Master thesis (Mette Lethan)

Identification and characterization of novel Chlamydomonas flagellar tip proteins

Master Thesis by Mette Lethan

Department of Biology University of Copenhagen

Denmark 2009

Supervisor Lotte Bang Pedersen

F A C U L T Y O F S C I E N C E U N I V E R S I T Y O F C O P E N H A G E N

Master thesis by Mette Lethan

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Preface This master thesis represents the final part of my education and is based on

experimental work carried out from May 2008 to June 2009 at Department of Biology,

Section of Cell and Developmental Biology, University of Copenhagen.

First of all I wish to thank my supervisor and daily mentor associate professor, Ph.D.

Lotte Bang Pedersen, for letting me join in on a very interesting project, thereby

introducing me to the cilia and a very smart model organism called Chlamydomonas,

and also for always being ready with help and support all the way through the project. A

special thanks to Ph. D student Jacob M. Schrøder for helping with the NIH3T3

fibroblast cells and always being ready with a great humour and a good story. Special

thanks to technician Søren L. Johansen for helping in the lab, and always providing

what you seem to be missing. A great thanks to the entire cilia group which comprises a

gathering of amasing people. A special thanks to Dorte L. Egeberg, Sonja K. Brorsen

and Tue S. Jørgensen for daily inspiration and discussions. A big thanks to the entire 5.

Floor especially to the entire ”Grøn Stue”, you know who you are, for supplying all the of

non-”lab” related activities. Without you this year would not have been the same. I am

very grateful to Niovi Santama for generously providing me with Nubp1 antibodies as

well as the Anna Akhmanova group for collaboration in the search of IFT172 and EB1

binding partners. I also thank the Chlamydomonas Genetics Center for strains. Lastly I

wish to thank my family and all my friends, for understanding that time is scarce.

Parts of the results obtained in this project were presented with a poster at the Gordon

Research Conference on Cilia, Mucus and Mucociliary Interactions in february 2009 in

Lucca, Italy.

Copenhagen, august 2009

__________________________

Mette Lethan


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Table of Contents

Preface .....................................................................................................................................2

Abstract ...................................................................................................................................5

Dansk resume.......................................................................................................................6

Abbreviations .......................................................................................................................8

1. Aim of study ...................................................................................................................11 1.1. Introductory remarks............................................................................................ 11 1.2. Specific aims........................................................................................................ 11

2. Introduction ....................................................................................................................13 2.1. Cilia and flagella structure ................................................................................... 13 2.2. Ciliopathies .......................................................................................................... 16 2.3. Ciliogenesis and the cell cycle............................................................................. 17 2.4. Intraflagellar transport.......................................................................................... 18

2.4.1. Anterograde IFT ............................................................................................20 2.4.2. Retrograde IFT..............................................................................................22 2.4.3. Tip turnaround...............................................................................................23 2.4.4. IFT particle polypeptides ...............................................................................24 2.4.4.1. IFT172 .................................................................................................... 25

2.5. EB1...................................................................................................................... 26 2.6. Nucleotide-binding protein 1 (Nubp1) .................................................................. 27 2.7. Kinesins ............................................................................................................... 28

2.7.1. Kinesin-2 family proteins ...............................................................................30 2.7.2. Kinesin-5 family proteins ...............................................................................31 2.7.3. Kinesin-14 family proteins .............................................................................31

2.8. Chlamydomonas as a model organism for ciliary functions ................................ 32

3. Results and discussion............................................................................................37 3.1. Introductory notes................................................................................................ 37 3.2. Chlamydomonas reinhardtii Nubp1 is present in the flagella .............................. 38 3.3. CrNubp1 localizes to the soluble membrane plus matrix compartment .............. 40 3.4. The flagellar level of CrNubp1 is unaffected by mutations affecting assembly of the main axonemal substructures: outer dynein arms, inner dynein arms, radial spokes and the central apparatus........................................ 42 3.5. CrNubp1 localizes to the basal bodies and the tip of the flagella ........................ 43 3.6. Mammalian Nubp1 localizes to the centrioles and the nucleus in NIH3T3 fibroblast cells...................................................................................................... 46 3.7. Identification of possible binding partners to CrEB1 and IFT172 C-term in Chlamydomonas wild type (CC-124) flagella .................................................. 47 3.8. Construction and purification of MBP-ARFA1A, MBP-FAP20 and MBP-Eg5 motor domain fusion proteins.............................................................. 52


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3.9. MBP pull-down analysis of ARFA1A, FAP20 and Eg5 motor domain ................. 52 3.10. Further testing of the function of ARFA1A......................................................... 56

4. Conclusions and future directions.....................................................................59

5. Materials and Methods..............................................................................................63 5.1. Eukaryotic cell cultures ........................................................................................ 63 5.2. Preparation of flagella and cell body extracts ...................................................... 65 5.3. PCR and cloning procedures............................................................................... 66 5.4. Expression of MBP fusion proteins...................................................................... 69 5.5. Purification of fusion proteins on amylose resin .................................................. 69 5.6. MBP pull-down assays ........................................................................................ 69 5.7. Protein quantification ........................................................................................... 70 5.8. Sodium Dodecyl Sulphate PolyacrylAmide Gel Electrophoresis (SDS-PAGE) ... 70 5.9. Western Blot analysis (WB)................................................................................. 71 5.10. Antibodies and affinity purification ..................................................................... 71 5.11. Immunofluorescence microscopy analysis (IFM) .............................................. 72

5.11.1. IFM on Chlamydomonas cells..................................................................... 74 5.11.2. IFM on NIH3T3 cells ................................................................................... 75

6. References ......................................................................................................................77

7. Appendices.....................................................................................................................84 Appendix A: Culturing media ...................................................................................... 84 Appendix B: Preparation of flagella and cell body extracts ........................................ 85 Appendix C: cDNA sequences and multiple sequence alignments............................ 88 Appendix D: Vector map............................................................................................. 93 Appendix E: Primers ................................................................................................... 94 Appendix F: Procedure for PCR ................................................................................. 95 Appendix G: Agarose gels .......................................................................................... 96 Appendix H: Transformation of DH10α E. coli cells ................................................... 96 Appendix I: Protein quantification ............................................................................... 97 Appendix J: Solutions for SDS-PAGE and western blotting ....................................... 99 Appendix K: Affinity purification of CrNubp1............................................................. 100 Appendix L: IFM........................................................................................................ 102 Appendix M: Overview of potential binding partners of EB1/IFT172 from Chlamydomonas flagella .......................................................................................... 103

*Picture on front page from: http://rydberg.biology.colostate.edu/Phytoremediation/2003/Boczon/chlamydomonas02.jpg


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Abstract Cilia and flagella are microtubule (MT)-based organelles protruding from the cell surface

of most eukaryotic cell, which play important roles in motility and sensory signaling.

Lack of normal functioning cilia can a number of diseases and developmental defects

including polycystic kidneys, blindness and polydactyly. Assembly and maintenance of

cilia are mediated by intraflagellar transport (IFT) a highly conserved bidirectional MT-

based transport system. IFT transports flagellar precursors from the flagellar base to the

tip for assembly (anterograde transport) and returns turnover products from the tip back

to the base (retrograde transport). The IFT system consists of anterograde (kinesin-2)

and retrograde (cytoplasmic dynein 2) motor complexes, and ca. 17 different IFT

particle proteins separated in two large complexes, A and B. The molecular

mechanisms by which these different components are coordinated and regulated at the

flagellar base and tip are unclear.

The unicellular green alga Chlamydomonas reinhardtii is a well-established

model organism for studying cilia and IFT. IFT turnaround at the flagellar tip involves:

inactivation/down-regulation of kinesin-2, activation/upregulation of cytoplasmic dynein

2, unloading of flagellar precursors and loading of flagellar turnover products. It has

previously been shown that EB1, a small MT plus-end tracking protein (+TIP) localizes

to the flagellar tip in Chlamydomonas where it interacts with IFT172 possibly regulating

IFT particle turnover. The aim of this project was to characterize and identify flagella tip

proteins, which are presumed to play central roles in IFT regulation and/or cilia

assembly and function. To this end, I used Chlamydomonas as a model organism and I

employed two different strategies. First, using an antibody generated against the small

nucleotide-binding protein 1 (Nubp1) prior to the onset of this study, I show using

western blotting and immunofluorescence microscopy that Nubp1 is localized to the

flagella in Chlamydomonas and is specifically enriched at the flagellar tip. Second, I set

out to identify binding partners of EB1 and IFT172 C-terminus using GST pull-down of

isolated flagella. This part of my thesis work was done in collaboration with Anna

Akhmanova and her group in Rotterdam, The Netherlands. Akhmanovas group

executed the GST pull-down experiments in Chlamydomonas using isolated flagella and


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GST-EB1/IFT172 fusion proteins and identified putative binding partners by mass

spectrometry. I subsequently cloned and characterized three of the potential

EB1/IFT172 binding partners identified.

Dansk resume Cilier og flageller er mikrotubuli (MT)-baserede organeller der udgår fra celleoverfladen

hos de fleste eukaryote celler, hvor de spiller vigtige roller i bevægelse og sensorisk

signalering. Hvis normalt fungerende cilier mangler kan det føre til forskellige

sygdomme og udviklingsmæssige defekter inklusiv cystenyre, blindhed og polydaktyli.

Dannelse og vedligeholdelse af cilier er medieret af intraflagellær transport (IFT) et

meget konserveret bi-direktionelt MT-baseret transport system. IFT transporterer de

flagellære byggesten fra flagellets base og til tippen til dannelsen (anterograd transport)

og returnerer ”turnover” produkterne fra tippen og tilbage til basen (retrograd transport).

IFT systemet består af anterograd (kinesin-2) og retrograd (cytoplasmic dynein 2) motor

komplekser samt ca. 17 forskellige IFT partikel proteiner, delt i to store komplekser

kaldet A og B. De molekylære mekanismer hvorved disse forskellige komponenter bliver

koordineret og reguleret ved flagellets base og tip er usikkert.

Den encellede grønne alge Chlamydomonas reinhardtii er en veletableret

modelorganisme til studiet af cilier og IFT. IFT ”turnaround” i flageltippen involverer:

inaktivering/nedregulering af kinesin-2, aktivering/opregulering af cytoplasmisk dynein 2,

aflastning af flagellære byggesten og lastning af flagellære ”turnover” produkter. Det har

tidligere været vist at EB1, et lille MT plusende associeret protein (+TIP), lokaliserer til

flagellets tip i Chlamydomonas hvor det interagerer med IFT172 muligvis i reguleringen

af IFT partikel ”turnover”. Formålet med dette projekt var at karakterisere og identificere

flagel tip proteiner, der formodes at spille centrale roller i IFT regulering og/eller

ciliedannelse og funktion. Til dette brugte jeg Chlamydomonas som en modelorganisme

og benyttede to forskellige strategier. Først, ved at bruge et antistof genereret mod det

lille nukleotidbindende protein 1 (Nubp1) før starten af dette studie, viser jeg ved brug af

western blotting og immunofluorescens mikroskopi at Nubp1 er lokaliseret til flagellerne


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I Chlamydomonas og er specielt beriget ved flageltippen. Derudover ville jeg identificere

bindingspartnere til EB1 og IFT172 C-terminal ved brug af GST “pull-down” fra isolerede

flageller. Denne del af mit speciale arbejde blev gjort i samarbejde med Anna

Akhmanova og hendes gruppe i Rotterdam, Holland. Akhmanovas gruppe udførte GST

”pull-down”forsøgene i Chlamydomonas ved brug af isolerede flageller og GST-

EB1/IFT172 fusionsproteiner og identificerede formodede bindingspartnere ved

massespektrometri. Jeg klonede og karakteriserede derefter tre af de potentielle

EB1/IFT172 bindingspartnere der var blevet identificeret.


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Abbreviations +TIP Plus-end tracking protein

AcTub Acetylated alpha-tubulin

Arf ADP-ribosylation factors

Arl Arf-like protein

BBS Bardet-Biedl syndrome

BCIP/NBT 5-bromo-4-chloro-3-indoylphosphate/Nitroblue tetrazolium

BSA Bovine serum albumin

C. reinhardtii Chlamydomonas reinhardtii

cDNA Complementary DNA

CGC Chlamydomonas Genetics Center

CrNubp1 Chlamydomonas reinhardtii Nubp1

DDT Dithiothreitol

DMSO Dimethylsulfoxide

EB End binding protein

E. coli Escherichia coli

EtOH Ethanol (CH3CH2OH)

FAP20 Flagella Associated Protein 20

GTP Guanosine triphosphate

GST Glutathione S-transferase

IC Intermediate chain

IFM Immunofluorescence Microscopy


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IFT Intraflagellar transport

Ig Immunoglobulin

KCBP Kinesin-like Calmodulin binding protein

KIF Kinesin superfamily protein

MAP MT-associated protein

MeOH Methanol (CH3OH)

MBP Maltose binding protein

MmNubp1 Mammalian Nubp1

mRNA Messenger RNA

MTOC MT organizing center

MT Microtubule

NIH3T3 National Institute of Health 3T3

Nubp1 Nucleotide-binding protein 1

OD Optical densities

PBS Phosphate buffered saline

PCD Primary cilia dyskinesia

PFA Paraformaldehyd

PKD Polycystic kidney disease

PCR Polymerase chain reaction

RPE Retinal pigment epithelial

RT-PCR Reverse transcription polymerase chain reaction

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis


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SLB Selective Lhx3/4 Lim-homeodomain transcription factor

binding protein

SN Supernatant

SOFA Site of axonemal severing

TAE Tris-acetate-EDTA

TAP Tris-acetate-phosphate

TBS Tris buffered saline

TBST TBS Tween-20

TFIIB Transcription factor IIB

WB Western blot analysis


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1. Aim of study

1.1. Introductory remarks Motile and the non-motile primary cilia are assembed and maintained by intraflagellar

transport (IFT), a highly conserved bidirectional microtubule-based transport system.

IFT transports flagellar precursors from the flagellar base to the tip for assembly

(anterograde transport) and returns turnover products from the tip back to the base

(retrograde transport). The IFT system consists of anterograde (kinesin-2) and

retrograde (cytoplasmic dynein 1) motor complexes, and ca. 17 different IFT particle

proteins separated in two large complexes, A and B. The molecular mechanisms by

which these different components are coordinated and regulated at the flagellar base

and tip are unclear. The molecular mechanisms of IFT turnaround in Chlamydomonas

involves: inactivation/down-regulation of kinesin-2, ativation/upregulation of cytoplasmic

dynein 2, unloading of flagellar precursors, and the loading of flagellar turnover

products. EB1 is a MT plus-end tracking protein (+TIP) and localizes to the flagellar tip

in Chlamydomonas reinhardtii (Pedersen et al., 2003), where it interacts with IFT172

possibly regulating IFT particle turnover (Pedersen et al., 2005).

1.2. Specific aims The aim of this project was to use Chlamydomonas reinhardtii as a model, to identify

and characterize novel flagellar tip proteins, that is presumed to play central roles in the

building of the cilia as well as the regulation of cilia mediated signal transduction and the

cell cycle. First, based on the results for CrEB1 in the flagella proteome analysis

(Pazour et al., 2005), CrNubp1 was chosen, based on the fact that it had the same

properties as CrEB1 in having fex peptides and all in the membrane plus matrix

fractions. Furthermore, the mouse kinesin-14 family member KIFC5A is a minus-end-

directed kinesin involved in regulation of centrosome duplication and the cell cycle.

KIFC5A interacts directly with nucleotide-binding protein 1 (Nubp1) and the related

protein Nubp2, and inactivation of either KIFC5A or Nubp1 in mouse fibroblasts results

in the presence of supernumerary centrosomes and an increase in the proportion of bi-


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and tri-nucleated cells (Christodoulou et al., 2006). Prior to the onset of this project, a

Chlamydomonas homolog of Nubp1 was cloned and an antibody generated against this

protein. My aim was to test this antibody and potentially characterize the protein.

Secondly, as an alternative approach to identifying novel tip proteins, I set out to

identify binding partners of C. reinhardtii EB1 and IFT172 C-terminus using GST pull-

down of isolated flagella from C. reinhardtii cells. This part of my thesis work was done

in collaboration with Anna Akhmanova and her lab in Rotterdam, The Netherlands.

Akhmanovas group executed the GST pull-down experiments in Chlamydomonas using

isolated flagella and GST-CrEB1 fusion protein and identified putative binding partners

by mass spectrometry. My aim was to clone three of the potential EB1/IFT172 binding

partners identified and retest this potential binding as well as to potentially a

characterized them.


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2. Introduction

2.1. Cilia and flagella structure Cilia and flagella (the terms are equivalent1) are long, thin organelles projecting like hair

from the surfaces of most eukaryotic cells, where they play important motile and

sensory functions (Christensen et al., 2007). The core of these organelles is composed

of a microtubule (MT)-based skeleton called the axoneme. The axoneme extends from

a modified centriole, the basal body, which anchors the axoneme in the cell. The

axoneme is surrounded by an extension of the plasma membrane continuos of the cell

body but the flagellum membrane is selectively different from the cell membrane in

overall composition, containing a different complement of membrane receptors and ion

channels (Christensen et al., 2007; Satir and Christensen, 2007). The axoneme is

composed primarily of MTs which are hollow cylinders built of heterodimers of α- and ß-

tubulin. The heterodimers bind together in a head-to-tail manner to form a protofilament

and the protofilaments bind side-by-side to form the MT wall. In this way the MTs

become polarized, with ß-tubulin at the fastest growing end called the plus end, and the

slowest growing end, the minus end, finishing with α-tubulin (reviewed in Desai and

Mitchison, 1997). MTs are very dynamic and are constantly polymerizing and de-

polymerizing. This occurs preferentially at the plus end, while the minus end is less

dynamic. The MT plus-ends are highly unstable switching rapidly between growth,

pause and shrinkage. This phenomenon is known as dynamic unstability and can be

modulated by MT-associated proteins (Howard and Hyman, 2003). The axoneme

consists of MT doublets, an A and B tubule, where the A tubule is a complete cylinder of

13 protofilaments and the B tubule an incomplete cylinder consisting of 10

protofilaments, attached to the A tubule (Figure 2.1.E). In the axoneme the MTs are

arranged such that the minus ends are embedded in the basal body while the plus ends

are oriented towards the tip of the cilium (Allen and Borisy, 1974). This means that the

axoneme is assembled at and constantly turning over at its tip which requires continous

transport of axonemal precursors from the cell body to the tip (Marshall and 1 The terms will be used interchangeably throughout this thesis.


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Rosenbaum, 2001; Rosenbaum and Witman, 2002). In general however, axonemal MTs

are very stable: stable MTs are characterized by acetylation and detyronisation of α-

tubulin, which is important for cilia maintenance and function. Further, antibodies

against acetylated or detyrosinated tubulin are frequently used as markers for cilia and

flagella (Poole et al., 2001).

Cilia and flagella are classified, depending upon their axonemal structure, as

motile cilia or non-motile cilia. Motile cilia and flagella contain an axoneme with nine

outer doublet MTs, held together by nexin links, as well as a central pair of MT. They

therefore have a ”9+2” structure (Figure 2.1.E, left). The motile cilia usually play a role in

moving fluids over a cell layer or in movement of single cells (Marshall and Kintner

2008; Ginger et al., 2008), and contain accesory components involved in motility,

including outer and inner dynein arms and radial spokes (Figure 2.1.E, left). Motile cilia

(”9+2”) can be found in multiple copies per cell like in the respiratory epithelia (Figure

2.1, A), mammalian oviduct and brain ventricles. Here they are designed to move the

fluid and mucous overlaying the ciliated epithelium by the coordinated beating of the

cilia. When motile cilia (”9+2”) are found in one or two copies per cell e.g in mammalian

sperm cells (Figure 2.1.B) and in the green alga Chlamydomonas reinhardtii (Figure

2.1.C; see section 2.8), they are often known as flagella. Here the flagella are important

for the movement of the cell (Marshall and Kintner 2008; Ginger et al., 2008).

Non-motile cilia, also known as primary cilia, only exist in one copy per cell and

are present in vertebrate cells when these are in growth arrest (Schneider et al., 2005;

Figure 2.1.D). Their axoneme structure consists of a ”9+0” structure, which means that

they lack the central pair of MT. Furthermore they also lack the accessory components

involved in motility, that is seen in the motile cilia (Figure 2.1.E). Primary cilia have been

shown to be involved in coordination and regulation of a variety of crucial cellular and

developmental processes (Christensen et al., 2007). Modified primary cilia also exist

and are present on differentiated cells of the eye and olfactory organs and are essential

for the senses of sight, equilibrium and hearing (Singla and Reiter, 2006).

However there are examples of cilia, which do not clearly fit into either of these

two groups. The nodal cells of developing mammalian embryos have cilia sharing


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features from both motile as well as primary cilia. These cilia have a ”9+0” structure, but

also posses outer arm dyneins. They generate a propeller-like motion that creates a

directional flow across the node required for establishment of the left-right asymmetry

axis (Hirokawa et al., 2006). And a novel 9+4 axoneme with four central MTs, have also

been identified on the notochordal plate of the rabbit embryo, thus indicating that some

degree of variation of axonemal structures exist (Feistel and Blum, 2006).

Figure 2.1: Cilia and flagella. A: Motile cilia on lung epithelia (http://www.newscientist.com/data/images/ns/cms/dn11602/dn11602-2_585.jpg). B: Spermatozoa approaching an egg (http://z.about.com/d/civilliberty/1/5/u/-/-/-/spermegg.jpg). C: Scanning electron micrograph of the unicellular, biflagellated green alga, Chlamydomonas reinhardtii (Pan et al., 2005). D: Scanning electron micrograph of renal epithelial cells in a kidney collecting tubule. Each cell has a primary cilium (Ci) (Pan et al., 2005). E: Axonemal structure. Cross section of motile 9+2 cilia (left) and immotile 9+0 cilia (middle). The axoneme is constructed of 9 doublet MTs connected via nexin links. Motile cilia also have a central placed MT-pair. The basal body consists of triplet MTs and no central pair (right). Axoneme figure modified from Dawe et al., 2007.


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The ciliary axoneme is anchored to the basal body, which is a modified centriole. The

basal body differs in structure from the ciliary axoneme, by consisting of MT triplets

(Figure 2.1.E, right). Separating the membrane compartments of the cilia and the cell

body, at the ciliary base, is a region known as the ”ciliary necklace” or ”ciliary pore”,

which is connected by fibers to the transition zone of the basal body (Figure 2.2; Gilula

and Satir, 1972, see section 2.3).

2.2. Ciliopathies Cilia are on almost every cell in the human body, where they play important motile and

sensory functions (Christensen et al., 2007), and it is therefore not surprising that

various human disorders can be related to defects in cilia. The phenotypes of these

disorders, the so-called ciliopathies, reflect the many roles cilia play in the human body.

Some examples are listed in table 2.1 (Badano et al., 2006; Marshall, 2008; Pan, 2008).

Defects are seen in both motile cilia and the non-motile primary cilia and the diseases

can either be linked to completely missing cilia or defects in or mis-localization of ciliary

proteins.

Defects in ciliary motility can lead to immotile cilia syndrome, also known as

primary cilia dyskinesia (PCD) (Bisgrove and Yost, 2006). The disease can be caused

by defects in multiple proteins involved in motility such as the dynein arms, the radial

spokes or the central pair MTs, and thereby only affects motile cilia (Afzelius, 2004). It

was a study of Chlamydomonas motility mutants defective in dynein that facilitated the

first identification of the genetic basis for PCD in patients (Pennarun et al., 1999).

Defects in motile cilia can also cause hydrocephalus (accumulation of water in the

brain) and altered left-right axis patterning during embryonic development as well as

infertility in male patients (Afzelius, 2004). Defects in the non-motile primary cilia can

lead to diseases caused by defects in signalling or assembly of the cilium. Examples

are polycystic kidney disease (PKD), Bardet-Biedl syndrome (BBS), polydactyly, obesity

and other more rare diseases. It was the study of flagellar assembly in Chlamydomonas

mutant ift88 that provided the first link between PKD and cilia (Pazour et al., 2000).


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Human disease Gene Cellular function Protein localization

Disease pathology

PCD DNAH5 DNAI1

Ciliary motility Outer dynein arms

Respiratory infections, anosmia, male infertility, otitis media and situs invertus

Meckel-Gruber syndrome

Cep290 MKS1; MKS3

For Cep290 unknown; Ciliogenesis

Basal body and IFT complexes

Brain malformation, polydactyly, kidney and liver cysts

PKD PKD1-2; PKHD1

Mechanosensing; PKHD1 unknown

Cilia and basal body

Polycystic kidney

Nephronophtisis NPHP!-5 Uncertain Basal body and cilia

Kidney cysts, liver fibrosis, retinal dysplasia

Joubert syndrome

Central nerve system abnormalities, kidney cysts, brain and retina malformations

Retinitis pigmentosa

RPGR Retinal transport Basal body Retinal degeneration

BBS including BBS1-12 Ciliogenesis Basal body and IFT complexes

kidney cysts, obesity, anosmia, retinal dystrophy, male infertility, situs invertus, diabetes

Oral-facial-digital syndrome type I

OFD1 Ciliogenesis Basal body Malformations of the face, oral cavity and digits, kidney cysts

Table 2.1. Human ciliary disease genes and their cell biological functions. Table modified from Marshall, 2008; D’Angelo and Franco, 2009.

2.3. Ciliogenesis and the cell cycle As mentioned above the ciliary axoneme is anchored to the basal body, which is a

modified centriole, and assembly and disassembly of the cilia is therefore tightly

coupled to centriole duplication and the cyclic nature of the centrioles during the cell

cycle. This means that the formation of a primary cilium, ciliogenesis, is a regulated

process and closely connected to the cell cycle in proliferating cells forming a primary

cilium (Figure 2.2; Quarmby and Parker, 2005). The cilium is assembled during G1 by a


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process called intraflagellar transport (IFT), after docking of the centrosome at the

plasma membrane and formation of the ciliary necklace (see section 2.3). The cilium is

most abundant in G0, and retracted in many cells at the entry into mitosis. Throughout

the cell cycle, the centrosome functions as a MT organizing center (MTOC), from where

the spindle poles are formed during mitosis and the primary cilium is nucleated during

growth arrest (G0) (Doxsey, 2001; Santos and Reiter, 2008). Centrosomes only

duplicate once per cell cycle and failure to do so correctly can result in e.g. multipolar

mitotic spindles and chromosomal missegregation. Several centrosomal proteins have

been determined to be essential for assembly of vertebrate primary cilia (Pedersen et

al., 2008). Others are linked to both cell cycle progression and resorption of the cilium

(Santos and Reiter, 2008). Of note is the mitotic regulatory kinase aurora A which

interacts with an adhesion scaffolding protein to control cilia disassembly (Santos and

Reiter, 2008). Aurora A is a member of Ipl family of kinases, and is modestly related to

CALK, a kinase involved in Chlamydomonas flagellar retraction (Pan et al., 2004) and

overactivity of aurora A and HEF1 has been associated with supernumerary

centrosomes and multipolar spindles (Pugacheva and Golemis, 2005).

2.4. Intraflagellar transport The structure of cilia and flagella presents a transport problem since there is no protein

synthesis in the ciliary compartment. Cilia and flagella are assembled at the distal tip

(Johnson and Rosenbaum, 1992; Marshall and Rosenbaum, 2001), and therefore the

building blocks, which are synthesized in the cell body, must be transported to the tip to

assemble and maintain the flagella. This is done via IFT, a process essential for

assembly and maintenance of cilia and flagella (Kozminski et. al., 1993; Rosenbaum

and Witman, 2002; Cole, 2003). IFT was first observed in Chlamydomonas reinhardtii,

by Joel Rosenbaum´s group in 1993 as a transport system unrelated to ciliary beating

(Kozminski et. al., 1993), and it was later shown that IFT is an evolutionary conserved

process for building and maintaining cilia and flagella in such evolutionary distant

organisms as Caernorhabditis elegans and humans (Cole et al., 1998; Rosenbaum and

Witman, 2002).


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Figure 2.2. Assembly and disassembly of primary cilia in the vertebrate cell cycle. In most cells, primary cilium formation first occurs during G1, as the mother centriole docks to the membrane. Assembly is mediated by IFT-dependent addition of ciliary precursors as the cilium extends directly from the mother centriole’s triplet MTs. During G1 and G0, the cilium functions as a cellular antenna. In the S-phase, the centrioles and DNA replicate, and at entry to G2, the cilium is disassembled, so that the matured centrioles can be ready for mitotic spindle formation. Once mitotic (M) cell division is complete, the centrioles can proceed to ciliary re-assembly in G1. Figure from Pedersen and Rosenbaum, 2008.


20

IFT is a bidirectional process that involves the movement of large protein complexes,

known as IFT particles, by two MT motor complexes responsible for anterograde (base

to tip) and retrograde (tip to base) transport. Cargo, such as building blocks and

turnover products, is coupled to the IFT particles, which are either classified as complex

A or complex B. Proteins destined for the cilium are assembled with the IFT particles

and motor complexes near the transition fibers, at a docking zone (Figure 2.3; Gilula

and Satir, 1972; Deane et al., 2001) and in this way enter the cilium. The transition

zone, also known as the “ciliary pore”, has been hypothesized to function as a barrier

controlling access of molecules to the cilium. The ciliary pore is thought to function, not

unlike the nuclear pore, as a regulated gate of entry where ciliary precursors and IFT

proteins accumulate prior to entering the ciliary compartment (Rosenbaum and Witman,

2002). Proteins destined for the cilium have signal targeting motifs, locating them there,

e.g. has the N-terminal RVxP motif been implicated in the localization of polycystin-2 to

the ciliary membrane (Geng et al., 2006). After assembly, the anterograde motor moves

along the B-tubules just underneath the ciliary membrane. When the complex reaches

the ciliary tip the cargo is unloaded and the IFT complex is reorganized. The retrograde

motor complex then transports new cargo back to the cytoplasm (Figure 2.3). Only one

motor complex is activate at a time and therefore the other is transported as cargo in an

inactivated form (Pedersen and Rosenbaum, 2008).

2.4.1. Anterograde IFT Anterograde transport is reliant on kinesin-2 motor proteins, which can exist as either a

heterotrimeric kinesin-II or a homodimeric kinesin-2 (see section 2.7) In

Chlamydomonas, the heterotrimeric kinesin-II consists of two motor subunits FLA10 and

FLA8 of 90 and 85 kDa. respectively, and the non-motor subunit FLA3 (100 kDa),

known as the kinesin-2-associated protein or KAP (Kozminski et al., 1995; Scholey,

2003; Miller et al., 2005; Mueller et al., 2005). In human and mouse the motor domains

are called KIF3A and KIF3B, respectively. Furthermore a third motor subunit called

KIF3C exists and has been found to associate with KIF3A (Scholey, 2008; Pedersen et

al., 2008). Kinesin-II associates with IFT particles at the transition zone and this huge


21

motor complex transports axonemal precursors and other cargo molecules to the ciliary

tip.

Anterograde IFT has been extensively studied in Chlamydomonas. Analysis of a

temperature sensitive Chlamydomonas mutant, fla10ts, which has a temperature-

sensitive mutation in the FLA10 gene (Adams et al., 1982; Vashishta et al., 1996), gave

the first indication that kinesin-II is required for anterograde IFT. When these

Figure 2.3: Assembly of cilia via intraflagellar transport (IFT). Ciliary proteins are transported in Golgi-derived vesicles along cytoplasmic MTs to the ciliary base. Here the ciliary proteins enter the cilium via the “ciliary pore” and the proteins are transported anterogradely along the axoneme by kinesin-II. Ciliary turnover products are, in turn, transported retrogradely along the ciliary axoneme by cytoplasmic dynein 2 for recycling or degradation in the cytoplasm. From Pedersen and Rosenbaum, 2008.


22

mutant cells were placed at the restrictive temperature of 32°C, IFT ceased and the

flagella began to shorten. If the flagella were isolated from the mutant at the restrictive

temperature, new flagella failed to form. These experiments have shown that kinesin-II

and IFT are required for assembly and maintenance of the flagella. In Chlamydomonas,

kinesin-II is the only anterograde motor, but this is not the case for C. elegans, where a

homodimeric kinesin-2 consisting of OSM3, has been observed to play a role in

anterograde IFT, in part by working in concert with kinesin-II (Scholey et al., 2004).

Kinesin-II is the core anterograde IFT motor in virtually all ciliary systems studied to

date. However other accesory motors may cooperate with it, as seen with OSM3

(Scholey, 2008).

2.4.2. Retrograde IFT Retrograde IFT is motored by an isoform of cytoplasmic dynein called cytoplasmic

dynein 2, previously known as cytoplasmic dynein 1b (Figure 2.4; Pfister et al., 2005;

Pedersen and Rosenbaum, 2008). Dyneins are minus-end directed multiprotein

motorcomplexes consisting of one or more heavy chains and several associated

proteins (Pedersen et al., 2008). In Chlamydomonas the motor complex consists of a

heavy chain, DHC1b (belonging to the AAA+ family of ATPases; Pazour et al., 1999;

Asai and Koonce, 2001), a light intermediate chain, D1bLIC (Hou et al., 2004), an

intermediate chain (IC)/WD repeat protein, FAP133, which may be specific for motile

cilia (Rompolas et al., 2007) and a light chain, LC8/FLA14 (Figure 2.4; Pazour et al.,

1998). In human and mouse, a motor heavy chain, DHC2, a light intermediate chain

D2LIC, and an intermediate chain, WD34, have been identified. However, the precise

function of the individual subunits of the cytoplasmic dynein 2 complex during

retrograde IFT is still unclear (Pedersen and Rosenbaum, 2008).

The cytoplasmic dynein 2 motor subunit, DHC2, was originally identified as a

dynein heavy chain in sea urchin embryos (Gibbons et al., 1994), and has subsequently

been studied further in Chlamydomonas and C. elegans (Pazour et al., 1999; Signor et

al., 1999).


23

Figure 2.4: Chlamydomonas cytoplasmic dynein 2. The heavy chains bind the outer double MT, and the light chains bind cargo. Modified from Rompolas et al., 2007.

2.4.3. Tip turnaround Both anterograde and retrograde IFT seem to occur at a constant rate along the cilium

(Kozminski et al., 1993), with sligth pauses at the base and tip, so the main points of

regulation of IFT are presumably at the ciliary base and tip (Pedersen and Rosenbaum,

2008). Cilia and flagella are assembled and continously turnover at their distal tip

(Johnson and Rosenbaum, 1992; Marshall and Rosenbaum, 2001). IFT transports

flagellar precursors from the flagellar base to the tip for assembly and returns turnover

products from the tip back to the base. The molecular mechanisms by which these

different components are coordinated and regulated at the flagellar base and tip are

unclear. The molecular mechanisms of IFT turnaround in Chlamydomonas involves: the

inactivation/down-regulation of kinesin-2, activation/upregulation of cytoplasmic dynein

2, unloading of flagellar precursors, and the loading of flagellar turnover products

(Pedersen and Rosenbaum, 2008). The timing and mechanisms of these events are

unknown, although some clues have recently emerged.

Regulation of kinesin-2 motor activity appears quite complex involving a variety

of different regulatory mechanisms (e.g. phosphorylation, comformational changes,

tubulin modifications) and molecules. To mention a few, analysis of mutants or

biochemical inhibitors affecting ciliary length, have revealed a number of kinases as


24

potential regulators of kinesin-II activity, including MAP kinases and NIMA-related

kinases (Pedersen and Rosenbaum, 2008). Also, kinesin-II activity may be regulated via

conformational changes in the KAP subunit, because KAP is required for localization of

kinesin-II at the flagellar base as well as for movement of the motor complex along

flagella in Chlamydomonas (Mueller et al., 2005). The mechanisms by which

cytoplasmic dynein 2 is regulated are virtually unknown. This could be due to the fact

that it has been difficult to purify biochemically, and the complex may contain additional

unidentified subunits (Pedersen and Rosenbaum, 2008).

At the flagellar tip, IFT particle turnover seems to be regulated by IFT172

(Pedersen et al., 2005; Tsao and Gorovsky, 2008) possibly in conjunction with the small

MT-associated protein EB1 (Pedersen et al., 2005), which localizes to the flagellar tip

and basal bodies in Chlamydomonas (Pedersen et al., 2003; also see section 2.4.4.1

and 2.5).

2.4.4 IFT particle polypeptides Associated with to kinesin-II and cytoplasmic dynein 2 are IFT particles, which have

multiple protein-protein interaction motifs serving as docking sites for cargo proteins,

such as ciliary building blocks (Cole, 2003; Blacque et al., 2008;). IFT particle proteins

were first identified in, and isolated from Chlamydomonas flagella, using the fla10ts

mutant (Kozminski et al., 1993; Piperno and Mead, 1997; Cole et al., 1998; Also see

section 2.2.1). Sucrose density gradient centrifugation was used to fractionate the

membrane plus matrix, allowing comparison of flagellar proteins, extracted under mild

conditions (Piperno and Mead, 1997; Cole et al., 1998). This led to the identification of approximately 17 different IFT particle proteins which can bee separated into two large

complexes, A and B (Cole et al., 1998; Piperno et al., 1998). Cloning and sequencing of

Chlamydomonas IFT particle polypeptide genes showed that both complex A and B

components have several domains and amino acid repeats typically involved in

transient protein-protein interactions (Cole, 2003). The IFT particle polypeptides all have

apparent molecular masses between 20 and 172, and are named IFT20 through IFT172

(Cole et al., 1998). Complex A comprises the following IFT particle proteins: IFT144,


25

IFT140, IFT139, IFT122A, IFT122B, and IFT43 and their overall function is primarily

associated with retrograde IFT. Complex B comprises the following IFT particle

proteins: IFT172, IFT88, IFT81, IFT80, IFT74/72, IFT57 (also known as IFT57/55),

IFT52, IFT46, IFT27, IFT25, IFT22 and IFT20. Complex B is required for anterograde

IFT and loss of any of the complex B proteins results in shortened or absent cilia. For

some complex B proteins, additional or specific functions related to flagellar assembly

have been described. For example, IFT20 is involved in transport of vesicles from the

Golgi to the ciliary base (Follit et al., 2006; Omori et al., 2008), IFT27 functions as G-

protein in the cell cycle (Qin et al., 2007), IFT46 is involved in transport of outer dynein

arms into the flagella (Hou et al., 2007), and IFT172 functions as a regulator of the

transition from anterograde to retrograde IFT in the tip of the cilia (Pedersen et al.,

2005; Tsao and Gorovsky, 2008).

2.4.4.1. IFT172 IFT172 is encoded by FLA11 and is the protein of complex B with the highest molecular

mass (172 kDa) (see section 2.4.4; Cole et al., 1998). The Chlamydomonas fla11

mutant has a point mutation in IFT172, which results in short or missing cilia as well as

accumulation of IFT particles in the ciliary tip (Pedersen et al., 2005). IFT172 has been

shown to contain a N-terminal WD repeat domain (WDD) composed mainly of β-sheets

and a C-terminal repeat domain (RPD) composed mainly of α-helices. In between the

RPDs a LIM interaction domain (LIM-ID) is located (Figure 2.5; Pedersen et al., 2005;

Tsao and Gorovsky, 2008). These structures have been shown to be very conserved

among different organisms and are involved in protein-protein interaction. Studies of

IFT172 in Tetrahymena, where the different domains had been selectively deleted,

showed that both the N- and C-terminal domains are essential for localization of IFT172

to cilia and for the assembly of cilia. A mutant with a partially truncated C-terminal

accumulated IFT particles in the ciliary tip, indicating failure of motor switching or

retrograde transport (Tsao and Gorovsky, 2008).

The IFT172 orthologue in rats is called SLB (Selective Lhx3/4 Lim-homeodomain

transcription factor Binding protein), and the LIM-binding domain has been shown to


26

interact specifically with members of the LIM homeodomain family of transcription

factors Lhx3 and Lhx4. Binding inhibits Lhx3 and Lhx4, indicating a possible role of

IFT172 as a transcription regulator (Howard and Maurer, 2000).

Mutant experiments in C. elegans have shown that mutations in the IFT172

orthologue OSM-1 gives a defect in sensory cilia (Perkins et al., 1986; Bell et al., 2006).

Furthermore, a screening in zebrafish mutants with kidney cysts, identified a mutation in

IFT172 (Sun et al., 2004). Finally, IFT172 dissociates easily from the rest of the IFT

complex B, which could indicate that it is in the periphery of the complex and thereby

ideally could be positioned to play a regulatory role in IFT (Pedersen et al, 2005).

Figure 2.5: IFT172 in Tetrahymena. The beta-sheats and alpha-helices are known for protein-protein interaction. LIM indicates, the Tetrahymena IFT172 domain homologous to the LIM-transcription factor domain in rats (SLB/IFT172). Modified From Tsao and Gorovsky, 2008.

2.5. EB1 EB1 is a MT plus-end tracking protein (+TIP) and belongs to one of the most conserved

families among the +TIPs, the EB family. EB proteins contain highly conserved N- and

C-terminal domains, which are separated by a less conserved linker sequence. The N-

terminal domain is necessary for MT binding and the C-terminal domain has a coiled-

coil region that mediates the parallel dimerization of EB protein monomers and at the

same time forms a surface for binding of various partners (Lansbergen and Akhmanova,

2006). EB1 is a relatively small MT-binding protein and it preferentially localizes to the

plus end of cytoplasmic MTs where it is involved in regulating MT dynamics.

Furthermore it is indirectly involved in linking the plus end with the cell cortex, mitotic


27

kinetochores and different cellular organelles by directing other MT-associated proteins

(MAPs) towards the plus tip. EB1 also localizes to the centrosomes and is required for

centrosomal MT anchoring. In addition, EB1 has been shown to localize to the ciliary tip

and the proximal part of the basal bodies in Chlamydomonas (Pedersen et al., 2003),

and centrosomal EB1 is required for assembly of primary cilia in mouse fibroblasts, by

Interacting with p150Glued in cilia formation (Schrøder et al., 2007). However, the exact

mechanism by which this occurs is unknown. Vertebrates contain two additional EB1-

like proteins (EB2 and EB3), and it is possible that EB2 and/or EB3 also contribute to

ciliogenesis.

2.6. Nucleotide-binding protein 1 (Nubp1) The Chlamydomonas protein Nubp1 (Figure 2.6), which was identified as part of the

Chlamydomonas genome sequencing project (Merchant et al., 2007), is homologous to

NBP1 in humans that belongs to the NUBP/MRP subfamily (Figure 2.7; Nakashima et

al., 1999). Nubp1 proteins are very conserved in different eukaryotes and contain a

MRP domain, a P-loop containing nucleotide triphosphate hydrolases (ATP/GTP-

binding) site, as well as an α- and ß-motif (see figure 2.6; 2,7; Nakashima et al., 1999).

In mammals, Nubp1 is closely related to Nubp2; however Nubp1 has a unique N-

terminal extension containing four cysteine residues, lacking in the shorter form, Nubp2

(Nakashima et al., 1999).

Figure. 2.6: Schematic presentation of Chlamydomonas Nubp1. It has highly conserved domains belonging to the NUBP/MRP subfamily: The ATP/GTP binding domain (Blue) and NUBP/MRP consensus pattern (Green) as well as the α-(red) and ß-(orange) motif; Also see Figure 2.7 for conservation of the motifs in different eukaryotes.


28

Nubp1 proteins have extensive similarity to the prokaryotic division-site-

determining membrane ATPase protein MinD (Nakashima et al., 1999). In bacteria,

FtsZ, the bacterial homologue of tubulin, assembles to a cytoskeletal element, the Z-

ring, that recruit other proteins to carry out cytokinesis. The positioning of the Z-ring is

determined by a gradient of negative regulators. MinD together with the min operon

proteins MinC and MinE cooperatively position the Z-ring, thereby determining the

separation site for cell division (reviewed by Lutkenhaus 2007).

The Chlamydomonas Nubp1 homolog has not previously been characterized

biochemically or functionally and studies on Nubp1 homologs in other organisms are

also scarce. However, in mouse fibroblasts, Nubp1 has been shown to interact directly

with the related protein Nubp2 as well as the minus-end directed kinesin KIFC5A.

Inactivation of either KIFC5A or Nubp1 in mouse fibroblasts results in the presence of

supernumerary centrosomes and an increase in the proportion of bi- and tri-nucleated

cells (Christodoulou et al., 2006). Whether these phenotypes in any way are coupled to

cilia is unknown.

2.7. Kinesins Kinesins constitute a superfamily of ATPase motor proteins that travel along MT tracks.

The family mediates diverse functions in the cell, including the transport of vesicles,

organelles, chromosomes and protein complexes (Hirokawa et al., 1998; Dagenbach

and Endow, 2004; Miki et al., 2005). The motor domain of the kinesin superfamily is

very conserved, and differences in the ca. 350 amino acid sequence is basis for the

classification of the motor proteins (Kashina et al., 1997). So far 17 different kinesin

families have been described (Wickstead and Gull, 2006) and a general kinesin

nomenclature was introduced in 2004 (Lawrence et al., 2004).

Usually a kinesin motor protein also comprises a regulatory neck domain

following the motor, and a tail region that interacts with cargo or other subunits (Figure

2.8). In contrast to the motor region, the tail region can be highly diverse among

kinesins, even within a family, and appears to bind cargo via adaptor or scaffolding

molecules (Hirokawa and Noda, 2008; Wickstead and Gull, 2006).


29

Figure 2.7: Multiple sequence alignment of Nubp1 homologs. The highly conserved domains of the NUBP/MRP subfamily include: The ATP/GTP binding domain (blue) and NUBP/MRP consensus pattern (green) as well as the α-(red) and ß-(orange) motif; See Figure 2.6 for a schematic presentation of Chlamydomonas Nubp1. Data obtained from Nakashima et al., 1999.


30

In most kinesins the motor domain is located in the N-terminus and such kinesins move

in the MT plus end direction whereas kinesins with the motor domain in the C-terminus

are minus end directed (Hirokawa and Noda, 2008). Normally kinesins are associated

as di- or trimers with the tails coiled together (Miki et al., 2001) and they walk in a cyclic

”hand-over-hand” manner of which many models exist. Basically, when the front motor

subunit binds ATP a conformational change displaces the weakly MT-interacting rear

head towards the MT-plus (or minus) end ahead of the other subunit and rebinds tightly

to ATP and the MT. The now rear head hydrolyses ATP causing a conformational

change releasing it from MT (Gennerich and Vale, 2009). I will shortly mention a few of

the kinesin families of interest to this thesis.

Figure 2.8. Schematic structure of conventional kinesin. Kinesin consists of a motor domain (head), a coiled-coil stalk region, and a cargo binding region (tail). Figure from Kikkawa, 2008.

2.7.1. Kinesin-2 family proteins

Kinesin-2 family members are known to participate in organelle transport, IFT and

spermatogenesis (Miki et al., 2005). Kinesin-II, the motor for anterograde transport, has

been described in section 2.4.1. However, kinesin-II also has non-cilia related functions

such as vesicle transport in neuronal axons (Hirokawa and noda, 2008)

Members of this family have not yet been described in fungi or higher plants that lack

cilia/flagella and sperm.


31


The kinesin-5 family proteins, also known as the BimC family, where given the family

number 5 because of the name of one of its most well known members, the mammalian

kinesin Eg5. The kinesin-5 family is the most conserved, monophyletic family and

kinesin-5 members are characterized by a characteristic BimC box domain (Kashina et

al., 1997). Kinesin-5 family members are found in mammals, yeast and higher plants.

They usually form homotetramers and are known to be mitotic motors functioning in

formation of the bipolar spindle during cell divison (Valentine et al., 2006). It is

hypothesized that they act in concert with minus-end-directed dyneins and other plus-

end-directed kinesins as well as serve to control the position of centrosomes and thus

play global roles in establishing and maintaining bipolar mitotic spindle structure. In all

known cases kinesin-5 family proteins prove to be localized to spindle MT (Kashina et

al., 1997). Mutations in the yeast bimC gene appear to block the separation of

duplicated centrosomes or spindle pole bodies resulting in the formation of defective

”monastral” mitotic apparati at early stages of mitosis. Furthermore, immunodepletion of

Eg5 in Xenopus oocytes causes defects in spindle formation at early stages of mitosis

(Kashina et al., 1997; Kapoor et al., 2000). Eg5 has also been shown to be expressed in

rodent postmitotic neurons. Here Eg5 is believed to be involved in organizing MT in the

devolping neurons (Ferhat et al., 1998). A specific inhibitor, called Monastrol, is known

to act specifically on the motor domain of human Eg5 arresting cells in mitosis (Cochran

et al., 2005).


Kinesin-14 family members are minus-end directed motors that cross-link MTs and play

key roles during spindle assembly. This family acts to regulate spindle length during

mitosis by cross-linking and sliding between parallel microtubules (Christodoulou et al.,

2006; Cai et al., 2009). Structurally Kinesin-14s have a conserved kinesin-like motor

domain at the C-terminus, a central coiled-coil stalk, and an N-terminal globular domain.

The mouse kinesin-14 member KIFC5A is involved in regulation of centrosome

duplication. Overexpression causes formation of aberrant, non-separated MT asters


32

and mitotic arrest in a promethaphase-like state. It is believed that the C-terminal minus-

end directed kinesins can produce forces that oppose the bimC-driven forces in the

mitotic spindle (Kashina et al., 1997), and knockdown of KIFC5A has been shown to

partly relieve the effect of the Eg5 inhibitor monastrol, indicating involvement in the

balance of forces determining the bipolar spindle during mitosis (Christodoulou et al.,

2006). KIFC5A interacts directly with Nucleotide-binding protein 1 (See section 2.6), and

inactivation of KIFC5A in mouse fibroblasts result in the presence of supernumerary

centrosomes and an increase in the proportion of bi- and tri-nucleated cells

(Christodoulou et al., 2006).

Chlamydomonas KCBP is a unique kinesin of the kinesin-14 family in that has a

calmodulin-binding domain. Close homologs are found in plants and there is also less

wellconserved member in sea urchins (Miki et al., 2005). Cytoplasmic dyneins are

lacking in plants and this could explain the abundance of this family member here, as

both are MT minus end-directed motors. Recently a KCBP kinesin has been shown to

localize to flagella and near the base of the flagella in Chlamydomonas. Although direct

functional data are lacking, this kinesin has been suggested to play a role in flagellar

assembly/disassembly as well as cell division (Dymek et al., 2006).

2.8. Chlamydomonas as a model organism for ciliary functions Chlamydomonas reinhardtii is a motile single-celled green alga about 10 µm in diameter

and with two similar flagella of approximately 12 µm in length, which it uses for motility.

Chlamydomonas has become a model of great importance in the world of biology.

Chlamydomonas possesses properties from both the animal and plant kingdom, showing similarity to animal cells by having centrioles and flagella, but at the same time also a relation to the plants, because it contains a chloroplast. These and other

properties make it the perfect model to study fundamental processes such as motility,

photosynthesis, cell cycle defects, responses to external stimuli such as light, and cell-

cell recognition (reviewed by Harris, 2001).


33

Chlamydomonas belongs to a very small group of model organisms where it is possible

to combine biochemical, genetic, and biological approaches to investigate the basic

biology and functions of ciliary and

basal body proteins (Badano et al.,

2006; Pan, 2008). Flagella of

Chlamydomonas are typical of

eukaryotic cilia and flagella, in that

they are composed of MT arranged

in the "9 + 2" structure (Figure 2.9).

Since different organisms solve

similar problems in similar ways,

studies on how MT assembly is

regulated in Chlamydomonas may

reveal mechanisms that are shared

by most other ciliated organisms.

IFT can be visualized in vivo without

the aid of fluorescence tagged

proteins and a large number of IFT mutants are available (Figure 2.9). Not surprisingly,

the protein components of the IFT machinery were first isolated and identified in

Chlamydomonas (Kozminski et al., 1993; Piperno and Mead, 1997; Cole et al., 1998;

section 2.4) and its flagella have been used extensively in the studies of IFT and other

cilia-related processes. Many known mutants of Chlamydomonas exist and make useful

tools for studying a variety of biological processes, including flagellar motility,

photosynthesis or protein synthesis. Since Chlamydomonas species are normally

haploid, the effects of mutations are seen immediately without additional timeconsuming

backcrossing (Figure 2.10; Pan and Snell, 2000). For instance a study of

Chlamydomonas mutants defective in dynein facilitated the identification of the genetic

basis for the disease PDC in human (Pennarun et al., 1999), and another study of a

mutant with defects in flagellar assembly (mutant ift88) provided the first link between

PKD and cilia (Pazour et al., 2000).


34

Figure 2.10: The Chlamydomonas cell cycle. 1-7: sexual reproduction 8: asexual reproduction. 1. Gametogenesis is induced, when the N-source is removed. 2. Adhesion of gametes of opposite mating types. 3. The cell walls are released and the mating structures activated. 4. Fusion of mt+ fertilization tubule with mt- mating structure. 5. Complete cell fusion. 6. Zygote (2n) maturation. 7. When the environment is optimal the zygote undergoes meiosis/germination and new vegetative cells (1n) are formed. 8. Cells undergo mitosis by resorbing the flagella and divide inside the cell wall of the mother cell wall (not shown). For C. reinhardtii 2 mating types, mt+ and mt-, exist. The figure was kindly provided by Ph.D. student Jacob M. Schrøder.


35

Chlamydomonas has a relatively short reproduction time of approximately 12-18 hrs,

depending on the temperature, light and media. Exposure to sunlight in an appropriate

medium produces uniform cultures containing large numbers of motile cells. The

Chlamydomonas cell cycle can be synchronized with cycles of light and dark, which is a

major technical advantage. Synchronization of the cells gives a higher yield of

flagellated cells for experimental purposes and the assembly and resorption of flagella

can more easily be studied, because the flagella are resorbed during entry into the

mitotic cycle and then reassembled after completion of the cycle. When

Chlamydomonas is grown with a 12:12, 14:10 or 16:8 light:dark cycle it can be fixed in

the G1 phase during the entire light

phase (Figure 2.11; Harris, 2001).

Chlamydomonas flagella can

readily be amputated and regrown,

observed, and measured. They can

be induced to gradually shorten

their flagella lengths, resulting in

complete loss, making it easy to

study the kinetics of flagellar

assembly and disassembly.

Chlamydomonas is one of the few

organisms from which cilia can be

isolated and purified in large

quantities, and at different stages of flagellar growth and shortening. A wide range of

chemical and physical stimuli can induce flagellation. The immediate response to an

acid shock produces intracellular acidification that induces an influx of calcium and

starts a signalling cascade resulting in activation of the severing machinery. The nine

outer doublet MT are broken at the distal end of the flagellar transition zone, the site of

axonemal severing (SOFA) (Sander and Salisbury, 1989; Quarmby and Hartzell, 1994;

Quarmby, 1996). Shedding of the flagella with a pH shock thus gives a very simple

biochemical method for isolating (and purifying) flagella.


36

By now, the genome sequence (Merchant el al., 2007), flagellar proteome

(Pazour et al., 2005), and flagellar transcriptome (Stolc et al., 2005) of Chlamydomonas

are known, which makes it easy to obtain the bioinformation needed for further research

with Chlamydomonas.

Since all organisms are related by evolution, the knowledge acquired from

studies of Chlamydomonas allows researchers to learn more about regulation of gene

expression in more complex plants and animals. Usually, finding the gene responsible

for a particular mechanism in human tissue without studying simpler model organisms is

nearly impossible. In this thesis, I have used Chlamydomonas as a model organism for

identifying and characterizing new proteins that localize to the flagellar tip, and which

may potentially be involved in regulating IFT and/or flagellar assembly/disassembly.


37

3. Results and discussion

3.1. Introductory notes Axonemal MTs are oriented with their plus end towards the flagellar tip, where constant

assembly and turnover takes place (Johnson and Rosenbaum, 1992; Marshall and

Rosenbaum, 2001; Marshall et al., 2005). It has previously been shown that EB1, as

one of few proteins, localizes to the flagellar tip in C. reinhardtii (Pedersen et al., 2003).

CrEB1 interacts with IFT172 at the ciliary tip, where they may regulate IFT particle

turnover (Pedersen et al., 2005). In the known proteomic analysis of the C. reinhardtii

flagellum (Pazour et al., 2005), EB1 was represented with very few peptides (2 unique

peptides) and all in the membrane plus matrix fraction.

Prior to the onset of my project, to identify other potential flagellar tip proteins,

proteins also found in the flagella proteome with approximately the same peptides and

exclusively in the membrane plus matrix fraction were identified by the Pedersen

laboratory, and a protein homologous to mouse Nubp1 (CrNubp1) was selected as an

interesting potential flagellar tip protein, because mouse Nubp1 is known to interact with

KIFC5A, a minus end-directed kinesin of the kinesin-14 family (Christodoulou et al.,

2006). The N-terminal cDNA coding region of CrNubp1 (GenBank accession,

gi:159485046; nt 1-619) was cloned and sequenced by the lab, and a polyclonal

antibody against the N-terminus of CrNubp1 was produced prior to the onset of this

project. I affinity purified the antibody, tested it and used it in my project to characterize

CrNubp1. These results are presented in sections 3.2-3.5. In addition, I obtained two

different antibodies specific for mouse Nubp1 (provided by Niovi Santama, University of

Cyprus), and I used these antibodies for immunofluorescence microscopy (IFM)

analysis of mouse NIH3T3 cells to determine whether Nubp1 localized to primary cilia or

basal bodies in these cells (section 3.6).

As an alternative approach to identifying novel tip proteins, I set out to identify

binding partners of CrEB1 and IFT172 C-terminus using GST pull-down of isolated

flagella from C. reinhardtii cells. This part of my thesis work was done in collaboration

with Anna Akhmanova and her lab in Rotterdam, The Netherlands. Akhmanovas group


38

executed the GST pull-down experiments in Chlamydomonas using isolated flagella and

GST-CrEB1 fusion protein (Pedersen et al., 2005), and identified putative binding

partners by mass spectrometry. The flagella were isolated and purified by me (see

section 5.2) and the resulting data was analysed by my supervisor Lotte Pedersen. I

subsequently cloned and characterized three of the potential EB1/IFT172 binding

partners identified (section 3.7-3.10).

3.2. Chlamydomonas reinhardtii Nubp1 is present in the flagella To characterize C. reinhardtii Nubp1 (CrNubp1), an antibody against the N-terminal part

of this protein was affinity purified and tested using western blotting (see section 5.10)

to see if it was present in the flagella. CrNubp1 is predicted to encode a 40.6 kDa

protein (Table 3.1). Immunoblot analysis with the affinity purified CrNubp1 antibody

detected a single ~55 kDa band in isolated C. reinhardtii flagella (Figure 3.1.A). The

apparent molecular weight of this protein is higher than predicted, which could be due to

the acidic nature of CrNubp1 (predicted pI of 4.95; Table 3.1) resulting in slower

migration during SDS-PAGE.

To compare the relative abundance of CrNubp1 in flagella and cytoplasm, blots

of wild type (CC-124) de-flagellated cell bodies were compaired with flagella isolated

from an equal number of cells (Figure 3.1.B). CrNubp1 is enriched in the flagella, similar

to IFT139 (IFT complex A protein). The majority of the cellular pool of CrEB1 is in the

cell body consistent with published data (Figure 3.1.B; Pedersen et al., 2003).

Table 3.1: Theoretical values for CrNubp1. The MW and pI values were calculated from the polypeptide sequences using the pI/MW tool at Expasy.ch. ”Peptides” refers to the number of peptides by which this protein is represented in the C. reinhardtii flagellar proteome (Pazour et al., 2005).

MW (kDa) GenBank ID pI Peptides Amino acids

CrNubp1 40.6 gi: 159485046 4.95 1 1215


39

Figure 3.1: Presence of CrNubp in C. reinhardtii flagella. Western blot analysis with affinity-purified CrNubp1 antibody detects a single band in wild type (CC-124) isolated flagella (A). In (B) protein samples prepared from de-flagellated wild type (CC-124) cell bodies and flagella were analyzed by western blot with antibodies against IFT139 (IFT complex A protein), CrNubp1 or CrEB1. The bottom panel shows a Coomassie-blue-stained gel run in parallel. Note that CrNubp1 is enriched in the flagella, similar to IFT139, while the majority of the cellular pool of CrEB1 is in the cell body.

Cell equivalents 4

25 -

35 -

45 -

55 -

70 - 95 -

130 - 170 -

Coomassie

CrEB1

CrNubp1

IFT139

cell body flagella

B A

70 -

55 -

45 -

35 -

25 -

kDa

95 - 130 - 170 -

80 3 2 1 8 4 2 1


40

3.3. CrNubp1 localizes to the soluble membrane plus matrix

compartment To determine where in the flagella CrNubp1 is present, wild type (CC-124) flagella were

disrupted by freezing and thawing in a detergent buffer. ATP was added, and the lysate

sucked through a 27 gauge needle, followed by centrifugation to sediment axonemes

and detergent-insoluble membranes (see section 5.2). This resulted in the release of all

of the total flagellar CrNubp1 into the supernatant/soluble fraction (Figure 3.2). This is

consistent with the finding that the one unique peptide of CrNubp1 found in the

Chlamydomonas flagellar proteome analysis was in the detergent-soluble membrane

plus matrix fraction (Pazour et al., 2005). Most of CrEB1 was also released in this

fraction. Upon further extraction of the pellet with high salt buffer, a small fraction of

CrEB1 was also associated with the salt extract (Figure 3.2). Thus, most of the flagellar

CrEB1 is soluble as consistent with previously published data (Pedersen et al., 2003;

Pazour et al., 2005). In contrast, several IFT components (IFT139, Fla10, IFT172,

D1bLIC) as well as the kinesin KCBP and Lis1-like protein CrLis1 were also present in

extracted axonemes in addition to the membrane matrix and high salt extract fractions

(Figure 3.2).


41

Figure 3.2: In fractionated wild type (CC-124) flagella, CrNubp1 localizes to the soluble membrane plus matrix compartment. Flagella protein samples from wild type cells were fractionated as described in materials and methods and immunoblotted with antibodies against IFT139 (IFT complex A protein), KCBP (Kinesin-like Calmodulin-binding protein; Dymek et al., 2006), FLA10 (Kinesin-II motor domain), CALK (Aurora protein kinase; Pan et al., 2004), IFT72 (IFT complex B protein), D1bLIC (Cytoplasmic dynein 2 light intermediate chain; Hou et al., 2004), CrNubp1 (this study), CrLis1 (Lissencephaly protein Lis1; Pedersen et al., 2007), and CrEB1 (Pedersen et al., 2003). Bottom panel shows a Coomassie-blue-stained gel run in parallel. Note that CrNubp1 is in the membrane plus matrix fraction only. Most of CrEB1 is also present in this compartment consistent with previously published results (Pedersen et al., 2003).


42

3.4. The flagellar level of CrNubp1 is unaffected by mutations

affecting assembly of the main axonemal substructures: outer dynein

arms, inner dynein arms, radial spokes and the central apparatus. To determine if CrNubp1 is affected by the lack of axonemal components necessary for

flagella motility, flagella from different mutant strains were isolated and immunoblotted

with antibodies against CrNubp1, IFT139 (IFT complex A protein) and CrEB1 (Figure

3.3). CrNubp1 is found in flagella of all Chlamydomonas mutants examined here,

including those with flagella that lack radial spokes (pf14) and the central pair (pf18), as

well as mutants that lack the inner (ida1/ida4) and outer (oda2) dynein arms (Figure

3.3). Therefore CrNubp1 most likely does not localize to any of these axonemal

structures. This is consistent with the flagellar fractionation data indicating that CrNubp1

is only found in the soluble membrane plus matrix fraction of wild type (CC-124) flagella

(Section 3.3; Figure 3.2; Pazour et al., 2005). The same is seen for CrEB1 also

consistent with Pazours proteome analysis. Only a small band is visible for IFT139 in

the ida4 mutant strain (Figure 3.3). The explanation for this observation is unclear and

cannot be the lack of flagella inner dynein arms, because no effect is seen in the ida1

mutant. It is possible that the ida4 mutant strain harbors some additional mutation that

affects IFT139, but further experiments are needed to clarify this.

Figure 3.3: The flagellar level of CrNubp1 is unaffected by mutations in genes affecting motility-related axonemal structures. Wild type flagella (wt; CC-124) and flagella isolated from different Chlamy-domonas mutant strains were analyzed by western blotting using antibodies against CrNubp1, IFT139 and CrEB1, as indicated. ida1 and ida4: lack inner dynein arms; oda2: lacks outer dynein arms; pf14: lacks radial spokes; pf18: lacks entire flagellar central apparatus. For details about these strains, see www.chlamy.org.


43

3.5. CrNubp1 localizes to the basal bodies and the tip of the flagella It was found by western blotting that CrNubp1 is present in the flagella, specifically in

the soluble membrane plus matrix compartment (Figure 3.1.A and Figure 3.2). To study

the localization pattern of CrNubp1 in whole C. reinhardtii wild type cells, IFM using the

polyclonal antibody directed against the N-term of CrNubp1 and a monoclonal anti-

acetylated alpha tubulin antibody to stain the flagella and basal bodies, was performed.

Using a methanol (MeOH) fixation method (see Section 5.11.1) this analysis showed

that CrNubp1 localized to the basal bodies in Chlamydomonas wild type cells (Figure

3.4). This result, however, shoul be interpreted with some caution because in contrast to

isolated flagella, western blotting of de-flagellated cell bodies or whole cells using the

CrNubp1 antibody failed to detect a band of the appropriate size (Figure 3.1.B and data

not shown), and therefore we do not know the specificity of the CrNubp1 antibody in the

cell body. However, similar analysis in mouse NIH3T3 cells strongly suggest that Nubp1

localizes to the basal bodies (see Section 3.6). In addition to possible basal body

localization of CrNubp1, in some cases, weak fluorescence was detected at the flagellar

tip on the MeOH fixed cells (data not shown).

To explore this possible tip localization further, IFM using an alternative fixation

protocol was performed. Interestingly, when cells were fixed using a fixation buffer with

Glutaraldehyde/NP40 (Lechtreck et al. 2009; Section 5.11.1) flagellar tip localization of

CrNubp1 in Chlamydomonas wild type cells was clearly observed (Figure 3.5). This is

consistent with the hypothesis that CrNubp1 is a flagellar tip protein and is also

consistent with my results indicating that CrNubp1 co-fractionates, at least in part, with

the known flagellar tip protein CrEB1 (see Section 3.3). Since mouse Nubp1 is known to

interact directly with the minus-end-directed kinesin KIFC5A (Christodoulou et al.,

2006), it is tempting to speculate that CrNubp1 similarly interacts with a minus-end-

directed kinesin at the flagellar tip in order to regulate flagellar disassembly and/or

transport of flagellar turn over products from the tip towards the cell body. However,

attempts to identify interaction between CrNubp1 and the known flagellar minus-end

directed kinesin KCBP (Dymek et al., 2006) were unsuccessful. It is of interest, though,


44

that minus-end-directed kinesins of the kinesin-13 family were identified at the flagellar

tip in Leishmania (Blaineau et al., 2007) and Giardia (Dawson et al., 2007).

Figure 3.4: IFM, using a methanol fixation method, shows basal body localization of CrNubp1 in C. reinhardtii wild type (CC-124) cells. Cells were grown at 20°C and subjected to IFM using a polyclonal antibody specific for CrNubp1 (red). To detect the flagella and basal bodies an antibody specific for acetylated alpha tubulin (green) was used. The IFM indicates that CrNubp1 (red) localizes to the basal bodies of the cells. Asterisks mark the basal bodies, shown enlarged in the insets.


45

Figure 3.5: IFM, using a Glutaraldehyde/NP40 fixation method, showing flagellar tip localization of CrNubp1 in C. reinhardtii wild type (CC-124) cells. Cells were grown at 20°C and subjected to IFM using a polyclonal antibody specific for CrNubp1 (red). To detect the flagella and basal bodies an antibody specific for acetylated alpha tubulin (green) was used. The two bottom panels show the tips of flagella in focus. The bottom panel in the middle was a control were no primary CrNubp1 antibody was added. The IFM shows that CrNubp1 (red) localizes to the tip of the flagella, and no tip localization is seen when no CrNubp1 primary antibody is added. The strong fluorescence of the cell body is due to autofluorescence of the cells and was also observed when no primary antibodies were added (not shown). Scale bar 10 µm.

CrNubp1!

No primary antibody!

AcTub! Merge!


46

3.6. Mammalian Nubp1 localizes to the centrioles and the nucleus in

NIH3T3 fibroblast cells To study the localization pattern of Nubp1 in NIH3T3 mouse fibroblast cells, IFM using

two different antibodies against mouse Nubp1, generously provided by Niovi Santama,

University of Cyprus (Christodoulou et al., 2006), was performed. The first antibody is

an affinity-purified anti-peptide antibody made in guinea pig against the C-terminus of

Nubp1 and the second antibody is an affinity-purified antibody made in rabbit against

bacterially expressed recombinant Nubp1 (both unpublished). In addition, cells were

stained with a monoclonal anti-acetylated alpha tubulin antibody to stain the flagella and

basal bodies. Using the antibody against the C-term of Nubp1, the IFM analysis of

serum starved NIH3T3 mouse fibroblast cells showed that Nubp1 appeared to

concentrate mainly in the nucleus (Figure 3.6; Bottom panel; Data not shown). This was

also seen in cells in interphase (Data not shown). However, when using the antibody

against recombinant Nubp1, localization to the basal bodies was also observed (Figure

3.6; Three top panels). These results supports my observations in the IFM on

Chlamydomonas using CrNubp1 antibody (Figure 3.4) despite the fact that I was unable

to detect CrNubp1 in cell bodies by western blot analysis. It is possible that so little

Nubp1 exists in the cell so that it is not detectable with our particular antibody. Taken

together, the results indicate that Nubp1 is found at the flagellar tip (Section 3.5 ,Figure

3.5) as well as in the basal bodies (Section 3.5, Figure 3.4; Figure 3.6). What is the

function of CrNubp1 at these sites? As mentioned above, CrNubp1 at the flagellar tip

could function as a regulator of flagellar disassembly or transport between the tip and

the basal bodies by affecting minus-end-directed kinesins. In mouse 3T3 fibroblast cells,

Nubp1 has been shown to interact directly with Nubp2 as well as KIFC5A. The mouse

kinesin-14 family member KIFC5A is a minus-end-directed kinesin involved in regulation

of centrosome duplication. Inactivation of either KIFC5A or Nubp1 in mouse fibroblasts

results in the presence of supernumerary centrosomes and an increase in the

proportion of bi- and tri-nucleated cells (Christodoulou et al., 2006). The results

presented here suggest that Nubp1 at the flagella tip and basal bodies might be

important for regulating centriole duplication and cell cycle progression via modulation


47

of the activity of specific minus-end-directed kinesins. It has been shown that a member

of the kinesin-14 family, called kinesin-like Calmodulin-binding protein (KCBP), does

exist in Chlamydomonas flagella and is localized near the base of the flagella in

interphase. Although direct functional data are lacking, this kinesin has been suggested

to play a role in flagellar assembly as well as cell division (Dymek et al., 2006). I have

so far been unable to detect association between KCBP and CrNubp1 using

immunoprecipitation and MBP pull-down assays (data not shown), but it might be worth

to re-examine possible interactions between these proteins using alternative

approaches. In addition, it would be worthwhile to examine the localization of Nubp1 at

different stages of the cell cycle and to look for additional binding partners.

3.7. Identification of possible binding partners to CrEB1 and IFT172 C-

term in Chlamydomonas wild type (CC-124) flagella To identify novel tip proteins, binding partners of IFT172 C-term and CrEB1 were

identified using Glutathione S-transferase (GST) pull-down of isolated flagella from wild

type (CC-124) cells. IFT172 plays a central role in the regulation of the transition

between anterograde and retrograde IFT at the tip of the flagellum, and a point mutation

in the C-terminus of IFT172 leads to accumulation of IFT particles at the flagellar tip

(Pedersen et al., 2005). IFT172 also interacts, at least indirectly, with CrEB1 (Pedersen

et al., 2005), which in turn is necessary for the formation of cilia in mouse fibroblast cells

(Schrøder et al., 2007). Finding binding partners to IFT C-terminus and CrEB1 might

therefore provide new insight into the mechanisms involved in IFT turn-over at the tip of

the flagella, as well as the regulation of flagellar MT elongation or disassembly.

GST pull-down experiments in Chlamydomonas using isolated flagella were

executed by Anna Akhmanovas group in The Netherlands. They ran the pull down

products on a SDS-PAGE gel (Figure 3.7) and identified putative binding partners using

mass spectrometry. The data was analysed by Lotte Pedersen, and a number of

potential binding partners of IFT172 C-term and CrEB1 in Chlamydomonas flagella

were identified (Appendix M). Three potentially interesting binding partners were chosen


48

for further analysis: an ARF-like protein (ARFA1A), a flagellar-associated protein

(FAP20) with homology to transcription factor IIB, and an Eg5-like kinesin motor protein.

Figure 3.6: IFM showing centriolar and nuclear localization of Nubp1 in growth-arrested NIH3T3 fibroblast cells. Cells were starved for 48 hours and subjected to IFM using an antibody specific for acetylated alpha tubulin (red) to detect the flagella and centrioles. In the three top panels an antibody specific for mouse Nubp1, made in rabbit, was used (green). In the bottom panel an antibody specific for the C-terminus of mouse Nubp1, made in guinea pig, was used (green). Nuclei were stained with DAPI (blue). Inserts: enlarged, shifted images of the centrioles and cilia. The IFM shows that Nubp1 (green) localizes to the centrioles as well as the nucleus of the cells. Scale bar 10 µm.

AcTub! MmNubp1! Merge!DAPI!

Ra

bb

it a

nti-N

ub

p1

G

uin

ea

pig

an

ti-N

ub

p1


49

Figure 3.7: Coomassie-stained gel showing the products obtained by pull-down assay of wild type (CC-124) flagella with GST, GST-CrEB1 and GST-IFT172 C-term, respectively. Image provided by Anna Akhmanova.

The small ARF (ADP-ribosylation factors)-related GTPase, ARFA1A, was pulled

down with both GST-CrEB1 and GST-IFT172 C-term. The ARF protein family

compromises structually and functionally conserved members of the Ras superfamily of

regulatory GTP (Guanosine triphosphate)-binding proteins with many proposed

functions in mammalian cells, including the regulation of several steps of membrane

transport (Figure 3.8). Recent results suggests that several ARL (ARF-like) proteins may

be involved in different aspects of MT-dependent functions as well as activation of

phospholipase D (Kahn et al., 2005). Some ARLs have been implicated in ciliopathies:

mutations in the cilia gene ARL13B lead to Joubert syndrome (Cantagrel et al., 2008)

and ARL6 has been identified as one of the genes underlying Bardet-Biedl syndrome

(BBS) (Fan et al., 2004). Chlamydomonas ARFA1A is most closely related to the human

ARF members ARF1 and ARF3-5, which have been implicated in effects on Golgi and


50

endosome morphology (Kahn et al., 2005). In a parallel study by the Akhmanova and

Pedersen groups, ARF4 was identified as a possible binding partner of human EB1, and

other (unpublished) lines of evidence by our group suggest that transport of vesicles to

the ciliary compartment is impaired when EB1 is inactivated. The potential binding of

ARFA1A to EB1 or IFT172 is therefore very interesting.

Figure 3.8: Conserved domains of Chlamydomonas ARFA1A. ARFA1A belongs to the family of small GTPases. The ARF family is a part of the superfamily Ras GTPases. The ARFA1A sequence was blasted for conserved domains on ncbi.nlm.nih.gov.

Flagella associated protein (FAP20) was also pulled down with both GST-CrEB1

and GST-IFT172 C-term. It belongs to the DUF667 superfamily highly similar to

vertebrate transcription factor IIB (TFIIB) (Figure 3.9). Accurate transcription of a gene

by RNA polymerase II requires the assembly of transcription factors at the promoter.

TFIIB localizes in the nucleus with transcription factors IID and IIA where it forms a pre-

initiation complex of RNA polymerase II. TFIID interacts specifically with the TATA box,

TFIIA with RNA polymerase II and TFIIB functions as a bridge linking the complex

(Deng and Roberts, 2007). It is not known whether Chlamydomonas FAP20 has similar

functions or how it localizes in the flagella or the nucleus. Apart from the flagellar

proteome analysis (Pazour et al., 2005) and Gli transcription factors of the Sonic

hedgehog signaling pathways (Haycraft et al., 2005), transcription factors have not

previously been shown to localize to the flagellum, and if FAP20 functions here it would

be a novel system for transcriptional control.


51

Figure 3.9: Conserved domains of Chlamydomonas FAP20. Fap20 belongs to the DUF667 superfamily. The FAP20 sequence was blasted for conserved domains on ncbi.nlm.nih.gov.

Eg5 motor domain was pulled down with GST-IFT172 C-term. This kinesin

belongs to the Kinesin-5 family (BimC family). The rate of bipolar spindle assembly

depends on the MT-gliding velocity of the mitotic kinesin Eg5 in mammals. During

mitosis they have an essential role in pushing the spindle poles to opposite sides of the

cell by pushing the astral MT in opposite directions (Valentine et al., 2006). The N-

terminal region containing the motor domain of Eg5 was the only part of the protein

whose sequence was known when this project started, and therefore I only cloned and

expressed this region to use in the following pull-down experiments. It is intriguing if an

Eg5-like protein interacts with IFT172 and the possible functional implications of such

an interaction are unclear. Nevertheless, we decided to pursue this further.

Figure 3.10: Conserved domains of Chlamydomonas Eg5. Eg5 belongs to the kinesin-5 family of kinases. The kinesin-5 family has a characteristic BimC box domain. The Eg5 sequence was blasted for conserved domains on ncbi.nlm.nih.gov.


52

3.8. Construction and purification of MBP-ARFA1A, MBP-FAP20 and

MBP-Eg5 motor domain fusion proteins

To assess if the three chosen proteins, ARFA1A, FAP20 and Eg5 motor domain,

identified in the pull-down analysis indeed interacted with CrEB1 and/or IFT172 C-term,

I set out to test whether the three proteins could bind to either IFT172 or CrEB1 in MBP

pull-down assays. The cDNAs corresponding to the three proteins were first cloned into

the pMalC2 vector (See vector map in appendix D) and transformated into DH10α E.coli

cells. This resulted in fusion of maltose-binding protein (MBP) in-frame to the three

genes and MBP-ARFA1A, MBP-FAP20, and MBP-Eg5 motor domain was constructed.

The sequences were verified for correct insertion. To express the fusion proteins the E.

coli cells were induced with IPTG and the MBP fusion proteins purified on amylose

beads. To check the purity of the MBP-fusion proteins, the beads were run on a SDS-

PAGE gel, and Coomassie stained (Figure 3.11). On the gel there is some, but not a lot

of contamination with E.coli proteins and the apparent molecular weights of the purified

fusion proteins are very close to the theoretical values (See table 3.2). Due to time

constraints, I decided to use these protein preparations for pull-down analysis without

further purification.

3.9. MBP pull-down analysis of ARFA1A, FAP20 and Eg5 motor

domain

To investigate the potential protein-protein interactions, a MBP pull-down experiment, in

which a Chlamydomonas wild type (CC-124) flagella lysate was mixed with either MBP,

MBP-ARFA1A, MBP-FAP20 or MBP-Eg5 motor domain bound to amylose beads, was

performed. The fusion proteins as well as the bound proteins were analyzed by SDS-

PAGE and immunoblotting using antibodies specific for either IFT172, CrEB1 or alpha-

tubulin (Figure 3.12). The results showed no interaction between FAP20/ARFA1A and

IFT172, EB1 or α-tubulin in this assay (Figure 3.12, lanes 3 and 4).


53

Figure 3.11: Coomassie-stained gel of purified MBP-fusion proteins. Fusion proteins were purified on amylose beads using affinity chromatography as described in materials and methods. Flow through (FT) was collected to check how successfully the procedure extracted the MBP fusion proteins from the input.


54

MW

(kDa)

GenBank ID pI Apparent

MW (kDa)

Peptides Amino

acids

MBP 50.8 5.21 ~60

FAP20 22.2 gi: 159468654 9.59 ~72 11 190

ARFA1A 20.6 gi: 159465365 6.43 ~67 2 181

Eg5 40.4 gi: 159475595 7.62 ~90 1 367

Table 3.2. Theoretical values for ArfA1A, FAP20 and Eg5 motor domain. The values were calculated from the polypeptide sequences using the pI/MW tool on expasy. MBP (MalE) sequence was obtained on New England Biolabs webpage as an excerpt from the entire pMalC2 plasmid sequence. The apparent molecular weight was estimated from a Coomassie stained gel (Figure 3.8). The apparent molecular weights indicated include the MBP fusion construct. ”Peptides” refer to the number of peptides by which the proteins were identified in the Chlamydomonas flagellar proteome (Pazour et al., 2005). Note that the Eg5 fusion protein only contains the N-terminal motor domain.

FAP20 and ARFA1A could have been false positives from the initial pulldown mass

spectrometry experiment, but a number of other factors, can have interfered with this

assay. It is possible that the MBP fusion causes the proteins to misfold thereby not

exposing their binding sites, or the proteins could be lacking post translational

modifications. ARFA1A is a GTPase, so the presence of GTP could have a great effect

of the folding and activity of this protein. Furthermore, the N-terminus of ARF proteins is

known to be critical for their function (Casanova, 2007; Liu et al., 2009) so adding MBP

to the N-terminus of ARFA1A likely interferes with its binding to other proteins.

An interaction of Eg5 motor domain was seen with both IFT172, IFT139 (though

a very weak band) and alpha tubulin (Figure 3.12, lane 5). Repetitions of the analysis

confirmed the association between IFT172 and MBP-Eg5 motor domain (data not

shown). Since MBP-Eg5 contains a motor domain with conserved MT binding sites

(Figure 3.10; multiple sequence alignment in Appendix C) it is not surprising that an

interaction with alpha tubulin is observed.


55

Figure 3.12. Western blot of pull-down analysis of MBP fusion proteins mixed with wild type (CC-124) flagella extract. Purified MBP fusion proteins immobilized on amylose resin (see Figure 3.11) were mixed with flagellar extract and bound proteins analyzed by SDS-PAGE and western blot with antibodies against flagellar proteins, as indicated. Note that MBP-Eg5 motor domain co-precipitates with IFT172, IFT139 and alpha tubulin.

It is more surprising that this domain would interact with IFT172, because if IFT172

were bound to the motor domain of Eg5, Eg5 would likely be inactive. It is possible that

IFT172 interacts with Eg5 via microtubules or that Eg5 is inactive when bound to

IFT172. Further experiments are needed to investigate this further. The interaction seen

between Eg5 and IFT139 could be due to the interaction between IFT172 and IFT139,

since IFT139 and IFT172 are both IFT particle proteins, and this could cause the co-

precipitation. To verify the possible binding of Eg5 to IFT172 and tubulin, it would be

useful to clone and sequence the whole Eg5 coding region. My supervisor Lotte

Pedersen recently obtained a full-length Eg5 cDNA clone and sequenced the entire

coding region. To further examine the potential association of Eg5 with tubulin and

IFT172, and potentially the function of Eg5 in Chlamydomonas, it would be highly

relevant to produce an antibody against the C-terminal region of Eg5, which, in contrast

to the motor domain, displays little sequence similarity to other known kinesins (see Eg5

multiple sequence alignment in Appendix C) and therefore would be more appropriate

for antibody production.


56

A specific inhibitor, called Monastrol, is known to act specifically on the motor

domain of human Eg5 (Cochran et al., 2005). Since the motor domain of

Chlamydomonas and human Eg5 are highly conserved (Figure 3.10; multiple sequence

alignment in appendix Appendix C), it is possible that this specific inhibitor will work on

Chlamydomonas Eg5. A few pilot tests have been conducted to see whether Monastrol

has an effect on Chlamydomonas flagellar length or motility, but so far none have been

observed (data not shown). It would be interesting to test the effect of Monastrol on cilia

assembly or disassembly in mammalian cells where Monastrol is known to inhibit Eg5

(Cochran et al., 2005). If an effect is seen, it will provide some clues as to the function of

Eg5 in Chlamydomonas flagella.

3.10. Further testing of the function of ARFA1A

Although no interaction was observed between ARFA1A and IFT172 or EB1 (Figure

3.12) in the MBP pull-down assay, this is a very interesting possible binding partner

given that an interaction between ARF4 and EB1 was observed in parallel studies in

ciliated Retinal pigment epithelial (RPE) cells (see Section 3.7). The interaction was

probably not seen because, as noted above, adding MBP to the N-terminus of ARF

proteins likely inhibits their association with other proteins (Casanova, 2007; Liu et al.,

2009). Chlamydomonas ARFA1A is most closely related to the human ARF members

ARF1 and ARF3-5. A polyclonal antibody raised against the C-terminus of ARF1 of

human origin was purchased, and tested to see if it was able to detect ARFA1A in

Chlamydomonas flagella. Flagella from wild type (CC-124) cells, along with RPE cells in

interphase or after 72 hours of starvation (kindly supplied by fellow student Tue S.

Jørgensen), as well as HeLa cells supplied by the company as a positive control, were

loaded on an SDS-PAGE gel and immunoblotted with the antibody against ARF1

(Figure 3.13). The ARF antibody does not seem capable of detecting ARFA1A in the

Chlamydomonas flagella. However a single band of the appropriate size is seen in

starved RPE cells, but not seen in the RPE cells in interphase. This could indicate that

ARF1 and related proteins are up-regulated during growth arrest, which is typical for

cilia-associated proteins.


57

Figure 3.13: ARF is detected in RPE cells after 72 hours of starvation. Western blot of Chlamydomonas wild type flagella (wt; CC-124), RPE cells starved for 72 hours, non-starved RPE cells, and non-starved HeLa cells shows that a single band corresponding to the size of ARF proteins is seen in starved RPE cells.

To further test the antibody, to see if it would be able to detect ARF localization in

Chlamydomonas wild type (CC-124) cells, an IFM was conducted, using the antibody

against ARF and a monoclonal anti-acetylated tubulin antibody to stain the flagella

(Figure 3.14). The antibody revealed weak punctate staining along the entire flagellar

length. This distribution is similar to that seen for several IFT components. Due to time

constraints I have not been able to test this possible co-localization further.


58

Figure 3.14: IFM, using a Glutaraldehyde/NP40 fixation method, showing localization of ArfA1A in C. reinhardtii wild type (CC-124) cells. Cells were grown at 20°C and subjected to IFM using a polyclonal antibody specific for human ARF (red). To detect the flagella and basal bodies an antibody specific for acetylated tubulin (green) was used. The bottom panels show the flagella tip in focus. IFM showed that ArfA1A (red) localizes with weak punctate staining along the entire flagellar length. The strong fluorescence of the cell body is due to autofluorescence of the cells and was also observed when no primary antibodies were added (not shown). Scale bar 10 µm.

Merge!ARF!AcTub!

No primary antibody!


59

4. Conclusions and future directions The purpose of this master’s project was to identify and characterize novel flagellar tip

proteins. First, based on the results for CrEB1 in Pazours flagella proteome analysis

(Pazour et al., 2005), CrNubp1 was chosen, based on the fact that it had the same

properties as CrEB1 in having few peptides and all in the membrane plus matrix

fraction. I set out to test this hypothesis and potentially characterize the protein.

Secondly, using pull-down experiments, I set out to identify binding partners of both

CrEB1 and IFT172 C-terminus, both shown to either localize to or be involved in flagella

tip-related processes (Pedersen et al., 2003;2005). To briefly summarize my results:

• By using an antibody specific for the N-term of CrNubp1, I showed by

immunoblotting that CrNubp1 is present in the flagella of the green alga

Chlamydomonas reinhardtii and that it is found in the membrane plus matrix

fraction, which was the basis for choosing to look at this particular protein to

begin with (Figure 3.1.A; Figure 3.2).

• I showed that CrNubp1 does not associate with the axonemal parts necessary for

flagellar movement, i.e. the outer and inner dynein arms, the radial spokes and

the central apparatus (Figure 3.3).

• I showed that CrNubp1 localizes to the basal bodies and the tip of the flagella in

C. reinhardtii wild type (CC-124) cells (Figure 3.4; Figure 3.5) and that an

antibody specific for mammalian Nubp1 localizes to the centrioles in the mouse

NIH3T3 fibroblast cells (Figure 3.6).

My supervisor and I, in collaboration with Anna Akhmanovas lab in Holland, identified

several potential binding partners of CrEB1 and/or IFT172 in Chlamydomonas flagella.

After retesting the binding of three of these (ARFA1A, FAP20, and Eg5 motor domain)

by MBP-pulldown analysis no positive binding was seen for FAP20 or ARFA1A with that


60

specific assay. However, I showed that Eg5 does indeed associate, at least indirectly

with IFT172, and with alpha-tubulin (Figure 3.12).

• With a commercial ARF antibody, I showed detection of a single band of

appropriate size in serum-starved RPE cells (Figure 3.13), and a localization

pattern in Chlamydomonas reinhardtii, similar to that observed of several IFT

components (Figure 3.14).

Together these findings show that Nubp1 is indeed a protein in the tip of the flagella.

What the exact function is remains unknown. However I believe that CrNubp1 could

function as a regulator of basal body duplication during mitosis mediating the transport

between the tip and the basal bodies, by interacting with a minus-end directed kinesin-

14 family member such as KCBP (Dymek et al., 2006). This is based on the findings in

mouse fibroblast cells, were Nubp1 has been shown to interact directly to the minus-end

directed kinesin KIFC5A, and inactivation of either resulted in the presence of

supernumerary centrosomes and an increase in the proportion of bi- and tri-nucleated

cells (Christodoulou et al., 2006).

The next step would be trying to identify binding partners of CrNubp1. I have

started some preliminary studies, by constructing a full length MBP fusion protein for

pull down analysis, but so far no binding partners have been identified. It might be

worthwhile to try alternative methods such as sucrose density gradient centrifugation,

gel filtration, chemical cross-linking, or yeast-two-hybrid analysis to identify binding

partners of CrNubp1.

It would be also be interesting to investigate the possible association of CrNubp1

with the IFT machinery, e.g. by employing a Chlamydomonas strain with a temperature-

sensitive mutation in the FLA10 gene, which encodes the 90 kDa motor unit of the

heterotrimeric kinesin-2 that powers anterograde IFT (Pedersen and Rosenbaum,

2008). If grown at 22°C, fla10 cells can form flagella of normal length that are virtually

indistinguishable from wild-type flagella. When the temperature is raised to 32°C, fla10

flagella become depleted of IFT particle proteins within the first 1-2 hours as a result of


61

stalled anterograde IFT but still active retrograde IFT (Adams et al., 1982; Vashishta et

al., 1996), Due to time constraints it has not been possible to test this, but it would be

interesting to do in the future.

The findings in the MBP pull-down experiments identified a likely interaction

between Eg5 motor domain and IFT172. Recently the first example of a regulator of the

cytoskeleton that tracks along MT plus ends in Drosophila has been identified (Rogers

et al., 2004). It could be possible that the plus-end-directed motor protein Eg5, is

tracking along the MT interacting with IFT172, thereby regulating IFT turnover at the tip

of the flagellum. It would be highly relevant to continue to test the inhibitor Monastrol on

Chlamydomonas as well as mammalian cells. If it is able to specifically inhibit Eg5 it will

give an inexpensive and quick indication of whether this hypothesis is correct. The

whole Eg5 sequence has also been obtained, and the next step would be to produce a

specific antibody against Eg5, thereby being able to not only confirm the Eg5-IFT172

binding, but also provide a good tool for characterizing Eg5 in Chlamydomonas cells.

In the pull-down experiments no interaction was identified between

FAP20/ARFA1A and IFT172 and/or CrEB1. This could be due to the lack of post

transcriptional modification in the fusion proteins, caused by the expression of

eukaryotic genes in the procaryote E. coli. To avoid this, maybe a yeast two-hybrid

system could be used, thereby using a eukaryote to test the protein interactions.

A commercially ARF antibody has been obtained and tested on Chlamydomonas as well

as RPE cells. Via immunoblotting ARF was not detected in Chlamydomonas, but a band

of appropriate size was detected in serum-starved RPE cells, indicating a possible

association with cilia. Fellow students in the laboratory are currently using this antibody

for immunoprecipitation studies in order to determine whether ARF proteins interact with

EB1 in mammalian cells. Further, a GFP-tagged ARF4 construct has recently been

created in the lab and in future experiments, this construct will be used to analyze the

dynamic localization of ARF4 in mammalian cells, which should reveal possible co-

localization/interaction with EB1. Preliminary IFM localization of ARF in

Chlamydomonas, using the commercial ARF antibody, suggested possible co-

localization with IFT components, but this localization needs to be confirmed in future


62

work. Nevertheless, given that mammalian EB1 localized at the basal body is important

for cilia formation in NIH3T3 cells (Schrøder et al., 2007) one can speculate that EB1

plays a role in targeting ARF-associated vesicles to the ciliary base where IFT proteins

subsequently take over to mediate transport of ARF-associating components towards

the ciliary tip.


63

5. Materials and Methods In this section I will shortly account for the methods used in this project.

More detailed protocols as well as recipes for solutions can be found in the appendix.

5.1. Eukaryotic cell cultures In this project I primarily used Chlamydomonas reinhardtii as a model organism for

studying flagellar tip proteins. All strains used were obtained from the Chlamydomonas

Genetics Center (CGC) and are listed in Table 5. 1. Further information on the different

strains can be obtained from the CGC website (www.chlamy.org).

C. reinhardtii was grown at 20°C either on TAP

(Tris-acetate-phosphate) 1.5% agar plates or in

TAP or M1 liquid media in 250 ml or 2000 ml glass

flasks (see Appendix A for recipes of culture

media). The cells growing in liquid media had a

constant supply of air. The cells were placed in an

incubator with 14 hours of high intensity white

fluorescent light (≈200 µE m-2s-1) followed by 10

hours of darkness (Figure 5.1). Cells were

harvested by centrifugation at 2,400 g for 10 min at

20°C and resuspended in 10 mM Hepes pH 7.5,

then allowed to recover at 20°C with constant light

for approximately 1-2 hours prior to temperature shift and/or flagellar isolation. For

temperature shift experiments, cultures in Hepes were placed at 32oC for 2 hours prior

to flagellar isolation (see below).

I also conducted some immunofluorescence microscopy (IFM) on NIH3T3

(National Institute of Health 3T3) fibroblast cells. NIH3T3 cells are an established cell

line of contact inhibited mouse embryonic fibroblasts with a generation time of about 22

hours. Upon 24 hours of serum starvation more than 90% of NIH3T3 cells form primary


64

cilia of approximately 6 µm in length (Schneider et al., 2005). The NIH3T3 cells were

cultured by Ph.D. student Jacob M. Schrøder, but I had the chance to watch and see

how it was conducted.

Strain Name Locus/Allele Phenotype/description CC-124 wild type mt- 137c (137C (mt-))

Allele: agg1; nit1-137; nit2-137

Wild type. Cannot grow on nitrate as sole N source

CC-503 cw92 mt+ (cw92)

Allele: cw92 Cell wall deficient.

CC-1032 pf14 mt+ (pf14)

Locus: PF14 Allele: pf14

Lacks radial spokes. Paralyzed flagella.

CC-1036 pf18 mt+ (pf18)

Locus: PF18 Allel: pf18

Lacks entire flagellar central apparatus. Paralyzed, rigid flagella.

CC-1919 fla10 mt- (fla10)

Locus: FLA10 Allele: fla10

Cannot regenerate flagella at 32 degrees.

CC-2230 oda2 mt+ (oda2)

Locus: ODA2 Allele: oda2

Outer dynein arm heavy chain gamma deficient. Lacks outer dynein arms. Swims slower than wild type and lacks the backward swimming response to stimulation with intense light.

CC-2664 ida1 mt- (ida1)

Locus: PF9 Allele: ida1

Inner dynein arms deficient. Impaired motility.

CC-2670 ida4 mt+ (ida4)

Locus: IDA4 Allele: ida4

Flagellar inner arm dynein light chain deficient. Mutants swim more slowly than wild type. Impaired motility.

Table 5.1. Chlamydomonas strains used in this project. mt-: mating-type minus. mt+: mating-type plus. For further details, see www.chlamy.org.


65

5.2. Preparation of flagella and cell body extracts Flagella can be removed from the Chlamydomonas cell bodies in various ways. The

stimulus I have given the cells was a shift to low pH. The acid shock causes the flagella

to detach at the distal end of the transition zone (Sander and Salisbury, 1989; Quarmby,

1996). Cells in 10 mM Hepes were subjected to pH shock by lowering the pH of the cell

culture to pH 4.5 with 1 M acetic acid followed by neutralization with 1 M potassium

hydroxide. Next, 1 mM DTT, 4% sucrose, 1:1000 plant protease inhibitor (Sigma), 5 mM

MgSO4, 0.25 mM EDTA were added and the flagella were isolated and purified by a

series of centrifugation steps as detailed in Appendix B. The purified flagella were

dissolved in flagella lysis buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1% Triton X-100, 1

mM DTT,1/100 plant protease inhibitor from Sigma, cat. no. P9599) or a HMDEK buffer

(10 mM Hepes, 0.5 M MgSO4, 2.5 mM EDTA, 0.25 M KCl, 1 mM DTT) + 0.5% NP40,

the flagella protein content was measured using the BioRad DC Protein Assay (section

5.7) and flagella were stored at

–80oC until use.

For biochemical fractionation of flagella, frozen flagella in lysis buffer were

thawed and ATP added to a final concentration of 10 mM. The mixture was sucked

through a 27 gauge needle several times and after centrifugation at 10,000 g for 5 min,

the SN (consisting of the membrane plus matrix fraction) was removed and the pellet

extracted with an equivalent volume of high salt buffer (20 mM Tris pH 7.5, 0.6 M NaCl,

1 mM DTT, 1/100 plant protease inhibitor from Sigma). After centrifugation as above the

salt extract SN was removed and the pellet (salt-extracted axonemes) was resuspended

in lysis buffer without Triton-X-100. All fractions were subjected to protein concentration

measurements using BioRad DC Protein Assay (section 5.7) and subsequently

analyzed by SDS-PAGE (section 5.8).

For analysis of isolated cell bodies, after harvest and resuspension in 10 mM

Hepes, cells were stained with Lugols solution (volume 1:1) and counted using a bürker-

Türk 0.0025 mm2 counting chamber. The cells had a density of 3x106 cells/ml. The cells

were deflagellated by pH shock, the flagella isolated as described above, and the cell

bodies treated according to Ahmed et al., 2008, and as described in the following (the


66

volumes of cell body and flagella preparations were monitored closely so as to keep

track of the cell equivalents).

Twentyfive ml of cells in 10 mM Hepes (3x106 cells/ml) were harvested by

centrifugation at 1,500 g for 5 min at 4°C and the pellet dissolved in 1 ml ddH2O. 100 µl

aliquots were then mixed with 500 µl Acetone. The tubes were centrifuged 6 min at

3,000 g, the supernatants (SN) removed, and the pellets dried. Pellets were

resuspended in 50 µl 0.5% SDS and boiled for 2 min. The lysate was spun for 6 min at

3,000 g and the SN saved for protein quantification and SDS-PAGE (sections 5.7 and

5.8). 5.3. PCR and cloning procedures For PCR amplification and cloning of cDNA corresponding to Chlamydomonas Nubp1

(full-length), FAP20 (full-length), ArfA1A (full-length) and Eg5 (motor domain) primers

were designed on the basis of the published or predicted cDNA sequences for these

proteins (Appendix C). The primers (listed in Table 5.2) contained restriction sites to

match the restriction sites of the multiple cloning site of the pMAL-c2 plasmid from new

England Biolabs (Appendix D). The pMAL-c2 plasmid is constructed such that the

multiple cloning site allows for insertion of genes under the control of the lac operon,

and such that the maltose binding protein (MBP) gene is automatically fused to the 5’

end of gene insert.

Using cDNA clones for each of the four target genes as template (cDNA clones

obtained via the CGC see appendix C for accesion number), the target genes were

amplified by PCR as detailed in Appendix F. PCR is a technique that uses thermal

cycling and enzymatic replication to amplify a single or few copies of a piece of DNA.

The PCR products were run on an 1% agarose gel (Appendix G) and the PCR products

purified using E.Z.N.A Cycle-Pure kit (Omega Biotek cat. no. D6492-01). The PCR

products, as well as the vector pMal-c2, were cut by the restriction enzymes specified in

Table 5.2 (all enzymes from Roche) for 1 hour at 37°C, and run on an 1% agarose gel

(Appendix G). The bands were excized from the gel and each PCR product band mixed

with one vector band. The mixed bands were purified using E.Z.N.A. Gel Extraction kit


67

(Omega Biotek cat. no. D2500-01), and ligated overnight at 16°C using a T4 DNA ligase

from Saveen-Werner.

Competent E. coli DH10α cells (prepared as detailed in Appendix H) were

transformed with 5 µl of each ligation mix (Appendix H) and incubated overnight at

37°C. The next day individual colonies were picked with a sterile toothpick, inoculated in

3 ml LB broth with ampicillin (100 µg/ml) and incubated overnight at 37°C. Plasmid mini-

preps were prepared with E.Z.N.A. Plasmid Mini Kit (Omega Biotek cat. no. D6942-01),

5 µl of each plasmid cut with the appropriate restriction enzymes (Table 5.2) and

analyzed on an 1% agarose gel (Appendix G). Colonies with the right inserts were

streaked on agar plates with ampicillin (100 µg/ml) and incubated overnight at 37°C. For

preparation of plasmid midi-preps colonies were transferred to 200 ml LB broth with

ampicillin (100 µg/ml) and incubated overnight at 37oC until an OD600 of approximately

1.6 was reached. An midi-prep was done using a kit from Macherey Nagel (cat. no. 740

420.50). The purified plasmids were sequenced by MWG-biotech. The sequences were

checked both by me and by my supervisor Lotte Pedersen.


68

Target gene* Restriction sites: Primer sequences:

Nubp1 EcoRI; PstI;

HindIII

Primer forward (EcoRI): 5´GTGAATTCATGGCGTCGTCCGCGAGCGC Primer forward (PstI): 5’GAGCACATCACCATCACACAGTGCCTGCAGGGTGCG Primer reverse (HindIII): 5ÁCAAGCTTCTACTTCGCCGCCGCTGCCTTC Primer reverse (PstI): 5’CGCACCCTGCAGGCACTGTGTGATGGTGAT

ArfA1A EcoRI; HindIII Primer forward (EcoRI) 5ĆTGAATTCATGGGTCTGATGGTTTCTAAGGC Primer reverse (HindIII) 5ĆTAAGCTTTCACGACTTGTTCTGGATGTTTTGG

FAP20 EcoRI; HindIII Primer forward (EcoRI) 5ĆTGAATTCATGTTCAAGAACGCCTTCCAATCC Primer reverse (HindIII) 5ĆTAAGCTTCTACGACTTCTGGATCGGCAG

Eg5 EcoRI; HindIII Primer forward (EcoRI) 5ĆAGAATTCATGGCCGAACCAAAGCCTGGAC Primer reverse (HindIII) 5ĆAAAGCTTCTTCTGGTTGACCTCGGGCC

Table 5.2. PCR primers used in this study. The cDNA sequences and accession numbers are in Appendix C. Primer sequences: Coding sequences are bold, overhang normal, restriction sites underscored. *All are full-length except Eg5. After confirming that the sequences of the recombinant plasmids were correct, freeze

cultures were made by mixing bacterial log-phase cultures (grown in LB medium with

100 µg/ml ampicillin) with sterile glycerol (33% vol/vol) and freezing them at -80°C. The

strains generated in this study were named as follows: LP123 (pMal-c2 with Nubp1 full-

length), LP128 (pMal-c2 with ArfA1A full-length), LP129 (pMal-c2 with FAP20 full-

length) and LP132 (pMal-c2 with Eg5 motor domain). All strains are ampicillin resistant.


69

5.4. Expression of MBP fusion proteins Recombinant E.coli strains were streaked out on LB agar plates with 100 µg/ml

ampicillin and incubated overnight at 37°C. The colonies were transferred to Erlenmeyer

flasks containing 500 ml LB media plus 100 µg/ml ampicillin, and incubated with

shaking at 30°C until an OD600 of 0.6-0.9 was reached. The cultures were cooled down

to ca. 18°C and after 30 min, IPTG was added to a final concentration of 1 mM. IPTG

induces the cells to produce the fusion proteins. After an overnight incubation at 18°C

the cells were harvested by centrifugation at 2,800 g for 13 min, resuspended in ice cold

PBS and centrifuged again at 2,500 g for 10 min. The pellets were stored at -80°C until

use.

5.5. Purification of fusion proteins on amylose resin Frozen pellets of recombinant E.coli cells were resuspended in PBS with 0.05% Triton

X-100 and lysozyme added 0.1 mg/ml. After incubating 15 min on ice, the solution was

sonicated until cells were completely lysed. The cell lysates were centrifuged twice at

10,000 g for 20 min to remove all cell debris and the cleared SN loaded onto pre-

washed colums with Amylose-Resin High Flow (New England BioLabs cat. no.

E8022S). After 3 washes, 1 column volume each, with PBS + 0.05% Triton X-100 the

column material was resuspended in PBS with 30% glycerol, transferred to Eppendorf

tubes and stored at -80°C until use. Purity of the fusion proteins was assessed by SDS-

PAGE and staining with Coomassie Blue (section 5.8).

5.6. MBP pull-down assays Frozen flagella in a HMDEK buffer (10 mM Hepes, 0.5 M MgSO4, 2.5 mM EDTA, 0.25 M

KCl, 1 mM DTT) + 0.5% NP40 were thawed on ice and centrifuged two times at 10,000

g for 10 min to remove axonemes. 100 µl flagella extract (ca. 5 mg/ml) and 80 µl fusion

protein (on beads) was mixed and incubated for 1-2 hours on a rotator at 4°C. The mix

was washed three times 10 min with HMDEK + 0.5% NP40 and the washed pellets

mixed with loading dye and DTT prior to analysis by SDS-PAGE (section 5.8).


70

5.7. Protein quantification To assess protein concentrations in flagellar extracts, a BioRad DC Protein Assay was

used. 100 µl Working Reagent, consisting of a mixture of reagent S (BioRad, cat. no.

500-0115) and Reagent A (BioRad, cat. no. 500-0113) in the ratio 1:50, was added to

the samples as well as to the Bovine Serum Albumin (BSA) standard. Next, 800 µl

Reagent B (BioRad, cat. no. 500-0114) was added and the samples whirly-mixed. The

samples were then kept out of light exposure for 15 min and the ODs were measured in

a Beckmann Coulter DU® spectrophotometer at 750 nm. The protein concentrations

were then obtained through linear regression of the OD values of a set of standards

(Appendix I).

5.8. Sodium Dodecyl Sulphate PolyacrylAmide Gel Electrophoresis

(SDS-PAGE) Chlamydomonas or E. coli proteins were separated using SDS-PAGE, which is a

technique that separates cellular proteins on a gel according to their mass. In this

project, the NOVEX/NuPAGE SDS-PAGE system from Invitrogen was used and all gels

and buffers for SDS-PAGE were from Invitrogen. Prior to SDS-PAGE analysis, protein

samples were mixed with 4xLDS loading dye (Invitrogen, cat. no. NP0007) to a final

concentration of 1x and DTT added to 25 mM final concentration. The samples were

heated at 95°C for 3-5 min and centrifuged at 5,000 g for 30 sec. The samples were

then loaded, along with 5 µl of a molecular size marker (PageRuler, Prestained Protein

Ladder from Fermentas, cat. no. SM 0671), onto a NuPAGE precast gel (3-8% NuPAGE

Tris-Acetate gel, 10% NuPAGE Bis-Tris gel or 4-12 % NuPAGE Tris-Acetate gel) that

was placed in a NOVEX system electrophoresis chamber. The electrophoresis was

carried out essentially as recommended by Invitrogen (100 min at 140 V) using a

denaturing running buffer appropriate for the type of gel used and which contained 0.25

% (vol/vol) NuPAGE antioxidant (cat. no. NP0005) in the inner chamber (see Appendix J

for solutions).

Gels were stained with coomassie blue or transferred to a nitrocellulose

membrane for western blotting (see section 5.9).


71

5.9. Western Blot analysis (WB) Western blotting is a technique where proteins separated by SDS-PAGE are transferred

(electro-blotted) to a membrane and subsequently identified by antibodies directed

against these proteins. Proteins were transferred from the gel to a nitrocellulose

membrane by placing it between filter paper and blotting pads, all soaked in a transfer

buffer (1x NuPAGE Transfer buffer (Invitrogen), 10% EtOH) and blotting, using a

NOVEX System, at 25 V for 2 hours. When transferring to check for ArfA1A protein, the

transfer buffer contained 20% Ethanol and the blot was transferred for 1 hour. The

effectiveness of the transfer was checked by staining with Ponceau S solution (Sigma,

Cat. No. P7170). After staining the membranes were washed in H2O and incubated in

blocking buffer for 30 min. The membranes were incubated with primary antibodies

(See section 5.10, Table 5.3) in blocking buffer overnight at 4°C. The following day the

blots were washed four times for 15 min with Tris-Buffered Saline Tween-20 (TBST)

before probing with secondary antibodies (Table 5.5) in blocking buffer for one hour at

room temperature. The membranes were washed again, four times for 10 min with

TBST, before developing with 5-bromo-4-chloro-3-indoylphosphate/Nitroblue tetrazolium

(BCIP/NBT) (KPL, Cat. No 50-81-08).

(See Appendix J for solutions).

5.10. Antibodies and affinity purification Antibodies or immunoglobulins (Ig) are proteins found in blood used by the immune

system to identify foreign objects. The principle in probing with antibodies in

immunofluorescense microscopy or western blotting analysis is to incubate samples

with primary antibodies raised in host animals against the specific protein of interest.

The primary antibodies are visualized with secondary antibodies, which are directed

against the host animal and conjugated to enzymes or fluorophores, which makes

visualization possible. Antibodies are produced by immunization of a rabbit, mouse or

other animal with the antigen to which an antibody is desired. The antigen for Nubp1 N-

term (nucleotide 1-594) was immunizated to a rabbit by Yorkshire Bioscience Ltd. When

an animal is immunized with a protein, the animal produces a variety of antibodies,


72

which recognize different parts of the protein. Nubp1 protein was run on a SDS-PAGE

gel and transferred to a nitrocellulose membrane (Section 5.8 and 5.9). After

visualization with Ponceau S. solution (Sigma, cat. No. P7170), the bands of interest

were cut into small pieces and incubated, rotating overnight at 4°C, in a BSA buffer (5

ml PBS, 3% BSA, 0.05% NaAzide). After 6-8 washes in PBS, the membranes were

incubated 2 days, rotaing at 4°C, with a buffer containing the Nubp1 antiserum (1.8 ml

PBS added 0.2 ml rabbit 63A 2. Bleed). After a series of washing steps (see appendix K

for detailed protocol), the antibody was extracted by two times adding 100 µl 0.2 M

glycine-HCl pH 2.5, whirlymixing for 1 min, transferring the liquid to a new eppendorf

tube, and immediately adding 50 µl KPO4 pH 9.0 with 5% BSA.

The OD of the antibody was measured to determine the concentration. The

antibody was tested on a flagella lysate using SDS-PAGE and western blotting

(sections 5.8 and 5.9).

A complete list of primary antibodies is given in table 5.3 whereas secondary antibodies

for IFM and western blot analysis are listed in table 5.4 and 5.5 respectively.

5.11. Immunofluorescence microscopy analysis (IFM) Immuno Fluorescence Microscopy is a method used to localize and quantify specific

components in the cell. It takes advantage of fluorephores which upon exposure to light

from a defined light source are excited by absorption of photons.

In a fluorescence microscope the exciting light is filtered so only the wavelength to be

absorbed is permitted to reach the preparation with fluorophores. Another filter allows

only the wavelength of the emitted light to pass through the occular/camera where it is

seen as bright colours depending on the wavelength.


73

Antigen Abbr. IgG Producer/ Cat. No.

Dilution (WB)

Dilution (IFM)

Target Mw

Monoclonal Anti-α-tubulin

α-tb Mouse Sigma Aldrich/ T 5168

1:2000 50 kDa

Monoclonal anti- Tubulin acetylated

AcTub Mouse Sigma Aldrich/ T 6793

1:200

Polyclonal ARF (H-50)

Arf1/arf Rabbit Santa Cruz/ Sc-9063

1:200 1:50 21 kDa

Aurora protein kinase

CALK Rabbit Pan et al., 2004 1:500 ~95 kDa

Dynein 1ß Light Intermediate Chain

D1bLIC Rabbit Hou et al., 2004 1:1000 46.6 kDa

ChlamydomonasEnd Binding protein 1

CrEB1 Rabbit Pedersen et al., 2003

1:1000 34 kDa

Kinesin-II motor domain (KII-neck)

FLA10 Rabbit Cole et al., 1998 1:500 90 kDa

IFT72 IFT72 Rabbit Qin et al., 2004 1:500 72 kDa Monoclonal IFT139

IFT139 Mouse Cole et al., 1998 1:500 139 kDa

IFT172 IFT172 Mouse Cole et al., 1998 1:200 172 kDa Kinesin-like Calmodulin-Binding Protein

KCBP Rabbit Smith, Elizabeth Department of Biological Sciences, Darthmouth college, Hanover, New Hampshire, USA

1:5000 140 kDa

Chlamydomonas Lissencephaly protein Lis1

CrLis1 Rabbit Pedersen et al., 2007

1:1000 ~37 kDa

Polyclonal Clamydomonas Nucleotide binding protein 1

CrNubp1 Rabbit This project 1:2000 1:500 40.6 kDa

Nucleotide binding protein 1 (mammal) (C-terminus)

mmNubp1 C-term

Guinea pig

Santama, Niovi University of Cyprus and Cyprus Institute of Neurology and Genetics, Cyprus

1:300

Nucleotide binding protein 1 (mammal)

mNubp1 Rabbit Santama, Niovi University of Cyprus and Cyprus Institute of Neurology and Genetics, Cyprus

1:300

Table 5.3. Primary antibodies used in this study.


74

Conjugated to IgG Producer Cat. No. Dilution DyLight 488 Donkey-Anti-Guinea pig Jackson

ImmunoResearch 706-485-148

1:1000

Alexa Flour 488

Donkey-Anti-Mouse Molecular Probes A 21202 1:1000

Alexa Flour 568

Donkey-Anti-Mouse Molecular Probes A 10037 1:1000

Alexa Flour 488

Donkey-Anti-Rabbit Molecular Probes A 21206 1:1000

Alexa Flour 568

Donkey-Anti-Rabbit Molecular Probes A 10042 1:1000

Alexa Flour 488

Goat-Anti-Rabbit Molecular Probes A 11070 1:1000

Alexa Flour 568

Goat-Anti.Rabbit Molecular Probes A 21069 1:1000

Alexa Flour 488

Goat-Anti-Rat Molecular Probes O 6382 1:1000

Alexa Flour 568

Goat-Anti-Rat Molecular Probes A 11077 1:1000

Table 5.4. Secondary antibodies used in this study for Immunofluorescence microscopy.

Conjugated to IgG Producer Cat. no Dilution Alkaline phosphatase

Goat-anti-mouse Sigma Aldrich A-1293 1:5000

Alkaline phosphatase

Goat-anti-rabbit Sigma Aldrich A 3687 1:5000

Table 5.5. Secondary antibodies used in this study for western blotting.

5.11.1. IFM on Chlamydomonas cells Cells in 10 mM HEPES, pH 7.5 were fixed for 20 min at -20°C in 100% methanol. The

fixed cells were centrifugated at 1,000 g for 2 min, 4°C, resuspended in PBS, and

allowed to adhere to polyethyleneimine-coated microscope slides for 10 min at room

temperature. The slides were immersed in 100% methanol for 5 min at -20°C and

allowed to dry. After rehydration with PBS + 1% NP40 for 10 minutes at room

temperature, the slides were immersed in PBS. The slides were then incubated for 30


75

min at room temperature in IFM blocking buffer (2.5% BSA in PBS), and incubated

overnight at 4°C in IFM blocking buffer with the primary antibodies (Section 5.10, table

5.3) wished to be visualised. After three 10 min washes in PBS, the slides were

incubated for 1-2 hours at room temperatur in IFM blocking buffer with the secondary

antibodies (Section 5.10, table 5.4) directed against the chosen primary antibody. After

tree times 10 min washes in PBS, the slides were mounted with mounting media and

viewed on a Nikon microphot-FXA epifluorescence microscope.

Another fixation method used a fixation buffer (1x HMEK, 1%NP40, 6% glutaraldehyd,

56% H2O) were CC-124 cells was mixed 1:1 with the fixation buffer and allowed to

adhere to PEI-coated microscope slides for 15-20 min. The slides were washed one

time in PBS, and incubated for 30 min in IFM blocking buffer. The slides were incubated

overnight at 4°C in IFM blocking buffer with the selected primary antibodies (Section

5.10, table 5.3). A control without primary antibodies were also made. After three 10 min

washes in PBS, the slides were incubated for 1-2 hours at room temperatur in IFM

blocking buffer with the secondary antibodies (Section 5.10, table 5.4) After three times

10 min washes, the slides were incubated 1 min with DAPI (4´,6-diamidino-2-

phenylindole, dihydrochlorid; Molecular Probes, Eugen, OR) 1:1000 in PBS. The slides

were mounted with mounting media.

Images were captured with a Nikon microphot-FXA epifluorescence microscope using

Elements 3.0 and processed by my superviser Lotte Pedersen using Adobe Photoshop.

The IFM with the glutaraldehyd/NP40 fixation was done mostly by my supervisor Lotte

Pedersen.

5.11.2 IFM on NIH3T3 cells

Cells in suspension on coverslips in a 6 well plate had been grown to 80% confluence in

growth medium or, to induce growth arrest, in serum free medium. The Cells were fixed

for 15 min. in 4% paraformaldehyde (PFA) in PBS. After fixation the coverslips with cells

placed in the wells, were washed 2 times 5 min. in PBS and incubated 10 min. in

permeabilization buffer (1% BSA, 0.2% Triton x-100, in PBS). The coverslips were

transferred to humid chambers and incubated 30 min. in IFM blocking buffer (2.5% BSA


76

in PBS) followed by incubation with primary antibodies (Section 5.10, table 5.3) in

blocking buffer for 1-2 hours at room temperature. After three times 5 min washes in

blockingbuffer, the coverslips were incubated for 45 min. at room temperature in IFM

blocking buffer with secondary antibodies (section 5.10, table 5.4) followed by a 5 min.

wash in blocking buffer, a 5 min. wash in PBS and a 1 min. nuclear staining with a

1:1000 solution of DAPI (Molecular Probes, Eugen, OR) in PBS. After another 5 min.

wash in PBS the coverslips were mounted cell facing down in droplets of mounting

media on an object glass.

Images were captured with a Nikon microphot-FXA epifluorescence microscope using

Elements 3.0 and processed by my superviser Lotte Pedersen using Adobe Photoshop.


77

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7. Appendices

Appendix A: Culturing media

Hutner Trace Elements for TAP:

For 1 L of trace elements, each compound is dissolved in the volume of water indicated

below. EDTA is dissolved in boiling water and FeSO4 is prepared last to avoid oxidation.

50 g EDTA in 250 ml H2O

22 g ZnSO4 in 100 ml H2O

11.4 g H3BO3 in 200 ml H2O

5.06 g MnCL2 in 50 ml H2O

1.61 g CoCl2 in 50 ml H2O

1.00 g CuSO4 in 50 ml H2O

1.10 g (NH4)6Mo7O24 in 50 ml H2O

2.74 g FeSO4 in 50 ml H2O

All solutions are mixed except EDTA. Bring the mixture to boil and add the EDTA

solution. When everything is dissolved, cool to 70°C and adjust the pH to 6.7 with 80-90

ml hot KOH (20%). Bring the final solution to a total volume of 1 L.

Tris-Acetate-Phosphate (TAP) media: 20 ml Tris (100x) (Saveen Werner AB, Cat. No. T1000-1)

20 ml TAP salts (100x) (37.5 g NH4Cl, 10 g MgSO4, 5 g CaCl2, dH2O to 1 L)

2 ml Phosphate (54 g K2HPO4, 27 g KH2PO4, dH2O to 500 ml)

2 ml Hutner Trace Elements

2 ml Glacial acetic acid

Adjust to 2 L with dH2O, autoclaving


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TAP agar plates:

1 L TAP

15 g/l of Plant agar (Duchefa Biochemie, Cat. No. P1001.1000)

Carbendazim Pestanal, diluted 1:500 for a final concentration of 0.2 µg/µl (stock 100

ng/µl in Methanol, Sigma Aldrich, Cat. No. 45826)

M1 media: 2 ml Trace elements (1000x) (H3BO3 1g/l, ZnSO4 1 g/l, MnSO4 0.3 g/l, CoCl2 0.2 g/l,

Na2MoO4 0.2 g/l, CuSO4 0.04 g/l)

2 ml Na citrate (2H2O)

2 ml FeCL3 (6H2O)

2 ml CaCl2

2 ml MgSO4

2 ml NH4NO3

1 ml KH2PO4

1 ml K2HPO4

Adjust to 2 L with dH2O, autoclaving

Appendix B: Preparation of flagella and cell body extracts

HMDEK (10 ml): 100 µl 1 M Hepes (Duchefa Biochemie, Cat. No. H1504.1000)

50 µl 1 M MgSO4

10 µl 0.5 M EDTA

25 µl 1 M KCl

10 µl 1 M DTT

100 µl of 10 % NP40


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Lugols solution (Made by technician Søren L. Johansen):

5 g I2 10 g KI Mixed with 85 ml distilled water to make a brown solution with a total iodine content of 130 mg/ml

Procedure for isolation of flagella from Chlamydomonas:

1. Check cells in a microscope, to determine that they have flagella.

2. Harvest cells: Spin 2400 rpm, 10 min, 20°C in a Hermle centrifuge.

3. Carefully discard supernatant and resuspend pellet in 10 mM Hepes, pH 7.5

(Duchefa Biochemie, Cat. No. H1504.1000), and transfer the cells to a beaker.

The cells are incubated until they have flagella (~ 1 hour). Cells are checked in a

microscope.

4. Give the cells a pH shock to deflagellate: While stirred, 1 M AcOOH are added

drop by drop, while the pH is monitered by a pH meter. When pH reaches 4.5 an

aliquot is taken and the cells are checked in the microscope for loss of flagella.

The cells are neutralized to a pH of 7.5 by adding 1 M KOH drop by drop.

5. While stirring add (for a final concentration of): 1 mM DTT, 4% sucrose, 1:1000

plant protease inhibitor (Sigma), 5 mM MgSO4, 0.25 mM EDTA

6. Cells are now stored on ice. Remove cell bodies by a Hermle centrifuge, 1500 rcf,

5 min at 4°C. The supernatant (flagella) is transferred to 30 ml centrifuge glasses

on ice and centrifuged again at 1500 rcf, 5 min, 4°C, to succesfully remove the last

of the cell bodies. Supernatant # 2 is transferred to new 30 ml centrifuge glasses

on ice.

7. The flagellas are pulled down by centrifuging in a Sorvall SS34 rotor, 12,000 g for

10 min, at 4oC. Discard supernatant and dissolve the flagella in HMDEK+4%

sucrose.

8. Transfer the dissolved supernatant to an eppendorf tube and centrifuge for 10 min


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at 10.000 g, 4oC.

9. The supernatant is removed and the pellet dissolved in the wanted buffer. A lysis

buffer (20 mM Tris, 100mM NaCl, 1% Triton, 1/100 Plant protease (Sigma), 1 mM

DTT) or a HMDEK buffer with 0,5% NP40 was used. The flagella are contained at

-80°C if not directly used for experiments.

Procedure for whole cell protein for SDS-PAGE received by David Mitchell (Ahmed et al., 2008):

1. Spin down cells and resuspend in water (maximum of 2x107 cells per 100 µl

water). For a dense culture, 2x106 cells/ml, can use 100 µl water per 10 ml

starting culture.

2. Add 5 volumes of acetone. Mix thoroughly.

3. Spin 3.000 g, 6 min, 4 °C. Discard supernatant, save pellet.

4. Allow most of acetone to dry, then resuspend pellet in 1X high SDS sample buffer

without DTT (minimum of 1 ul per 5x105 cells).

5. Set tube in boiling water bath 2 min.

6. Spin as in step 3, but save the supernatant to a fresh microfuge tube.

7. Add 5% (vol/vol) DTT. Heat sample in boiling water again before loading on gel.


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Appendix C: cDNA sequences and multiple sequence alignments.

cDNA clones Accession number Nubp1 1030073 B02 FAP20 1024 105 B03 ArfA1A 1024 079 C09 Eg5 137882

Table 7.1. Accesion numbers of the cDNA clones used in this project. (Primers in yellow or bold, see appendix E for primer sequences) C. reinhardtii Nubp1 (Chlamy ID: C_740040) 5’ForwardATGGCGTCGTCCGCGAGCGCAGCACCCACCGGCGAGGTGCCGGACAACGCCAACCAGCACTGCCCAGGCACCGCCAGCGACCAGGCGGGCAAGTCCGCCGCCTGCGCCGGCTGCCCTAACCAGAGCATATGCGCCACTGCGCCGAAGGGGCCGGACCCAGACCTGGCTGCAATTGCGGCACGCATGTCGCGCGTGAAGCACAAGCTGCTGGTGCTGAGTGGCAAGGGTGGCGTGGGCAAGAGCACGGTGTCGGCGCAACTGGCCTTTGCGCTGGCGCGGCGCGGCTTCGAGGTTGGGCTGCTCGACATTGACATCTGCGGCCCCAGTGTGCCCAAGATGCTGGGGCTGGAGGGGCAGGAGATCCACTCCTCGGGCGCTGGCTGGAGCCCCGTGTACGTGGAGGACAACCTGGCGGTCATGAGCATTGGCTTCATGCTGCCTAACCCCGACGAGGCCGTCATCTGGCGTGGACCACGCAAGAATGGCCTGATTAAGCAGTTCCTCAAGGATGTGGACTGGGGCGAGCTGGACTACCTGGTGGTGGACGCGCCTCCCGGCACCAGTGACForward-GAGCACATCACCATCACACAGTGCCTGCAGGGTGCGReverseGCGCTAGGCGATG GCGGCAGTGGCGGCGCGGCGGCAGTGATTGTGACCACGCCGCAGGATGTGGCCATCATCGACGTGCGCAAGGAGGTCAACTTCTGTCGCAAAGTGGGACTGCCGGTGCTGGGTGTGGTGGAGAACATGGCGGGCCTGGTGCTGCCAGTGGAGCGGGCGGCATTCAGCGCCGTGCGCAGCAAGCGGGACGGCGCGGCTGGGGCTGGGGCTGGGGCTGGGGCTGCCGAGGCGGCAGTCGAGGAGGAGGAGGAGGAGGACGTGACGGCCGCAGTGCTGGCGCTGTTGGCGGAGCGCTTCCCGGGGGCGCAGCTGCGGCTGCGGGCCGAGGTGTTTCGTGAGGGCGGCGGTGCGGCGCGCATGTGTGCCGACATGGGTGTGCCGCTGCTGGGCCGCCTGCCGCTGGACCCAGGTCTAGGCGCCGCAGCGGACGCGGGCCGCAGCGTGCTGCCGGAGGCGGCTGGGGCGGCGGGGGCGGATGCGGTTAAGGGAGGGCTGGAGGGCGTGGCGCCTCAGGCGTGCATTGCGCCGCTGATGGCGATTGTGGGGAAGGTGGTGGAGCAAACGACGGGCGCGGACGGGAAGGCAGCGGCGGCGAAGTAGReverse-3’


89

C. reinhardtii small ARF-related GTPase ARFA1A (gi|159465364:159-704) 5’ForwardATGGGTCTGATGGTTTCTAAGGCCCTCAGCCGGCTCTTTGGCAAGAAGGAGATGCGCATTCTGATGGTCGGTCTGGACGCCGCTGGTAAGACTACCATCCTCTACAAGCTGAAGCTGGGCGAAATCGTGACGACTATTCCCACCATCGGCTTCAACGTGGAGACTGTGGAGTACAAGAACATCAGCTTCACCGTGTGGGATGTCGGTGGCCAGGACAAGATCCGCCCTCTGTGGCGGCACTACTTCCAGAACACTCAGGGCCTCATTTTCGTCGTGGACAGCAATGACCGCGATCGTATTGGGGAGGCCAAGGATGAGCTGCACCGCATGCTCAACGAGGACGAGCTGCGGGACGCCGTGCTGCTGGTCTTCGCCAACAAGCAGGACCTGCCCAACGCCATGAACGCGGCGGAGATCACGGAGAAGCTGGGCCTGCACGGCCTGCGCCAGCGCCACTGGTACATCCAGTCCACGTGCGCCACCAGCGGCGAGGGCCTGTACGAGGGCCTGGACTGGCTGAGCCAAAACATCCAGAACAAGTCGTGAReverse-3’

Figure 7.1: Multiple sequence alignment of CrARFA1A homologs.


90

C. reinhardtii flagellar associated protein, FAP20 (>gi|159468653:127-699) 5’ForwardATGTTCAAGAACGCCTTCCAATCCGGCTTCCTGTCGGTGCTGTATAGCATTGGCTCGAAGCCCCTAGAGATTTGGGACAAGCAAGTTAGCAATGGCCACATCAAGCGCATCACAGACGCGGACATCCAGTCTTCGGTGCTGGAGATAATGGGGCAGAACGTCTCCACAACATACATCACCTGCCCAGCGGATCCCAACAAGACGTTGGGGATCAAGCTGCCGTTCCTGGTGCTCATCATTAAGAACCTGAACAAATACTTCAGCTTCGAGGTACAAGTTCTGGACGACAAGAACGTGCGGAGACGCTTCCGCGCGTCGAACTATCAGTCGACAACGCGCGTTAGCCGTTCATCTGCACCATGCCCATGCGCCTGGACAGCGGGTGGAACCAGATTCAGTTCAACCTGTCGGACTTCACGCGCCGCGCTTACGGGACGAACTACATCGAGACGCTGCGGGTCCAGGTGCACGCGAACTGCCGCATCCGCCGCATCTACTTCTCCGACCGCCTGTACTCGGAAGAGGAGCTGCCCGCCGAGTTCAAGCTCTTCCTGCCGATCCAGAAGTCGTAGReverse-3’

Figure 7.2: Multiple sequence alignment of CrFAP20 homologs.


91

C. reinhardtii Eg5 full length (underscored: the N-terminal motor domain cloned for this project; red: stop codon) 5’ForwardATGGCCGAACCAAAGCCTGGACCTGGGCCTGGCGAGGGCAATGATGGAGTCAACGTGAATGTGTTGCTTCGCTGCAGGCCTCTGAGCGACAAGGAAATCGCTGAGAGGACCCCGCAAGTTATTTCCTGCAATGAGGCTCTGCGCGAGGCGACCCTCTATATCCAGAGCGTCGGAGGAAAACAGACGTCCAAGACGTTCCGCTTTGACAGGGTCTTCAGCCCCGAGTCCTCTCAGGAGAAGCTGTTTAAGCAGGCCATCGTGCCCATCGTCCAGGAGGTCATGGAGGGCTTCAACTGCACTATCTTTGCCTATGGCCAGACGGGCACGGGCAAGACCTACACCATGGAGGGCGGGCCGCGCCGCAGCGACGACGGCAAGGTGCTGTCCGCCGAGGCCGGTGTCATCCCGCGCTCCATCAAGCAGATCTTTGACACCATCGAGGCCAATAACACCGACTCCACCGTCAAGGTCACCTTTCTTGAGCTGTACAATGAGGAGCTAACGGACCTGCTGTCGACCTTCGACGACGGCAAAGAGGACGGCAAGCGCCTGCGGCTTCTGGAGGACCGCAGCGGTGTGGTGGTGCAGGGCCTGGAGGAGGTGGTGGTCAAGAGCGCCGCGGAAATCTACCAGGTGCTGGACCGCGGCACGGCCAAGCGCCGCACCGCCGAGACGCTGCTAAACAAACGCTCCAGCCGCAGCCACTCGGTGTTCTCCATCACCATCCACATGCGTGAGGTCACGCCCGAGGGCGAGGATGTTGTTAAGGTGGGCAAGCTGAACCTGGTGGACCTGGCGGGCAGTGAGAACATCTCTCGCTCCGGTGCCAAGGACGGCCGCGCGCGTGAGGCGGGCTCCATCAACCAGTCGCTGCTGACGCTGGGCCGCGTCATTACCGCGCTGGTGGAGCACTCGGGCCACGTGCCCTACCGCGACTCTAAGCTGACGCGCCTGCTACGCGAGTCGCTGGGTGGCAAGACCAAGACCTGCATCATCGCCACCATCGCGCCCACGGTGCAGTGCCAGGAGGAGACCATCAGCACGCTGGACTACGCGCACAGGGCCAAGAACATCCGCAACCGGCCCGAGGTCAACCAGAAG-ReverseATCTCCAAGACCGCCATGATCAAGGAGATGTCCA GTGAGATGGAAAAGCTGCGCATGGAGCTGATTGCGCAGCGCGAGAAGAATGGCATCTACATCTCCACCGACAAGTTCCAGGCGGACGAGCTGGAGCGCGTGCAGCTGCGCGACACCGTCAAGGCCCTGCGCGAGGAGATGAAGCTGGAGCAGGAGGAGTTCGCCGCCACGCTGGAGCAACAGAAGTCCGAGTCGGACAAGGCCATCAGCCGCCTGCAGTCGGAGCTGTCGGCCGCCAAGGCGGACCTGGAGTACATGGACGCGCGGCTGCAGGAGGCCAGCCGCAGCATCCACGAGCGCCAGTACATCATCACCGCGCAGCGGACGGCGGAGCAGGACGTGGCGGAGGCCGCCGAGGCGGTGCGCACCGAGTTGGCGGCGGCAGCGCAGGACGTGGGCGTGCTGTTTGCCTCGCTGGAGGAGCTGTCGGACGTGCACAGCGGCGACCGTGACACCATCAAGCACGTGCAGTCGCTGGTGGCGGAGCGGCTGGGCGGACTGGGCTCTAAGCTGACTGGCGCGGCGGAAGGCCAGCGCCGGCAGCTGGCCGGCCTGGGTGAGTCGCTGGCCTCGTTCAAGGCGCAGAAGGCGCAGGACCTGCAGCAGCTGCTGGGCCGCGTGGCGGCGGTGCAGGACACCGTGGCGGCCATGCGCGACGCCGTGGAGCGCCAGGCCCAGGCCGCCGAGGCCGCCGCGTCCGCTGCTCTGGGCCGCATTAACGATGCCACCGGCTCGCATGTGGCGGTGGCGGTTGCGGCGGCGCAGGGCCTGGAGGCGGCCACGCGCGGCGCCGTCGACGCGCTGGCGCAGGCGATGGAGCAGCAGTCGCAGCAGCTGGCGGCTTTTACTCAGCAGCAGGCGGCGTCTTCAGAGGCGGCGTGCAAGGCGCTGCATGAGGCCATGGGCCGCCTGTCGGGCCGCTTCGAGGGCGTGCAGGCGGCGGCGGACCAGGCGGGGACTGTGGTGGAGGAGCGCACGGCAGCTCTGGGCGAGGGGATGGGCTCGTTCGCGGAGCGCTACAAGGAGTCGTGCGCGGCGCAGCAGGCGGTGCTGATGGCGCAGATTACGGCGCTGGTGGCGGCGTTTGCGGAGGAGCGCGCGGGCGAGGTGGCGCGCGAGGTGACGGCGCTAAAGCAGCAGGCGGTGGAGGGCGGCAAGGTGGTGCGGCGCCAGCTGGGCGGCATCGCTGGCACTGCCGCGTCCGCTCGCCAGGAGGTGCAGGAGGCGGAGGCGTCCCTGGCGAGTGGCATGCAGTCGCAGCAGGGCCGCGTGCAGGAGGCCACCACCAGCCTCACGGCCTCGCTGCGCAGCACGCACGACCAGGCGCGGGCCATCCACGCCGGCGTAGCGGGCCAGCTGGCCACCAGCATCACCGCGCAGGAGTGTTTCAACAGGGCATGGGTCAGGCGTGCAGCAGTGGCATTGAGGCGGTGCAGGGTGCCGTGCGTGCCTGCGTGACGGGCGCCTATGAGGCGGCGGCGTCCGCGGCGTACTCTGCACGGA-3’


92

Figure 7.3: Multiple sequence alignment of CrEg5 homologs. Notice how the N-terminal motor domain are highly conserved, whereas the C-terminal region, in contrast to the motor domain, display little sequence similarity to other known kinesins.

C r _E g 5 1 - M A EP K P G PG P G E GN D G V NV N V L LR C R P LS D K E IA E R T PQ V I S CN E A L RE A T L YI Q S V GG K Q T SK T F R FD R V F SP E S S QEA t 1 - - - -- - - M SS R H D KE K G V NV Q V L LR C R P FS D D E LR S N A PQ V L T CN D L Q RE V A V SQ N - I AG K H I DR V F T FD K V F GP S A Q QKH s _K i f 1 1 1 - M A SQ P N S SA K K K EE K G K NI Q V V VR C R P FN L A E RK A S A HS I V E CD P V R KE V S V RT G G L AD K S S RK T Y T FD M V F GA S T K QID m _6 1 F 1 M D I SG G N T SR Q P Q KK S N Q NI Q V Y VR V R P LN S R E RC I R S AE V V D VV G P R EV V T R HT - - - LD S K L TK K F T FD R S F GP E S K QC

C r _E g 5 8 0 K L F KQ A I V PI V Q E VM E G F NC T I F AY G Q T GT G K T YT M E G GP R R S DD G - - KV L S A EA G V I PR S I K QI F D T IE A N N TD S T V KVA t 7 3 D L Y DQ A V V PI V N E VL E G F NC T I F AY G Q T GT G K T YT M E G EC R R S KS A P C GG L P A EA G V I PR A V K QI F D T LE G Q Q AE Y S V KVH s _K i f 1 1 8 0 D V Y RS V V C PI L D E VI M G Y NC T I F AY G Q T GT G K T FT M E G E- R S P NE E Y T WE E D P LA G I I PR T L H QI F E K LT D N G TE F S V KVD m _6 1 F 7 8 D V Y SV V V S PL I E E VL N G Y NC T V F AY G Q T GT G K T HT M V G N- E T A EL K S S WE D D S DI G I I PR A L S HL F D E LR M M E VE Y T M RI

C r _E g 5 1 5 8 T F L EL Y N E EL T D L LS T F D DG - - - -- K E D GK R L R LL E D - RS G V V VQ G L E EV V V K SA A E I YQ V L D RG T A K RR T A E TL L N K RSA t 1 5 3 T F L EL Y N E EI T D L LA P E D LS R V A AE E K Q KK P L P LM E D G KG G V L VR G L E EE I V T SA N E I FT L L E RG S S K RR T A E TF L N K QSH s _K i f 1 1 1 5 9 S L L EI Y N E EL F D L LN P S S D- - - - -- V S E RL Q M F DD P R N KR G V I IK G L E EI T V H NK D E V YQ I L E KG A A K RT T A A TL M N A YSD m _6 1 F 1 5 7 S Y L EL Y N E EL C D L LS - - T D- - - - -- D T T KI R I F DD S T K KG S V I IQ G L E EI P V H SK D D V YK L L E KG K E R RK T A T TL M N A QS

C r _E g 5 2 3 2 S R S HS V F S IT I H M RE V T P EG E D V VK V G K LN L V D LA G S E NI S R S G- A K D GR A R E AG S I N QS L L T LG R V I TA L V E HS G H V PYA t 2 3 3 S R S HS L F S IT I H I KE A T P EG E E L IK C G K LN L V D LA G S E NI S R S G- A R D GR A R E AG E I N KS L L T LG R V I SA L V E HL G H V PYH s _K i f 1 1 2 3 3 S R S HS V F S VT I H M KE T T I DG E E L VK I G K LN L V D LA G S E NI G R S G- A V D KR A R E AG N I N QS L L T LG R V I TA L V E RT P H V PYD m _6 1 F 2 2 9 S R S HT V F S IV V H I RE N G I EG E D M LK I G K LN L V D LA G S E NV S K A GN E K G IR V R E TV N I N QS L L T LG R V I TA L V D RA P H V PY

C r _E g 5 3 1 1 R D S KL T R L LR E S L GG K T K TC I I A TI A P T VQ C Q E ET I S T LD Y A H RA K N I RN R P E VN Q K I SK T A M IK E M S SE M E K LR M E L IAA t 3 1 2 R D S KL T R L LR D S L GG R T K TC I I A TV S P A VH C L E ET L S T LD Y A H RA K N I RN K P E VN Q K M MK S T L IK D L Y GE I E R LK A E V YAH s _K i f 1 1 3 1 2 R E S KL T R I LQ D S L GG R T R TS I I A TI S P A SL N L E ET L S T LE Y A H RA K N I LN K P E VN Q K L TK K A L IK E Y T EE I E R LK R D L AAD m _6 1 F 3 0 9 R E S KL T R L LQ E S L GG R T K TS I I A TI S P G HK D I E ET L S T LE Y A H RA K N I QN K P E VN Q K L TK K T V LK E Y T EE I D K LK R D L MA

C r _E g 5 3 9 1 Q R E KN G I Y IS T D K FQ A D E LE R V Q LR D T V KA L R E EM K L E QE E F A AT L E Q QK - - - -- - S E SD K A I SR L Q S EL S A A KA D L E YMA t 3 9 2 S R E KN G V Y MP K E R YY Q E E SE R K V MA E Q I EQ M G G QI E N Y QK Q L E EL Q D K YV G Q V RE C S D LT T K L DI T E K NL S Q T CK V L A STH s _K i f 1 1 3 9 2 A R E KN G V Y IS E E N FR V M S GK L T V QE E Q I VE L I E KI G A V EE E L N -- - - - -- R V T EL F M D NK N E L DQ C K S DL Q N K TQ E L E TTD m _6 1 F 3 8 9 A R D KN G I Y LA E E T YG E I T LK L E S QN R E L NE K M L LL K A L KD E L Q NK E K I FS E V S MS L V E KT Q E L KK T E E NL L N T KG T L L LT

C r _E g 5 4 6 5 D A R LQ E A S RS I H E RQ Y I I TA Q R T AE Q D V AE A A E AV R T E LA A A A QD V G V LF A S L EE L S D VH S G D RD T I K HV Q S L VA E R L G-A t 4 7 2 N E E LK K S Q YA M K E KD F I I SE Q K K SE N V L VQ Q A C IL Q S N LE K A T KD N S S LH Q K I GR E D K LS A D N RK V V D NY Q V E LS E Q I S-H s _K i f 1 1 4 6 5 Q K H LQ E T K LQ L V K EE Y I T SA L E S TE E K L HD A A S KL L N T VE E T T KD V S G LH S K L DR K K A VD Q H N AE A Q D IF G K N LN S - - --D m _6 1 F 4 6 9 K K V LT K T K RR Y K E KK E L V AS H M K TE Q V L TT Q A Q EI L A A AD L A T DD T H Q LH G T I ER R R E LD E K I RR S C D QF K D R MQ D N L EM

C r _E g 5 5 4 4 - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - G L GS K L T GA A E G QR R Q L AG L G E SL A S F KA Q K A QD L Q Q LL G R V AA V Q D TVA t 5 5 1 - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - N L FN R V A SC L S Q QN V H L QG V N K LS Q S R LE A H N KA I L E MK K K V KA S R D LYH s _K i f 1 1 5 4 1 - - - -- - L F NN M E E LI K D G SS K Q K AM L E V HK T L F GN L L S SS V S A LD T I T TV A L G SL T S I PE N V S TH V S Q IF N M I LK E Q S LAD m _6 1 F 5 4 9 I G G SL N L Y QD Q Q A AL K E Q LS Q E M VN S S Y VS Q R L AL N S S KS I E M LK E M C AQ S L Q DQ T N L HN K L I GE V M K IS D Q H S- - Q A FV

C r _E g 5 5 9 3 A A M RD A V E RQ A Q A AE A A A SA A L G RI N D A TG S H V AV A V A AA Q G L EA A T R GA V D A LA Q A M EQ Q S Q QL A A F TQ Q Q A AS S E A ACA t 6 0 0 S S H LE A V Q NV V R L HK A N A NA C L E EV S A L TT S S A CS I D E FL A S G DE T T S SL F D E LQ S A L SS H Q G EM A L F AR E L R QR F H T TMH s _K i f 1 1 6 1 5 A E S KT V L Q EL I N V LK T D L LS S L E MI L S P TV V S I LK I N S QL K H I FK T S L TV A D K IE D Q K KE L D G FL S I L CN N L H EL Q E N TID m _6 1 F 6 2 7 A K L ME Q M Q QQ Q L L MS K E I QT N L Q VI E E N NQ R H K AM L D S MQ E K F AT I I D SS L Q S VE E H A KQ M H K KL E Q L G- A M S LP D A E EL

C r _E g 5 6 7 3 K A L HE A M G RL S G R FE G V Q AA A D Q AG T V V EE R T A AL G E G MG S F A ER Y K E SC A - - -- - - A QQ A V L MA Q I T AL V A A FA E E R AGA t 6 8 0 E Q T QE M S E YT S T F FQ K L M EE S K N AE T R A AE A N D SQ I N S II D F Q KT Y E A QS K - - -- - - S DT D K L IA D L T NL V S S HI R R Q HEH s _K i f 1 1 6 9 5 C S L VE S Q K QC G N L TE D L K TI K Q T HS Q E L CK L M N LW T E R FC A L E EK C E N IQ K P L SS V Q E NI Q Q K SK D I V NK M T F HS Q K F CAD m _6 1 F 7 0 6 Q N L QE E L A NE R A L AQ Q E D AL L E S MM M Q M EQ I K N LR S K N SI S M S VH L N K ME E - - -- S R L TR N H R ID D I K SG I Q D YQ K L G IE

C r _E g 5 7 4 7 E V A RE V T A LK Q Q A VE G - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - --A t 7 5 4 L V D SR L H N FK D A V SS N K T FL D E H VS A V N NL T K D AK R K W ET F S M QA E N E AR E G A DF S A A KH C R M EL L L Q QS V G H AE S A F KHH s _K i f 1 1 7 7 5 D S D GF S Q E LR N F N QE G T K LV E E S VK H S D KL N G N LE K I S QE T E Q RC E S L NT R T V YF S E Q WV S S L NE R E Q EL H N L LE V V S QCD m _6 1 F 7 8 2 A S Q SA Q A E LT S Q M EA G M L CL D Q G VA N C S ML Q V H MK N L N QK Y E K ET N E N VG S V R VH H N Q VE I I C QE S K Q QL E A V QE K T E VN

C r _E g 5 7 6 3 - - - -- - - - GK V V R RQ L G G IA G T A AS A R Q EV Q E A EA S L A SG M Q S QQ G R V QE A T T SL T A S LR S T H DQ A R A IH A G V AG - - - --A t 8 3 4 C K I TH E S L KE M T S KQ V T D VS S L V RS A C D SN E Q H DA E V D SA R T A AE K D V TK N S D DI I Q Q IE R M S ED E K A SV S K I LE N V R SHH s _K i f 1 1 8 5 5 C E A SS S D I TE K S D GR K A A HE K Q H NI F L D QM T I D ED K L I AQ N L E LN E T I KI G L T KL N C F LE Q D L KL D I P TG T T P QR K S Y LYD m _6 1 F 8 6 2 L E Q MV D A R QQ L I T ED R Q R FI G H A TV A T D LV Q E S NR Q F S EH A E H QR Q Q L QI C E Q EL V R F QQ S E L KT Y A P TG T T P SK R D F VY

C r _E g 5 8 3 0 - - Q LA T S I TA Q E C FN R A W VR R A A VA L R R CR V P C VP A - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - --A t 9 1 4 E K T LE S F Q QD Q C C QA R C I ED K A Q ET F Q Q QY M E Y EP T G A TP T K N EP E I P TK A T I ES L R A MP I E T LV E E F RE N N S YE S F A TKH s _K i f 1 1 9 3 5 P S T LV R T E PR E H L LD Q L K RK Q P E LL M M L NC S E N NK E E T IP D V D VE E A V LG Q Y T EE P L S QE P S V DA G V D CS S I G GV P F F QHD m _6 1 F 9 4 2 P R T LV A T S PH Q E I VR R Y R QE L D W SD L D T TA T I D EC S E G EH D V S MH S V Q EL S E T ET I M N ST P I E PV D G V TV K R G CG T T R NS

C r _E g 5 - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - - -- - - -A t 9 9 4 E T K PQ Q L T RS P L S QL K F S YS N R S SQ R R F VL L L H NA N L G PF L V M MV C D M CM H I P LD H F L YM T Q PH s _K i f 1 1 1 0 1 5 K K S HG K D K EN R G - -- I N T LE R S K VE E T T EH L V T KS R L P LR A Q I NL - - - -- - - - -- - - - -- - - -D m _6 1 F 1 0 2 2 N S N AL K P P VA T G G KR S S S LS R S L TP S K T SP R G S PA F V R HN K E N VA - - - -- - - - -- - - - -- - - -


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Appendix D: Vector map

Figure 7.4: pMAL™-c2 Vector (New England BioLabs). 6646 base pairs. Has an exact deletion of the malE signal sequence. Arrows indicate the direction of transcription. Unique restriction sites are indicated.


94

Appendix E: Primers Proteins to be sequenced

Restriction sites

Primers Melting temperature with/without overhang (°C)

Nubp1 Full length (1215 bp)

EcoRI HindIII PstI

Primer forward (EcoRI): 5´GTGAATTCATGGCGTCGTCCGCGAGCGC Primer forward (PstI): 5’GAGCACATCACCATCACACAGTGCCTGCAGGGTGCG Primer reverse (HindIII): 5ÁCAAGCTTCTACTTCGCCGCCGCTGCCTTC Primer reverse (PstI): 5’CGCACCCTGCAGGCACTGTGTGATGGTGAT

67/62 71 67/62 67

ARFA1A (560 bp)

EcoRI HindIII

Primer forward (EcoRI) 5ĆTGAATTCATGGGTCTGATGGTTTCTAAGGC Primer reverse (HindIII) 5ĆTAAGCTTTCACGACTTGTTCTGGATGTTTTGG

62/55 62/56

Eg5 (1120 bp)

EcoRI, HindIII

Primer forward (EcoRI) 5ĆAGAATTCATGGCCGAACCAAAGCCTGGAC Primer reverse (HindIII) 5ĆAAAGCTTCTTCTGGTTGACCTCGGGCC

59/64 58/64

FAP20 589 bp

EcoRI, HindIII

Primer forward (EcoRI) 5ĆTGAATTCATGTTCAAGAACGCCTTCCAATCC Primer reverse (HindIII) 5ĆTAAGCTTCTACGACTTCTGGATCGGCAG

62/56 63/56

Table 7.2: Primer sequences. mRNA coding sequences are bold, overhang normal, restriction sides underscored. Vector map see appendix C


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Appendix F: Procedure for PCR

Mastermix: 25 pmol Forward primer

25 pmol Reverse primer

10 µl 1 % Triton x100

1-5 µl cDNA template

10 µl Herculase 10x buffer

2 µl dNTP mix (10 mM/dNTP → 200 µM final pr. DNTP)

5 µL DMSO (5 % final)

ddH2O up to 100 µl

Samples are heated at 98°C for 5 min then cooled down. After a quick spin in a

centrifuge, 1 µl of Herculase® Enhanced DNA Polymerase (Stratagene, cat. no.

600260) is added. The samples are divided into 5 tubes, put in a thermocycler and run

at an appropiate program depending on fragment size and melting temperature of

primers.

Temperature (°C) Time (Minutes)

Number of cycles

Initiating denaturing 98 5 1x

Denaturing

Annealing

Elongation

98

temp. gradient

72

1

1

1.5

10x

Denaturin

Annealing

Elongation

98

temp. gradient

72

1

1

1.5

25x

Final Elongation 72 10 1x

Table 7.3. PCR thermal cycling program. temp. gradient = temperature gradient.


96

Appendix G: Agarose gels

50x TAE (Tris-acetate-EDTA) buffer: 242 g Tris base

57.1 ml Acetic Acid

100ml 0.5 M EDTA (shake vigorously before use)

Add ddH2O to 1 L

1% agarose gel: 0.5 g of agarose is heated in 50 ml 1xTAE buffer until completely melted. The gel is

cooled to app. 40°C, and 1.5 µl of ethidium bromide is added. The gel is poured into a

10 well chamber. When solid, samples can be loaded. Mix samples with DNA load and

load 5-10 µl pr. well with 5 µl of a molecular marker, 1 kb DNA ladder. The gels were

run in 1xTAE buffer for 1-1.5 hrs. at 90V and visualized.

Appendix H: Transformation of DH10α E.coli cells LB Broth:

20.6 g LB broth EZMixTM Powder (Sigma Aldrich, Cat. No. L7658)

up to 1 L of ddH2O

LB agar plates:

20.6 g/l of liquid LB Broth

15 g/l of Plant agar (Duchefa Biochemie, Cat. No. P1001.1000)

Ampicillin (100 µg/ml)

up to 1 L of ddH2O


97

Preparation of competent of DH10α E.coli cells:

1. Streak DH10α cells onto an agar plate without antibiotics and inoculate overnight

at 37°C.

2. Transfer one colony from the plate to 50 ml of liquid LB media until a OD600 of

app. 0.35 is reached.

3. Centrifuge at 1500g, 5 min. Resuspend pellet (cells) in 5 ml of icecold 50 mM

CaCl2 and let it rest on ice for 30 min. Store at -80°C in aliquots of 100 µl.

Transformation of DH10α E.coli cells: 1. Add 5 µl ligated vector or 1 µl vector (positive control) or nothing (negative

control) to 100 µl competent DH10α E.coli cells.

2. Incubate on ice for 20 min.

3. Heatshock for 2 min in a 42°C waterbath. Let it cool on ice.

4. Transfer to 0.5 ml of LB media and recover 1 hour at 37°C while shaking.

5. 150 µl of each solution is streaked on agar plates with ampicillin and incubated

overnight at 37°C.

6. A number of colonies are picked and restreaked on a fresh agar plate with

ampicillin. After incubation at 37°C single colonies can be picked for further

analysis.

Appendix I: Protein quantification Bovine Serum Albumin standard (Pierce, cat. No. 23209; 2 µg protein/µl Bovine) was

diluted with lysis buffer to desired concentration and a protein standard curve was made

using the BioRad DC Protein Assay as exampled below.


98

µg BSA OD750 (1) OD750 (2) OD750 (Mean value)

0 0 0 0

5 0.0870 0.0843 0.0857

10 0.1614 0.1611 0.1613

20 0.2719 0.2899 0.2809

30 0.3938 0.4084 0.4011

40 0.4768 0.4779 0.4774

50 0.5899 0.5246 0.5573

Linear regression: 0,011071741

The linear regression is used to determine the protein content of the lysate.

CC-124, Flagella lysate OD750 µg protein/µl

4 µl 0.3266 7.37

6 µl 0.3848 5.79

Mean value: CC-124: 6.58 µg protein/µl

The optical density of the flagella lysate is measured at 750 nm, and this OD value is

divided with the linear regression coefficient of the standard curve to obtain a value for

the protein quantum in the sample. All samples are diluted with ddH2O so the same

volume and concentration can be loaded to a polyacrylamide gel.

y = 0,0074x + 0,0064 R² = 0,99225

0

0,05

0,1

0,15

0,2

0,25

0 5 10 15 20 25 30 35

OD

75

0

µg protein


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Appendix J: Solutions for SDS-PAGE and western blotting

Gel Cat. No NuPAGE buffer

(Invitrogen)

Cat. No

10 %

Bis-Tris Gel

NP0302BOX MOPS SDS

Running buffer

(20x)

NP0001

4-12 %

Bis-Tris Gel

NP0322BOX MES SDS

Running Buffer

(20x)

NP0002

3-8 %

Tris-Acetate

Gel

EA03752BOX Tris-Acetate SDS

Running Buffer

(20x)

LA0041

Running buffer: 30 ml NuPAGE Running Buffer 20x stock (Invitrogen)

570 ml ddH2O

Transfer buffer 10% Ethanol: 30 ml NuPAGE Transfer Buffer 20x stock (Invitrogen) (Cat. No. NP0006-1)

60 ml 96% EtOH

510 ml ddH2O

Transfer buffer 20% Ethanol: 30 ml NuPAGE Transfer Buffer 20x stock (Invitrogen) (Cat. No. NP0006-1)

120 ml 96% EtOH

450 ml ddH2O


100

Tris-Buffered Saline Tween-20 (TBST):

10 ml 1 M Tris/HCl pH 7.5

80 ml 1,5 M NaCl

1 ml Tween 20

ddH2O up to 1 L

Blocking buffer: 50 ml TBST

2.5 g non-fat dry milk

Coomassie staining: Coomassie in destain (Serva, Cat. No. 17524)

Appendix K: Affinity purification of CrNubp1

Phosphate Buffered Saline (PBS): 8 g NaCl

0.2 g KCL

1.15 g Na2HPO4

0.2 g KH2PO4

Add to 1 L ddH20, pH 7.4, autoclaving

Tris-Buffered Saline (TBS): 10 ml 1 M Tris/HCl pH 7.5

80 ml 1.5 M NaCl

ddH2O up to 1 L


101

Procedure for affinity purification:

1. The CrNubp1 protein (500µl protein added 166µl NuPage LDS sample buffer

(4x), Invitrogen, Cat. No. NP0007) and 16.7 µl DTT (AppliChem, Cat. No. 27565-

41-9) is loaded onto a 10% NuPage gel (see section 5.9).

2. The Gel is transferred to a nitrocellulose blot and the protein bands visualized

using Ponceau S solution (Sigma, Cat. No. P7170). The band of interest was cut

into small pieces.

3. Incubate the pieces in a BSA buffer (5 ml PBS, 3% BSA, 0.05% NaAzide (Sigma,

Cat. No. S-2002)) with rotation overnight at 4°C.

4. Wash the filters 6-8 times with PBS.

5. Incubate filters in a buffer containing the CrNubp1 antiserum (1.8 ml PBS added

0.2 ml rabbit 63A 2. Bleed) for 2 days with rotation at 4°C.

6. Wash filters 3 x 5 min. in TBS

7. Wash filters 2 x 5 min. in TBS added 0.1% NP40.

8. Wash filters 3 x 5 min. in TBS

9. Add 100 µl 0.2 M glycine-HCl pH 2.5. Mix for 1 minute. The liquid (antibody) is

transferred to an eppendorf tube.

10. Immediately add 50 µl KPO4 pH 9.0 with 5% BSA to the eppendorf tube. Mix well.

11. Repeat step 9 and 10.

12. The OD of the antibody is measured usin TBS as a blank. The concentration of

the antibody can be calculated as:

[Antibody] = C (mg/ml) = (A280/ε) · 10 ; ε= 14 (antibody)


102

Appendix L: IFM

Paraformaldehyde stock, 20%, 10 ml (PFA): 2 g paraformaldehyd is placed in a 20 ml beaker glass and added 8 ml ddH2O and 3

drops of 1 M NaOH. A magnetic stirrer is placed in the glass beaker and the solution is

stirred at 60°C for 10-30 min. or until the PFA has dissolved. The solution is filtered

through a Millipore filter and ddH2O is added up to a total volume of 10 ml. The PFA is

stored at 4°C.

Blocking buffer for IFM: 25 ml PBS

0.5 g Bovine Serum Albumin (BSA, 2.5 % final) (Fluxa cat. No. 05480)

Permeabilization buffer for IFM: 25 ml PBS

50 µl Triton X 100 (0.2 % final)

0.25 g Bovine Serum Albumin (1% final)

Mounting media/antifade (5 ml): 4 ml glycerol

500 µl 10 x PBS

500 µl ddH2O

0.1 g N-propylgallate (Sigma-Aldrich; 2% final)

The solution is stirred for 1-2 hours until the N-propylgallate is dissolved


103

Appendix M: Overview of potential binding patners of EB1/IFT172

from Chlamydomonas flagella.

Protein

found

GenBank

accesion

number

Known of the protein Described in

Chlamy ID

Pulled

down by

Small ARF-related GTPase: ARFA1A/ ARFA1B

gi|159465365 Present in flagellar proteome

C.reinhardtii 186126 EB1

IFT172

Eg5

gi|124359634 Kinesin, motor region. This kinesin belongs to the Eg5_BimC family (kinesin-5).

Medicago truncatula

137882 IFT172

Flagella Associated Protein

FAP20: 1. gi|159468654 2. gi|32400772 FAP261: 3. gi|159477961 FAP295: 4. gi|11230985 5. gi|11230987 6. gi|159479618 Nitrogen Starv-ed gametogene-sis1 (NSG1): 7. gi|159487699 Unnamed: 8. gi|159481253 9. gi|159481973

1. DUF667 superfamily, highly similar to verte-brate transcription factor IIB of unknown function 2. transcription factor TFIIB 3. Contains con-served SMC domain and TFIIIC B-block do-main involved in tran-scription regulation 4.5.6. Cyclic nucleotide dependent protein ki-nase; cyclic nucreotide dependent protein ki-nase II; Found in fla-gellar and basal body proteome. 7. Probably chlamy specific 8. Contains ankyrin re-peats 9. Contains mul-tiple an-kyrin repeats, similar to unc-44

All in C. reinhardtii Except 2: Triticum aestivum

4/5/6: 131695

2/4/5/6/7/

8/9:

EB1

1/3:

IFT172

Flagellar Adenylate Kinase

gi|159488592

Flagellar adenylate kinase

C. reinhardtii EB1

NAP1 gi|159477313 nucleosome assembly protein

C. reinhardtii EB1

Phragmo- gi|145338697, microtubule motor may be in flagellar proteome

Arabidopsis thaliana

114085 EB1/


104

plast Orienting Kinesin 2 POK2

v. 2 ID=C_1350007

IFT172

kinesin (centromeric protein)-like protein

gi|9280323 kinesin (centromeric protein)-like protein


114085 EB1

cGMP-dependent protein kinase

gi|159487245

CGK2, found in flagel-lar proteome, involved in signalling initiated by flagellar agglutination during mating

C. reinhardtii 181794 EB1

prefoldin, putative (ISS)

gi|116000618

prefoldin, putative (ISS) stabilizes polypeptides prior to folding with CCT chaperonin, in particular tubulin. Potentially interesting.

Ostreo-coccus tauri

Similar to Chlamy PFD6 ID: 188584

EB1

putative phragmo-plast-asso-ciated kinesin-related protein 1

gi|47848099

similar to KLP-2 like kinesin involved in mitosis

[Oryza sativa Japonica Group]

114085 IFT172


gi|9280323



114085 IFT172

Vsf-1 protein

gi|18698670

bZIP transcription factor,

Oryza sativa 188572 IFT172

putative vsf-1 protein

gi|42408548

Oryza sativa Japonica Group

IFT172

Os08g0543900

gi|115477615

[Oryza sativa (japonica cultivar-group)]

188572 IFT172

Ankyrin repeat protein (ISS)

gi|116059081

Top homologs are sea urchin and vertebrates. Cilia specific?

Ostreo-coccus tauri

Similar to Chla-my ID 113356

IFT172

Hypo-thetical proteins

1. gi|159476560 2. gi|147845616

1. CHLREDRAFT_142416 weakly similar to Nu-clear mitotic apparatus

1: C. reinhardtii

1: 142416 2:

IFT172


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protein 1 (NuMA pro-tein). Top hits are cili-ates and vertebrates. May be ciliary. 2. Conserved apolipo-protein A-I-binding pro-tein YjeF. Role in cell cycle regulation and spermatogenesis

2: Vitis vinifera

103444

Predicted Proteins

1. gi|168045411 2. gi|159487695 3. gi|159491346

1.Contains conserved ADP-ribosylation and E3 binding sites, Top homologs are verte-brates, not plants. Cilia-associated? 2. Possibly Flagella-associated, similar to NSG1 (nitro-gen-starved gametoge-nesis 1) 3. Calcium-de-pendent protein kinase

1: Physcomi-trella patens subsp Patens 2./3: C. reinhardtii

1. Homo-logous to Chla-my ID 188957 3: 123538

1/3:

IFT172

2:

EB1

Unnamed protein products

1. gi|159490770 2. gi|116056040 3. gi|10178250 4. gi|30688628 5. gi|18378889 6. gi|18378889 7. gi|168032367

1. centriole proteome protein Similar to Cep290 2. Protein with predicted involvement in meiosis (GSG1) (ISS) Top hits are vertebra-tes. TRAPP (transport protein particle), fusion of ER-to-Golgi transport vesicles 3. PHD Zinc finger family protein with BROMO domain 4. PHD Zinc finger family protein 5/6. Arma-dillo/beta-catenin repeat family protein 7. predicted protein

1: C. reinhardtii 2: Ostreo-coccus tauri 3/4/5/6: Arabidopsis thaliana 7: Physcomi-trella patens subsp patens

1: 169091 2: 146428 3: 187405 4: 187405 Homo-logous to Chlamy ID: 5: 140706 6/7: 140706

1/2/3/

4/5:

IFT172

6/7:

EB1

Arabidopsis thaliana (Higher Plant); C. reinhardtii (Green alga); Medicago truncatula (Higher Plant); Oryza sativa Japonica Group (Rice); Ostreococcus tauri (Smallest Free-living Eukaryote); Physcomitrella patens subsp. Patens (species of moss); Triticum aestivum (Common wheat); Vitis vinifera (Common Grape Vine).

Master thesis (Mette Lethan)

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