concentrations of dantrolene on cell density are unlikely to be due to effects on RyRs.

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Future studies could use similar assays to test the concentration dependency of the effects of tetracaine and other RyR antagonists/agonists (eg. ryanodine, caffeine, CmC).

References

1. Senaratne, S.G., et al., Bisphosphonates induce apoptosis in human breast cancer cell lines. Br J Cancer, 2000. 82(8): p. 1459-68.

2. Hammoud, Y., Rice, T., and Mackrill, J.J., Oxysterols modulate calcium signalling in the A7r5 aortic smooth muscle cell-line. Biochimie, 2012.

Personal Statement

Partaking in this summer research project has been a wonderful experience for me. Not only has it allowed me a chance to work with a respected member of the scientific community, Dr. John Mackrill, from whom I received priceless guidence and advice, but it has granted me insight into the vast world of scientific research. Until now, my main experiences working in a lab took place in the 3 hour practical classes that exist as part of the curriculium of my degree. This project allowed me to gain a much more detailed understanding of the workings of a lab and of the field of research as a whole. I am thoroughly pleased with my experience and thank the Biochemical Society for making such an incredible experience possible.

Report on Biochemical Society Funded 6 week Summer Vacation Studentship ‘Do subtle changes to the ribosome alter translational recoding driven by 2A peptides?’

Studentsip carried out in Dr. Jeremy Brown’s laboratory at Newcastle University by Dina Bastola.

Background: Protein synthesis is carried out by ribosomes, which faithfully decode the information in mRNA into protein. However, on occasion, ribosomes are prompted to deviate from the genetic code, in events termed programmed translational recoding. Examples of this are stop codon read-through, used in a variety of situations to produce proteins extended beyond the first stop codon in the RNA, and frame-shifting, where the ribosome changes reading frame on the RNA. Events such as these are most often encountered in viruses, which use them to maximise the coding capacity of their compact genomes and to provide regulation of the level of expression of certain proteins.

The focus of my summer project was to be the 2A peptide of Foot and Mouth Disease Virus, which drives another recoding event. In this, termed Stop-Carry On recoding, ribosomes that have translated to the end of the peptide terminate translation on a sense codon and then reinitiate protein synthesis. This leads to the generation of 2 proteins from one open reading frame. The reaction is driven entirely by the peptide, which presumably interacts in specific ways with the ribosomal peptidyl transferase centre and exit tunnel to do this. While significant data has been accrued on importance of specific amino acids within 2A, requirements in the ribosome for the 2A reaction have not been probed.

Aims: The initial aims of the project were to examine yeast strains in which either the ribosomal RNA or a specific protein, Rpl17, that lines the exit tunnel were altered. Ribosomal RNA alterations were to be generated by removing modifications that normally occur on it (methylation and pseudouridylation), whereas the ribosomal protein was to be studied by random mutagenesis. In each case the aim was to look to see if the changes affected the 2A reaction using a reporter system developed previously in the laboratory.

Work on ribosomal RNA was not carried out, as the yeast strains available for this did not have the correct set of markers for the experiments. However, progress was made towards analysing Rpl17. In particular, several plasmids were generated into which mutations could be inserted into RPL17. This entailed several PCR steps to amplify portions of RPL17 and its promoter and terminator and then clone them into a yeast expression vector. Initial attempts at this failed, but by ‘trouble shooting’ the protocol and trying the PCR under several different conditions using alternative DNA polymerases, amplification of the desired products was achieved. These were subsequently ligated into the yeast expression vector.

As the ribosomal RNA work was not carried out, I became involved in a project examining another translational recoding event, which the laboratory started a new collaboration on during the summer. This is a coupled termination-reinitiation event that takes place in human respiratory syncitial virus (RSV). In this, translation of the first open reading frame on

one of the viruses RNAs is necessary for translation of the second, and this relies on specific sequences in the RNA around the stop codon of the first open reading frame. The aim of the experiments was to confirm whether or not the reinitiation reaction could be recontituted in yeast, which would then allow genetic analysis. I was provided with plasmids containing the wild type sequence from RSV driving reinitiation on a chloramphenicl acetyltransferase (CAT) gene, the expression of which was monitored using a commercial sandwich elisa assay. Control plasmids contained CAT alone (a positive control for CAT expression) or mutant versions of the RSV sequence (negative control) which should not have yielded expression of CAT. Yeast strains containing these plasmids were grown and the assay for CAT expression carried out on lysates from them. The results were positive, and I could show, through repeated experiments, that the RSV sequence that drives coupled termination-reinitiation in human cells also works in yeast. This has opened up a new area for research in the laboratory, and is the most positive thing to have come out of my vacation studentship.

This research project gave me a great opportunity to gain valuable experience in a laboratory environment. I have learnt various skills and methods, which I wouldn’t have had the chance to in my degree work. I have also gained skills on time management. This project has helped me be independent and apply my knowledge from lectures into the laboratory. I have really enjoyed my time doing the summer project and would definitely recommend it to anyone who’s looking to gain experience and skills.

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Beatrice Viviane Vetter The Biochemical Society Studentship Report 2012

A Biophysical Investigation of Protein-Drug Binding in Mycobacterium tuberculosis using 2D-IR Spectroscopy

Supervisor: Dr Paul A Hoskisson University of Strathclyde, Glasgow, UK

Background Tuberculosis (TB) is a global health problem with an estimated 8.8 million cases and 1.5 million deaths reported worldwide in 2010 (WHO, 2011) and is often associated with poverty, immunodeficiency and underdeveloped nations or centres of urban decay. The etiological agent of this pulmonary disease, Mycobacterium tuberculosis (MTB), has regained interest due to a recent increase of multi-drug resistance strains. The long-chain enoyl-acyl carrier protein reductase InhA, which catalyses the biosynthesis of mycolic acids in the cell wall of MTB, has been found to be the main drug target of the key frontline drug Isoniazid (INH) which has been used since the 1950s. INH is activated by the oxygenase KatG producing an active adduct with NADH in the cell which inhibits InhA and hence stopping the growth and survival of MTB. Therefore, elucidating the detailed molecular mechanisms in resistance and the action of established anti-TB drugs can aid drug design. To examine the structural dynamics involved in the protein-drug binding of InhA and INH the novel technique called 2-dimensional infrared spectroscopy (2D-IR) is used.

Aims The aim of this project was to investigate InhA activity in specific site-directed mutants (SDM) by replacing single amino acid residues with alanine, to measure the activity of these mutant proteins in enzyme assays and employing ultrafast 2D-IR spectroscopy to examine protein-ligand interactions.

Description of Work The plasmid pETTOPO-TEV containing inhA wild-type was extracted from Escherichia coli DH5α and used as a template for mutagenesis. Site-Directed Mutagenesis (SDM) of specific amino acids in the INH-NADH adduct binding pocket of InhA was carried out using Stratagene QuikChange® II Kit. Hereby, single amino acids residues suspected to be interacting with the ligand were replaced with alanine (see Fig.1). The mutant plasmid was transformed into competent E.coli DH5α cells for nick repair. Overexpression of InhA was carried out in E.coli BL21 with the WT and the SDM mutants. Cultures were induced with isopropyl-β-D-1-thiogalactopyranoside (IPTG), the cell pellet harvested by centrifugation and re-suspended in Sodium-Phosphate Buffer pH 7.5 with 200mM NaCl, 20mM imidazole and 10% glycerol and stored at -80°C. InhA-His6 was purified by immobilized metal ion affinity chromatography with a GE Healthcare ÄKTA Purifier using GE Healthcare HisTrap FF crude affinity columns with Sodium-Phosphate Buffer and imidazole gradient. The purified fractions were analysed by SDS-PAGE and further concentrated with Merck Millipore Amicon Ultra centrifugal filter units. In the physics department, under the supervision of Dr Neil Hunt, Fourier-Transform Infrared Spectra were taken with a Bruker Vertex 70 FTIR Spectrometer of INH-NADH adduct in different concentrations, deuterium sodium-phosphate buffer in various concentrations and molarities, as well as DMSO in various concentrations with D2O to investigate possible effects on INH-NADH adduct spectra.

Figure 1. Molecular contact of INH-NADH adduct and InhA. Red: INH derived isonicotinic acyl group; Blue: NADH; Green: InhA side chains; Magenta: Serine94 confers INH resistance. Figure taken from Rozwarski et al., 1998, p. 100, Fig.4.

Beatrice V Vetter Biochemical Society Studentship Report 2012

2

Protein Ladder A1 A2 A3 A4 A5 A6 A7 A8 A9

kDa 212 158 116 97.2 66.4 55.6

42.7

34.6 27.0 20.0

14.3

Figure 2. SDS-Page Gel of purified InhA S94A (28.5 kDa) fractions A1-9 with concentrations between 0.54 and 0.94 𝒎𝒎

𝒎𝒎.

Results Five SDM mutants of InhA (S94A, F149A, M155A, P193A and W222A) were obtained and verified by sequencing. Overexpression of InhA WT and mutants worked, but precipitation of InhA was observed during purification and concentration. This was problematic, since at least ≈25 𝑚𝑔

𝑚𝑙 protein is needed for Fourier-Transform

Infrared (FTIR) and 2D-IR spectroscopy. Various compounds were tried to keep InhA in solution during concentration. Usage of Urea to recover precipitated protein was unsuccessful. Sarkosyl FTIR Spectra showed that it is unsuited to aid protein solubility as it has peaks around 1600 cm-1 which is the area of interest when investigating INH and InhA interactions. However, increasing the concentration of glycerol to 20% in the Sodium-Phosphate Buffer was successful for InhA WT and FTIR and 2D-IR spectra at the Rutherford-Appleton-Laboratories could be obtained (see Fig.3). 2D-IR spectra (Fig.3) offer access to new information on the dynamics of a system with a sub-picosecond time-resolution by showing so-called vibrational coupling patterns. Hereby, every peak in a 1D-FTIR spectrum results in a peak pair diagonally across the spectrum, but if a vibrational mode of the molecule interacts with another, these peak pairs will appear in the so-called “off-diagonal region” (see Fig.3 A). The high time-resolution enables us to observe changes in peak amplitudes (see Fig.3 B) showing clearer which groups of INH are interacting. By comparing the 2D-IR spectra of InhA WT with INH-NADH adduct with its mutants the structural dynamics involved in the protein-drug binding and molecular mechanisms in resistance can be elucidated.

Figure 3. 2D-IR Spectra. A+B: Isoniazid (INH) concentration 300 mM at 0.25 ps and 1.50 ps. C+D: Purified wild-type InhA which was estimated UV-absorption of ca. 26 mg/ml at 0.25ps and 1.5ps. Dashed lines show vibrational coupling patterns. Data obtained at Rutherford-Appleton-Laboratories. Pictures courtesy of Dr Daniel Shaw, University of Strathclyde.

Beatrice V Vetter Biochemical Society Studentship Report 2012

3

Departure from original Proposal Due to time restriction and solubility problem of InhA WT and mutants, no comparison of wild-type and SDM mutant enzyme activities could be obtained, as well as comparing FTIR and 2D-IR thereof.

Future Work - Enzyme assays of InhA mutants - Further SDM of the INH-NADH binding pocket of InhA - FTIR and 2D-IR spectroscopy of mutant InhA with INH-NADH adduct

Value of Studentship Working in the Hoskisson lab was a very enjoyable experience and greatly beneficial for myself in terms of lab skills and increasing my technique repertoire. I was also given the opportunity to gain an insight into biophysics as part of a dynamic multi-disciplinary team which I found highly stimulating. This studentship was an invaluable educational experience and helped finalise my decision of pursuing a PhD in a multi-disciplinary research environment after my degree.

Value to the Laboratory The contribution to laboratory was through the production of mutant proteins, which are integral to an on-going project in the Hoskisson and Hunt laboratories that is funded by the Leverhulme Trust. These mutants will form the basis of on-going studies through the remaining 2 years of the project. More widely I contributed to the laboratory through integrating in to the work environment and helping with the daily tasks within the laboratory that all members undertake.

References Adamczyk, K., Candelaresi, M., Robb, K., Gumiero, A., Walsh, M. A., Parker, A. W., Hoskisson, P. A., Tucker, N. P. and Hunt, N. T. (2012) Measuring protein dynamics with ultrafast two-dimensional infrared spectroscopy. Meas. Sci. Technol. 23, 1-16.

Hunt, N. T. (2009) 2D-IR spectroscopy: ultrafast insights into biomolecule structure and function. Chem Soc Rev 38, 1837-1848.

Rozwarski, D. A., Grant, G. A., Barton, D. H. R., Jacobs Jr. and W. R., Sacchettini, J.C. (1998) Modification of the NADH of the Isoniazid Target (InhA) from Mycobacterium tuberculosis. Science 279, 98-101.

World Health Organisation (2011) Global Tuberculosis Control: WHO Report. ISBN 978 92 4 156438 0

Biochemical Society Summer Studentship Report 2012 Regulation of the mitochondrial uncoupling protein 3 expression and function in hypoxia

Student: Beatriz Soto Huelin Supervisor: Dr Susana Cadenas, Department of Molecular Biology, Universidad Autónoma de Madrid

Background

Uncoupling proteins (UCPs), such as UCP1, UCP2 and UCP3, are mitochondrial transporters present in the inner membrane of mitochondria. It is well known that UCP1 is involved in adaptive thermogenesis in mammals. However, the function of UCP2 and UCP3 seems to be related to protection against oxidative stress, although this is controversial. UCP3 expression is restricted to skeletal muscle, brown adipose tissue and heart. It has recently been reported that hypoxia increases UCP3 expression levels.

Aims of the project

Our aim was to confirm the increased expression levels of UCP3 in cells subjected to hypoxia and to study the effect of the oxidant hydrogen peroxide (H2O2) on UCP3 expression. An increased expression in this condition would suggest a possible antioxidant function of the protein. We also studied the possible implication of the transcription factors Nrf2 (NF-E2-related factor 2) and ATF-1 (active transcription factor-1) in the regulation of UCP3 expression. Finally, we analyzed the functional effects of UCP3 in these situations using mRNA interference techniques.

Description of the work carried out

During my stay in the lab, experiments were conducted in two different cell lines: HL-1 (from mouse heart) and C2C12 (from mouse skeletal muscle).

To try to reach our goals we used varied techniques, some of which were new for me:

• Immunoblotting of nuclear and mitochondrial fractions for Nrf2 and UCP3, respectively, of cell cultures treated with H2O2 at different times.

• Immunoblotting of nuclear and mitochondrial fractions for ATF-1 and UCP3, respectively, of cell cultures subjected to hypoxia (1% O2) or normoxia.

• Gene silencing (UCP3 and Nrf2) using RNA interference in HL-1 cell cultures, which later were treated with H2O2.

• Techniques to extract RNA from the silenced and H2O2-treated cell cultures. Extracted RNA was subjected to RT-PCR to obtain cDNA and to qPCR to analyze the amount of cDNA of different genes of interest, like UCP3, UCP2, Nrf2 or HO-1.

• Functional studies of silenced cultures using the XF24 Seahorse Analyzer (Seahorse Bioscience), to measure the oxygen consumption rate and the extracellular acidification rate. Different reagents (electron transport chain inhibitors, uncouplers, etc.) were added during the experiments to study the effect of H2O2 on the cellular bioenergetic profile.

The work carried out also required the maintenance of cell cultures and the separation of mitochondrial, nuclear and cytoplasmic fractions from the cells.

Assessment of the results and outcomes

Immunoblot analysis of cell cultures treated with H2O2 for different times showed an increase of both UCP3 and Nrf2 expression levels, reaching the maximum at 3 and 6 h. Similar results were observed in the case of immunoblots of cells subjected to hypoxia (1% O2).

RT-PCR and qPCR analysis were used to determine the degree of interference achieved through the use of siRNA specific for UCP3 or Nrf2. The results indicated that the amount of cDNA for these genes was very low compared to controls and, consequently, the interference procedure was very efficient.

The results of the functional bioenergetic analysis showed clear differences in cellular oxygen consumption between UCP3 silenced and control (scrambled) cells after the treatment with H2O2 both in basal respiration and after the addition of oligomycin (ATPase inhibitor).

All these results indicate that UCP3 might play a role in response to oxidative stress. The most relevant result obtained in this project is that the increase in UCP3 expression levels seems to be related to increases in Nrf2 expression so that Nrf2 could be one of the transcriptional factors that participate in the regulation of UCP3. This conclusion is also based on other results obtained in the laboratory using Nrf2 silenced samples, in which UCP3 expression did not increase in the presence of H2O2, and on the analysis of UCP3 gene sequences that seem to contain Nrf2 binding sites known as antioxidant response elements.

Future directions

Taking into account the results obtained in this project and other results from Dr. Cadenas’ lab, future studies include ex vivo experiments in hearts from mice lacking UCP3 (UCP3 knockout) and wild-type mice using the Langendorff perfusion system, in order to determine whether UCP3 participates in protection against damage produced during cardiac ischemia-reperfusion, a situation in which reactive oxygen species are generated. It is also important to study the signalling pathways involved in the cardioprotective effect of ischemic preconditioning and the possible involvement of UCP3.

Departures from the original proposal

Although the studies were planned in HL-1 cells only, we performed experiments in both HL-1 and differentiated C2C12 cells. All the techniques used were carried out in both cell lines except for functional studies and RNA interference, which were performed exclusively in HL-1. Moreover, our studies mainly focused on Nrf2 instead of ATF-1, as initially planned.

Value of the studentship to the student

I think this experience has been a good opportunity to learn from practice a lot of things that I have learnt during the lectures of my Degree in Biochemistry. In addition, it helped me to be self-confident with my work and I felt comfortable carrying out the work thanks to the research group. I think seminars and meetings have been useful to learn how to argue and explain the results of my experiments. And, of course, I have learnt a lot about oxidative stress, mitochondrial uncoupling proteins, the electron transport chain and more topics related with their research.

Value of the studentship to the lab

This studentship has helped to confirm results obtained previously in the laboratory and to complement studies of the research group performed in different conditions. It has also helped members of the laboratory to gain experience in training and in project design. Beatriz made rapid progress and perfectly integrated in the research team contributing to create a good research atmosphere in the laboratory.

Photo: the research team. From left to right: Andrea Anedda, Beatriz Soto, Susana Cadenas, Elia López-Bernardo and Cristina Vaca.

Validation of novel Rx3 targets during early eye morphogenesis

Biochemical society Summer studentship report 2012

Student: Ciarán Kennedy

Supervisor: Dr. Breandan Kennedy, School of Biomolecular and Biomedical Science, University College Dublin

Background:

Our eyes provide us with the means to explore and experience the world around us in a way that no other sense can come close to. Despite the importance of vision in our everyday lives, we are only now in the early stages of understanding the early stages of eye development. There are many different processes involved in eye formation and as such it is controlled by many different pathways. (Chow and Lang, 2001) One pathway of particular interest uses the Rx (retinal homeobox) transcription factors to control early eye development. The importance of the Rx gene to vertebrate eye development was first discovered over 15 years ago, and mutations in the human paralogue RAX have since been linked to eye malformations such as micropthalmia and anopthalmia. (Lequeux et al., 2008) Of the three zebrafish identified RAX paralogues, (Rx1, Rx2, Rx3) Rx3 has been shown to be the most critical in early eye development. Dr. Kennedy has identified an Rx3-/- mutant, known as chok or eym, which fails to form eyes during early development and also have an expanded forebrain. (Kennedy et al., 2004) Very few targets of Rx3 have been identified to date, so this eym mutant remains an important model to better understand this critical pathway.

Aims:

The aim of this project was to try and confirm if specific genes which were found to be down-regulated in the eym mutant were in fact downstream targets of the Rx3 gene. This was to be done using a combination of in-situ hybridisations and RT-PCR performed at various time points during eye morphogenesis on both wild-type and eym embryos.

Description of work carried out:

Wholemount in-situ hybridisation was performed at various points of development on both wild-type zebrafish embryos and on the embryos produced by mating two confirmed carriers of the eym mutation. As the mutation is recessive, 1 in 4 of the offspring of two carriers should display the eym phenotype. The genes looked at were six7, hmx1 and aldh1a3. Embryos displaying the eym phenotype were identified after the technique was completed by looking any differences in staining between the potential mutant and their siblings. Wild-type embryos tend to have a “shovel” shaped head where the developing eye fields can be discerned, the eym mutants however lack this characteristic shape. (fig 1)

Digoxigenin-labelled riboprobes for several of the genes being looked at were synthesised. This was done by first amplifying the target gene, or a specific area of the gene, by PCR, cloning this into a suitable vector and transforming this into competent bacterial cells. These were then used along with a digoxigenin-labelling kit to produce the riboprobe for the in-situ hybridisations.

cDNA was also synthesised for use in RT-PCR. This was done by extracting RNA from whole embryos and then converting this to cDNA. Both of these steps were done using commercially available kits.

Results:

The spatial expression of the six7 gene was shown successfully at 17, 13, 11 and 9 hours post fertilisation in phenotypically normal embryos. The spatial expression of hmx1 was successfully shown at 11 and 9 hours post

fertilisation in phenotypically normal embryos. Mutant expression for several of these time points was also shown.

Unfortunately the aldh1a3 probes that we had did not seem to produce specific staining and there was not enough time to synthesise new probes.

Fig 1: In-situ hybridisation results for six7 at 17 hours post fertilisation. Note the difference in head shape between the eym phenotype (eym) and the phenotypically normal sibling (sib) visible in the dorsal view. Also note the absence of staining in the eym visible best in the lateral view. This implies that at this time point, six7 is expressed at much lower levels in eym embryos than in the normal siblings, or not at all.

Future directions:

This project has a lot of scope for further research, to date the riboprobes for some of the potentially down-regulated genes in the eym mutant have not been successfully synthesised, so the spatial expression of these genes is not yet known in the mutants. As well as this, the expression of some of these genes in wild-types has also not been well documented.

Departures from the proposed project:

Due to the length of time it takes to do each in-situ hybridisation, and the fact that there were no stocks of probe or expression vector for some of the genes, it was decided that the focus should be on the genes for which there were probes already made.

Value of the studentship to the student:

For me, the studentship was a great experience and has been a great opportunity to develop research skills which will be of great use to me in the years to come. I have gained practical knowledge of many techniques and skills used in labs such as in-situ hybridisation, molecular cloning, riboprobe synthesis, working with DNA and RNA, PCR and zebrafish husbandry and breeding. I believe that these skills are likely to help me in completing my final year lab-based research dissertation. It was a pleasure to work with Dr Kennedy and with everyone else in the lab.

Value of the studentship to the lab:

It was a pleasure to be able to host Ciaran Kennedy in our lab under the Biochemical Society Summer studentship. Ciaran was central to our re-establishing the in situ hybridisation technique in the lab. The work that Ciaran completed will underpin additional work to determine gene expression in mutant and drug-treated zebrafish larvae and help complete PhD theses and publications. Ciaran was a pleasure to have in the group. He is very bright and hard working.

References:

Chow, R.L., Lang, R.A., 2001. Early eye development in vertebrates. Annu. Rev. Cell Dev. Biol. 17, 255–296.

Lequeux, L., Rio, M., Vigouroux, A., Titeux, M., Etchevers, H., Malecaze, F., Chassaing, N. and Calvas, P. 2008. Confirmation of RAX gene involvement in human anophthalmia. Clinical Genetics, 74: 392–395.

Kennedy BN, Stearns GW, Smyth VA, Ramamurthy V, van Eeden F, Ankoudinova I, Raible D, Hurley JB, Brockerhoff SE (2004) Zebrafish rx3 and mab21l2 are required during eye morphogenesis. Dev Biol 270:336-349.

Conor Hennessy Biochemistry society summer studentship 2012

The effect of plakoglobin knockdown on the expression and localization of connexin 43 Aims of the project: Arrhythmogenic right ventricular cardiomyopathy is characterised by progressive loss of cardiomyocytes of the right ventricle, which are replaced by fibro-fatty tissue. The main clinical symptoms are arrhythmia, heart failure and sudden death. It has a prevalence of 1:1000 to 1:5000 and is a leading cause of sudden cardiac death in people under 35. Two mutations in plakoglobin have been associated with ARVC and as such the aim of this project was to investigate what effect the knockdown of the plakoglobin gene in zebrafish had on both cardiac morphology and function and localisation of connexin 43, a protein of gap junctions which are important in propagation of the electrical signal in the heart. Because the localization of connexin 43 had already been completed by the time the project started, the focus was changed to refining of a morphological scoring system and examination of beta-catenin localization in the heart. Beta catenin is an armadillo protein which can substitute for plakoglobin in adherens junctions. Work carried out: To achieve the above aims the following work was carried out in the process of the studentship. Fish were paired for spawning and embryos collected 2-3 times a week for 4 weeks. These embryos were injected with Plakoglobin morpholino (5 ng) at the once cell stage, control embryos received a 5-base-pair mismatch control morpholino (~30 embryos per treatment each day). Phenol red-injected and un-injected controls were also collected and observed. Embryos were monitored and scored for defects/changes in cardiac morphology and function at 24, 48 and 72 hours post fertilisation. When they reached 72hpf they were dehydrated and stored. They were then rehydrated and embedded in cryo-embedding medium for cryosectioning. They were then cryosectioned and placed on glass slides. Sections were incubated with anti beta-catenin polyclonal antibody (Sigma) followed by Alexa 546 secondary antibody. Assessment of results and the outcomes of the studentship: Effects of plakoglobin knockdown on zebrafish cardiac morphology and function: The scoring scheme of Brannen et al., 2010 was modified for this study, and zebrafish were scored, at 24, 48 and 72 hours post fertilization for changes in morphology. A score of zero corresponds to normal morphology, with defects in head, tail, yolk, and heart getting scores from 1-3 in order of severity. Heart defects noted were, pericardial oedema, heart structure, and blood pooling. Figure 1 represents the results of scoring at 72 hpf of four groups (morpholino, morpholino control, phenol red-injected and uninjected control), representing injections of 30 embryos per treatment per day and four separate experimental days. As is evident in the graph the most deformed and affected embryos were in the morpholino groups, with almost no effect seen in any of the control groups. There was considerable variation in the defects due to the inexperience of the experimenter, in addition to variation in effects of the morpholino. However, plakoglobin knockdown had an adverse effect on cardiac morphology and function.

Box plot of score in each treatment group

Treatment group

Scor

e

UIC MoMoCo

Ph Red

0

5

10

15

Figure 1: Box plot of scores (median, interquartile range, max and min) seen across the different treatment groups. The graph shows that the higher scores, indicating the most damage, was in the morpholino group, while there was little if any effect seen in any of the control groups. Beta-catenin localisation in zebrafish heart sections: Embryos were cryosectioned and heart sections (10 μm) were with anti-β-catenin antibody and fluorescent secondary antibody (ALEXA 546), sections were examined under a fluorescent microscope to determine the localisation of beta catenin in the embryos. Beta catenin localized to the membrane and the cytosol as seen in Figure 2. While the procedure was successful, no differences between control and morphant embryos were seen. Given more time slides could be examined by confocal microscopy to achieve better visualization of beta-catenin localization.

Figure 2:Beta-catenin in membrane (honeycomb pattern) and cytosol of zebrafish heart sections at

72 hpf.

Future directions in which the project can be taken: Following my project there are a number of things that could be researched further. Firstly examining the sections using a confocal microscope would be desirable as it would provide a much more decisive and clear image of beta-catenin localization. It would be useful to examine the effects on sodium channel expression and localization, to further investigate arrhythmogenic effects. Any departures from the original proposal: The initial project proposed that I examine the effect of plakoglobin knockdown on connexin 43 localisation and expression, however upon commencing my studentship this task had already been completed by the resident post doctoral researcher Dr Elke Rink. Thus my project supervisor decided that I would examine the effect of knockdown on beta-catenin instead.

A comparison of the activity of Dimethylarginine dimethylaminohydrolase (DDAH) isoforms and evaluation of the effects of novel substrate analogues on their active sites

Corinne Morfill, Biochemical Society Summer Vacation Studentship 2012

School of Life Sciences, University of Westminster. Supervisors: Dr Caroline Smith and Monika Dowejko PhD

Introduction Dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) are enzymes that breakdown methylarginines. More specifically for this project; methyl-L-arginine (L-NNMA) and asymmetric dimethylarginine (ADMA). Nitric Oxide synthases (NOSs) catalyse the production of Nitric Oxide (NO) which is an important cellular signalling molecule involved in the regulation of ‘normal’ endothelial function. ADMA and L-NMMA are known inhibitors of NOS and ADMA is an emerging biomarker to predict the risk of heart disease. Overexpression of DDAH reduces ADMA concentrations and increases angiogenesis, the formation of new blood vessels. However, little is known about the differences in activity and specificity between the two DDAH isoforms. The studentship is designed to determine the differences in the activity and substrate specificities of the DDAH isoforms using recombinant expression techniques. Dr Caroline Smith and her team have developed ADMA based compounds to be tested on the DDAH isoforms. Aims The aim of the studentship is to gain an understanding of the specific activities of the two isoforms for the potential to design novel inhibitors. The two main objectives of this project are:

1: To compare the activities of the two DDAH isoforms and characterise their substrate specificity for both ADMA and L-NMMA. 2: To test novel substrate based inhibitors in the activity assay and to determine their effects on cultured endothelial cells. Work carried out Human DDAH 1 and 2 (QIAgenes construct, Qiagen) were expressed in E.coli and induced with IPTG for four hours, as this was determined as the optimum expression point. The proteins were purified using His-IDA (Machery Nagel). DDAH 1 and 2 metabolise ADMA to form citrulline in vivo, this was then measured using colorometic citrulline assay based on Knipp which was optimised in this project. The assays contained ADMA or L-NMMA as substrates and SDMA was used as a blank in sodium phosphate buffer. All experiments were carried out in duplicate, repeated on many occasions, to produce conclusive results. The data was analysed using two-way ANOVA. Raw164.2 cells were stimulated with and without LPS (1ug/ml) in Dulbeccos modified medium and the ADMA based compounds were added at 0.5-500 µM to determine if there were non specific effects on nitrite production from these cells (4 replicates per plate n=3). Nitrite was measured using Greiss assay2 and sodium nitrite in DMEM medium was used to generate a standard curve. Cell viability in the presence of the ADMA based compounds( 0.5-500 µM) was determined using MTT assay briefly MTT 0.1mg/ml in DMEM was added to cells for 30 minutes, medium was removed and the cells were resuspended in DMSO 100ul, colour changes were read at 590nm.

Results Active recombinant protein was successful expressed and purified. The method described by Knipp (2000) was adapted to be used with the recombinant DDAH 1 and DDAH2. Of the five novel substrate based inhibitors, the data produced suggests that the novel cyclic NG, NG-(N-methylpiperazinyl) arginine analogue, inhibits DDAH 1 without eliciting effects on nitrite production and cell viability. The other four compounds either showed high toxicity to the cells or low ADMA inhibition. Through the results gained, it is possible that this particular compound; NG, NG-(N-methylpiperazinyl) arginine analogue, can form the basis of a potential drug therapeutic, therefore, we have submitted an abstract to the British Pharmacological Society, in the hope to present our findings at the 2012 winter conference.

Future directions in which the project can be taken

We have identified that the NG, NG-(N-methylpiperazinyl) arginine analogue specifically interacts with DDAH1 and is an interesting lead chemical to design further DDAH interacting compounds. Use a bioinformatics approach to look for potential sites of modification in the DDAH1 and 2, data generated qualified using the recombinant proteins and assay developed in this project. Further research may also be carried out to investigate the inhibition and biological effects of dimethyl arginine dimethylaminohydrolase (DDAH) in diseases where nitric oxide (NO) production is dysregulated.

Value of the studentship to student

Through this studentship I gained a valuable insight into the reality of lab research. I had the opportunity to perfect techniques I have already come across through my undergraduate studies, as well as new techniques. I gained a further understanding of Nitric Oxide and the importance of this molecule throughout the body and the diseases that occur though the inhibition of this molecule. Through this fascination of NO and DDAH, I have a strong desire to further my knowledge by embarking onto a PhD program within this area of research. I learned how to trouble shoot when problems arose … especially through the protein purification stage of the project, as well as taking into consideration time limitations, designing experiments and analysing data. This knowledge of a variety of different techniques, both practical and theory will assist me in areas of scientific research I will be sure to endeavour throughout my career, but as a student, these techniques will unequivocally aid me in my final year project.

Value of studentship to lab

This project was generated within the on-going research being carried out by Dr Caroline Smith and her team. There were many ways in which the project moved the research along, specifically; through the purification of the DDAH1 and DDAH2, we were able to compare the activities of the two DDAH isoforms and characterise their substrate specificity and testing the novel substrate based inhibitors to determine their effects on cultured RAW264.7 cells. This data was sent to the medicinal chemist, Dr Sharon Rossiter, to further design compounds based on our results.

Report on Biochemical Society Funded 6 week Summer Vacation Studentship ‘Do subtle changes to the ribosome alter translational recoding driven by 2A peptides?’

Studentsip carried out in Dr. Jeremy Brown’s laboratory at Newcastle University by Dina Bastola.

Background: Protein synthesis is carried out by ribosomes, which faithfully decode the information in mRNA into protein. However, on occasion, ribosomes are prompted to deviate from the genetic code, in events termed programmed translational recoding. Examples of this are stop codon read-through, used in a variety of situations to produce proteins extended beyond the first stop codon in the RNA, and frame-shifting, where the ribosome changes reading frame on the RNA. Events such as these are most often encountered in viruses, which use them to maximise the coding capacity of their compact genomes and to provide regulation of the level of expression of certain proteins.

The focus of my summer project was to be the 2A peptide of Foot and Mouth Disease Virus, which drives another recoding event. In this, termed Stop-Carry On recoding, ribosomes that have translated to the end of the peptide terminate translation on a sense codon and then reinitiate protein synthesis. This leads to the generation of 2 proteins from one open reading frame. The reaction is driven entirely by the peptide, which presumably interacts in specific ways with the ribosomal peptidyl transferase centre and exit tunnel to do this. While significant data has been accrued on importance of specific amino acids within 2A, requirements in the ribosome for the 2A reaction have not been probed.

Aims: The initial aims of the project were to examine yeast strains in which either the ribosomal RNA or a specific protein, Rpl17, that lines the exit tunnel were altered. Ribosomal RNA alterations were to be generated by removing modifications that normally occur on it (methylation and pseudouridylation), whereas the ribosomal protein was to be studied by random mutagenesis. In each case the aim was to look to see if the changes affected the 2A reaction using a reporter system developed previously in the laboratory.

Work on ribosomal RNA was not carried out, as the yeast strains available for this did not have the correct set of markers for the experiments. However, progress was made towards analysing Rpl17. In particular, several plasmids were generated into which mutations could be inserted into RPL17. This entailed several PCR steps to amplify portions of RPL17 and its promoter and terminator and then clone them into a yeast expression vector. Initial attempts at this failed, but by ‘trouble shooting’ the protocol and trying the PCR under several different conditions using alternative DNA polymerases, amplification of the desired products was achieved. These were subsequently ligated into the yeast expression vector.

As the ribosomal RNA work was not carried out, I became involved in a project examining another translational recoding event, which the laboratory started a new collaboration on during the summer. This is a coupled termination-reinitiation event that takes place in human respiratory syncitial virus (RSV). In this, translation of the first open reading frame on

one of the viruses RNAs is necessary for translation of the second, and this relies on specific sequences in the RNA around the stop codon of the first open reading frame. The aim of the experiments was to confirm whether or not the reinitiation reaction could be recontituted in yeast, which would then allow genetic analysis. I was provided with plasmids containing the wild type sequence from RSV driving reinitiation on a chloramphenicl acetyltransferase (CAT) gene, the expression of which was monitored using a commercial sandwich elisa assay. Control plasmids contained CAT alone (a positive control for CAT expression) or mutant versions of the RSV sequence (negative control) which should not have yielded expression of CAT. Yeast strains containing these plasmids were grown and the assay for CAT expression carried out on lysates from them. The results were positive, and I could show, through repeated experiments, that the RSV sequence that drives coupled termination-reinitiation in human cells also works in yeast. This has opened up a new area for research in the laboratory, and is the most positive thing to have come out of my vacation studentship.

This research project gave me a great opportunity to gain valuable experience in a laboratory environment. I have learnt various skills and methods, which I wouldn’t have had the chance to in my degree work. I have also gained skills on time management. This project has helped me be independent and apply my knowledge from lectures into the laboratory. I have really enjoyed my time doing the summer project and would definitely recommend it to anyone who’s looking to gain experience and skills.

Biochemical Society Vacational Studentship Report 2012

Using Fluorescent Probes to Investigate the Link Between TDP43 Misfolding and Amyotrophic Lateral Sclerosis In Vivo Student: Georgina Kontou, University College London (UCL) Supervisor: Dr. Leila Luheshi, Department of Chemistry, University of Cambridge INTRODUCTION Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder that results in the deterioration of the motor neurons in the brain and the spinal cord. Studies have shown that toxic inclusions of TAR DNA binding Protein-43 (TDP43) have been found in the cytoplasm of neurons in patients suffering from ALS. TDP43 is a 43kDa nuclear protein that is expressed by the TARDBP gene on chromosome 1. Its main function is to regulate the transcription of DNA into mRNA. TDP43 is involved in alternative splicing and stability of the mRNA (1). Thus, TDP43 has the ability to interact with both DNA and RNA (2). In ALS patients, TDP43 inclusions are present both in the nucleus and the cytoplasm of the neurons. The protein is often phosphorylated, ubiquitinated, and truncated, usually to a 25kDa C-terminal fragment (3). Such modifications can severely affect the protein’s function, resulting in gain or loss of function mutations. Consequently, it is important to investigate whether the structure of the protein is affected by mutations that can possibly alter the 3D arrangement of the protein in vivo. The investigation was carried out by considering the following substitutions: (a) an alanine to threonine substitution at residue 315 (A315T)(1) and (b) a methionine to valine substitution at residue 337 (M337V)(4). These mutations employed are examples of errors commonly found in patients suffering from familial ALS. They are both found on the C terminal domain of TDP43. AIMS The objective of this research was to investigate whether mutations in the C-terminal domain of TDP43 in ALS affected patients influence the aggregation of the TDP43 inside cells. DNA constructs possessing these mutations were generated for the full-length protein, and a 14kDa C-terminal domain of TDP43. The constructs were modified to contain a tetracysteine tag (CCXXCC) in the C-terminus, which facilitates site-specific fluorescent protein labeling. The tetracysteine sequences used were composed of 6 (TC6) and 12 (TC12) amino acids. To investigate the localisation and distribution of the mutated C-terminal fragment inside cells a specific dye known as FlAsH was added. FlAsH is a bi-arsenical analogue of fluorescein that is widely used to monitor protein

folding and protein aggregation (5). The FlAsH dye fluoresces when it is bound to four thiol groups present in the tetracysteine motif (fig 1), which can be detected using fluorescence microscopy.

MATERIAL AND METHODS Site directed mutagenesis pcDNA3.1 plasmids containing the mutated C-terminal fragment (CTF) and mutated full-length protein with TC6 (CCPGCC) and TC12 (FLNCCPGCCMEP) tags have been constructed, using side directed mutagenesis (fig 2).

The oligonucleotide primers possessing the desired mutations were added to the TC6 and TC12 pcDNA3.1 templates and the whole plasmid was amplified in a thermocycling reaction using pfu polymerase. Plasmids that do not contain the DNA insert are not methylated and therefore are digested by Dpn1. Bacteria Transformation The plasmids were used to transform xl10 gold ultracompetent E.coli cells. The bacteria were plated out in 2XYT growth media plates containing the antibiotic ampicillin. The plasmids containing the DNA constructs were selected because of their resistance to ampicillin. Plasmid Extraction The plasmids carrying the TDP43 constructs were extracted from E.coli using a miniprep commercial kit. In order to confirm the presence of the TDP43 insert in the vector, the plasmids were digested with EcoR1 and Xba1 restriction endonucleases. The DNA was then run on an agarose gel along with DNA markers of known molecular weight (fig3).

Fig 1) The above diagram illustrates the detection approach employed, using the FlAsH dye. Adapted from Roberti, M. J., Bertoncini, C. W.,

Tetracysteine motif

Mutation

Size

Protein TDP43

43kDa

A315T

TC6 TC12

M337V

TC6 TC12

14kDa

A315T

TC6 TC12

M337V

TC6 TC12

Fig 2) DNA constructs used in the experiments.

Biochemical Society Vacational Studentship Report 2012 Additionally, samples were sent for DNA sequencing and the results were aligned to the original nucleodite sequence of the protein using Basic Local Alignment Search Tool (BLAST), in order to confirm that the mutation has been introduced to the sequence. When the mutations were successfully introduced, a larger amount of the plasmids needed to be produced. The pcDNA3.1 vectors containing the insert were amplified by transforming DH5α competent E.coli. The plasmids were then extracted using a midiprep commercial kit in order to obtain a more concentrated solution in order to have enough DNA to transfect the cells. Tissue Culture and Transfection Human embryonic kidney cells (HEK293T) were cultured in T75 culture flasks. HEK293T cells were transfected with the mutated CTF of TDP43. Additionally, cells were transfected with Green Fluorescent Protein (GFP), with an α-synuclein construct containing TC12 motif (fig 4), and the transfection reagent on its own. HEK293T lysis The cells were lysed with RIPA and NP40 lysis buffers and were run on an SDS-PAGE. A western immunoblot was performed to detect β-actin at 488nm. β-actin is an abundant protein in the cell, therefore, high levels of β-actin indicated that the cells were healthy (fig 5). In-cell FlAsH detection and Fluorescence Microscopy HEK293T cells were transfected with FlAsH dye that binds to the tetracysteine motif. α-synuclein has been previously successfully detected using FlAsH experiments (6). HEK 293T cells were transfected with wild type and mutated CTFs at two different concentrations of FlAsH, 1μM and 2.5μM respectively. HEK293T cells that were transfected with the CTF of TDP43 were observed under a fluorescence microscope. The images were processed using ImageJ processing software. RESULTS AND DISCUSSION The A315T and M337V mutations were successfully

introduced in the sequence of the TDP43 C-terminal fragment, using side directed mutagenesis. The pcDNA3.1 vectors contained the mutated TDP43 constructs (fig 3). These TDP43 constructs were expressed and

detected inside HEK293T cells using the FlAsH labeling approach. Before transfecting the HEK293T cells with the TDP43 constructs, they were transfected with GFP to evaluate the effectiveness of the transfection process and with α-synuclein to ensure that the FlAsH dye works (fig 4). In previous experiments, an α-synuclein construct containing the TC12 motif fluoresced very well when bound to the FlAsH dye in vivo (6). HEK293T were also transfected with the transfection reagent only to assess its toxicity to the cell. The western immunoblot was performed to ensure that the cells were healthy, eliminating possible factors that might influence the accuracy of the results (fig 5).

The key objective was to determine whether the wild type C-terminal domain forms aggregates within cells and whether the mutant CTFs that have been expressed behave similarly, or exhibit differences in their behavior. The mutated CTFs were expected to localize in the cytoplasm. Visible inclusions were only observed in the cells that have been transfected with CTFs possessing the M337V mutation and the TC6 tag (fig 6).

Fig 6) A. HEK293T cells transfected with M337V-TC6 CTF. The arrow points at the nucleus B. In-cell detection of M337V-TC6 CTF using FlAsH labeling C. HEK293T cells expressing M337V-TC6 CTF construct. The arrow points at the cytoplasm.

Fig 3) DNA gel of Xba1 and EcoR1 digested plasmid containing TC6 and TC12 CTF of the TDP43 constructs. pcDNA vector is 5427 base pairs and CTFs are 273base

Fig 5) Western immunoblot for the detection of β-actin on NP40 and RIPA lysed HEK293T cells

Biochemical Society Vacational Studentship Report 2012 In contrast, snapshots of other HEK293T cells, transfected with the TDP43 constructs, showed no visible inclusions (fig7). The identity of the inclusions is unknown. They can possibly be aggregations of the mutated CTF. Further experiments are required to investigate whether these are actual aggregations and not just vesicles that have taken up the dye. Also, it is not known whether the FlAsH dye stains other cellular proteins. In order to confirm the identity of the inclusions, they must be extracted from the cytoplasm and assess their solubility in detergent. Additonally, TDP43 constructs containing an HA tag in the N-terminus will be detected using immunohistochemistry. In order for the results to be consistent, the constructs with the HA tag should co-localise with the constructs detected using FlAsH staining. Unfortunately, a western immunoblot for detecting TDP43 in lysed cells was not performed, due to time constrains. Additionally, the cells have not been transfected with the full-length protein and mutated forms. Deviations from Original Plan and Future Decisions: The original plan involved the use of the SHYSY5Y neuronal line. However, human embryonic kidney (HEK293T) cells were preferentially used as they have high transformation efficiency and are easier to work with. Once the procedure is optimized, neuroblastoma cells will be used instead, to study TDP43 aggregations. Additionally, experimental use of FlAsH has not yet been optimized. In the future, FlAsH labeling might be able to provide a quantitative measure of the TDP43 aggregations inside the cell. This is possible by evaluating whether there is a change in fluorescence intensity when FlAsH dye binds to full length TDP43, or to the 14kDa C-terminal fragment, using fluorescence microscopy. REFERENCES 1. Guo W, Chen Y, Zhou X, Kar A, Ray P, Chen X, et al. An ALS-

associated mutation affecting TDP-43 enhances protein aggregation, fibril formation and neurotoxicity. Nat. Struct. Mol. Biol. 2011 Jul.;18(7):822–30.

2. Chen-Plotkin AS, Lee VM-Y, Trojanowski JQ. TAR DNA-binding protein 43 in neurodegenerative disease. Nat Rev Neurol. 2010 Apr.;6(4):211–20.

3. Gregory JM, Barros TP, Meehan S, Dobson CM, Luheshi LM. The aggregation and neurotoxicity of TDP-43 and its ALS-associated 25 kDa fragment are differentially affected by

molecular chaperones in Drosophila. PLoS ONE. 2012;7(2):e31899.

4. Tamaoka A, Arai M, Itokawa M, Arai T, Hasegawa M, Tsuchiya K, et al. TDP-43 M337V mutation in familial amyotrophic lateral sclerosis in Japan. Intern. Med. 2010;49(4):331–4.

5. Scheck RA, Schepartz A. Surveying protein structure and function using bis-arsenical small molecules. Acc. Chem. Res. 2011 Sep. 20;44(9):654–65.

6. Roberti MJ, Bertoncini CW, Klement R, Jares-Erijman EA, Jovin TM. Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein. Nat Meth. Nature Publishing Group; 2007 Mar. 11;4(4):345–51.

The Value of the Studentship The 8-weeks placement at the University of Cambridge has been an amazing experience for me and I would really like to thank Biochemical Society for giving me this opportunity. I was able to apply many of the techniques that are widely used in molecular biology labs. More importantly, I have been introduced in a new way of thinking that cannot be developed during the course of an undergraduate degree. I have learned how to evaluate the effectiveness of my methods and seek new ways of improving them in order to obtain better results. After this experience I am definitely sure that I would like to pursue graduate studies and eventually obtain a PhD degree, and involve myself in research. Acknowledgments I would like to thank Dr. Leila Luheshi, Dr. Teresa Pereira de Barros and Janice Ng for their guidance and support throughout the project. I would also like to thank everybody in 290 and UB7 for their help and constructive conversations.

Fig 7) HEK293T cells transfected with A315T-TC6 CTF.

Localisation of the flagellar glycosylation machinery in the human pathogen Aeromonas caviae

Student: Hannah Southam

(3rd year Microbiology Student at the University of Sheffield) Supervisor: Jennifer Parker

(Post-doctoral Research Associate at the University of Sheffield) Introduction and Aims of Project

The opportunistic pathogen Aeromonas caviae has been implicated in a variety of intestinal and extra intestinal human diseases (Parker and Shaw, 2011). Swimming motility, a major virulence factor in A. caviae, is dependant upon expression of a single polar flagellum, in which the flagellin subunits (FlaA/FlaB) are glycosylated with pseudaminic acid (Pse) residues (Parker et al., 2012). Motility Associated Factors (Maf proteins) are considered the candidate glycosyltransferases responsible for transferring Pse onto Ser/Thr residues in the central domain of the flagellin. Maf1, the single maf gene homologue in A. caviae, has been identified as a putative glycosyltransferase and a maf1 disruption mutant is non-motile, lacks polar flagellum and produces non-glycosylated FlaA (Parker et al., 2012). It is currently unknown whether Maf1 localises to the cell pole in order to transfer Pse acid residues onto flagellins prior to their export via the dedicated type III secretion system. To investigate whether Maf1 has a specific subcellular localisation, the aim of this project was to express a gfp-tagged version of the maf1 gene using the produced Maf1-GFP protein as a probe to follow its subcellular localisation in vivo inside live A. caviae cells using fluorescent microscopy. Using a distinct lateral flagellin system, A. caviae also display swarming motility across solid surfaces. Encouraging A. caviae to swarm across laboratory media is difficult to achieve, and therefore as a side project; the aim was to develop a recipe for solid media that would enable A. caviae to display swarming phenotype. Materials and Methods Generation of gene constructs. DNA fragments for gene constructs were first generated by PCR with genomic DNA as the template for maf1. PCR products were checked for the correct size (GFP; ~700bp, maf1; ~2kb, FliF; 1.4kb) on agarose gel and purified. The individual fragments were then used as templates to generate the gene fusions by Overlapping Extension PCR. The PCR product was checked for correct size (GFP_Maf1, Maf1_GFP; ~2.7kb, FliF_GFP; ~2.4kb) on agarose gel and purified. Cloning of constructs into A. caviae. The gene constructs generated by overlapping extension PCR were ligated into pGem cloning vector using standard T/A cloning in E. coli DH5α cells. Those vectors containing the correct inserts (as checked by analytical restriction digest) were then used as templates in PCR to generate gene constructs containing appropriate restriction sites for ligation into pSRK (multi-host cloning vector). Appropriate restriction enzymes were used ligate the constructs into pre-cut pSRK Kmr. After transformation into E. coli DH5α cells and antibiotic selection, correct clones (pSRK Kmr::FliF_GFP) were isolated and transferred into E. coli S17-1 ʎpir cells, and then subsequently transferred in to A. caviae Sch3N (wild type) cells by conjugation. Fluorescence Microscopy. Slides for microscopy were prepared using the protocol described in Green et al., 2009. Sch3N cells containing pSRK::FliF_GFP were grown to A600 = 0.8 in LB broth supplemented with appropriate selection antibiotics (50μg/mL) and 1mM IPTG. An agarose mount was prepared by pipetting 90μl of 1% agarose containing 30% LB onto a glass slide and then covering with a cover slip, using the protocol developed by Srivastava et al., 2006. Once the mount had set, the cover slip was removed and 3μl of the culture was pipetted onto the agarose then covered with a fresh cover slip.

Figure 1: In vivo localisation of FliF_GFP in live cells Figure 1a: Confocal (i) and fluorescence microscopy using GFP filter (ii) of freshly prepared cells. Figure 1b: Confocal (i) and fluorescence microscopy using GFP filter (ii) of cell preparation that had been stored for an additional 4 days at 4◦C.

Prepared slides were then stored in the fridge (4◦C). The slides were mounted onto a microscope stage heated to 37◦C, and then visualised by confocal fluorescence microscopy using a GFP filter set. Assessment of swarming motility. 2μL of liquid broth was transferred onto the centre of plates containing the swarming agar (0.8% (w/v) Difco Nutrient Broth, 0.6% (w/v) Eiken Agar, 0.5% (w/v) Nacl – made up in tap water). The plates were incubated face up at room temperature. Swarming was assessed by migration of the bacterial population across the agar surface from the central spot of inoculation towards the outer edge of the plate.

Figure 1: In vivo localisation of FliF_GFP in live cells

Results Generation of PSRK::FliF_GFP that can be visualised by fluorescence microscopy when expressed in Sch3N cells. FliF_GFP was constructed as a control protein as FliF is the earliest flagellar structural component and therefore localises to the cell pole (Green et al, 2009). After transfer of the (pSRK::FliF_GFP) construct into A. caviae, via conjugation from E. coli S17-1 ʎpir, the correct sequence of FliF_GFP in pSRK was confirmed by sequencing analysis. Confocal and fluorescence microscopy of Sch3N cells expressing FliF_GFP was performed on fresh cell preparations (Fig 1a) and on cell preparations that had been stored at 4◦C for four days (Fig 1b). Figure 1a(ii) shows that there is no detectible fluorescence in freshly prepared cells. Figure 1b(ii) shows that stored cells showed fluorescence under the GFP filter, but that this fluorescence is dispersed throughout the cytoplasm of the cell. The reasons for the above observations are discussed later, however they demonstrate that the GFP is functional and detectible, and that further optimization is required. Expression of Maf1_GFP and GFP_Maf1 gene constructs in pGem. To investigate subcellular localisation of Maf1 in A. caviae, both N-terminal and C-terminal GFP fusions were generated so that at least one would be functional in vivo. Gene fragments for GFP_Maf1 and Maf1_GFP were generated by overlapping extension PCR and ligated into pGem, however we had no further success in cloning the construct into pSRK for subsequent expression in A. caviae. Demonstration of swarming motility in A. caviae Sch3N cells. As Figure 2 demonstrates, the recipe developed in this project enables A. caviae Sch3N to display the swarming phenotype.

Discussion The aim of this project was to express a GFP-tagged version of the Maf1 gene in A. caviae cells, enabling it to function as a probe to follow its subcellular location in vivo, to investigate whether Maf1 localises to the cell pole alongside its cognate substrates (the flagellins). Unfortunately, due to time constraints and technical difficulties, it was not possible to clone the GFP_Maf1 and Maf1_GFP into pSRK and subsequently express them in A. caviae maf1-. Despite the unsuccessful cloning of maf1 gfp gene

Figure 2: Swarming motility in A. caviae Sch3N cells Swarming was assessed by migration of the bacterial population across the agar surface from the central spot of inoculation (3μl of liquid Sch3N cell culture) towards the outer edge of the plate.

into A. caviae, we have successfully constructed and expressed the control (FliF_GFP) into Sch3N cells and examined the subcellular localisation of FliF_GFP using confocal fluorescence microscopy. Results showed that cell preparations incubated at 4◦C for four days allowed visualisation of the GFP signal at a higher level that that of fresh preparations (Fig 1). The four day old cell preparation showed fluorescence dispersed throughout the cytoplasm. This could possibly be due to over expression of FliF_GFP, so that the excess of protein no longer solely localised to the cell pole. Alternatively it is possible that the GFP tag affected localisation of FliF, however, a previous study by Green et al., 2009 showed that a C-terminal GFP tag in Vibrio cholerae did not affect localisation. It was also not possible to detect fluorescence in the freshly prepared cells despite the addition of IPTG during growth of the cells. The presence of fluorescence in the four day old preparation suggests that the GFP tagged protein does fluoresce at detectable levels but requires a period of incubation. Despite further optimization required, to our knowledge this is the first time fluorescence microscopy has been performed in live A. caviae cells.

Figure 2: Swarming motility in A. caviae Sch3N cells

Value of Studentship to Student and Acknowledgments Receiving funding from the Biochemical Society has enabled me to embark on a rewarding, inspiring and thoroughly enjoyable research project, which has provided me with the laboratory experience and determination to apply for PhDs and pursue a carrier in scientific research. I am now well practiced in a range of microbiological and molecular biology techniques such as: growth of bacterial strains, PCR reactions, restriction digests, running agarose gels, ligations and transformations. I also had the opportunity to use techniques which I would not have had the chance to use as part of my undergraduate studies, such as: swimming/swarming motility assays, Overlapping Extension PCR, conjugations and fluorescence confocal microscopy. I would like to thank my supervisor Jennifer Parker for all her help and support throughout the project, the rest of the research group for their help in the laboratory and Colin Gray for his assistance with microscopy. Thanks also to the organisers of the University of Sheffield Medical School SURE scheme for their hard work in giving myself and fellow students the opportunity to take part. Finally I would like to thank the Biochemical Society for providing the funding which made this invaluable experience possible. Value to Supervisor, Research Group and Future Directions The progress that I have made during my summer placement, such as developing the swarming assay protocol, and generating and expressing the control (pSRK Kmr::FliF_GFP) into A. caviae cells, will be continued to be used by post-graduate students and post-doctoral researchers within the research group. Using the methodology developed in this project, future work in the research group will further optimize the protocol for preparing cells for microscopy and use alternative cloning methods to express a Maf1-GFP fusion in A. caviae maf1- cells, to continue the project and move towards confirming whether or not Maf1 has a subcellular localisation.

Deviations from Original Proposal Due to time constraints and cloning difficulties, it was not possible to clone the GFP_Maf1 and Maf1_GFP into pSRK and subsequently express them in A. caviae maf1-. As an additional project I helped to develop a protocol for swarming, which provided me with the additional opportunity to learn more practical techniques. References

• Green, J.C.D., Kahramanoglou, C., Rahman, A., Pender, A.M.C., Charbonnel, N., and Fraser, G.M. (2009). Recruitment of the Earliest Component of the Bacterial Flagellum to the Old Cell Division Pole by a Membrane-Associated Signal Recognition Particle Family GTP-Binding Protein. Journal of Molecular Biology 391, 679-690.

• Parker, J.L., Day-Williams, M.J., Tomas, J.M., Stafford, G.P. and Shaw, J.G. (2012) Identification of a putative glycosyltransferase responsible for the transfer of pseudominic acid onto the polar flagellin of Aeromonas caviae Sch3N. MicrobiologyOpen 1(2), 149-160.

• Parker, J.L., and Shaw, J.G. (2011). Aeromonas spp. clinical microbiology and disease. Journal of Infection 62, 109-118.

• Srivastava, P., Fekete, R.A., and Chattoraj, D.K. (2006). Segregation of the Replication Terminus of the Two Vibrio cholerae Chromosomes. Journal of Bacteriology 188, 1060-1070.

Probing Conformational Flexibility in H2B C-terminal Helix Using FRET

Jonathan Faherty,

Centre for Chromosome Biology, Department of Biochemistry, National University of Ireland Galway, Ireland.

Introduction

Objectives

We proposed to assemble histone mutants with altered proline residues and develop an assay using FRET-capable fluorescent dyes attached to the αC helix and nucleosomal DNA so that we can monitor the αC helix conformation to look for evidence of proline isomerisation. We also proposed to express and purify candidate proline cis-trans isomerases to test their effect.

Optimisation of Histone Mutant Expression

Histone mutants H2B P103A, H2B P103A K116C and H3 C96A C110A were previously grown in large scale 2YT media cultures and induced with 0.4mM IPTG for four hours. While these mutants expressed well following this protocol the histone H2B K116C mutant showed low expression in 2YT media. A revised method including more aeration and a more continuous log phase was unsuccessful so test expressions of H2B K116C were carried out in 9 different media to optimise its expression (See Figure 2). LBM broth Lennox media was found to give the best expression for the mutant.

Figure 2: Test expressions of H2B K116C in L-Broth, 2YT-Broth, LB-Broth Lennox, LBM-Broth Lennox, LB-Broth Miller, LBM-Broth Miller, Terrific Broth, SOC-Broth and SOB-Broth at T0, T4 and T22.

Histone Mutant Preparative Expression and Purification

The histone mutants were extracted from inclusion bodies and purified by cation exchange chromatography using an AKTA express chromatography system. A revised protocol for inclusion body preparation was implemented for histone H2B K116C which helped increase the protein yield by minimizing protein loss during sonication.

Figure 1: Proline 103 isomerisation could lead to a reorientation of the αC helix. A fluorophore dye attatched to the lysine 116 residue after it has been mutated to cysteine can be used indicate if this reorientation takes place.

The dynamic properties of nucleosomes are crucial to enable chromatin function. Histone H2B contains an additional αC helix which is orientated in the opposite direction to the preceding histone fold α3 helix by a beta turn about a proline residue. Isomerisation of the proline could lead to a reorientation of the αC helix, placing it close to the dyad region of the nucleosome. Mutation of this proline has strong phenotypes, structures equivalent to the isomerised state are seen for other proteins, and proline isomerases with histone chaperone activity have been identified.

A comparison of a chromatogram of H2B K116C expressed in LBM media and purified from inclusion bodies using the revised protocol (Figure 4) with one of 2YT media using the previous protocol (Figure 3) shows a much higher yield of protein with 0·9mg protein expressed and purified with the old protocol and ~10mg with the new protocol.

RNA Folding Prediction

Figure 5: H2B wt RNA Folding prediction Figure 6: H2B K116C RNA folding prediction

PCR Optimisation

Summary

With the inclusion of an optimised protocol for H2B K116C expression this project can now be advanced in the future by refolding octamers of the histone mutants that have been labelled with an Alexa-594 maleimide dye. Nucleosomes can then be reconstituted using these modified histones and Alexa-488 end labelled DNA produced from large scale PCR. These nucleosomes would then be analysed by FRET to investigate the conformational flexibility of the H2B αC helix.

Figure 4: Chromatogram of H2B K116C expressed in LBM Lennox media and purified using a revised protocol.

Figure 3: Chromatogram of H2B K116C expressed in 2YT media and purified using a standard protocol.

Due to the difficulties in expression and purification of the H2B K116C mutant, RNA folding predictions of the mutant were estimated using the mfold web server (RNA Institute, University of Albany) to see if this problem had a structural basis. Comparisons of the folding form of a region of 100bp surrounding the K116C codon site shows increased C-G base pairing and increased stability around the K116C codon site in the mutant (Figure 6), with a higher initial free energy value of ~ 40kJ/mol compared to ~32kJ/mol for the wildtype (Figure 5), possibly contributing to the difficulties in expressing the mutant.

Figure 7: Agarose Gel electrophoresis of trial PCR

A trial PCR of the 147bp DNA sequence (A0A) that can be tagged with a fluorophore for FRET analysis shows that a final primer concentration of .4µM gives the optimal yield of the DNA fragment (see Row 4 of Figure 7).

Biochemical Society Summer Vacation Studentship Report, 2012

An investigation into the inflammatory potential of Propionibacterium acnes in prostate cells in vitro. Student: Kathryn McAvoy (Supervisor: Dr. Lorraine Martin)

School of Pharmacy, Queen’s University, Belfast

Background and Description of Project Prostate cancer is one of the leading causes of cancer death in men in Western countries and is the most common tumour that afflicts older men (Fehri et al., 2011).

Several studies have proposed that chronic prostatic inflammation is an important factor in the development of this malignancy. A Gram-positive bacterium; Propionibacterium acnes (P. acnes) has been reported to be frequently present in various prostatic diseases and its presence has been correlated to inflammation in prostate cancer specimens (Alexeyev et al. 2007 and Cohen et al. 2005). Presently, four P. acnes phylotypes are known to exist, denoted type IA, IB, II and III (McDowell et al., 2005; McDowell et al., 2008) and have been demonstrated to vary with regard to their immunostimulatory properties.

P. acnes is suspected of playing an important role in the pathogenesis of prostate cancer as it satisfies several fundamental criteria: it is able to elicit a significant inflammatory response that is imperative for triggering carcinogenesis; it is slow growing, of low virulence and can withstand destruction by immune cells. Finally, P. acnes is sufficiently prevalent in society to account for the high incidence of prostate cancer (Shannon et al., 2006).

Aims of Project The initial aim of this project was to evaluate the inflammatory potential of the individual P. acnes phylotypes (type IA, IB, II and III) against normal prostate epithelial cells in culture. This work involved the examination of protease activity within P. acnes culture supernatants prior to the establishment of a co-culture system. These latter experiments involved the culture of normal prostate epithelial cells in the absence and presence of soluble bacterial-derived mediators and heat-inactivated bacteria. Cell lysate and conditioned media were then assessed for changes in the expression/activity of proteases, protease-activated receptor 2 and inflammatory mediators, such as IL-8.

Procedures carried out The initial weeks were spent familarising myself with the topic through reading of the background literature. This was balanced with training in essential techniques such as tissue culture and receiving an induction to the microbiology laboratory protocol.

I then went on to gain experience in performing cell counts and the anaerobic culture and growth of P. acnes strains on blood agar before culture in broth/media.

I became proficient in co-culture experiments investigating the affects of inoculating RWPE-1 cells (normal phenotype) with different strains of P. acnes and conducted a variety of assays to investigate protein levels (Western blots) and their expression (PCR) as well as functional activity assays e.g. steady state protease assays, zymography and calcium mobilization assays.

Results Immunodetection of PAR-2 The level of PAR-2 in cell lysate samples was investigated via Western blots. An initial experiment was carried out to become familiar with the technique and to ensure the protocol used was suitable to provide reproducible results. It investigated PAR-2 expression in RWPE-1 and PC-3 lysate samples and the Western blot image was then quantified using ImageJ software (Figure 1). The results showed, as expected, that the cancerous PC-3 lysate samples express more PAR-2 than the RWPE-1 (normal prostate epithelial) cells.

RWPE-1 cells were then infected with the four different strains of P. acnes, at an MOI of 10:1 incubated for 24 hours and 48 hours and lysate samples were prepared. Western blots were then preformed on these samples. Figure 2 shows the Western blot image obtained from the 48 hour lysate samples. The protein levels of the respective samples were then quantified using ImageJ software and the results were expressed as a percentage of the control (Figure 3). The lysate samples infected with the various strains of P. acnes show much greater PAR-2 expression than the control of non-infected RWPE-1 cell lysates. Increased PAR-2 expression was particularly evident in prostate cells infected with P. acnes types IA and II, and to a lesser extent IB, indicating some differences in the inflammatory potential of the various phylotypes,

Zymography Gelatin zymograhy was carried out to investigate protease activity, predominantly matrix metalloproteinase (gelatinolytic) activities, within co-culture conditioned supernatants.

Figure 4 shows the gelatin zymogram obtained after running 24 and 48 hour supernatant samples on the gel. The top bands on the gel correspond to a molecular weight of 92 kDa which could be representative of MMP-9 (Matrix metalloproteinase 9). To further confirm the presence of MMP-9 a next step would be to conduct a Western blot using an anti-MMP-9 antibody. The control used in the experiment was untreated prostate cells, suggesting that there is a basal level of expression of gelatinolytic activity from prostate epithelial cells but a more pronounced activity was observed in all of the treatment wells. In addition, the samples treated with P. acnes strains IA and IB showed increased activity in the 48 hour samples. All of the P.

Figure 1: PAR-2 expression in RWPE-1 and PC-3 determined using immuno-detection with an anti-human PAR-2 antibody

Figure 2: RWPE-1 PAR-2 levels using 48 hour lysate samples

Figure 3: PAR-2 expression in 48-hour lysate samples expressed as a percentage of the control sample

acnes strains showed increased activity compared to control. A lower band of 64 kDa was also observed which could be due to active processed fragments of either MMP-2 or MMP-9.

Calcium Mobilisation The effect of P. acnes infection on PAR-2 activation was investigated via calcium mobilisation. Calcium mobilisation was used as an indirect way of looking at PAR-2 expression, as if there is greater PAR-2 expression there should be greater activation. PAR-2 activation was assessed using a PAR-2 specific agonist substrate. Activation of PAR-2 results in a rise in calcium and this can be measured using a calcium sensitive dye.

Calcium mobilisation was preformed on a co-cultured 96 well plate. Relative to the control wells (non-infected RWPE-1 cells) there was a more pronounced effect in the wells treatment the four P. acnes strains (IA, IB, II and III). This would suggest that there is more activation and thus expression of PAR-2 in the wells containing prostate cells infected with P. acnes. As before some differences were observed between the different P. acnes phylotypes.

Departures from the original proposal The original aims of the project were closely followed. There were small deviations from the initial timetable, allowing me to move through the induction process quicker than expected, leaving a greater proportion of time available for experiments and investigations.

In addition to the stated aims, the effect of P. acnes infection on PAR-2 activation was also evaluated via calcium mobilization. These experiments were included as a further indicator of increased levels of PAR-2 in the presence P. acnes.

Future directions in which the project can be taken There are a number of directions in which this project can be taken. For example, confocal microscopy using P. acnes specific antibodies would confirm the presence of P. acnes intracellularly within the co-culture system. This technique could also be used to confirm the expression of cell surface proteins in the presence of P. acnes.

Further work may include examining other immune responses associated with P. acnes infection, to investigating the inflammatory pathways that contribute to the chronic inflammation seen prior to prostate cancer development.

It is hoped that studies such as these will contribute to the long-term goal of potentially developing anti-infective therapies for prostate cancer and a vaccination

Figure 4: Gelatin zymogram of RWPE-1 (non-infected and infected with P. acnes) conditioned supernatant samples taken at 24hours and 48hours

strategy, which could potentially diminish the incidence of this devastating disease in men.

The value of the studentship World-leading work has already been conducted at Queen’s University on the identification, typing and classification of P. acnes isolates (Dr McDowell) and I was keen to assist Dr Martin’s studies in comparing the inflammatory potential of these various P. acnes phylotypes. The project allowed me to gain a unique insight into cutting edge breakthrough science. Working in the field of prostate cancer and in studies which will help further our understanding and knowledge of the role of P. acnes in this disease was ideal way to spend my summer.

The length of the project provided the opportunity for expert training in laboratory techniques and gave me a much more expansive learning experience than is normally available as part of the MPharm degree program. I have gained considerable benefits, both personally and professionally from this summer studentship programme and feel that I have a much greater awareness of laboratory research. I am looking forward now to exercising my skills within my final year project over the next number of months.

This project challenged me to apply knowledge I had already gained throughout my Pharmacy degree whilst allowing me the opportunity to learn within a more focused environment through literature searches and informal discussions with staff and postgraduate students. Spending time with the postgraduate students allowed me to benefit from their experiences. I am particularly indebted to Mr Darragh McCafferty (PhD student) who provided me with training and direct laboratory supervision on a day-to-day basis.

Overall the project has broadened my horizons in terms of appreciating and understanding the opportunities that are available in terms of a future scientific career either within academia or industry.

References Fehri L., Mak T., Laube B., Brinkmann V., Ogilvie L. et al. (2011) Prevalence of Propionibacterium acnes in diseased prostates and its inflammatory and transforming activity on prostate epithelial cells. International Journal of Medical Microbiology 301: 69-78.

Alexeyev O., Marklund I., Shannon B., Golovleva I., Olsson J. et al. (2007) Direct Visualization of Propionibacterium acnes in Prostate Tissue by Multicolor Fluorescent In Situ Hybridization Assay J Clin Microbiol 2007, 45(11):3721-3728.

Cohen R.J., Shannon B., McNeal J., Shannon T., Garrett K. (2005) Propionibacterium acnes associated with inflammation in radical prostatectomy specimens: A possible link to cancer evolution? Journal of Urology 173: 1969-1974.

McDowell A., Valanne S., Ramage G., Tunney M., Glenn J. et al. (2005) Propionibacterium acnes Types I and II Represent Phylogenetically Distinct Groups. Journal of Clinical Microbiology 43: 326-334.

McDowell A., Perry A., Lambert P., Patrick S.(2008) A new phylogenetic group of Propionibacterium acnes. Journal of Medical Microbiology 57: 218-224.

Shannon B., Garrett K., Cohen R.J. (2006) Links between Propionibacterium acnes and prostate cancer. Future Oncology 2: 225-232.

Summer Vacation Studentship Report The differential role of p110α and p110δ in B-cell signalling

Katharina Franziska Becker Introduction For B-cell development and survival, expression of and tonic signalling through a functional B-cell receptor (BCR) is required. Phosphatidylinostol-3-kinases (PI3Ks) are key enzymes in this pathway. Upon activation, they catalyse the phosphorylation of the 3-hydroxyl group of phosphatidylinositol, generating phosphatedyl-inositol-3,4,5-trisphosphate (PIP3). Class IA PI3Ks are heterodimeric proteins composed of a regulatory (p85) and catalytic subunit (p110). Of the three isoforms of the catalytic subunits, p110α and p110β are expressed ubiquitously whereas the expression of p110δ is mainly restricted to lymphocytes. Both p110α and p110δ are essential for B-cell maturation. In mature B-cells, signalling through the BCR seems to rely almost entirely on p110δ, even though p110α is still expressed at a functional level in those cells. The aim of the studentship project was to look into the differential roles of p110α and p110δ in both antigen-induced and tonic signalling to establish whether tonic signalling can be achieved via either isoform. The production of PIP3 leads to the membrane recruitment and activation of signalling enzymes containing a PH-domain, most notably the kinase Akt. Through a different pathway, the activation of a MAP-kinase-cascade leads to the phosphor-rylation of the MAP-kinase ERK. To analyse the isoform-specific kinase activities, B-cells were isolated out of p110α-knock-out mice and knockin mice expressing a catalytically inactive p110δD910A mutant protein. The complete lack of p110α expression in the knock-out-strains might lead to a higher compensation through p110δ, whereas the presence of the catalytically inactive D910A-p110δ spatially obstructs compensation through p110α. Hence, knock-out and knock-in should not directly be compared. The analysis of the mutant mouse strains was therefore complemented by the use of isoform-specific PI3K-inhibitors and a pan-PI3K-inhibitor. To look into the differential role of the PI3K-isoforms in antigen-induced and tonic signalling, B-cells were stimulated with antibodies against the F(ab')-fragment for 1,2 or 5 minutes and compared to unstimulated cells. Inhibitor Specificity The p110δ-inhibitor IC87114 was used and known to be specific at 3µM. The specificity of two p110α-inhibitors (GSK-2222734A at 300nM and INK 1437-1146-229 at 6µM) was to be compared. To do this a system in which the phosphorylation of Akt relies mainly or

exclusively on p110α-activity would be ideal. The MCF10 breast epithelial cell line is thought to be very p110α-dependent and was therefore used for this experiment. MCF10 cells were incubated with the respective inhibitors and stimulated with EGF. Expression of p110α and p110δ was measured as well as phosphorylation of Akt. As can be seen in figure 1, MCF10 cells express both p110α and p110δ, the latter at a lower level. Treating the cells with the p110δ-inhibitor IC at 3µM reduced Akt phosphorylation. This leads us to believe, that at least some of the pAkt-production is due to previously unappreciated p110δ-activity and is therefore lost if treated with a p110δ-inhibitor. Incubating the cells with the p110α-inhibitor GSK did not reduce Akt phosphorylation whereas the p110α-inhibitor INK completely reduced the phosphorylation-signal. Based on the assumption that at least some of the pAkt is produced by p110δ, we suspect that the complete inhibition of phosphorylation by the INK compound is due to off-target inhibition of the δ-isoform. This is further confirmed by the later experiments. From this experiment no conclusions can be drawn as to the potency of the GSK inhibitor. The fact that we see no change in Akt phosphorylation with and without the drug could be due to a p110δ-dependence of this signal or an insufficient inhibitory effect. Previous titrations of the GSK-compound have shown effects at concentrations much lower than used in this experiment and a complete lack of inhibition of p110α at 300nM seems very unlikely.

p110α- and p110δ-Inhibition in B-cells B-cells were isolated out of WT, p110α-KO and p110δD910A-mice using CD43 microbeads. Cell purity was checked via flow-cytometry and over 95% purity could be shown in all experiments. The cells were subsequently incubated in medium containing either the p110δ-inhibitor IC, the

Figure 1 MCF 10 cells were stimulated with EGF and treated with isoform-specific PI3K-inhibitors. Phosphorylation of the downstream-signalling enzyme Akt was used to indicate kinase activity

p110α-inhibitor INK or a DMSO-control and stimulated at 37°C for 5 minutes. Comparing Akt and ERK phosphorylation in these cells with unstimulated controls shows a high increase in signalling-activity upon stimulation. The p110δ-inhibitor IC reduced the signal in the WT and lead to a complete loss in the p110α-KO. Upon inhibition of p110δ in WT-mice the p110α-isoform compensates functionally but not completely. In p110α-KO-mice the inhibition of p110δ cannot be compensated for and the signal is therefore lost completely. p110δ-inhibition had no effect on both stimulated and unstimulated p110δD910A cells, showing that the p110δ they express is indeed catalytically inactive and that IC had no off-target effects on the PI3K pathway at the concentration used. The p110α-inhibitor caused a loss of pAkt signal in stimulated and unstimulated p110δD910A B cells – as expected – because these cells with their catalytically inactive p110δ-isoform rely completely on p110α for their signal-transduction. The fact that we can see a signal reduction in the p110α-KO-B cells treated with the INK inhibitor supports our earlier findings that INK might have off-target effects on the p110δ-isoform. Unexpectedly, the stimulated p110δD910A-B cells had a pERK signal almost as strong as the stimulated WT and p1110α-inhibition did not reduce this signal completely as it did with the pAkt-signal. This suggests that the MAPK-pathway that leads to ERK phosphorylation might upregulated by compensatory mechanisms in p110δD910A B cells. Comparing the basal signal in the three genotypes, it seems that inhibiting both isoforms (D910A+INK or αKO+IC) led to a loss of the basal signal whereas inhibiting just one isoform did not reduce the signal significantly. This supports the hypothesis, that both p110α and p110δ can relay basal signalling in unstimulated cells. Mass Spectrometry A second objective of this project was to directly measure PIP3-production via mass spectrometry. Ideally this could replace Western-Blot analysis of Akt-phosphorylation as a more direct and quantitative method to measure PI3K-activity. I successfully isolated and stimulated B-cells for this purpose and extracted the membrane lipids for further derivatisation. However, although we

could measure PtdIns(4,5)P2 in these samples, PtdIns(3,4,5)P3 was not detectable. This may be because PtdIns(3,4,5)P3 levels are much lower in B cells than in neutrophils. The lab is therefore exploring different methods to increase the sensitivity of the assay. Conclusion During the six weeks of the summer project I could show that the MCF10 breast epithelial cell line is not an ideal model to assess the specificity of p110α-inhibitors since p110d also contributes to Akt phosphorylation in these cells. INK, the p110α-inhibitor used during the experiments seems to have off-target effects on the δ-isoform and all experiments should therefore be repeated with a more specific p110α-inhibitor. The experiments conducted with B-cells of different genotypes support the theory, that both the α- and δ-isoform of the p110 PI3K can be involved in tonic signalling whereas antigen-induced signalling relies mainly on p110δ. Inhibiting both isoforms leads to a complete loss of Akt phosphorylation. Replacing the Western Blot analysis with mass spectrometry would be desirable to get a more direct and quantifiable measure of PI3K-activity. However, establishing the optimal conditions for the mass spectrometry to get a PIP3-signal proved to be more difficult than expected. The presence of a PIP2-signal shows that the lipid-extraction has been successfully performed and by adjusting other parameter such as cell number and stimulation it should be possible to measure a PIP3-signal in the future. Acknowledgments I thank Dr Klaus Okkenhaug for hosting me in his group and Dr Fabien Garcon and Amy MacQueen for their support and patients in answering my questions. Bibliography Bilancio et al. (2006) Blood, 107: 642-650 Clark et al. (2011) Nature Methods, 8: 267-272 Clayton et al. (2002) J. Exp. Med. 196, 6, 753-763 Kurosaki (2002) Curr Opin Immunology, 14:341-347 Monroe (2004) Curr Opin Immunology, 16:288-295 Okkenhaug et al. (2002) Science, 297: 1031-1034 Okkenhaug, Vanhaesebroeck (2003) Nature Reviews

Immunology, 3:317-330 Pauls et al. (2012) Frontiers in Immunology, 3:224 Porto et al. (2004) Molecular Immunology, 41 599-613

Figure 2 Stimulated and unstimulated cells of different genotypes were treated with the p110δ-inhibitor IC (left pannel) or the p110α-inhibitor INK (right pannel) and phosphorylation of Akt and ERK was measured to indicate signalling-activity. Levels of totalAkt serve as a loading control.

Kristen A. Johnson IpgD in DC: My Summer Vacation Studentship with The Biochemical Society and Dr. Miller

Figure 1: With Dr. Miller in his Lab

This summer I worked on a project that involved two phosphatases: ipgD and

SopB. Both of these enzymes are bacterial effector proteins that modify inositol signaling in mammalian epithelial cells in order to ensure virulence of their parent bacteria. IpgD and SopB share 47% identity and 86% amino acid similarity but differ in their substrate preference and specificity. IpgD removes the 4-phosphate from phosphatidylinositol 5-phosophate 4,5-biphosphate, while SopB is less specific and hydrolyzes phosphates from the 4- and 5-positions of the inositol ring. Both enzymes modulate inositol signaling so vital cell processes like apoptosis are controlled during the infection process.

The goal of my project was to determine if the difference in promiscuity between ipgD and SopB is due to different mechanisms to recognize their substrates after they are bound to the active site or if they recognize and bind different molecules such that non-substrates are precluded from entering the active site altogether. In the lab this meant I needed to successfully clone ipgD and SopB, purify soluble protein products, perform phosphatase activity assays, and test the enzymes substrate specificities for different inositol molecules. An additional goal was to test for ipgD binding to non-substrate ligands and determining the KD using isothermal titration calorimetry. Due to time and technical challenges, I spent the summer working mainly with ipgD, and I have uncovered new experimental information to help me tie up the loose ends of this research project.

Starting out, I used PCR to clone ipgD and SopB into a plasmid that would allow expression of the enzymes as SUMO fusions, which was intended to increase solubility of the expressed proteins. I performed the cloning using two different methods: the Gateway method (GW) (Invitrogen) and using restriction endonucleases (RE) and ligase. Successful clones were identified and were transformed into E.coli (Rosetta (DE3) cells, Novagen) for expression. I began using a small-scale expression system to determine if ipgD could be expressed successfully. I inoculated 2.5 ml cultures of TB with ampicillin then induced the culture with 1 mM IPTG. A soluble protein product was observed using SDS PAGE (Figure 2). Larger scale cultures of 1 to 8 liters were only performed for ipgD after successfully reproducing the small scale tests. After scaling up the experiment, I was able to use column chromatography using glutathione sepharose beads

to purify ipgD. The GST tag on the ipgD construct made it possible to use this type of column as the tag binds to the column while other proteins do not. The protein was eluted using a phosphate buffer with 50 mM glutathione as an elution buffer. I collected 500 μl fractions and tested for the presence of GST-ipgD using a Coomassie protein assay. SDS PAGE was also used to identify fractions containing GST-ipgD and to assess its purity (Figure 2A).

To test the activity of the purified ipgD and identify its dependence on pH and other parameters, I made 18 different buffers with a range of pH values and additives. Using a 96 well plate reader I tested a verity of substrate concentrations, and enzyme concentrations with the buffer set to identify optimal reaction conditions. To determine the activity levels of the ipgD, I first used BioMol green reagent to stop the reaction after a given time and then read the plate at 620 nm to measure phosphate released during the reaction. I also performed a kinetic assay and read the continuous formation of product at 405 nm.. After comparing the activity of the ipgD to a positive control I observed detectable, but low levels of activity. As the ipgD still had its GST fusion tag, I hypothesized that this tag was limiting the enzymes activity. To test this hypothesis, I used TEV protease to remove the GST tag (Figure 2B). Fortunately I am continuing this project this semester with Dr. Miller, which will give me the time to optimize the reaction conditions and complete testing of the substrate specificity and inositol phosphate binding.

Figure 2A: SDS PAGE of ipgD Purification. From left to right: MW standards in kDa, total lysate, soluble fraction, flow through, eluted fractions 1-6. 2B: Cleaved ipgD 1. MW standards in kDa, 2. ipgD+GST, ipgD, GST.

Overall, the eight weeks I spent in the lab this summer was a fantastic summer research program. I had great guidance from my research advisor Dr. Greg Miller at The Catholic University of America in Washington, D.C., and I also had a great deal of independence in the lab. The delicate balance of guidance and independence helped me grow in my scientific reasoning and expand my understanding of how to work in a laboratory. There is something about having your own set of pipettes and unlimited supply of chemicals that really allows one to grow into a young scientist. In addition, this experience exposed me to all of the behind-the-scene prep work of experimentation. Making gels, buffers, and autoclaving solution are all aspects of scientific research that provide one with experimental options that I never knew before this experience. Working in Dr. Miller’s lab was no cookie cutter laboratory class; this was real scientific research! The amazing research experience provided by the Summer Vacation Studentship helped me in deciding to pursue a PhD in biochemistry. Looking back, I cannot imagine spending my summer in any other way (or lab)!

Characterisation of disease pathology in a zebrafish transgenic model of Parkinson’s Disease Background: Parkinson's disease (PD) is a neurodegenerative disease involving loss of dopaminergic neurons of the substantia nigra pars compacta as well as other neuronal types, as well as the accumulation of α-synuclein in Lewy bodies. It is the second most common of the neurodegenerative diseases affecting humans so is of interest to investigate. Mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are the most frequent known cause of autosomal PD in patients, as well as appearing in 'sporadic' cases. The clinical symptoms of LRRK2 PD are identical to idiopathic PD, with formation of Lewy bodies. This variable pathology suggests that LRRK2 acts upstream in the PD neurodegeneration process. In previous work, the Fleming lab has investigated the role of LRRK2 in protein homeostasis. They observed that α-synuclein and ubiquitin aggregated in LRRK2 expressing cells as well as in vivo LRRK2 zebrafish model. This work was done with transient over-expression of LRRK2 in zebrafish embryos which gives rise to mosaic expression of the over-expressed protein which is transient; not lending itself to the observation of long term effects of the over-expression. The lab was given 4 transgenic zebrafish lines expressing human LRRK2 by a collaborator; 2 expressing wild-type and and 2 with G2109S (GS) mutant forms of the LRRK2 transgene. My 8 week project hoped to characterise some of the phenotypes observed in the fish with confirmed LRRK2 transgene (and the GAL4 promoter required to activate it), possibly developing this with screening small molecules that would reduce or rescue the observed pathology. Methods: Firstly I learnt to sex zebrafish in order to set up breeding pairs to collect eggs. It was important to ensure that the fish were both correctly sexed so that they could be returned to their respective holding tanks afterwards. The fish were set up in tanks with barriers between the males and females and the barriers had to be removed at a specific time of day in order to encourage them to breed before eggs could be collected from the breeding tanks. As I was crossing a heterozygous Gal4 line with a heterozygous Lrrk2 line, only 25% of the offspring had the desired genotype and so therefore there was a limited number of the embryos that I could utilise. This along with the fact that not all breeding pairs mated each day meant that we had to repeat the cross multiple times to procure enough samples. For all of my study I had to do PCR on a portion of each embryo/larva before I could group them into their genotypes and perform further analysis on them. Given that the 2-6 day old offspring are only 2mm in length, this was a rather fiddly process but I got more efficient at it with practice. The PCR itself was very time consuming as I had to do a tissue digest first as well as be particularly careful in setting up the Gal4 PCR as it was easily contaminated. With a large number of samples required to get enough Gal4/Lrrk2 fish, this took up most of my time in the first half of the project. When it came to investigating any potential pathology of the fish, I learnt a variety of techniques to fix, section or stain the samples so that they could be compared with each other. I sectioned the embryos using both a paraffin embedding and a cryosectioning method to get the samples thin enough (between 5-10µm). I used antibody staining on the fixed and sectioned or wholemount fish and then the fluorescent microscope in order to analyse the results. I had to use the digital image capture with varying resolution and exposures to take pictures on both the brightfield and fluorescent microscope images. This included mounting the samples which had to be done with both whole fish and those with missing tails from PCR. The majority of mounting was done in glycerol but I tried agarose in some more difficult cases to ensure a correctly orientated fish. Results: Having observed several clutches of eggs of different crosses of the ubiquitous Gal4 promoter and the wt and mutant type LRRK2 strains, it was clear that one of the GS lines gave rise to a strong phenotype similar to that arising from transient over-expression. There was a marked increase in both mild and severe development of embryonic-stage defects as well as other obvious physical deformities, these were observed in approximately 10% in the surviving fish. There was also a marked increase seen in dead embryos at the 24hour stage. This is compared to an anticipated 1-2% abnormality and death rate in wt crosses. When genotyped, most of these severe defects correlated strongly with fish that contained both the LRRK2 and Gal4 genes simultaneously, highly implicating them as the cause. The knowledge that Gal4 can be toxic itself when expressed strongly could explain the increased pathology seen in the fish that did not have both genes as this was also the case, though not as severe or

common as in fish with both transgenes. These results were difficult to confirm due to time constraints and the complexity of obtaining enough PCR results across the genotypes but I am relatively confident in the data obtained thus far. We analysed the neurons in the fish at early embryonic stages so as to try to note any abnormalities (see figure). This proved difficult again due to lack of large sample numbers that could be processed in my short project. My pilot data shows that some of the neurons observed were imperfectly orientated or showed similar features to those seen in the transiently expressing fish, though this should be compared to both earlier and later developmental stages to be sure. In learning techniques to section and stain the dopaminergic neurons with tyrosine hydroxylase (TH) I was able to see if they were also affected by the gene expression, with the fluorescence beautifully showing up in the location expected. However, the orientation of the fish made it difficult to score exact numbers of neurons since we were looking at cross sections and counting and comparing the number of neurons between samples would have been ambiguous and error prone. The best way to do this would be with whole-mount fish at an early developmental stage. We therefore did not observe any quantity difference in this preliminary analysis but would expect the numbers of dopaminergic neurons to be reduced in the LRRK2/Gal4 fish when analysed in wholemount. Analysis and conclusions: The GS line cross showed a rather marked increase in the number of fish showing pathology similar to that in that observed from transient over-expression of Lrrk2 (Lichtenberg et al., Cell Death and Disease, 2011). This suggests that the over-expression of the human LRRK2 gene in zebrafish results in defects that are relevant to PD and these transgenic lines will be a useful tool for further studies.. We did not have time to attempt to 'rescue' the phenotypes of the fish but this could be done at a later date to observe any improvement and thus prove that defects seen are caused by increased LRRK2 kinase activity. It would also be prudent to try to get better pictures of the neural system by removing the rest of the tissue surrounding it by digestion, giving much clearer images which could more easily be compared. I found a method for doing this towards the end of my project which can now be attempted in the lab to check its success. The lab is currently breeding the lines to ensure homozygous fish are available for subsequent crosses. This would remove the need to PCR every fish in order to establish whether any contain the gene. The fish would then be entirely intact and more large-scale analysis could be done, since PCR of each fish requires a small sample each time, this can affect the fish's staining and how easily it can be mounted for observation. Whole mount fish are easier to use than partial specimens. Moving forwards this would make it much more straightforward to do all of our analysis and get a large volume of samples in order to score them. In this lab I have learnt a wide range of incredibly useful techniques that I think have really

developed me as a scientist as well as my understanding of research. I was able to get some results, which should be really useful in the future, and also in determining the next direction in which this particular vein of the lab research would move. I also went to weekly lab meetings with the overall group which ensured that I saw how the little piece that I was doing fitted into a larger collaboration's work. Some parts of my work have also reached dead ends, this I believe is also important in order to realise that research is not always straightforward and that it can also be frustrating, but that it does not mean that the project is going nowhere.

I really enjoyed working with Dr Fleming and hope to complete my 3rd year degree project with her early next year, furthering my work on this topic. I am still very keen to pursue disease and neurology so am taking Part II neuroscience this year, with modules in neurodegeneration as well as neural development. I hope to further my studies by taking a PhD in the future as I can see myself enjoying lab work and finding a research-based career really fulfilling and so will endeavour to enter this vein of work. I am excited to see how the project will develop and hopefully all the hard work on this topic will come to fruition given the results so far and the next steps we will take.

Figure legend Acetylated tubulin staining of a zebrafish larva at 2 days old to show the developing motor neurons in the trunk.

Structural studies of a Cdc37-Pp5 complex; regulation of Cdc37 by dephosphorylation

Biochemical Society Summer Studentship Report 2012 Student: Laura Mariotti Supervisors: Dr. Cara Vaughan & Dr. Jasmeen Oberoi

The background and the aims of the project Hsp90 is a molecular chaperone which helps the folding and the activation of many proteins, including oncogenic kinases. One of its substrate is the phosphatase Pp5 composed of an auto inhibitory domain (which is displaced upon interaction with Hsp90) and a catalytic domain [1]. Once activated, Pp5 can dephosporylate Cdc37 at Ser13 (Hsp90 acting as a bridge holding the two proteins) [2]. Cdc37 is a cochaperone and intervenes in the interaction between Hsp90 and its client kinases. To better understand the molecular details of Cdc37 dephosphorylation by Pp5, the aims of the project were to clone, express and purify a chimeric protein of the N-terminus of Cdc37 (with the phosphoserine replaced by a phosphomimetic glutamate) joined by a glycine-serine linker to the catalytic domain of Pp5 (Fig.1). Two chimeric proteins were designed; a shorter one with the amino acids 5 to 20 of Cdc37 and a longer one with amino acids 1 to 30 of Cdc37. Crystallisation of the chimeric protein would allow the visualisation of the Cdc37 peptide bound in the catalytic site of Pp5.

A description of the work carried out There were no major departures from the original proposal.

1. Molecular cloning

The primers used for the PCR were designed to contain the glycine-serine linker and the S13E mutation. The catalytic domain of Pp5 and the phosphomimetic peptide of Cdc37 were amplified. For the short construct, the PCR fragment of the catalytic domain of Pp5 was used as a template for the second PCR. In contrast, for the long construct, the two PCR products were digested with the restriction enzyme NheI. The chimeric constructs were digested with restriction enzymes and ligated into digested PGEX6P1 (which contains a GST tag) and into PETM6T1 (which contains a NusA tag). The recombinant vectors were transformed into TOP10 E. coli cells. The presence of the insert was rapidly visualised by diagnostic PCR and confirmed by sequencing.

2. Small scale expression trials/Large scale expression

Two cell lines were used for the small scale expression trials, BL21* and T7 express Plys IQ at two different temperatures, 21°C and 37°C. 10 ml of E. coli cultures were grown and at an optical density of 0.4 were induced by 0.4mM IPTG. The Pp5 catalytic domain also required the addition of MnCl2 to the cultures. The chimera solubility was also tested by taking a sample from the pellet, the supernatant and from beads (glutathione sepharose beads for the GST tagged construct and TALON metal affinity resin for the NusA tagged construct). Using the BL21* cell line at 21°C showed the best expression and solubility (Fig.2). These conditions were used for the large scale expression. 4 litres of both the short and long NusA fused chimeric proteins were grown and 10 litres of the GST fused short chimera. The cells were cultured at 37°C until they reached an optical density of 0.4, 0.4mM MnCl2 was then added and they were then induced by 0.4mM IPTG. The temperature was reduced to 21°C

Fig 1. Schematic representation of the chimeric protein

Fig 2. Gel picture showing the expression of the protein without IPTG (1st column), at 21°C (2nd) and at 37°C (3rd)

Fig 3. Gel picture showing the fractions from the DEAE column after TEV cleavage

Fig 4. Gel picture showing the fractions from the S200 gel filtration column

Fig 5. Gel picture showing the fractions from the ResQ column

and the cultures were grown overnight. After centrifugation, the pellets were resuspended in lysis buffer, the cells were lysed and centrifuged again.

3. Purification protocol

A. GST-fused chimera

A GST batch column was first used to bind to the GST tagged protein. Overnight, the protein was cleaved from the column using PreScision tag enzyme. An S200 gel filtration column was then run, followed by a Resource Q column.

B. NusA-fused chimera

A DEAE column was first used to bind the protein. TEV protease was added overnight and another DEAE column was run (the protein flows through) (Fig 3). To separate our protein by size, we then used an S200 gel filtration column (Fig 4). Finally, a resource Q column separated our protein well enough for crystallisation trials (Fig 5).

4. Crystallisation

We used general screen plates with a wide range of conditions: pH Clear, Index, Classics and JCSG at 4°C and 16°C.

An assessment of the results Our chimeric proteins of 41kDa (long construct) and of 40 KDa (short construct) have been purified (as shown on Fig. 6 for the long construct) at a final concentration of: NusA-fused short construct: 11.97 mg/ml NusA-fused long construct: 13.53 mg/ml The total yield per liter was: NusA-fused short construct: 0.56 mg in 1L NusA-fused long construct: 0.57 mg in 1L Crystallisation was therefore possible. Preliminary results show a crystallisation condition which will have to be pursued. The GST-fused chimera had a very low yield and concentration which didn’t allow crystallisation trials. Furthermore, a contaminant of slightly higher molecular weight could not be separated from our protein.

Future directions in which the project can be taken

The crystallisation conditions should be optimised once crystals are detected in the general screen plates. Kinetic parameters of our protein could also be determined using phosphatase assays. Molecular distances between the Pp5 catalytic domain and the cdc37 peptide could be analysed using FRET or EPR. The binding affinity of our chimera to Cdc37 or Pp5 could be assayed using ITC.

Value of the studentship

This studentship gave an incredible opportunity to discover many techniques not learnt at university and understand the skills required for research: time management (experiments planning), organisation (maintenance of a lab book), scientific communication (presentation of results to colleagues).

[1] Yang, J. et al. (2005) EMBO Journal, 24, 1‐10. [2] Vaughan, C. K. et al. (2008) Molecular Cell, 31, 886‐95.

Figure 1- Immunofluorescence images to show an islet stained blue with dapi (a marker for the nucleus), red with antibodies for insulin and green with antibodies for smooth-muscle actin (SMA).

Biochemical Society Studentship Report 2012 Using mesenchymal stem cells (MSCs) to improve islet transplantation therapy for diabetes Student: Mahdieh Godazgar Supervior: Professor Peter Jones, King’s College London Introduction/ Aims The selective destruction of beta-cells by auto-immune antibodies in type 1 diabetes mellitus renders patients unable to control their glucose levels by the secretion of the hormone insulin. Most patients can gain glycaemic control via insulin therapy. However, for a small minority of patients achieving a balance between hyperglycemia and hypoglycaemia with insulin therapy is a difficult one to accomplish, and it is these patients for whom which islet transplantation is an alternative option. Despite the improvements made to islet transplantation in the year 2000 with the development of the Edmonton Protocol [1], islet transplantation still faces challenges as graft function progressively declines, leading to only 10-15% of patients remaining insulin-independent after 5 years [2]. However recent studies have shown that co-transplantation of Mesenchymal Stem Cells (MSCs) in mouse models improves the capacity of islet grafts to reverse hyperglycaemia and maintain graft morphology [3]. Data from animal models suggest that these effects may be mediated by the immunosuppressant and/or angiogenic properties of MSCs [3-4]. Delivery of MSCs with islets via the clinically preferred intravascular route will require a method for forming islet-MSC composites, and our preliminary studies using mouse islets suggest that the MSCs interact with, and possibly even enter the islets. However, MSCs do not have unique markers and the main aim of my studentship was to optimise an MSC staining techniques so that morphological studies can then be carried out.

Results Human islets were co-cultured with human MSCs and a time-lapse video was generated by capturing images every 15 minutes for 36-48 hours on a Nikon Biostation. Before the MSCs were co-cultured they were incubated with fluorescent nanobeads, which they engulfed, enabling them to be tracked by fluorescence microscope. The videos showed that MSCs were highly motile, interacting with the islets and potentially entering the islets. In order to investigate this further we fixed and immunostained the islet-MSC composites (Figure 1) using antibodies against smooth-muscle actin (SMA; a non-specific marker for MSCs), and insulin (a marker for beta-cells in the islets), with Dapi used to stain the nuclei.

The results from the staining showed some green fluorescence within the islets, suggesting that the MSCs had penetrated them. However the fluorescence images of the islets have a punctate appearance, which could be the fluorescent beads used to track the MSCs during the time-lapse video and not the immunofluorescent antibodies used to stain smooth-muscle actin. If the beads engulfed by the MSCs maintain their fluorescence when fixed, it would be a significant aid to specifically identifying the MSCs that were co-cultured with the islets.

In order to identify the origin of the green fluorescence, mouse islets and MSCs were co-cultured and then stained. All islet-MSC composites were stained for the nucleus and insulin, however one group of islets MSC composites was stained with SMA antibodies, whilst a second group was not. If the latter contained green fluorescence it would indicate that the beads maintain the fluorescence when fixed and stained. A third group of islet-MSC composites were stained with the secondary antibody for smooth-muscle actin, but not the primary antibody to check for non-specific staining.

The results of the immunofluorescence staining showed no green fluorescence in the group which was not stained for smooth muscle actin, but did show fluorescence in the group which had been stained for smooth muscle actin. Interestingly there also appeared to be green fluorescence in the group stained for non-specific staining, which indicates either non-specific staining or it could be beads that had maintained their fluorescence. Further staining will be required to clarify this.

Deviations from the original plan/ Future directions in which the project will be taken/ The original plan was to do functional studies on insulin secretion and content after mouse islets were co-incubated with MSCs. However, the studentship was used to carry out preliminary experiments to increase my confidence cell culture techniques and to optimise MSC staining techniques. I can now progress onto carrying out functional studies as part of my BSc laboratory project. Value of the Studentship to the Student: I feel extremely privileged to have been a part of the Diabetes Research Group this summer; being involved in group discussions and sharing an interest in this stimulating area of research. Having completed the studentship I now feel confident in carrying out experimental techniques such as tissue culture and immunohistochemistry and have enjoyed encountering techniques such as radioimmunoassay, PCR and western blotting. I have also had the opportunity to gain an insight into how research is carried out and was surprised to find that an on-going part of it is optimising protocols as new experimental challenges are faced and the patience and perseverance this requires. I now look forward to beginning my third year BSc. laboratory project with enthusiasm and have been inspired to pursue a career in this arena.

Value of the Studentship to the Lab: The Studentship benefitted both the Diabetes Research Group and Mahdieh. The Group gained an enthusiastic, motivated young research worker who spent hours setting up complicated experiments and analysing the data, developing an in vitro model which we will continue to use in future studies. In return, Mahdieh received training in modern biomedical research techniques, she experienced first-hand the pleasures and frustrations of research, she realised that she liked working in a laboratory environment, and she found out that she was rather good at doing experiments. Mahdieh is now in the final year of her BSc programme and she has started a final year research project with the Group, using the experimental model which she helped to develop during the summer. The research project accounts for about 1/3 of Mahdieh’s final year and the experience which she obtained during the summer has given her an excellent foundation for her practical project work. She will now assess in vitro the functional consequences of the anatomical association between MSCs and islets in terms of viability and insulin secretion. If, as expected, the outcome is positive we will go back to our in vivo model of diabetes and assess the function of MSC/islet composites as improved transplant material. This is a long-term project but Mahdieh will have made a significant contribution through her summer studentship and final year project.

References 1. Shapiro AM, Lakely JR, Ryan EA et al (2000) Islet transplantation in seven patients with type 1 diabetes mellitus using a

glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230-238 2. Ryan EA, Paty BW, Senior PA et al (2005) Five-year follow up after clinical transplantation. Diabetes 54:2060-2069 3. Rackham CL, Chagastelles PC, Nardi NB et al (2012) Co-transplantation of mesenchymal stem cells maintains islet organisation

and morphology in mice. Diabetologia 54:1127-1135 4. Kim YH, Wee YM, Choi MY et al (2011) Interleukin (IL)-10 Induced by CD11b+ Cells and IL-10-Activated Regulatory T Cells

Play a Role in Immune Modulation of Mesenchymal Stem Cells in Rat Islet Allografts. Mol Med 17:697-708

Investigation of antipsychotic drug interactions with the multidrug transporter ABCC1 and ABCG2

Student: Martin Feighan Supervisor: Dr Ian Kerr

Aim: ABC G1 is a member of the human ATP binding cassette transporter family and is found in the small intestine where they limit the absorption of the compounds from the gastrointestinal tract, in the bile duct canalicular membrane to promote the biliary drug excretion, and at the blood:brain barrier where they limit uptake of drugs into the brain. The investigation will be to determine whether the ABC G2 transporter has an effect on the uptake of antipsychotic drugs. This will be done by showing mitoxantrone accumulation in Mcf7-FLV cells, which overexpress ABC G2 proteins, is higher than non-treated MCF7 cells, implying that ABC G2 proteins have an effect on the uptake on the antipsychotic drugs.

Description of work: First step was to determine that the cell line MCF-7 FLV had overexpressed ABC G2 proteins compared to MCF-7 non treated cells, which was done by SDS page electrophoresis and western blot. Once this was determined both cell lines were treated underwent functional assays and cytotoxicity assays using the compounds phenothiazine, thioxanthene and butyrophenone, which are the molecular basis of a large range “typical” antipsychotics. For the functional assay cellular accumulation of fluorescent mitoxantrone was measured as an indirect way of telling the accumulation of the compounds. As a control Chrysin was used, as it is a known inhibitor of the ABC G2 transporter.

Alongside the MCf-7 lines, lines of COR L23 DFS and COR L23 ADR cells were set up to extend the invitigation to see if ABC C1 transporters also played a role in antipsychotic drug uptake, COR L23 ADR overexpressing the transporters. Western blot was used to show COR L23 ADR did overexpress ABC C1 and functional/cytotoxicity assays showed the effect of the compounds. For the functional assay Calcium –AM was used as a substrate and Probenicid as an inhibitor.

Functional assay for MCF-7 cells:

96 well plate seeded at 10-15k cells per well and incubated for 48 hours at 37 °C, 5% CO2. Substrates and inhibitors added in DMSO, final concentration not exceeding 0.5%. Incubated for 2 hours at 37 °C, 5% CO2 and washed thrice with cold PBS. Cells fixed with 4% w/v paraformaldehyde for 4 minutes followed by cold PBS wash. Plates read using fluorescence micro-plate reader.

Fig 1: molecular structure of phenothiazine, thioxanthene and butyrophenone (left to right).

Functional assay for COR L23 cells:

96 well plate seeded at 2k cells per well and incubated for 24 hours at 37 °C, 5% CO2. Substrates and inhibitors added in DMSO, final concentration not exceeding 0.5%. Incubated for 30 hours at 37 °C, 5% CO2 and washed twice with cold PBS. Plates read using fluorescence micro-plate reader.

Cytotoxicity assay:

Cells seeded at 2k per well and incubated for 24 hours at 37 °C, 5% CO2. Drug of interest added to each well on plate, concentrations ranging from 0um to 30um, including a DMSO control. Incubated for 4 hours at 37 °C, 5% CO2. Media pipetted off and replaced with 200ul DMEM +10% serum. Incubated at at 37 °C, 5% CO2 until cells with no drug (0um) reached confluence. 20ul MTT solution (5mg/ml thiazolyl blue tetrazolium bromide in sterile PBS) added to each well and returned to incubation for 3 hours. Media removed and replaced with 200ul DMSO to resuspend formazan (by product of MTT oxidation) and optical density read at 600nm.

Results: The results were inconclusive. Throughout the functional assay the florescent readings were never consistent enough to determine if ABC G2 and ABC C1 had a role to play in the uptake of antipsychotic drugs. Initially this was thought to be a fault with the cells, despite the western blots showing overexpression of the transporters in their respective lines, due to known inhibitors of the ABC G2 and ABC C1 not being consistent either. Only once my placement was finished was a fault discovered in the plate reader I was using.

The cytotoxicity showed that the molecules phenothiazine, thioxanthene and butyrophenone being nontoxic towards the COR L23 lines. High concentrations of thioxanthene showed slight toxicity towards the MCF-7 lines

Further directions: Until the uptake of antipsychotic drugs can be confirmed by a consistent set of data then there is very little I can suggest for future based work however the obvious extension to this would be to undertake similar assays with another branch of pharmaceutics, such as sedative drugs for example to see if overexpression of ABC transporters affects all kinds of drugs or if they are more specific. An alternative would be to extend the look at antipsychotics by looking as specific examples such as chloropromazine, fupenthixol or clozapine.

Personal statement: The lack of consistent data to give a conclusion due to the plate reader fault was disappointing and given more time I would like to have completed the investigation when the fault had been found.

Overall I enjoyed my time on the placement. I had a lot of freedom and independence compared to laboratory classes during term and this gave me a greater appreciation of what it is like to be a research scientist and greatly increased my interest in doing a PhD following my degree. I was able to get experience in procedures hadn’t undertaken before, such as in tissue culture and I noticed an improvement in my skills in more routine laboratory techniques which will be of great benefit for my final year research project.

Matthew Brash

Characterising CpG Island (CGI) Chromatin in Murine Mice ES Cells

Background CpG islands are distinct regions of DNA characterised by their high levels of non-methylated Cytosine/Guanine di-nucleotides. CpG islands serve as promoter sites for over two thirds of eukaryotic genes and play an important role in recruitment of specific regulators of the chromatin structure. Regulators are often directly or indirectly involved in histone modifications and can bind through Zinc-finger domains. It has been shown that the CxxC zinc finger domain of the KDM2 histone demethylases will associate only with non-methylated CpG regions, allowing specific targeting of proteins to these regions. The recruitment of such proteins leads to a change in the methylation state of target histone thus altering the local chromatin environment and the transcriptional likelihood of the gene in that region.1

Bio-CAP is an affinity chromatography technique used to isolate digested CpG island DNA or chromatin. Bio-CAP relies on the engineering of an AviTag to the KDM2B demethylase CxxC domain, which then is biotinylated using a biotin ligase. The modified CxxC domain is then attached to avidin beads which form the solid support of the column. The CxxC domain can then capture CpG island chromatin which is eluted with increasing salt concentrations; proteomic analysis is then carried out. 2 Aims

• Optimising protein expressing of the KDM2B CxxC domain by a Rosetta 2 strain of E.coli for use in future Bio-CAP experiments

• Optimising Bio-CAP • Carry out Bio-cap experiment using chromatin extracted from mice ES cells.

The project In an attempt to improve protein expression of the KDM2B CxxC domain we decided to compare codon optimisation against non-codon optimisation. To amplify the provided vector containing our codon optimised gene we transformed it into E.coli XL-10 strain and grew colonies on a kanamycin plate. The CxxC gene was PCR amplified and this was confirmed by running a 0.8% agarose gel which showed two clear bands indicating: the complete vector and amplified gene region. Our CxxC codon optimised gene was to be cloned into an expression pNIC28 vector which contained additional sequence to add a His-tag to the transcribed protein. We adopted ligation independent cloning (LIC) as our chosen method and so treated our CxxC insert with T4 DNA Polymerase (DNAP). We were provided with a T4 DNAP digested pNIC28 vector and attempted our ligation reaction. For ligation we mixed 1 µl of vector with µl of insert and this was added to 100 µl of XL-10 gold E.coli via a 1 minute heat shock treatment. The E.coli were then spread onto a kanamycin plate and left at 37°C overnight, however we did not see any colonies the next morning. In an attempt to overcome our problem of no colonies we: altered the ratio of insert to vector for the hybridisation step, plated different volumes of hybridisation mixture and made fresh insert and vector. Even after trying combinations of newly produced insert/vector with previously prepared vector/insert we were still seeing no colonies produced. We decided that the T4 digestion stage of our LIC was going wrong so: we took a fresh batch of T4 DNAP from another lab, produced new vectors/inserts, ligated and transformed into XL-10 and finally plated. We had produced XL-10 colonies with our recombinant codon optimised plasmid which we then purified through mini-prep and transformed into our E.coli expression strain of Rosetta 2. Codon optimised and non-codon optimised Rosetta strains were grown in 500ml of 2XTY at 37°C until we induced expression of CxxC with IPTG. The cells were incubated at 30°C for 3 hours, sonicated and then mixed with the nickel beads to bind the His-tag on the CxxC proteins. The CxxC proteins were eluted by an increasing salt concentration and analysed on a coomassie stained SDS-PAGE gel. For our Bio-CAP experiments we tested to see if we could get improved output of our chromatin by cross linking the protein and DNA. We had been growing mice ES cells in a medium specifically developed to allow sufficient growth but no differentiation. We harvested these cells 10 plates at a time and used this to extract chromatin for our Bio-CAP experiments. In order to cross link we used formaldehyde which forms polymers between DNA and protein. Our first aim was to get optimised cross linking conditions so we tested 0.1%, 0.25%, 0.5% and 1% formaldehyde, digested with Hinp1 and centrifuged to get

Matthew Brash supernatant with our chromatin. The chromatin from each formaldehyde treated cells had its cross links reverse and DNA purified which was then run on an agarose gel. We next wanted to look at the digestion step in the preparation of our chromatin which had been cross linked. In order to do this we ran an agarose gel of the DNA extracted from Mnase/Hinp1 digestion against 0.1%/0.25% formaldehyde. Hinp1 has a recognition site of GCGC whereas the Mnase will cut randomly. Finally we took the best optimised conditions of 0.1% formaldehyde and Hinp1 digestion and carried out a Bio-CAP experiment. From each of the elutions we ran a silver stain gel as a sensitive identification of proteins, western blots for histone H3, H3k4trimethylation and KDM2A all epigenetic markers and a qPCR. Results + Discussion Our coomassie stained protein gels appeared to show a slightly greater intensity for the CxxC band in the codon optimised compared to the non-codon optimised. However this cannot give us a conclusive answer to whether the CxxC domain expression has been increased by codon optimisation as this may simply be down to the amount of protein loaded and accuracy of coomassie staining. Altering the temperature of incubation or volume in which the cells where grown in may be a suitable future experiment to see if this gives us any difference between the codon and non-codon optimised cells. We chose 0.1% as the most suitable formaldehyde concentration as our protein gel showed this gave us the largest cuts of DNA, which would correspond to poly-nucleosomes being made. We want larger cuts as the CxxC bound beads bind linker regions and will not capture our CpG Island (CpGi) chromatin if they cut into mono-nucleosomes. To further optimise the preparation of chromatin we ran a gel with Mnase/Hinp1 digestest cross linked cromatin at 0.1%/0.25%, and this indicated that a Hinp1 digest with 0.1% formaldehyde was the most favourable conditions. To optimise the digestion step further we may want to attempt sonication of the chromatin and compare this to Hinp1 digested via a DNA gel. Our CAP experiment was not overly successful as we could only see histone H3 in our western blots and not H3K4trimethyl or KDM2A. We would have expected to see all of these epigenetics markers but the cross linking may be causing precipitation problems meaning a lot of the protein is not being captured. Our silver stain revealed many proteins and our qPCR verified that the DNA from our extracted chromatin was CpGi specific. Departures from plan Due to problems with our cloning we did not have time to send the Bio-CAP samples for mass spectroscopy analysis, however from our blots we were not entirely convinced it would have been beneficial. Benefit of the Studentship To me – The studentship was an excellent way for me to use my biochemistry knowledge from my first two years of study and implement this into real experimental situations. I have learnt many new techniques such as qPCR and western blots which has helped me to think for myself when planning an experiment. The most important thing I have learnt is that the majority of time you will not get the results you want first time however with perseverance even the smallest breakthrough feels like a major one. Bibliography 1 Blackledge NP, Zhou JC, Tolstorukov MY, Farcas AM, Park PJ, Klose RJ.. (2010). CpG Islands Recruit a Histone H3 Lysine 36 Demethylase.Molecular Cell. 38 (2), 179-190. 2Neil P. Blackledge, Hannah K. Long, Jin C. Zhou1, Skirmantas Kriaucionis, Roger Patient and Robert J. Klose,. (2011). Bio-CAP: a versatile and highly sensitive technique to purify and characterise regions of non-methylated DNA. Nucleic Acids Research. 40 (4), e32.

Matthew Jacobs – Biochemical society summer studentship report 2012

Superoxide anion production in algae and current generation in Bio-photovoltaic device

Brief background and overview Bio-photovoltaic (BPV) devices generate current by capturing exogeneous electron (e-) from photosynthetic organisms1. Understanding how electrons are released from the cells can help increasing the efficiency of the device. The overall purpose of this project was to improve understanding of the mechanisms by which e- are released from algae. Electrons are believed to be released from the cytosol into the apoplastic space in the form of superoxide anions (O2

●-) via the catalytic activity of plasma membrane (PM) localised NADPH oxidases (NOX). They catalyse the extracellular O2

●- production, a short lived species, believed to be a key charge mediator in BPV devices. Furthermore, the cell wall also includes important components in the pathway of O2

●- release, acting as a barrier between the apoplastic space and the extracellular space. Here we used Chlamydomas as a model organism to investigate whether cell wall is important in O2

●- generation in algae. Previous work in the lab has shown that O2

●- production positively correlates with power output from the BPV device in another algal species, Phaeodactylum tricornutum. Furthermore, different growth regimes showed different O2

●- production (light-dark grown cultures generates more O2●- than light-light grown cultures). Thus,

quantitative PCR (qPCR) was performed to measure NOX transcripts in algae cultures to see whether the transcripts had been affected. Overall, this work may shed light on the molecular level control of NOX activity. Bioinformatic analysis of diatom RBOL proteins As an initial exercise to gain experience of bioinformatic analysis and to provide information about the sequence and structure of NOX proteins in diatoms, protein sequences from Phaeodactylum tricornutum and Thalassiosira pseudonana were aligned with the canonical H.sapiens NOX protein sequence, homologous proteins in A.thaliana and C.reinhardtii. Sequences were also analysed for the presence of various domains2 and would also potentially be of use for qPCR later. The results of the analysis indicate that there is extensive conservation of structure between the different homologues and similar conservation of sequence within functional domains (e.g. NADPH binding domain). A part of the analysis is shown below as an example (Figure 1). Each enzyme has similar structure, with a similar pattern of TM domains and functional domains (Figure 1b). All enzymes have the same functional domains. The most strikingly unique proteins are those from P.tricornutum with an extra set of TM domains apparent. The exact numbers of domains are difficult to explicitly predict, as different software gives different predictions and a somewhat subjective conclusion must be drawn. In this sense, the bioinformatic analysis was a useful exercise personally.

Figure 1. Bioinformatic analysis of NOX sequences. (a) The overall results of the analysis were used to construct this diagram, showing the relative structures and conserved domains of the enzymes. The aligned positions of the domains are not absolute, as the proteins are of quite different sizes, but the arrangement helps to show the similarities. (b) Comparison of FAD binding domains of the different NOX isoforms, with conserved residues highlighted in Clustal colours. The first 5 RBOH entries are from Arabidopsis.

Superoxide anion production in C.reinhardtii strains grown in TAP In order to assess the role of the cell wall in the O2

●- production, different strains of C. reinhardtii were investigated – WT (possessing a ‘normal’ cell wall) and the cell wall deficient strains, cw15 and cw92. These cells were grown until mid-exponential phase and a O2

●- assay was performed using XTT, an organic compound which reacts with the unstable O2

●- to form a stable orange formazan which can be accurately measured

(a)

(b)


spectrophotometrically. The results suggest that cell wall deficient mutants (cw15 and cw92) produce more O2●-

than WT initially. However, the kinetic between WT, cw15, and cw92 becomes different. Over time, O2●-

production in WT increases exponentially whilst the cw mutant O2●- production becomes saturated (Figure 2:

blue, yellow and red data). Together, these data suggest that the cell wall does have a role in releasing O2●- into

the extracellular environment. Furthermore, O2

●- production was also measured for the sta6 mutant in C. reinhardtii. In this mutation, the C. reinhardtii fail to accumulate starch because it lacks the small subunit of a heterotetrameric ADP-glucose pyrophosphorylase3. However, it was discovered that this mutant also showed a decrease in NOX1 and NOX2 transcripts based on RNAseq data (unpublished data). Therefore we would predict that O2

●- production will be impaired in sta6 mutant compared to other strains. As predicted, we found that the O2

●- production is lowered significantly (Figure 2, green line).

Figure 2. Superoxide production time course for all four C.reinhardtii strains; wt, cw15, cw92, sta6. The relative kinetics of wt and the cw mutants suggests the cell wall is of importance, whilst the NOX deficient sta6 mutant shows little production, as predicted.

qPCR analysis of NOX transcripts in P.tricornutum Previous work from the lab has shown that P.tricornutum cells produce more or less O2

●- when grown in diurnal or constant light conditions respectively. This provides an opportunity to study control of O2

●- production at the transcriptional level, by quantifying relative expression of NOX genes in cells grown under the different light regimes. Significant time was devoted to optimising RNA extraction from P.tricornutum, with various protocols. Eventually, I successfully extracted RNA from Phaeodactylum (Figure 3a). Little time remained to perform cDNA synthesis and the qPCR reactions, although preliminary results show that the primers chosen and indeed all aspects of the qPCR seem to work well, with problems arising only in the RNA purification and cDNA synthesis stages. The melt curve shown below (Figure 3b), for a representative housekeeping gene (Histone 4; H4) shows single peaks, indicating a single product as a result of adequate specificity of primers.

Figure 3. RNA extraction and qPCR analysis of NOX transcript in Phaeodactylum. (a) RNA extraction from Phaeodactylum using Tri-reagent (left) or LiCl protocol (right). In both diagram, a DNA ladder is loaded in the left hand lanes, followed by 3 different RNA samples. (b) A typical melt curve from a preliminary qPCR run, for the product from the H4 primers. Other reference genes and the target genes showed a similar curve, suggesting that the primers are specific and only produce a single product in each case.

(a) (b)


Conclusions and possible future work Overall, each objective attempted has yielded some useful information. The bioinformatic analysis has highlighted remarkable conservation between NOX homologues, but also differences between more closely related species. Objective 2 has provided useful data for future work assaying C.reinhardtii strains grown in TP+, experiments which will indeed be performed in the near future in this laboratory. The XTT assays from TAP grown C.reinhardtii have yielded arguably the most important results, such as providing strong evidence for the role of NOX in O2

●- production and providing additional information regarding the function of the cell wall in O2

●- production. It would perhaps be valuable to conduct XTT assays in the presence of a NOX inhibitor, to further strengthen the role of NOX. Furthermore, O2

●- production of complemented sta6 mutant (sta6/STA6) should be investigated. Additionally, running XTT assays over a timescale more appropriate to BPV would be useful. Future qPCR work to complete objective 5 will be greatly assisted by what has been achieved so far. This project has provided a unique opportunity to experience a leading research environment and learn invaluable skills and techniques, whilst providing useful data for the laboratory. It has been challenging, rewarding and most importantly, enjoyable. Reference 1. Bombelli, P., Bradley, R.W., Scott, A.M., et al (2011) Quantitative analysis of the factors limiting solar power transduction by Synechocystis sp. PCC 6803 in biological photovoltaic devices. Energy Environ. Sci., 4, 4690-4698 2. Hervé, C., Tonon, T., Collén, J., et al (2006) NADPH oxidase in Eukaryotes: red algae provide new hints. Curr. Genet., 49, 190-204 3. Zabawinski, C., Koornhuyse, N.V.D., D’Hulst, C., et al (2001) Starchless mutants of Chlamydomonas reinhardtii lack the small subunit of heterotetrametic ADP-glucose pyrophosphorylase. J. Bacteriol., 183, 1067-1077

Biochemical Society Student Report 2012 Matthew Ratcliff

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Is the response of the zinc transporter ZnT5 zinc specific of promiscuous?

Supervisor: Professor Dianne Ford, Institute for Cell & Molecular Biosciences, Newcastle University

Background: Zinc is an essential micronutrient. ZnT5 (SLC30A5) is a bidirectional zinc transporter [1] present in the apical membrane of human enterocytes [2]. It has already been shown that the expression of ZnT5 is reduced in response to an increase in zinc. A recently discovered ZTRE sequence (zinc transcriptional response element - CACTCCC(CC)GGGAGTG) has been shown by EMSA and reporter gene experiments to be responsible for a reduction in expression by acting as a binding site for a repressor protein [3]. Other metals have not yet been tested to see if they can also affect expression of ZnT5 though the ZTRE sequence.

MTF1 is a transcription factor that increases expression of some genes in response to zinc, including those encoding many variants of the small intracellular zinc-binding protein metallothinein. Microarray analysis of mRNA identified a raft of genes that became more responsive to an increase in zinc concentration under conditions of MTF1 knockdown, which led to reduced expression of metallothinein. We hypothesised that this reduction in metallothinein expression reduced zinc buffering capacity within the cell and so increased the sensitivity of genes regulated through MTF1-idependent mechanisms to zinc. We thus predicted that the ZTRE would be over-represented in the panel of genes that become more responsive to zinc under conditions of MTF1 knockdown.

Aims: 1. To determine if transcription of ZnT5 is regulated by other metals, as well as by zinc, through

the ZTRE sequence 2. To determine if metals other than zinc can affect levels of ZnT5 mRNA in Caco-2 cells

(human intestine) using RT-qPCR 3. To search for the ZTRE sequence upstream of the coding sequence of genes that became

more zinc responsive when zinc buffering by metallothinein was reduced by MTF1 knockdown

Description of work and Results: Cell culture, transfection and metal treatments - Caco-2 Cells were maintained at 37ᵒC with 5% CO2

in air. Cells were grown in plastic flasks using Dulbecco-Modified Eagle’s Medium (DMEM) that included 4.5 g/l glucose and Glutamax. The medium was supplemented with 10% foetal calf serum, 1% non-essential amino acids and 60µg/ml gentamicin. Confluent cells were first digested using trypsin before being seeded into 6 well plates at a cell density of 3.5 x 105 cells per well (cell density calculated using a haemocytometer). Seeded cells were then transfected 24 hours after seeding using Genejamer reagent (Stratagene). The transfection mix per well was 4µl Genejamer, 2µg plasmid construct and 100µl serum-free medium. Metal treatments were applied in serum free-medium 24 hours after transfection. Plasmids used for transfection were a ZnT5 ZTRE promoter reporter-gene construct, mutant (non-functional) ZTRE promoter reporter gene construct and MT2A promoter reporter-gene construct (positive control).

Reporter gene assay – Lysis buffer (0.25M Tris-HCL, 0.25% (v/v) Nonident P40 and 2.5 mM EDTA) was used to prepare cell lysates for reporter gene assay. The cells plus lysis buffer were then frozen at -20ᵒC for 30 minutes then thawed at room temperature before being centrifuged to remove cell


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debris. Chlorophenol Red-β-D-galactopyranoside (CPRG) was used as the substrate to measure β–glactosidase enzyme (reporter gene) activity. Any change in absorbance due to the release of cholophenol red from enzyme activity was measured at 560 nm. Protein concentration of cell lysates was measured using Bradford reagent so absorbance measurements could be expressed relative to total protein concentration. Unfortunately transfection was unsuccessful on numerous occasions. However, follow on experiments by another student using fresh Caco-2 cells revealed that none of the metals tested have an effect on the ZnT5 promoter-reporter gene plasmid construct (Figure A).

RT-qPCR – Caco-2 cells were treated with zinc, copper, cobalt and nickel at concentration shown (Figure B) before RNA was extracted, reverse transcribed then analysed by RT-qPCR. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a reference gene. RNA purity was tested using a Nanodrop spectrophotometer and the presence of DNA after reverse transcription was confirmed using PCR. PCR products viewed on agarose gel stained with ethidium bromide. Analysis of data by one-way ANOVA indicated no statically significant differences (Figure B). These experiments however, were insufficient to allow us to conclude that ZnT5 does not respond to metals other than zinc because the positive-control response (a reaction in the mRNA level at higher zinc concentration) was not observed.

Bioinformatic analysis - 1000 bases upstream of the first exon plus the 5’UTR of each gene that became more responsive to zinc under conditions of MTF1 knockdown was searched for the ZTRE sequence (CACTCCC(CC)GGGAGTG) and alternatives that have been confirmed to bind the repressor protein by EMSA (C-(A/C)-C-(T/A/G)-C-C-(C/T)-(N)n-(G/A)-G-G-(A/T/C)-G-(T/G)-G with one mismatch per half of the palindrome allowed).The search was carried out using the algorithm Fuzznuc (http://www.hpa-bioinfotools.org.uk/pise/fuzznuc.html). As the number of bases that can be tolerated between each half of the palindromic ZTRE is unknown each half was used in a separate search then genes were then classified as having either both, one or none of the ZTRE halves. If candidate genes had transcript variants transcript variant 1 was used in the searches.

Out of 109 genes the full ZTRE sequence (present as two halves) was identified in 49. 46 genes had half the sequence and 14 had no identifiable ZTRE sequence (Table 1). The vast majority of genes (87%) had a full or half ZTRE. However, this number is no greater than expected by chance in 1000 bp of random DNA sequence. Based on this analysis it thus appears that the genes shown to be more responsive to zinc under conditions of MTF1 knockdown did not respond through a ZTRE-mediated mechanism. A more robust statistical analysis would be to search the same number of genes that did not become more responsive to zinc when MTF1 expression was reduced. It would then be possible to determine if this number was statistically lower than 49, or 46 in the case of genes with only half a ZTRE. An alternative refinement of the bioinformatics analysis could be to reduce the size of the upstream region in which the occurrence of the ZTRE is considered to indicate a likely functional role.

Future directions: Future experiments should determine if both halves of the ZTRE palindrome are required for functionality. If they are then maximum distance that can be tolerated between each half of the ZTRE palindrome whilst still retaining its functionality should then be identified. A refined bioinformatics analysis could then be carried out on the basis of this new information.

http://www.hpa-bioinfotools.org.uk/pise/fuzznuc.html


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Value to the student: I learned a lot and really enjoyed my summer placement. I am now much more able to read and understand scientific papers and working with the PhD students and researchers helped me learn a lot about future career pathways and what to do next. I feel much more confident in the lab following protocols, carrying out experiments and solving problems which no doubt will help me in my third year project next year. The placement let me gain lab experience and observe/carry out many molecular biology, cell biology and cell culture techniques which are essential to move forward onto a PhD course.

Value to the lab: The ZTRE searches identified the likely occurrence in 95 genes that can be revisited once more is known about the specific criteria that defines a functional ZTRE sequence. Also the 14 genes with no identifiable ZTRE sequence that were none the less affected by MTF1 knockdown could be used a s the basis to explore other potential metal-sensing mechanisms.

References: 1. Valentine, R.A., et al., ZnT5 variant B is a bidirectional zinc transporter and mediates zinc

uptake in human intestinal Caco-2 cells. J Biol Chem, 2007. 282(19): p. 14389-93. 2. Cragg, R.A., et al., Homeostatic regulation of zinc transporters in the human small intestine

by dietary zinc supplementation. Gut, 2005. 54(4): p. 469-78. 3. Coneyworth, L.J., et al., Identification of the human ZTRE (zinc transcriptional regulatory

element) - a palindromic protein-binding DNA sequence responsible for zinc-induced transcriptional repression. J Biol Chem, 2012.

Fig A) Reporter gene assay results for Caco-2 cells transfected with a ZnT5 promoter reporter plasmid. Activity of the promoter was measured as β-galactosidase (reporter gene) activity in cell lysates. Enzyme activity is expressed relative to protein concentration. Data are shown as mean ± SEM normalised to control condition (no metal treatment or transfection) n=3-9 [3].


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Fig B) ZnT5 mRNA levels in Caco-2 cells measured by real-time qPCR after different metal treatments and normalized to the "housekeeping gene" GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Data are expressed as mean ± SEM (n=5). Analysis of data by one-way ANOVA indicated no statically significant differences.

Full ZTRE Half ZTRE No ZTRE Half or Full ZTRE 49/109 46/109 14/109 95/109

Percentage 45 42 13 87

Table 1) Summary of identified frequency of occurrence of the ZTRE sequence in genes that become more responsive to zinc under conditions of MTF1 knockdown. “Full ZTRE” indicates both halves of a ZTRE present in either order;” Half ZTRE” indicates either half present, “no ZTRE” indicates no identifiable ZTRE sequence. Results are for all known possible ZTRE functional variants C-(A/C)-C-(T/A/G)-C-C-(C/T)-(N)n-(G/A)-G-G-(A/T/C)-G-(T/G)-G with one mismatch per half of the palindrome allowed and no limit applied to the allowed distance between the two half-sequences.

0

0.5

1

1.5

2

2.5

Untreated Zinc 3µM Zinc 100µM Copper20µM

Cobalt100µM

Nickel 20µM

mR

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f ZnT

5

Cell treatmeant

The Cross Talk Between Apoptosis & Autophagy Nicolette Moreau, Undergraduate at the University of Bristol

Supervisor: Dr Jon Lane, University of Bristol Additional Thanks to Tom MacVicar, PhD Student at the University of Bristol

Aims • To investigate the behaviour of RPE1 cells after mitophagy stimulation. • To examine the apoptotic response of the RPE1 cell line after mitophagy has occurred. • To determine the importance of mitochondria on autophagosome formation

Background

Mitochondria are organelles found within most cells. They have a number of diverse roles and are known to be significant in the production of energy, via oxidative phosphorylation, and cell survival. It is also known that they play a major role in various cell death mechanisms including autophagy and apoptosis, both of which aid in destroying unwanted cells or cytoplasmic constituents. Recent discoveries suggest that autophagy and apoptosis share some regulation mechanisms, allowing cell adaptation to changing environmental conditions [1].

Mitophagy is a highly specialised and critical form of autophagy that removes damaged or unwanted mitochondria from the cell, thereby preventing further disastrous impacts. It is likely that cells are able to monitor their levels of mitochondrial damage and regulate mitophagy in response to this. Mitophagy has also been shown to be important in some forms of Parkinson’s disease where a mutation in the Pink1/Parkin pathway decreases the effectiveness of mitophagy and leads to a diseased phenotype. The Lane lab has developed several cell lines that stably express Parkin and these remove their mitochondria when exposed to the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The following experiments use human retinal pigment epithelial cells with yellow fluorescent protein labelled Parkin (RPE1-YFP Parkin), as these respond particularly well to treatment with CCCP. Approximately 90% of the cells lose all their mitochondria after addition of CCCP for 24 hours. This cell line can then be maintained in culture once the mitochondria have been removed (as long as it is provided with glucose).

Initial Experiments

• Maintain RPE1-YFP Parkin cells using appropriate, sterile cell culture techniques.

• Confirm that Parkin is recruited to mitochondria after 1hr CCCP treatment (as found by others previously). The co-localisation between Parkin and mitochondria is shown in Figure 1.

• Live Cell Imaging of Parkin recruitment in CCCP treated cells.

Figure 1 – Co-localisation of Parkin and Mitochondria 1hr after treatment with CCCP. RPE1 cells expressing YFP-Parkin (green) were then fixed with anti-COXIV antibodies (red). Cell nuclei were labelled with DAPI (blue).

Determining the Importance of Mitochondria in Autophagosome Formation

Previous evidence has suggested that mitochondria may be important sources of membrane in autophagosome formation. Mitochondrial damage can also activate autophagy. The autophagic response in cells lacking mitochondria was tested to determine the requirement for mitochondria in autophagy induction.

Wells of cells, both with 1hr CCCP and without, were incubated under different conditions. Cells were incubated in full medium (fed) or starvation medium to induce autophagy, in the presence of Bafillomycin A1 to inhibit lysosomal degradation and block autophagy. Coverslips were then fixed and stained with anti-LC3 (an autophagosome marker) and puncta numbers counted. Results suggested that cells containing healthy mitochondria gave a more robust autophagy response, but these results will need to be validated. One way of doing this is to immunoblot for lipidated LC3. This was attempted, but results were inconclusive.

Determining the Effect of Mitophagy on Apoptosis

Various apoptotic inducers (Staurosporine, Anisomycin, Fas Ligand, and TNF) were used on cells not treated with CCCP to determine which methods of apoptotic induction were effective with the RPE1 cell line. It was hoped that this experiment would aid understanding of the method of apoptosis used by this cell line. PARP cleavage was originally used to see which cells were undergoing apoptosis but this was not successful, so nuclear morphology (DAPI staining) was used instead.

Staurosporine was the only apoptotic inducer that was effective. Control and CCCP treated cells were incubated with staurosporine for 4, 6 and 24hrs. They were then fixed and stained with HSP-60 to correlate cell death with mitochondrial content. Unfortunately too few apoptotic cells could be observed at any time point, for al conditions. This may be due to the unresponsiveness of the cell line to apoptotic inducers or the apoptotic cells being lost in the fixing process. It was concluded that the RPE1 cell-line may not be the best model to study mitochondrial apoptosis.

Figure 2 – Example fields of cells stained with HSP60 from the mitochondrial “repopulation” assay. Cells were labelled with anti-HSP60.

Recovery of the Mitochondrial Population after CCCP Addition

The aim of this experiment was to monitor cells depleted of mitochondria by CCCP treatment, over an extended period to observe whether they could be repopulated with mitochondria through biogenesis from a residual template. CCCP was added to the cell line and coverslips removed from this periodically and fixed in methanol. They were then treated with anti-HSP60 antibody, which labelled the mitochondria, and DAPI. New mitochondria were distinguished from mitochondria originally not removed by CCCP treatment as those not removed would not have Parkin recruited to them. Initial experiments looked promising as images taken at 9 days appeared to show vastly increased numbers of mitochondria in cells expressing Parkin. In addition, the mitochondria seemed to be assembling into networks within the cells (Figure 2). The experiment was repeated over 12 days to see if this phenomenon could be seen again. Although a similar effect was noted, it was concluded from the second experiment that it was most likely to be due to the non-parkin expressers repopulating the culture and not due to mitochondrial biogenesis after all. The network structure seen could be explained by the fact that the media may have acidified over time, indirectly causing changes in mitochondrial dynamics. There is potential for this to be repeated to confirm these results by changing the media frequently. This may provide interesting avenues for future research.

References: 1. Cheng, J. P. and Lane, J. D. (2010) Organelle dynamics and membrane trafficking in apoptosis and autophagy. Histol Histopathol. 25, 1457-1472

Value of the Studentship

The summer internship allowed me to gain experience researching and implementing methods and techniques I had not previously come across. It also gave me the opportunity to experience creating protocols from scratch, a vital experience that is not possible in an undergraduate laboratory situation. I was presented with the chance to use new equipment, work alongside postgraduates and pursue original research. Additionally, I was able to see the administration that is involved in running a lab, such as ordering stock and health and safety, and was also invited to attend presentations and journal meetings. I now see the importance of collaborations between laboratories and was thrilled to have my desire to work in research confirmed by this internship. I also believe that my presence aided the laboratory by providing an extra pair of hands and enabled a PhD student to gain experience in a teaching and supporting role.

The experience I had on this placement was fantastic and I am extremely grateful that I was given this opportunity.

Quantitative PCR to Determine Mitochondrial DNA Content of the Cells after CCCP treatment

Following on from the previous experiment, Q-PCR was used to determine whether CCCP treatment was removing the entire mitochondrial DNA content, or only most of it. Potentially it is very interesting as a small amount of mitochondrial DNA remaining may allow future mitochondrial biogenesis.

DNA isolated from CCCP treated and non-CCCP treated cells was ineffective. Time limitations meant that this could not be repeated.

Techniques

• Tissue Culture • Cell Imaging • Immunofluorescence • Western Blotting • Quantitative PCR • Metamorph Imaging • Widefield Microscopy

Analysis of novel Pleckstrin Homology domains

Introduction: Signaling pathways are essential for proper cell function by regulating protein transport and targeting to the correct place and time which is usually required for protein activation. Pleckstrin Homology (PH) domains are present in many proteins and facilitate membrane localization. The primary sequence of the PH domains is approximately 120 residues which are eventually folded into a 7 beta sheet sandwich capped by an alpha helix at the C-terminus. The variable loops formed between the beta sheets bind many ligands including Phosphatidylinositol Phosphate (PIP) lipids and are thus involved in pathways such as PI-3-P and PI3-K. Therefore it is important to identify PH domains and understand in detail their function. However, PH domains possess little primary sequence homology.

Research objectives/Aims: Our aim is to identify and study the basic cell biology of all the yet unrecognized PH domains in budding yeast (S. cerevisiae). Bioinformatics tools aligning primary sequence only (BLAST) have been effective up to this point in finding PH domains homologues. Here, other tools to calculate the secondary structure will be engaged in order to identify previously undetected PH homologues. Subsequently, the cell biology of these new domains will be studied in brief by observing the localization patterns of the GFP tagged PH containing proteins.

Description of the work carried out:

We used PFAM and SCOP databases to dissect the PH super family and classify PH-like domains into 15 categories each with quite separate sequence characteristics. We performed searches with each category of PH domain using many tools such as Psi-blast, HHpred, PHYRE, SMART, and FFAS to predict novel homologues PH domains. The most promising tool was HHpred, which was used as the primary tool. We cloned the new PH domains by standard techniques into a GFP-tagging plasmid. The plasmids containing the PH domain were then transformed to yeast cells and left to grow in an incubator for 48 hours. Finally, cells were grown in liquid culture to mid-log phase, before being imaged by confocal microscopy (Leica AOBS

Figure 1: modeled structure of a PH-like domain (3OHM)

SP2 confocal microscope). This procedure was repeated during the course of 8 weeks for all novel PH domains. In cases of possible weak intracellular targeting, PH domains were multimerised (dimers and/or tetramers), and re-observed for clear localization.

Results and discussion: Seeding HHpred with a classical PH domain and a representative for each class of PH domain we increased the number of PH-like domains in yeast from 71 to 90, with some of the new ones occurring in proteins already known to contain one or more PH domains already, but many appearing in proteins not known to contain PH domains. The most interesting occurrence of a new PH domain was in the membrane trafficking protein Vps13. We cloned all the new PH domains that might be involved in membrane trafficking and checked for their localization using confocal fluorescence microscopy (for example, Figure 2).

Deviations and future directions:

We had some deviations from the original plan, as we did not have time to recognize ligand sites of the PH domains, since our research expanded (from 9 to 19 new domains), and we began to focus on a new analysis of the whole PH domain super family. We have created I believe a scaffolding of the PH domain super family. This work opens the horizons for many questions to be researched, starting with the search for ligands, using single point mutagenesis. We have also found homologues of Gyp7 in the human genome, so that we can extend our knowledge from novel yeast PH domains to novel human PH domains, and find implications and roles for the PH domains in the human proteome and how they are involved in health and disease. This will increase our understanding and will give us a more complete and functional idea about PH domains.

Value of studentship: This was my first experience in real laboratory environments, it was something new, exciting and in many ways different than anything that I was taught at the university. Although I improved on many techniques I learned at the university labs, I also learned that techniques can be optimized, and adjusted according to your experiment. I believe

Figure 2: VPS13 PH domain dimer and tetramer (as indicated) tagged with GFP and observed by confocal microscopy. Punctate targeting (absent from monomer, not shown) is weak with the dimer and very strong with the tetramer.

DIMER TETRAMER

being around experienced people and especially my supervisor helped me widen my perspectives and changed a bit my perception of science. For now I know that it is not a world of undeniable facts proved by older generations, but rather a world of theories and proven experiments that can be challenged and changed anytime. So finally I would like to thank the Biochemical Society and Dr. Tim Levine for allowing me to have this great opportunity, because the biochemical student vacation experience will help me this year to ease my transition into my third year project, whilst encouraging me to start my research career by advancing directly to a PhD program.

Summer Studentship Report to the Biochemical Society

Student: Phil Rowell

Supervisor: Dr Julie Burns, Leeds Institute of Molecular Medicine (LIMM)

Validation and Characterisation of FGFR2 Activating Mutations in Human Urothelial Carcinoma

Introduction and Background

Fibroblast Growth Factor Receptor 2 (FGFR2) is a member of the Fibroblast Growth Factor Receptor family. It is located on the long (q) arm of chromosome 10 in the position indicated in figure 1 below.

Figure 1. Position of FGFR2 in the q arm of chromosome 10

Defects in FGFR2 are of clinical significance in a number of Craniosynostosis syndromes, such as Apert syndrome and Pfeiffer syndrome, and alterations in its expression levels are implicated in the progression of some cancers.

Project Aims

The Knowles lab at LIMM specialises in the investigation of human urothelial carcinomas. Much work has been done on the effects of mutations to FGFR1 and FGFR3, but the role of FGFR2 has been little studied. High Resolution Melts (HRM) carried out on DNA from tumor samples had identified a number of point mutations (E134K, Y376C, N550K and H683Y) in the FGFR2 gene. The aim of this project was to characterise these mutations as activating or non-activating. This was to be done by mutating wild type FGFR2 and inserting it into a retroviral vector construct, which was then to be used in the transduction of NIH3T3 mouse fibroblast cells and immortalised normal human urothelial cells (NHU-TERTs).

Work Carried Out

The first stage of the project involved cloning a wild type (WT) FGFR2 sequence. cDNA copies of wild type FGFR2 were produced from normal human urothelial cell mRNA, using Superscript II reverse transcriptase and oligo-dt primers. The cDNA samples were amplified by PCR with a high fidelity DNA polymerase (Stratagene’s Easy-A), and products analysed by agarose gel electrophoresis. Correct sized bands (~2600bp) were identified, gel-purified and ligated into Promega’s pGEM-T Easy plasmid vector. The ligated vector was then used to transform XL-1 Blue competent cells, then recombinants selected with ampicillin and IPTG/Xgal for blue-white colour screening.

Recombinant colonies were selected and grown overnight as mini-preps (5mL) in LB broth with ampicillin. Cell pellets were subsequently spun down, re-suspended, lysed, and plasmid DNA extracted. Small samples of each plasmid were digested with Not1 (a restriction enzyme that cuts sequences at either side of the pGEM insert site), and digest products run on agarose. Samples with inserts were subject to ethanol precipitation of their

FGFR2 comprises three extra-cellular Ig-like domains, a membrane spanning region and two cytoplasmic tyrosine kinase (TK) domains. On ligand binding, FGFR2 dimerises, becomes active through transphosphorylation, then mediates downstream signalling in the cell. The normal function of such signalling is important in processes that include cell division and growth, wound healing and embryonic development.

DNA, followed by capillary electrophoresis sequencing using 3.2μM primers and Applied Biosystems’ BigDye Terminator Cycle Sequencing kit. A wild type FGFR2 gene clone was identified.

The next step involved site directed mutagenesis (SDM) of wild type FGFR2 to generate the individual mutations. PCR reactions, using WT FGFR2 and primers containing the respective mutations, were carried out to produce mutated product, which was used to once again transform XL-1 Blue competent cells. Plasmid DNA was extracted from selected colonies and sequenced across the sites of expected mutation to check that SDM had been successful.

Once the mutated sequences had been confirmed, mutant and WT FGFR2 clones were subcloned from pGEM and inserted into a retroviral backbone vector (pFBneo) using Clontech’s In-Fusion HD Cloning methodology. After a final round of XL-1 transformations, colonies were screened for both plasmid and insert, then mini-preps, plasmid extracts and sequencing carried out to confirm integrity. Maxi-preps (150mL) of selected colonies were grown, then plasmids were extracted and used to transform a retro-viral packaging cell line, Phoenix Ampho (PhA). Retrovirus (based on Murine Moloney virus) was subsequently harvested from the PhA culture medium supernatant and frozen down ready to transduce the target cell lines, NIH3T3 and NHU-TERT.

Departures from Original Plan

There were no departures from the plan itself, but time constraints meant that the work was not fully completed.

Future Directions

NIH3T3s and immortalised normal human urothelial cells (NHU-TERTs) will be transduced with mutant FGFR2, WT FGFR2 and empty vector, then tested biochemically and phenotypically for activation. Unfortunately, time did not allow for me to carry out this work over the summer, but my ‘visiting research assistant’ status has been extended so I can return and continue the work during term time.

Characterisation of these mutations as activating should be followed proteomic analysis, comparing the protein content of mutant forms with wild type. Results from these investigations will then hopefully lead to the identification of new therapeutic targets for the treatment of urothelial carcinoma.

Outcomes

I enjoyed every moment of my time at LIMM. Aside from the techniques I learnt and mastered, the experience I gained and the knowledge passed on to me by my supervisor and the other scientists I worked with, I found out what it is really like to be a research scientist. Although the description above makes it sound as if the work was carried out in a neat, linear fashion, the reality was actually one of ‘three steps forward and two steps back’ all the way through. It was the ‘two steps back’ parts that were the most interesting – this was when unexpected results occurred, problems had to be solved, options explored, procedures optimised. I enjoyed the sense of overcoming obstacles and seeing the work progress. It has convinced me that I have the right temperament and problem solving mentality to follow up my undergraduate studies with a PhD, hopefully leading to a successful career in research. I would like to thank the Biochemical Society for funding me in this fantastic experience.

Biochemical Society Studentship Report – Summer 2012

Prerana Huddar, University of Edinburgh ([email protected])

Project conducted at the Human Genetics Unit, Edinburgh under the guidance of Professor Nick Gilbert

Project title:

Developing novel approaches for structure determination of DNA and chromatin

Background information:

In mammalian cells, DNA is wrapped around histone proteins to form nucleosomes, which are then further folded into solenoidal chromatin fibres. Although we know quite a lot about the fundamental structure of chromatin fibres, due to a lack of suitable techniques, we do not have a full understanding of how they are packaged into higher-order structures. The lab is really interested in developing new techniques for studying chromatin structure, and as a result, there is a collaboration with the Chemistry department who have made new molecules for investigating the supercoiling of DNA. In mammalian cells, DNA helices can become positively or negatively supercoiled (the winding is tighter and looser, respectively) due to the action of polymerases. The new molecules developed (trimethylpsoralen linked to biotin and fluorophores) intercalate preferentially into negatively supercoiled DNA and have been used to map topological domains within the human genome. The experiments conducted in this project use versions of the trimethylpsoralen molecule that has been conjugated to biotin and fluorescein.

Figure 1: Structure of the biotin trimethylpsoralen molecule (bTMP) developed to investigate DNA supercoiling

Aims:

- Establish any binding bias of trimethylpsoralen to a particular form or sequence of DNA

- Establishing and optimising conditions of crosslinking of fluorescein-trimethylpsoralen to DNA

Firstly, in order to determine whether the trimethylpsoralen had a bias towards a certain form of DNA, A, B and A/B intermediate forms of DNA were designed and annealed. The biotin-trimethylpsoralen molecule (bTMP) was then crosslinked to these sequences using UV light.

Similarly, in order to determine whether the trimethylpsoralen had a bias towards AT-rich or GC-rich sequences, both the FTMP and the bTMP were crosslinked to AT-rich and GC-rich sequences.

Methods:

Crosslinking was conducted by incubating DNA with FTMP or bTMP and mixing with TE buffer. The mixtures were then incubated in darkness for a period of time ranging from 20 minutes to 1 hour and then crosslinked in the presence of UV light for a period of time ranging from 10 to 45 minutes To remove the un-crosslinked FTMP from the crosslinked DNA, the mixtures were passed through G25 sephadex columns that were constructed in the lab.

Crosslinking of FTMP to DNA was detected by dot blotting and PAGE. Dot-blotting was conducted using an HRP-linked anti-fluorescein antibody in order to detect whether FTMP had crosslinked to DNA sequences. DNA FTMP cross linked samples were also run on a 15% PAGE gel and FITC fluorescence detected by a phosphoimager, using a filter wavelength of 473nm (to detect any FTMP crosslinked to DNA).

The FTMP molecule was also transfected into RPE1 cells using various approaches: lipofection, oligofection, “happyfection” (a biological polymer-based method) and by simply mixing in the FTMP with cells in order to see whether it would be transported across the nuclear membrane and into the chromatin effectively. This is important for any future work with the FTMP molecule to investigate chromatin structure. Photographs of the transfected cells were taken using a Nikkon Ti microscope.

Results and Discussion:

- To determine the optimum concentration of bTMP to use for crosslinking experiments, differing concentrations of bTMP were cross linked to GC and AT rich sequences. The crosslinked bTMP was detected by using Streptavidin-HRP and an ECL reagent. The amount of crosslinking was quantified using a low-light camera. The values were then plotted to obtain binding curves.

Fig 2: bTMP binding curve

- bTMP was UV-crosslinked to the AT-rich and GC-rich sequences for 10 minutes, as described previously in the “methods” section.

bTMP crosslinks preferentially to the AT-rich sequence and does not crosslink to the GC-rich sequence, establishing the trimethylpsoralen’s bias towards AT-rich sequences.

3mg/ml seems to be the optimum concentration of bTMP to use for crosslinking experiments with the AT sequence. A curve could not be obtained for the GC sequence, as bTMP binds preferentially to the AT sequence.

Fig 3: Dot blot to detect bTMP crosslinking to AT and GC sequences

- bTMP was crosslinked in the presence of UV light to the A, B and A/B intermediate forms of DNA for 10 minutes.

bTMP crosslinks preferentially to the A/B intermediate form, with minimal crosslinking to the A form and no crosslinking to the B form. Mass spectrometric analysis conducted in the lab indicates that psoralen only binds to thymine. Furthermore, the literature shows that psoralen has a preference for thymines that are next to adenines (hence the preferential binding to the AT-rich sequence in the previous result). The A/B intermediate structure has twice the number of thymines than the A and B form sequences do. These thymines are also next to adenines, which would explain the preferential binding of psoralen to the A/B intermediate sequence of DNA.

- FTMP was mixed with A, B, A/B intermediate, AT-rich and GC-rich DNA (in separate experiments) and the resulting mixtures were exposed to UV radiation for time periods ranging from 10 minutes to 45 minutes, in either dH20 or TE buffer, but crosslinking was unsuccessful.

- A new FTMP molecule with a longer linker chain was synthesised and was used for conducting the same experiments that were carried out with the old FTMP. These too were unsuccessful. Since the structure of the molecule was much improved, the inability to crosslink is unlikely to be due to a defect in the structure of the molecule itself; the crosslinking protocol probably needs to be tweaked a bit more.

- Attempts to transfect RPE1 cells with FTMP showed that the most efficient method of doing so was “happyfection” (a biological polymer based method), however happyfecting only managed to carry the FTMP into the cytoplasm, whereas it should ideally have crossed the nuclear membrane in order to crosslink into the chromatin. Since the controls were successful, the inefficiency of the transfection lies with the structure of the FTMP.

Future work:

Future work would consist of trying to develop a crosslinking protocol or a new FTMP molecule that would crosslink to DNA. It would also be interesting to establish why psoralen has such a strong preference for binding to thymines over cytosines, even though thymines are structurally similar to cytosines.

Value of the studentship to the student:

I have really enjoyed working in the Gilbert lab this summer; I have learned many new laboratory techniques and have honed my skills of planning and analysis. My experience of the studentship has also reinforced my desire to pursue an MD or a PhD after my undergraduate medical studies.

Value of the studentship to the lab:

This studentship has provided an invaluable contribution to our current efforts to understand the role of DNA supercoiling in human chromatin. Many of the properties of the psoralen-based molecules we have developed remain uncharacterised. Prerana’s work has established that our bTMP molecule has a strong bias for thymines, independent of DNA structure in the oligonucleotide model she used. Furthermore, a thorough analysis of our new FTMP molecules determined that they are unable to bind DNA. This important observation identifies that these FTMP molecules are currently inappropriate for studying chromatin structure.

UV Crosslinked Not UV Crosslinked

A B A/B A B A/B

Biotin control oligo

Biochemical Society Studentship Report - 2nd July – 10th August 2012 Student: Rachel Patel, 2nd Year Biochemistry undergraduate at the University of Oxford Supervisor: Professor Jerry Turnbull, Institute of Integrative Biology, University of Liverpool Daily supervision by: Chloe Williams, final year PhD student Aims of the Project The project aimed to introduce me to cell culture techniques within the first couple of weeks with the focus on culturing some newly acquired EpiSCs (mouse embryonic stem cells) carrying GFP labelled transcription factors (Pitx3, Lmx or Sox1). The intention was then to emulate a paper published by Jaeger et al in Development (2011, 138, 4363-4374). The authors had developed a procedure to enhance the production of dopaminergic neurons during a differentiation protocol developed by Ying et al (Nature Biotechnology, 2003, 21, 183-186). They observed enhanced production of dopaminergic neurons when an inhibitor of the FGF/ERK signalling pathway was applied for the first 2 days of differentiation. However, applying this inhibitor for 5 days of the protocol resulted in no significant enhancement. We wanted to attempt to reproduce this result and then to use chemically modified heparins in the place of the inhibitor to investigate whether they could produce a similar effect. Measurement of differentiation was to be done by fluorescence microscopy of the GFP markers as well as by immunostaining and qRT-PCR of additional factors. Work undertaken During the first two weeks of the project I developed cell culture techniques using standard mouse ES cells due to a difficulty in obtaining a clear protocol for the culturing of the EpiSCs. I gained experience of thawing ES cells and setting up initial dishes, sub-culturing them throughout the week and keeping a daily eye on their well-being as well as freezing vials of them down when many confluent dishes had been obtained. I also learnt immunostaining techniques using these ES cells, performing the procedure both with supervision and then on my own. This allowed me to put a technique often mentioned in a theoretical context in lectures into practice and the chance to perform the procedure on my own gave me experience of the importance of good timekeeping and of dealing with problems which arose. I stained dishes with primary antibodies against the transcription factors Oct4 and Nanog, which are markers of the pluripotent state. Using fluorescent secondary antibodies I obtained pictures using a fluorescence microscope, examples of which are presented below:

A B Fluorescence pictures overlaid using Photoshop. Nuclear staining by DAPI is in blue. A shows the nuclear expression of Oct4 which was stained using a red fluorescent 2o antibody. B shows expression of Nanog which was stained using a green fluorescent 2o antibody. By the third week of my project the protocol for the EpiSCs had been obtained. My PhD student supervisor and I thawed vials of the EpiSCs (obtained from the Li lab, Imperial College, London) and cultured them throughout the third week in order to increase their numbers. This gave me experience of a different form of cell culture, as the EpiSCs are trypsin-insensitive, and experience of recognising colonies of this alternative cell line. During this growth period I also continued to gain experience of methods using standard ES cells. I performed an RNA extraction protocol with ES cells followed by a cDNA synthesis protocol and set up a

couple of qRT-PCR runs using primers against Oct4 and Nanog. However I was unable to obtain sufficient data to analyse due to a limitation in the availability of the Taq polymerase. The Pitx3 labelled EpiSCs grew extremely poorly from the point of thawing and it proved impossible to encourage them to adhere to the culture dishes. However we grew up healthy colonies of the Lmx and Sox1 labelled cells. During this period we developed modifications to the culture protocol provided, for example we found that splitting the EpiSCs every other day did not allow them to proliferate efficiently, whereas only splitting them every 4 days allowed confluent colonies to emerge. We obtained sufficient dishes to start the 6 day differentiation protocol by the beginning of my fourth week – the intention was to count GFP labelled cells and to perform immunostaining and qRT-PCR on days 2 and 5 of the protocol. Two controls were planned - one which would be undifferentiated and one which would be differentiated without the FGF/ERK inhibitor - then some cells would only be inhibited for the first 2 days whereas others would be inhibited for 5 days of the protocol. Unfortunately all of the Lmx labelled cells had become infected by this time, but the experiment was planned using the healthy Sox1 labelled cells. During the first three days of the protocol we observed that several of the dishes seemed to become randomly infected – rather than just being restricted to one of the experimental conditions the infection was spread throughout all. Unfortunately by the day when the first results were due all dishes were carrying an infection except those we had left undifferentiated, leading to the conclusion that our differentiation medium had become infected. We therefore had to abandon this experiment and make up fresh medium whilst once again boosting the numbers of our remaining cells. However the EpiSCs all died throughout the latter half of my fourth week and so at the start of my fifth week I thawed some more vials and set up new dishes. During this fifth week I found that the Lmx labelled cells I had thawed perished whereas the Sox1 labelled ones did not, leading to the suspicion that the Lmx labelled vials of cells may have harboured an infection from the start. Despite my best attempts to grow up the remaining Sox1 labelled cells I found that they were all dead by the beginning of my sixth and final week. Outcomes and future directions This project provided me with my first real experience of daily lab work. I am really pleased to have gained some valuable experience of techniques such as cell culture which are too time-consuming to be included in the practical sessions of my undergraduate degree. Additionally spending 6 weeks in the lab has allowed me to soak up the atmosphere of research and to get a glimpse into the sort of day-to-day practices which I might undertake if I decide to continue my studies with a PhD. Despite the excellent aims of my project outline it was not possible to perform reliable experiments with the cell lines as they proved to be very difficult to grow successfully. If this project is to be continued by a member of the lab I suggest that fresh EpiSCs are requested from Imperial College as it would appear that at least some of the cells I was working with were carrying an underlying infection. Alternatively if the differentiation of EpiSCs is a line to be pursued I suggest that EpiSC lines could be developed in Liverpool using a protocol for their production from standard ES cells. Although I am still undecided as to the direction I will pursue after my degree, the project has given me a positive insight into what a PhD would be like. I do feel disappointed that the difficulties we encountered with the new EpiSC lines mean I have not gained more experience of data analysis. It would have been satisfying to obtain and interpret more experimental results – the experience has highlighted the important fact that success in research often takes a lot of time and if I had been present in the lab for a longer period more results may have been obtained. Acknowledgements I would like to thank Professor Turnbull for the opportunity to gain six weeks of experience in his lab group and also Chloe Williams for her enthusiasm and invaluable help.

Photo of Adam Whitaker (left) and Dr Tomlinson (right)

Investigation of the ADAM10 interaction with TspanC8 tetraspanin Supervisors Name: Dr Tomlinson My Name: Robin Adam Whitaker

Aim The aim of this project was to understand how ADAM10 interacts with TspanC8 tetraspanins and to map this interaction. A description of the work carried out ADAM10 is a transmembrane metalloprotease that cleaves the extracellular domains of transmembrane targets. ADAM10 is key in development and disease processes and so could be a therapeutic target to treat heart attacks, stroke, cancer and Alzheimer's disease (Saftig and Reiss 2011). Tetraspanins are a superfamily of 33 transmembrane proteins in humans that help co-ordinate transmembrane proteins by recruiting them into tetraspanin-enriched microdomains (Rubinstein 2011). TspanC8 tetraspanins interact with ADAM10 and are essential for ADAM10's enzymatic maturation and cell surface trafficking (Prox 2012: Haining et al 2012). For my project I used 2 main methods

• Biochemistry experiments: For these experiments I cultured the HEK293T cell line, which is a highly transfectable model cell line that is commonly used in the Tomlinson lab to study protein-protein interactions and transfected the cells with certain plasmids to express the proteins. After transfection I lysed the cells and immuno-precipitated proteins of interest out of solution, before western blotting.

• Flow cytometry experiments: For these experiments I cultured HEK293T cells and transfected them with

certain expression constructs before staining to map the epitope on which antibodies bind. An assessment of your results and the outcomes of the studentship As per the original application, the project has not really differed from its main goal. As well as the main project, I also worked on subsidiary projects, for example I tested the extraction of ADAM10 in different buffers and with different antibodies and tested ADAM10 mutant expression constructs. The experiment I will focus on for this report is the result for truncated ADAM10 expression constructs, which were designed to identify the minimal TspanC8 tetraspanin-

binding region. The HEK293T cells were transfected with FLAG epitope-tagged Tspan14 (a TspanC8 tetraspanin) or tetraspanin CD9 (a non-TspanC8 control), in the presence or absence of two HA-tagged ADAM10 truncation constructs, one for the cysteine-rich plus disintegrin domains. Figure 1 shows; that both truncated ADAM10 constructs interacted with Tspan14 but no interaction was observed with CD9. Since the cysteine-rich domain of ADAM10 is common to both constructs, this gives clear indication that the cysteine-rich domain in ADAM10 interacts with the TspanC8 tetraspanins. The whole cell lysate shows the correct expression of ADAM10 and tetraspanin constructs (Figure 1). In additional experiments, the ADAM10 cysteine-rich domain construct was found to interact with Tspan14 as efficiently as full-length ADAM10 (data not shown). Taken together the experiments strongly suggest that the cysteine-rich domain in ADAM10 which is the most membrane-proximal domain and thus with the most potential to interact with the smaller tetraspanins, does indeed interact with the TspanC8 tetraspanins. The next experiment shown aimed to map the epitopes of two anti-ADAM10 monoclonal antibodies on ADAM10 using the truncated constructs used above. The hope with this experiment is that if they bind to the cysteine-rich domain, they could potentially be used to disrupt ADAM10-tetraspanin interactions and therefore to determine the consequences on ADAM10 activity.

Fig. 1: Interactions of Truncated

HEK293T cells were transfected with the two ADAM10 truncation constructs and the cells stained with two different ADAM10 monoclonal antibodies (clone 11G2 from the Rubinstein Lab (Arduise, 2008) and a second commercially avaiable from R&D Systems). In addition, cells were stained with a myc antibody to detect the myc epitope engineered onto each construct. Figure 2 shows; that both ADAM10 antibodies bound to the cysteine-rich plus disintergrin construct and not to the cysteine-rich only construct, as detected by a positive “shoulder” representing the fraction of cells that were successfully transfected. Expression of both constructs was confirmed by myc staining (Figure 2). This data suggest that both antibodies bind to the disintegrin domain of ADAM10, or to an epitope created by the combination of disintegrin and cysteine-rich domains.

Future Directions The lab will carry on mapping the epitope of ADAM10 and its interactions with tetraspanins and has recently confirmed my results and extended them using additional ADAM10 mutant constructs. The lab will now generate soluble forms of the ADAM10 cysteine-rich domain and TspanC8 tetraspanins to test whether they interact with each other using BIACORE and potentially in crystals to obtain structural information. Using a high throughput binding assay the lab will hopefully identify a small molecule inhibitor from a library of 40,000 compounds. If successful they will then use lead compounds to try and affect the interaction between ADAM10 and tetraspanins. Any departures from the original proposal The only departure from the original proposal was the additional experiments aimed at mapping the epitopes where antibodies bound on ADAM10 using flow cytometry. Conclusion After eight weeks of working with Dr Tomlinson, in a fully functioning lab, I now understand what it is like to go into academia. I have learnt a great deal including a variety of techniques which I can now take forward and build upon in my final year and possibly my career. I conversed with a variety of people about their work and attended many lab meetings, this has led me to realise how much opportunity is out there for me. One of the most interesting days in my internship was visiting the animal house with a PhD student. I watched many experiments performed on mice and it has led to a deeper understanding of the experiments that I can do. This internship has opened my eyes to the opportunities out there for Biochemistry undergraduates and spurred me on to a new found enthusiasm for the subject. The data in this report will be used in a BBSRC project grant application that will be submitted in January, 2013. References

1. Haining E.J. et al (2012) 1. The TspanC8 subgroup of tetraspanins interact with A disintegrin and metalloprotease 10 (ADAM10) and regulate its maturation and cell surface expression. J Biol Chem. In press

2. Rubinstein E. (2011) the complexity of tetraspanins Biochem. Soc. Trans. (2011) 39, 501–505; doi:10.1 3. Arduise C. (2008) Tetraspanins regulate ADAM10-mediated cleavage of TNF-α and epidermal growth factor J

Immunol 181 7002-7013

Fig. 2 Flow cytometry for ADAM10 epitope

Ryan Petrucci

Biochemical Society Studentship Report 2012

Title: The Role of Hic-5 in the reorganisation of the Actin Cytoskeleton

Background

Cardiovascular diseases (CVD) including coronary heart disease and stroke are the leading causes of death worldwide. Pathological migration and contraction of vascular smooth muscle cells (VSMC) is known to contribute towards CVD. The actin cytoskeleton (AC), an essential component of VSMC, is involved in contractile responses. Hydrogen peroxide-inducible clone-5 (Hic-5) has been recently identified as a protein associated with and regulating the AC. However, little is known of its mode of activation.

Aims

The project aimed to further characterise Hic-5 tyrosine phosphorylation using a range of techniques to express, purify and explore cytoskeleton interactions of Hic-5 and its non-phosphorylatable construct Hic-5Y60F in mammalian cells.

The initial aim of the project was to quantify the difference in cell size and morphology of groups of transfected cells. This would produce data to strengthen the argument that a specific transfection caused a change in size and morphology. A secondary aim of the project was to extract DNA from cultured bacteria and use this as a plasmid for future transfections. This part of the placement would allow me to learn a new laboratory technique.

Description of work

As one of the main aims of the project was the quantify the size of the transfected MEF cells, I learnt how to use a computer program called ImageJ. This program would allow me to use saved immunofluorescent microscopy images and would map out the outline of each cell and then calculate the area. I would then log these results in Microsoft excel. Once there were sufficient data for each type of transfection I would then statistically analyse the data.

I analysed the data using a program called GraphPad Prism. This program can carry out statistical analyses of data and additionally present the data in graph format. I used this program to present the data to my supervisors during meetings as it visualised the data.

A different aspect of the project was to transfect more MEF cells. These could then be stained using immunofluorescence techniques and analysed. To transfect these cells DNA containing Hic-5, Hic+Src, Y43F and Y43F+Src needed to be produced. To do this, colonies of bacteria were transformed with necessary constructs. I was then shown how to extract the DNA using a MINI and MAXIprep protocol. These processes allowed me to extract the DNA, then subsequently analyse it for purity. The pure DNA could then be used by the team I worked with for future transfections of mammalian cells such as vascular smooth muscle cells.

To analyse the purity of the extracted DNA I used a spectrophotometre, which gave values for DNA content and protein content. A ratio of DNA:Protein was then calculated. A ratio above 1.85 was required so that the DNA was of satisfactory purity.

Results

Interestingly, the statistical analysis of the transfected MEF cell areas showed statistical significance. The group that had been transfected with Hic-5+Src were significantly larger in area and more spreading in morphology compared to the other 3 groups. This tantalising data shows that Src activation of Hic-5 causes a change in its activation status. This change then leads to the reorganisation of the actin cytoskeleton and allows the cell to spread and form lamellipodial extensions. Interestingly, the Hic-5+Src cells were larger and more spreading that the Y43F+Src cells. This showed that Y43 is probably a vital initial phosphorylation site of Hic-5.

The results of the DNA extraction showed the successful process of bacterial transformation and that satisfactory levels of DNA purity could be attained. This was important for the laboratory team as new PhD students will be using this technique to further investigate Hic-5.

Conclusion

My studentship in the Cardiovascular Research Laboratory at the University of Manchester was a fantastic experience. Having previously spent 7 months in the lab for my thesis project, I was very keen to get in to the lab for my summer placement. The team welcomed me warmly and I felt like I was a valued member of the team throughout my placement.

Although some aspects of my project could not be completed in the time frame of the studentship, many were. The cell immunostaining and analysis of cell area were vital aims that were accomplished. Additionally, learning DNA extraction on transformed bacteria was also a worthwhile skill because they can be used long after I have left the lab. Lastly, the studentship also enabled me to learn and use a statistical analysis program, which allowed me to visualise the data and relationships between different groups of data. I feel like that was an important aspect of the summership because it gave me an added appreciation of the importance of the data that I collected.

In conclusion, I thoroughly enjoyed the studentship. It gave me the opportunity to add to my previous lab work, gain new lab skills and feel part of a team. I would like to thank the Biochemical Society of Great Britain for their superb support, without which none of this would have been possible. I would also like to thank my supervisors, Dr Vasken Ohanian, Dr Jaqui Ohanian and Simon Forman for their guidance and support.

Biochemical society summer studentship report – 2012 Sam Crossman

Fig. 2 – The Gal4 UAS system allows for spatial expression of a target

sequence (PcmRNAi).

Novel roles for the exoribonuclease Pacman in growth and differentiation Student: Sam Crossman

Supervisor: Dr Sarah Newbury, Brighton and Sussex Medical School

Background

Differential gene expression at specific times during development is responsible for the transition of a single fertilized egg to a complex multicellular organism. The mechanisms employed by the cell to control gene expression have also been shown to play a significant role in a number of other important processes, including stem cell differentiation and cancer. For many years, it was thought that transcriptional regulation was the main form of gene expression control employed by eukaryotic cells. However, the importance of RNA stability has become increasingly apparent, with some recent studies suggesting that as much as 40-50% of alterations in gene expression come at the level of RNA stability (Cheadle et al, 2005).

In the model organism Drosophila there are two routes of RNA degradation: (i) the exosome; a multimeric complex responsible for degrading RNA in the 3’–5’ direction and (ii) Pacman; a highly conserved progressive exoribonuclease that degrades RNA in the 5’–3’ direction and is present in all eukaryotic cells. Previous work carried out in the Newbury lab has shown that Pacman is involved in a number of developmental processes, including the control of growth and differentiation of the wing imaginal discs which go on to form the adult wings.

In order to identify potential targets of Pacman, my supervisors group has previously used microarray analysis to compare gene expression in pacman mutants and wild type Drosophila. If the expression of a gene is upregulated in the mutant strain then it is possible that the mRNA transcript of this gene is a target of Pacman in the wild type line. One such potential target identified in these early screens is known as simjang.

Proposed objectives of investigation

(1) To build on previous work which used RNA interference to knock-down pacman at different larval stages in order to determine the time points where Pacman function is essential for correct wing development.

(2) To determine whether down-regulation of simjang can rescue the effects of pacman knock-down.

Departures from the original proposal

In the time between the submission of my application and the start of my studentship it became apparent that simjang was unlikely to be a direct target of Pacman and consequently the focus of the study was instead directed towards the primary objective; to determine the stages of development where Pacman function is critical.

Description of work

As aforementioned, work previously completed by members of the Newbury group has involved the knockdown of pacman in the wing discs at particular developmental time points in order to determine when Pacman activity is essential. Knockdown of pacman in this manner is achieved using the Gal4UAS system (Figure 2). Pacman knockdown occurs when an RNA interference construct (PcmRNAi) is expressed. In turn, the expression of this construct will only occur when a separate protein, Gal4, is bound to an upstream activator sequence (UAS). By using Gal4 drivers specific to the wing disc alone, it is possible to knock down pacman exclusively in these regions.

Temporal control is provided by the introduction of Gal80ts, a temperature sensitive inhibitor of Gal4 (Figure 3). When flies are raised at 19oC (the

permissive temperature), Gal80ts will repress Gal4, resulting in no knockdown of Pacman. However, at higher, restrictive temperatures (>29oC) the Gal80ts repressor is inactivated, resulting in PcmRNAi expression and subsequently pacman knockdown.

Fig. 1 – Pacman mutants display both smaller wings (left) and smaller imaginal

discs (right)

Fig. 3 – Gal80ts provides temporal control. At 19oC, Gal80ts is active and knockdown does not

occur. At 29oC, Gal80ts is inactive and knockdown is achieved as PcmRNAi is

Biochemical society summer studentship report – 2012 Sam Crossman

Fig. 4 – Results showing the presence of perdurance within the Gal4-UAS system, first with an actin driver (top) and then using

nubbin (bottom). Western blotting was used (right) to detect the amount of protein present at each time point compared to the Actin

loading control.

As it is known that Pacman function is critical for correct wing development, the above technique was used to knock down pacman in the posterior half of the wing imaginal disc at different stages of development in order to determine when its function is required. The results from these experiments suggest that Pacman is only essential from L2 onwards.

One issue with this technique is the presence of perdurance within the system. As there is an unknown time period between the inactivation of Gal80ts and the subsequent depletion of Pacman protein, it is impossible to determine the specific developmental time points where Pacman activity is required for correct wing growth. Consequently, the majority of my time in the lab was spent attempting to determine the length of this unknown time period to allow for the clarification of a number of previous results.

In order to do this, transgenic flies were moved from 19oC to 29oC before larval samples were taken at various time points following Gal80ts inactivation. Once all samples were collected, the protein was extracted before western blot analysis was used to determine the amount of Pacman present at each time point. First, an actin-GAL4 driver (act-GAL4) was used to knock down pacman across the entire larvae. This provided a basic overview of the time required for knockdown to occur that was used to help with the selection of suitable time points when the experiment was repeated using the nubbin-GAL4 driver which is specific to the wing pouch region of the wing imaginal disc.

Results

The results for my perdurance experiments are included in figure 4. When using the act-GAL4 driver a clear depletion in Pacman protein is observed within 31 hours of GAL80ts inactivation that is comparable to the two negative control samples (0h Gal80- and 24h Gal80-). As the time points used in this experiment provided such a clear trend, similar ones were used for the nubbin-GAL4 repeat. Again, a similar depletion in Pacman was observed, with the amount of protein decreasing to a level comparable to a control sample kept at the restrictive temperature for the entire experiment (29C) within 30 hours.

These results can be used to assist with the interpretation of previous results obtained in the lab. For example, if you take into account the fact that pacman knockdown requires at least 30 hours to occur; it is likely that Pacman is not actually required until the start of L3 and not from L2 onwards as was previously anticipated.

Value of studentship to the student

The entire summer project has been thoroughly enjoyable and has undoubtedly helped me a lot. Spending a prolonged period in an active research environment has given me the opportunity to greatly improve my general lab techniques whilst working with Drosophila has proven fascinating and provided me with a broad range of skills that could be easily transferred to any fly lab. Recommended reading has provided me with a detailed knowledge of a number of aspects of developmental biology as well as assisted me greatly in my ability to analyse and efficiently interpret scientific papers, a skill that I am sure I will find invaluable in years to come. Completing this studentship has provided me with confidence for my third year project and confirmed my desire to complete a PhD upon graduation.

Value of studentship to the lab

The results obtained from my set of experiments will be taken into consideration any time a member of the group uses the Gal4 UAS system to knock down pacman expression and can also be used to support a number of previous results in a similar manner to the example included above in the results section.

References

Cheadle C, Jinshui F, Cho-Chung YS, Werner T, Ray J, Do L, Gorospe M and Becker KG; 2005; “Stability regulation of mRNA and the control of gene expression”; Ann NY Acad Sci; 1058:196-204.

Biochemistry Society Summer Studentship Report 2012 Student: Sammi Allouni, Supervisor: Claire M Perks

Background The insulin-like growth factor (IGF) axis has been demonstrated to play a vital role in the establishment and maintenance of prostate cancer (CaP), the most common non-cutaneous cancer diagnosed in males. Cohort studies established strong links between circulating IGF-1 levels and CaP development and progression risks (1,2). The IGF binding proteins (IGFBP) IGFBP-1 and -2 are theorised to be heavily involved in the progression of cancers and can act to regulate cell function both dependently and independently of IGF1-R. This lab has shown that IGFBP-2 can act via the following pathway: IGFBP-2 interaction with β1-integrin receptor, which leads to inactivation of tumour suppressor gene PTEN (3), This results in inactivation of the pathway activated by IGFs and promotion of prostate cancer cell growth and progression and causes the intrinsic promotion of prostate cancer cell survival and growth. Additionally, β1-integrins are also required for IGF-1R localization in focal contacts and for its mitogenic effects. In the case of IGFBP-3 this lab (and others) have demonstrated that it may act to inhibit growth and encourage apoptosis, but may also act to stimulate growth and inhibit apoptosis in tumour progression (4-6). It was discovered that this action might be brought about partly via the interaction of IGFBP-3 with caveolin-1 (CAV-1) via its scaffolding domain (CSF) (7). CAV-1 is an integral membrane protein that regulates essential cell processes such as the PI3K/Akt transduction pathway controlling cell growth and survival. Research carried out in this group showed that binding to CAV-1 and β1-integrins regulated the ability of IGFBP-3 to exert intrinsic inhibitory effects by increasing association and promoting the recruitment of focal adhesion kinases to the cell membrane. CAV-1 has also been found to be implemental in the aberrant activation of the PI3K/Akt pathway (9). CAV-1 has been found to associate with IGF-1R (8) and bind PTEN and any reduction in this association leads to a decrease in PTEN levels at the membrane thus causing PI3K/Akt activation. Aims The overall aim of this project was to investigate the hypothesis that IGFBP-2 intrinsically inactivates PTEN via decreasing both its association with CAV-1 and its presence at the membrane and this causes alteration to the consequent association between CAV-1 and β1IR. We aimed to investigate the

prediction that this would result in PI3K/AKT activation and trigger the intrinsic ability of IGFBP-2 to promote cell growth and furthermore that IGF-1 and IGFBP-3 would have analogous and opposite effects respectively. Work Carried Out and Results Throughout the course of the project the techniques that were primarily used included tissue culture, treating cells and performing time course assays, lying cells, protein assays, immuno-precipitation assays (IP’s), western blotting and tritiated thymidine incorporation assays (TTI’s).

During week 1 we began by dosing DU145 cells of 2 different densities (0.04*10^6) and 0.1*10^6) with IGF-1 (100ng/ml) and running a time course (24hrs and 48hrs). Proliferation was then assessed using TTI. Controls were run at an IGF-1 dose of 25ng/ml. Nether the results of the TTI at 24hrs or 48hrs implied any significant increase in cell number leading us to conclude that the doses of IGF-1 used to treat the cells may not have been high enough (i.e. the threshold value may not have been crossed). During this week practice of some other techniques to be used such as western blotting were also carried out.

In week 2 we then plated 2x24 well plates of DU145 cells at a density of 0.025*10^6, one of which was to be used for a TTI assay and the other to make lysates to be used in an IP. At this stage we carried out identical IP’s using 2 different lysis buffers (triton lysis buffer and NP-40) in order to determine is the method of lysis had any effect on the result of the IP. We protein assayed the lysates and performed an IP on them using CAV-1 antibody to pull down with. The other 24 well plate was treated with different doses of IGF-1 (0ng/ml, 12.5ng/ml, 25ng/ml, 50ng/ml, 100ng/ml, 100ng/ml, 250ng/ml, 500ng/ml) and examined using a TTI assay after 48hrs. The results of this TTI implied that 100ng/ml was the optimum dose to treat the cells with.

We moved on to run a western blot of the IP’d lysates from week 2 in the 3rd week. We probed for both IGF1-Rβ and CAV-1. The results showed very strong CAV-1 signals in our samples, but only showed IGF1-R to be present in the positive control. This implied that CAV-1 and IGF1-R may not be linked or that it may be better to pull down with IGF1-R antibody. In addition we carried out a time course (0mins, 30mins, 60mins, 2hrs) on DU145 cells at a concentration of 0.8*10^6, dosed with 100ng/ml IGF-1. The samples were then lysed and


an IP carried out (in which IGF1-R was used to pull down with) ready to be run on a gel in week 4.

The results from week 3’s IP showed no bands present in our samples for either CAV-1 or IGF-1R. This implied that the antibody we used must not have been effective in pulling down. During week 4 we also prepared DU145 lysates (treated with 100ng/ml IGF-1, 30mins and 60mins). We then carried out a western blot on week 4’s IP’s probing initially for CAV-1 and IGF1-R. The results of this western blot showed that CAV-1 had been pulled down successfully but no IGF1-R was present in the samples despite being present in the positive controls. This, together with our previous results, seemed to confirm our suspicions that CAV-1 and IGF1-R were not in fact associated. Based on these results we decided to re-probe the same membrane for β1IR and PTEN. β1IR appeared to be present in the positive controls but not in the samples thus suggesting that it was not associated with CAV-1. In contrast however the results for PTEN appeared to show that it was very strongly associated with CAV-1.

For the remainder of the project we went on to investigate the main element of our hypothesis relating to IGFBP-2. In week 6 we carried out a time course on DU145 cells treated with IGFBP-2 (DU145 cells at concentration of 0.8*10^6 and dosed with 250ng/ml IGFBP-2, 0mins, 30mins, 1hr, 2hrs). The lysates produced were then IP’d (CAV-1 used for pull down) and run on gels the following week. We probed for CAV-1, IGF1-R, PTEN and β1IR. The results showed no association between CAV-1 and IGF1-R or β1IR but again inferred an association between CAV-1 and PTEN. In this initial experiment the results obtained appeared to go against our initial hypothesis in that there seemed to be no discernable increase in protein levels, thus implying that IGFBP-2 had no significant effect. Similarly, when ran the neat cell lysates from the same time course on gels there again seemed to be no discernable increase. In the final weeks we went on to repeat the same time course (with the same cell density, IGFBP-2 doses), but with both cultures of cells grown on plastic (as before) and fibronectin. The thinking behind this being that the media on which the cells were grown may have a bearing on our results. The results from this experiment however still displayed no significant patterns in protein abundance. Thusly we concluded that we had disproved this element of our hypothesis.

Departures From The Original Proposal and Future Directions of the Project By and large, the work carried out followed that suggested in the original proposal. The only way in which it departed was in that we were unable to carry out all that was suggested due to time constraints.

As time did not permit full investigation into our hypothesis this would first and foremost be the logical next step in which to take the project. With this in mind future directions may include analogous experiments to those already carried out but with the aim to investigate the effects that IGF-1 and IGFBP-3 have on the aforementioned pathway. The Value of This Studentship The primary value of the studentship to me was in giving me an insight into the realities of working in a laboratory environment, an insight that cannot be gained simply from laboratory sessions on my university course. The environment gave me a chance to be more independent in the laboratory than in university laboratories. This insight is one that will be hugely beneficial when considering the possibility of further study after the final year of my undergraduate degree. Another aspect in which the studentship was also of great value was in improving my laboratory skills (both practical and general problem solving and planning in the lab) and in learning the importance of keeping a detailed diary of the work carried out in the laboratory. During my time at the lab I also improved a great deal with regard to the reading an analysis of scientific papers. All of these skills will be of great benefit to me in my final year (particularly in my final year project) and potentially throughout my future career. With regard to the value of the studentship to the lab the work I carried out during my studentship has disproven a hypothesis that is of interest to the research group. The work was therefore of value to the lab in addition to myself.


References:

1. Roddam, A. W et al. (2008) Ann Intern Med 149, 461-471, W483-468

2. Rowlands, M. A et al. (2012) Cancer causes and control: CCC23, 347-354

3. Uzoh et al (2011) British journal of cancer 104, 1587-1593

4. Rajah, R et al (1997) J Biol Chem 272, 12181-12188

5. Burrows, C., et al (2006) Endocrinology 147, 3483-3500

6. Mehta, H. H., et al (2011) Cancer Res 71, 5154-5163

7. Singh, B., et al (2004) J Biol Chem 279, 477-487

8. Salina, B., et al (2010) PLoS One 5, e14157

9. Xia, H., et al The American Journal of Pathology 176, 2626-2637

Student: Steven Simmonds Supervisor: Prof Mike Geeves Bench Supervisor: Dr Anja Schmittmann Institution for placement: University of Kent

Biochemical Society Summer Studentship 2012 Obtaining an NMR spectrum for Tropomyosin

Background Tropomyosin (Tm) is a coiled coil linear protein. A single Tropomyosin molecule spans seven subunits of the protein actin. When the muscle is in a resting state Tropomyosin is held onto the actin by troponin, upon stimulation by calcium ions this structure is “unlocked” allowing myosin to bind. In vivo several different isoforms of the protein Tropomyosin have been identified; variation is increased as it exists as a dimer that can be either a heterodimer or a homodimer. Tropomyosin also polymerises end to end via post-translational acetylation that occurs in the cell. When tropomyosin is expressed in Escherichia coli this polymerisation is lost, as the protein product is not acetylated. This means that it will only bind weakly to actin molecules as the N terminus is positively charged if unacetylated. However this can be overcome by mimicking the acetylation by addition of an alanine serine construct at the N terminus of the molecule (1), moving the N-terminal positive charge further away from the interaction site. This Ala.Ser addition then restores the bind affinity to near wild type levels. Recently it has become possible to express high levels of both Homo and Hetero-Dimers that enable the start of high-resolution structural studies on the protein. Aims My project had several objectives. The first was the expression of tropomyosin in E. Coli with and without an N terminal His Tag. These proteins were used to create an N15 labelled heterodimer for NMR spectroscopy after assembly and purification. The second aim of the project was to express in E.coli both tropomyosin with the AlaSer acetylation mimic tag and tropomyosin without the AlaSer tag. These proteins were then used to measure the tropomyosin viscosity in varying salt concentrations and also the Tm binding affinity for actin using a sedimentation assay with and without the presence of troponin T. The viscometry assays are useful as they show the ability of the tropomyosin to polymerise in solution effectively with and without the alanine serine tag. The sedimentation assay shows the affinity of the two proteins for actin and the affect that the presence of troponin T has on this affinity. Heterodimer Assembly and Purification Initially the two proteins expressed for the creation of the heterodimer were α-tropomyosin without the His tag and without the alanine serine tag (Tm WT) and tropomyosin alpha with a his tag and an alanine serine extension (Tm AS His). This wasn’t ideal as for NMR the sample needs to be as un-viscous as possible and the presence of the alanine serine extension on the his tagged protein will increase the samples viscosity compared to a heterodimer where neither proteins have the extension. To overcome this a construct was made for the protein tropomyosin with a his tag and without the alanine serine extension

(His-Tm WT). Both His-AS-Tm and WT-Tm were cultured in unlabelled media first to gauge expression levels and to test the creation of a heterodimer using these particular proteins, with the possibility of using the single His-AlaSer tagged protein later in viscometry assays. The Tm WT had issues being expressed in light broth media, although after expressing the protein in NZY high expression media there were no problems. The proteins were then purified, both Tm AS His and Tm WT His were run on a nickel gravity flow through column, and the Tm WT was purified by a salt gradient on a FPLC machine. The first formation of heterodimer yielded a low product due to the ratio of tagged to untagged used as most of the His tagged protein formed a homodimer, the ratio of one tagged to two untagged was therefore upped to one tagged for six untagged proteins for the labelled heterodimer preparation. For the NMR heterodimer Tm WT and Tm WT His proteins were used. The Tm WT was grown as above and the Tm WT His was expressed in N15 labelled minimal media. The heterodimer was then purified on a cobalt based gravity flow through Talon column. Unfortunately due to time constraints I was unable to get any time on the NMR after the preparation and purification of the correct construct. Viscosity Assays The viscosity of the proteins AlaSer-Tm, His-AlaSer-Tm and WT-Tm WT were compared using a Cannon-Manning semi-microviscometer, each at a concentration of 20µM. The data collected shows that the presence of the His tag made the sample much more viscous than without the His tag and also less susceptible to changes in salt concentration. The protein without the alanine serine extension was the least viscous as expected, being more susceptible to smaller changes in salt concentration than tropomyosin with the extension (figure one). The viscosity was indistinguishable from buffer at ≥100 mM KCL. Further Investigation Unfortunately due to time constraints I was unable to complete actin binding affinity assay with and without troponin T and it would be beneficial to carry out this study. It would also be interesting conducting the same viscosity assays as above in the presence of troponin T as this binds in the overlap region of tropomyosin required for polymerisation. Value of the studentship To me the worth of this studentship has been unparalleled. Due to the support from both the biochemical society and all the staff in Professor M Geeves’ lab I have become very proficient in protein purification by both gravity flow through and using an FPLC column, dialysis, centrifugation and SDS-PAGE. As well as learning new skills such as these I have been able to build on basic lab techniques such as making buffers until they have become second nature. The most important thing that I have gained from this experience is the knowledge that a career in science is definitely one that I wish to pursue. I intend to use these skills during my final year project and hopefully later during a PhD.

Figure 1: A graph of time taken for meniscus of proteins at 20 µM concentration to drop in a Cannon-Manning semi-microviscometer over a range of potassium chloride concentrations. The proteins used were: Tropomyosin with Alanine serine extension without a His tag Tropomyosin with alanine serine extension with His tag Tropomyosin without alanine serine extension or His tag References: (1) Heald, R. W., and Hitchcock-Degregori, S. E. (1988) The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin, J. Biol, Chem. 263, 5254-5259.

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Tom Wilding-Steele Supervisor-Chen Bing, University of Liverpool, Department of Obesity and Endocrinology

Investigating the effect of vitamin D on glucose uptake and insulin signalling in human adipocytes

Introduction and Aims

Obesity and type 2 diabetes is characterised by chronic mild inflammation, adipose tissue plays a critical part in the development of this inflammation. It is thought that in obese adipose tissue chemokine’s (mainly MCP-1) are released in response to hypoxia caused by an increase in the density and mass of adipocytes (Trayhurn et al., 2006). This recruits macrophages to adipose tissue which along with pre-adipocytes secrete a range of pro-inflammatory cytokines in particular TNF-α, IL-6, IL-8 and the IL-1 family. IL-6 has been shown to bind to and inhibit the insulin receptor as well as marking IRS-1 for proteosomal degradation while TNF-a inhibits IRS-1 and reduces the concentration of anti-inflammatory adipokines for example adiponectin (Jager et al., 2007). Vitamin D has been shown to play an increasingly important role in the immune system and adipose tissue where it modulates adipogenesis and lipid metabolism. More importantly it has been shown to reduce the levels of MCP-1, IL-6 (Gao et al., 2012). Several studies have also shown that obese people are often deficient in vitamin D due to lifestyle choices associated with obesity or fat sequestering vitamin D (Scragg, 2008),(Husemoen et al., 2012). Although vitamin D has been shown to reduce the levels of several cytokines involved in reducing insulin resistance it is not known if vitamin D affects insulin sensitivity in adipocytes. This study was aimed to examine whether treating adipocytes with vitamin D would reduce the effect of macrophages on glucose uptake and the insulin signalling pathway.

Methods

Due to the time it takes to grow cells (almost 1 month) I was given mature human adipocytes, these has been grown from pre-adipocytes and at confluence differentiated into adipocytes. A model has been developed in the lab which involves incubating mature adipocytes with macrophages (macrophage conditioned media- MC) and this has been shown to reduce glucose uptake and reduce insulin signalling pathway protein levels.

Adipocytes were pre-treated with or without 10nM of vitamin D for 48 hours; cells were then treated with MC, while another group was treated with RPMI (the media macrophages are grown in) as a control for 24 hours. A glucose uptake assay was performed following stimulation with 10nM of insulin for 7 or 20 minutes. Cell lysates were collected, and western blotting was used to measure protein levels of GLUT-4, IRS-1, P-P85 and P-AKT. The concentrations of IL-6 in the cell medium were determined using ELISA.

Results

There was a trend of reduction in glucose uptake when treated with MC and a partial reversion to normal when cells were pre-treated with vitamin D but it was not statistically significant. Insulin seemed to have no effect on glucose uptake in all groups possibly due to too long a gap between administering insulin and measuring glucose uptake with a fluorescent plate reader.

The initial study has shown protein abundance of IRS-1, p-P85 and GLUT4 was decreased in response to MC media and this effect was partly ameliorated by pre-treatment with vitamin D. Insulin noticeably increased the abundance of the insulin signalling proteins especially in control and vitamin D treated groups.

Fig1. A sample of western blotting showing the protein expression of IRS-1, P-85, GLUT4 and GAPDH which was used to normalise the results. The protein abundance was determined again using western blotting in the secound study., relative ratio was determined by normalising it to GAPDH and data was analysed using one-way anova.As shown in Fig.2 , GLUT4 levels were dramatically reduced in respose to MC media (p=0.001) and were partially restored after


treatment with vitamin D and stimulated with insulin (p = 0.05). The changes in p-AKT levels were simliar to GLUT4 (Fig. 3). There was a significant reduction in response to MC (p=0.005); treatment with vitamin D partially reversed it in insulin-stimulated group (p= 0.005). There was also a significant difference between insulin and non insulin treated samlples in vitamin D treated cells (p=0.05). There was a similar trend in P-85 and IRS- 1 but they were not statistically significant.

IL-6 concentration was increased several hundred fold in response to MC media, and it was almost halved by pre-treatment wth vitamin D (p=0.005). There was also a significant difference between insulin and non insulin treated samples with IL-6 increased in respose to insulin for both MC and vitamin D treated samples (p=0.005). Fig 4. Showing mean and SE of IL-6 protein levels in cell media determined using ELISA and analysed using one-way anova (n=4 per group); **p<0.01.

Discussion The results of glucose uptake were inconclusive due to technical issues. However, analysis of the insulin signalling protein levels showed that pre-treatment with vitamin D did almost restore protein levels to normal especially when treated with insulin. Analysis of IL-6 levels in the cell media showed that vitamin D almost halved the effect of macrophages. These results suggest that vitamin D could be beneficial in adipose tissue, such as increasing insulin signal transduction and reducing the release of cytokines. Further research and clinical trials are needed to investigate whether vitamin D could help reduce insulin resistance in people at risk of developing type 2 diabetes or people with type 2 diabetes especially in obsese people as they are likely to already be deficient in vitamin D. Departures from original proposal Gene expression levels of GLUT4 was not determined using real-time PCR instead ELISA was used to determine IL-6 concentration.

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Fig 2. GLUT4 relative protein abundance determined using western blotting. Data are mean± SE, analysed using one-way anova (n=4 per group). *p<0.05; ***p<0.001.


Value of Studentship The studentship was immensly helpful for me as it helped me understand how a research lab operates and what would be required to undertake a phd making me certain that I want to pursea a research career. It has helped me to develop my lab skills for example I completed many western blots and ELISA’s by myself. As well as making me confident dealing with large data sets and performing detailed analysis on them and helped me to improve my scientific writing skills.There is also the possibility I could go to the Diabetes UK conference and present my research which would be immensly interesting and helpful. Value of studentship to the lab Tom was a brilliant student who has shown a strong commitment and worked very hard. Tom learned lab skills quickly that he was capable to perform experiments effectively. During this project Tom obtained several interesting result, which will be followed up by two MRes students. A number of antibodies that Tom has evaluated for western blotting will be very useful in future studies. The data on the potentially beneficial effects of vitamin D in adipose tissue will support the application for further funding to extend this research.

References Gao, D., Trayhurn, P., Bing, C., 2012. 1,25-Dihydroxyvitamin D3 inhibits the cytokine-induced secretion of MCP-

1 and reduces monocyte recruitment by human preadipocytes. International Journal of Obesity. Husemoen, L.L.N., Thuesen, B.H., Fenger, M., Jørgensen, T., Glümer, C., Svensson, J., Ovesen, L., Witte, D.R.,

Linneberg, A., 2012. Serum 25(OH)D and Type 2 Diabetes Association in a General Population A prospective study. Dia Care 35, 1695–1700.

Jager, J., Grémeaux, T., Cormont, M., Marchand-Brustel, Y.L., Tanti, J.-F., 2007. Interleukin-1β-Induced Insulin Resistance in Adipocytes through Down-Regulation of Insulin Receptor Substrate-1 Expression. Endocrinology 148, 241–251.

Scragg, R., 2008. Vitamin D and Type 2 Diabetes Are We Ready for a Prevention Trial? Diabetes 57, 2565–2566. Trayhurn, P., Bing, C., Wood, I.S., 2006. Adipose Tissue and Adipokines—Energy Regulation from the Human

Perspective. J. Nutr. 136, 1935S–1939S.

Investigating the localisation and complex formation of the Nek11 DNA damage response kinase

Supervisors: Professor Andrew Fry/ Miss Sarah Sabir Department of Biochemistry University of Leicester

Background Nek11 belongs to a family of eleven serine/threonine protein kinases. Of these, Neks1, 10 and 11 are implicated in the DNA damage response (DDR). Nek11 plays a central role in the DDR in response to ionizing radiation (IR). Briefly, after IR, Chk1 is activated by upstream kinases, it then phosphorylates Cdc25A on S76. Chk1 also phosphorylates S273 on Nek11 thereby activating it. Nek11 then phosphorylates Cdc25A at S82. These phosphorylation events target Cdc25A for degradation resulting in cell cycle arrest.

Aims Nek11 exists as four alternatively spliced variants: Nek11-Long (Nek11L), Nek11-Short (Nek11S), Nek11C and Nek11D. Previous work in our laboratory has shown that each of these isoforms shows distinct localisation patterns. This project aimed to build on this knowledge by investigating how these distinct localisation patterns are regulated and whether these isoforms respond differently to DNA damage. Specifically, my first objective was to investigate whether the Nek11 isoforms shuttle in and out of the nucleus. This would be done by comparing the localisation with and without treatment with a nuclear export inhibitor, leptomycin B (LMB). Secondly, I would investigate the motifs required for localization, by examining the localization of the isolated catalytic (1-287) and non-catalytic (288-645) domains of Nek11L. In addition, mutations of putative nuclear export sequences (NESs) would be generated in Nek11L and Nek11S to investigate whether these are important for localisation. The final objective was to examine whether Nek11 isoforms form a complex with either Cdc25A or Chk1 in response to DNA damage; this would be assessed by co-immunoprecipitation experiments after DNA damage induction.

Description of the work carried out To examine the dynamics of Nek11 localisation, we performed immunofluorescence microscopy with U2OS cells stably expressing each of the Nek11 isoforms as GFP-tagged proteins; these were untreated or treated with LMB for 3 hours prior to fixation. To identify regions of Nek11L required for its localization, U2OS cells were transiently transfected with GFP-tagged versions of the catalytic or non-catalytic domains alone. Additionally, NES mutants were generated by site-directed mutagenesis (Nek11L-L631A, I633A and L631A/I633A, and Nek11S-I455A) and transiently transfected into cells. Cells were stained with antibodies against GFP to detect tagged Nek11 proteins, while DNA was stained with Hoechst 33258.

To investigate whether Nek11 forms a complex with other DDR components, conditions required to activate the G2/M checkpoint needed to be optimised. To induce DNA damage etoposide was used. Briefly, U2OS cells were treated with 10 or 20 µM etoposide for 40 minutes before lysates were collected at 1, 3 and 4 hours post treatment. Lysates were Western blotted for phospho-Chk1 and Cdc25A to check for activation of the checkpoint. Optimised conditions were then used on U2OS:GFP-Nek11 stable cell lines. Lysates were subjected to precipitation using the GFP-Trap® system and bound proteins analysed by Western blot with GFP antibodies.

An assessment of the results and outcomes of the studentship In the absence of LMB, most Nek11 isoforms are predominantly cytoplasmic. However, upon addition of LMB, all isoforms showed a predominantly nuclear localisation. These data indicate that the Nek11 isoforms shuttle in and out of the nucleus. However, the percentage shift from mostly cytoplasmic to mostly nuclear differs between the isoforms suggesting differences in regulation and potential function. Both the catalytic and non-catalytic domains exhibited predominantly cytoplasmic localization; however, experiments with LMB will be required to see if these domains also shuttle. Two of the point mutants exhibited mainly nuclear localization suggesting that these residues lie within potentially functional NESs.

To test conditions required to induce the G2/M checkpoint, different concentrations of etoposide were used and phosphorylation of Chk1 was assessed at different time-points post-treatment. Results revealed that Chk1 was activated 2 hours after treatment with 20 μM etoposide. These conditions were then used on U2OS:GFP-Nek11L cell lines, however initial precipitation using GFP-trap beads was not successful and will need to be optimized.

Future directions in which the project can be taken Firstly, the localization of the isolated domains in the presence of LMB would reveal which domain contains the sequences that control nucleocytoplasmic shuttling. Secondly, further mutations could be made in the putative NESs to test their role in Nek11 localization. Thirdly, the Nek11-Cdc25A coprecipitation experiments will need to be optimized to look at complex formation in response to different forms of DNA damage. Finally, as this project has suggested different roles for each Nek11 isoform in the cell, future work can begin to explore this in the context of the DDR.

Any departures from the original proposal Due to time constraints, a few of the objectives were not completed. It was not possible to determine the localisation of all NES mutants, as two of the mutations were not in frame. It was also not possible to continue the GFP-Trap experiments to determine whether Nek11 forms complexes with Cdc25A or Chk1 following DNA damage by etoposide, although optimised conditions for DNA damage were obtained.

The value of the studentship to myself and the lab The studentship has provided an excellent opportunity for me to gain laboratory skills and become acquainted with current molecular cell biology techniques. Working on this project has developed my initiative and passion for Biochemistry and made me sure I would like to pursue a career in scientific research. In addition, the support from other members of staff in the lab has been invaluable and my results will provide preliminary data on which the current PhD student, Sarah Sabir, can continue to investigate regulation of the Nek11 protein.

Summer Studentship reports 2012

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Transcript of Summer Studentship reports 2012