1
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Systems Biology of the Cell Cycle
Assistant ProfessorUniversity of Amsterdam, Swammerdam Institute for Life Sciences
E-mail: [email protected]
ICYSB – 6th International Course in Yeast Systems BiologyGöteborg, Sweden – 4 June 2013
Matteo Barberis
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
A “Baby” of UNICELLSYS
(by Stefan Hohmann at the Final UNICELLSYS Meeting, Innsbruck, 4-6 March 2013)
2
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Thank to …
Stefan Hohmann Marija Cvijovic
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
(Oltvai Z.N. & Barabási A.L., Science, 2002, 298: 763-764)
Life’s complexity pyramid
UNIVERSITY OF AMSTERDAMSwammerdam Institute for Life Sciences
3
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Cellular Systems Biology
• … the study of the integrated and interacting network of genes, proteins and metabolites that are responsible for the normal and abnormal cellular functions as well as the emergent properties that create life
• … aims at mechanistic understanding of cellular functions through interactions of constituent molecules in space and time
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
(from the Multidisciplinary Centre for Integrative Biology (MyCIB), The University of Nottingham, United Kingdom)
Multidisciplinarity
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
4
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
(from the Multidisciplinary Centre for Integrative Biology (MyCIB), The University of Nottingham, United Kingdom)
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
The Cell Cycle
(Morgan D.O., The Cell Cycle: Principles of Control, 2007, pp. 210)
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
5
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
ICYSB, Göteborg Matteo Barberis
Unravel molecular mechanisms underlying
Cell Cycle Control in budding yeast
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
(Lodish H., Berk A. et al., Molecular Biology of the Cell, Sixth Edition, 2008, pp. 973)
6
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
4) Later in S phase, Clb3/4-Cdk1 activate replication origins finish S initiate the formation of mitotic spindle
5) As cells enter G2, Clb1/2-Cdk1 mitotic spindle formation trigger chromosome
segregation nuclear division
3) At the G1/S, Clb5/6-Cdk1 activate pre-replication complex
Cdk Activity during Cell Cycle Phases
(Lodish H., Berk A. et al., Molecular Cell Biology, Sixth Edition, 2008, pp. 973)
1) In G1 phase, Cln3-Cdk1 senses nutritional conditions drives cell growth
2) At the G1/S, Cln1/2-Cdk1 activate budding
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
DNA ReplicationDynamics
(computational analysis and testable predictions)
(Futcher B., Yeast, 1996, 12: 1635-1646;
Fitch I. et al., Mol. Biol. Cell, 1992, 3: 805-818)
Cdk/Cyclin and CkiRegulation
(integrating molecular biology, cell imaging and mathematical modeling)
SBI Seminar, Dublin Matteo Barberis 3.12.2012
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ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Oscillations of Cyclins
• The oscillatory behavior is widely known as “waves of cyclins”
(Morgan D.O., The Cell Cycle: Principles of Control, 2007, pp. 210)
(Lodish H., Berk A. et al., Molecular Cell Biology, Sixth Edition, 2008, pp. 973)
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
• Cdk1/Cyclins and Sic1 activities show alternate oscillations
Oscillations of Cdk1/Cyclins and Sic1
(Zachariae W. & Nasmyth K., Genes Dev., 1999, 13: 2039-2058)
Cdk1-Clb5,6
Cdk1-Clb3,4Cdk1-Clb1,2
Substrate/SubunitInhibitor of Cyclin-dependent kinase
8
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Mechanisms controlling S-Cdk Activation
(Morgan D.O., The Cell Cycle: Principles of Control, 2007, pp. 210)
Humboldt University BerlinInstitute for Biology
Max Planck Institutefor Molecular Genetics
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Failure of Proper Timing – Cancer
• Decrease in p27Kip1 levels, due to its degradation, occurs in ~ 50 % of carcinomas (aggressive, high-grade tumors and poor prognosis, increased apoptosis)
• p27Kip1 cytoplasmic localizationcorrelates to enhanced cancer aggressiveness
(Blain S.W. and Massagué J., Nat. Med. 2002, 8: 1076-1078)
9
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Sic1 and p27Kip1: Two Sides of the Same Coin
(Barberis M. et al., Biochem. J., 2005, 387: 639-647)(Lacy E.R. et al., Nat. Struct. Mol. Biol., 2004, 11: 358-364)
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
(Kaizu K. et al., Mol. Syst. Biol. 2010, 6: 415)
Interaction Map of S. cerevisiae Cell Cycle
10
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Overview of the Cell Cycle Control System
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Cln3Cdk1-Cln3Cdk1-Cln3-Far1
Cdk1Cdk1-Cln3-Far1-p
Degradation
SBF/MBF
SBF/MBF-Whi5-p
Clb5,6
Cdk1-Clb5,6Cdk1-Clb5,6-Sic1Cdk1-Clb5,6-Sic1-6p
Sic1
Sic1-6p
Degradation
Cdk1-Cln1,2
Cln1,2
Far1
Far1-p
CLB5,6mRNA
CLN1,2mRNA
Cdk1
Cln3
Far1
CLB5,6mRNA
CLN1,2mRNA
Cdk1-Cln1,2
Cdk1-Clb5,6-Sic1
Cdk1-Clb5,6
CYTOPLASMCYTOPLASM
NUCLEUSNUCLEUS
BUDDINGBUDDINGDNASYNTHESIS
DNASYNTHESIS
24
44
43
42
50
40
1
24
3
51
28
3841
47 32
10
14
11
15
19
20
16
13
12
18
26
SBF/MBF-Whi5
Whi5-p
Degradation
Whi5
5
7
6
9
46
37
845Whi5
172234
36
39
48
2125
49
27
29
33
53
30
35
31
Whi5-p52
23
(Barberis M. et al., PLoS Comput. Biol., 2007, 3: e64)
Kinetic Model
11
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Model Improvement
cytoplasm
nucleus
Compartmentalization
P0 PS
1
2
nucleus
cytoplasm
cyt
cyt
Vol
mcyt
ii
cyt
dt
dVolv
dt
dm
Cell size growth during the G1 phase
Volume Changes
r
jjij
i vndt
dm
1
nuc
cyt
Vol
Volcyttr
r
jjij
nuc mkvndt
dm
1
cyt
nucVolVol
nuctr
r
jjij
cytmkvn
dt
dm
1
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Model Description by Mass Action Kinetics
Systems equations
r – number of reactionsSi – metabolite concentrationsvj – reaction ratesnij – stoichiometric coefficients
Network properties Individual reaction properties
r
jjij
i vndt
dS
1
p,pSvv
S1
S2S4
S3
v1 v2
v3
v4
v5 ijnN
d[S1]/ dt = v[1] v[2] = p1 – p2S1
d[S2]/ dt = v[3] v[4] = p3S1S4 – p4S2
d[S3]/ dt = v[5] = p5S2
d[S4]/ dt = v[3] + v[4] = – d[S2]/ dt
p1 p2
p3
p5
p4
0 1 2 3 4 50
0.5
1
S[t]S1
S2
S3 S4
Time
12
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Modeling Pipeline
Time courses for: Cln3, Far1, Clb5, Sic1Localization of: Clb5 and Sic1
(Rossi, R.L. et al., Cell Cycle, 2005, 4: 1798-1807)
(Alberghina, L. et al., J Cell Biol, 2004, 167: 433-443)0
0,5
1
1,5
2
2,5
3
3,5
4
0 50 100 150 200 250 300 350
Time (minutes)
Pro
tein
lev
els
Sic1
Clb5
Clb5/Sic1
ETHANOL 2%
0
0,5
1
1,5
2
2,5
3
3,5
4
0 20 40 60 80 100 120 140
Time (minutes)
Pro
tein
lev
els
Sic1
Clb5
Clb5/Sic1
GLUCOSE 2%
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Modeling Pipeline
Kinetic constants (BIAcore analysis) for:binding of Sic1 to the Cdk-cyclin complex
(Barberis, M. et al., Biochem. J., 2005, 387: 639-647)
Cyclin A Cdk2
N-term
C-term
Sic1 (226-248)
13
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Monitoring of changes in mass concentration on the chip surface
How the Information is generated?
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Study properties of small modules, e.g. Cln3 activity, transcription factors regulation, Cln1-2 and Clb5-6 activities, Sic1 localization, …
Cln3 activity
0 20 40 60 80 100 120 1400
0.00002
0.00004
0.00006
Time (minutes)
Con
cent
ration
(M
)
Cdk1Cln3nuc
Cdk1Cln3-Far1nuc
Cln3 activity
Simulated Time Courses for Protein and Protein Complex Concentrations
14
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Study properties of small modules, e.g. Cln3 activity, transcription factors regulation, Cln1-2 and Clb5-6 activities, Sic1 localization, …
Transcription
factors regulation
0 20 40 60 80 100 120 1400
0.002
0.004
0.006
0.008
0.01
0.012
Time (minutes)
Con
cent
ration
(M
)
SBF/MBFnuc
SBF/MBF-Whi5nuc
Transcription factors
regulation
Simulated Time Courses for Protein and Protein Complex Concentrations
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Reproducing Experimental Data
Glucose Ethanol
Time (minutes)
Con
cent
ration
(M
)
0 20 40 60 80 100 120 1400
0.005
0.01
0.015
0.02
0
0.875
1.75
2.625
3.5
Sic1tot Clb5tot
0 50 100 150 200 250 300 3500
0.005
0.01
0.015
0.02
0
0.875
1.75
2.625
3.5
Time (minutes)
Con
cent
ration
(M
)
Sic1tot
Clb5tot
Experimental dynamics of Clb5
Experimental dynamics of Sic1
Simulated dynamics of Clb5
Simulated dynamics of Sic1
(Barberis M. et al., PLoS Comput. Biol., 2007, 3: e64)
15
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Model Validation
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Model Validation
16
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Model Validation
0 20 40 60 80 100 120 1400
0.01
0.02
0.03
0.04
0.05
0.06
Time (minutes)
Con
cent
ration
(M
)
OE-CLN3
cln3
WT
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
0 20 40 60 80 100 120 1400
0.01
0.02
0.03
0.04
0.05
0.06
0 20 40 60 80 100 120 1400
0.001
0.002
0.003
0.004
0.005
0 20 40 60 80 100 120 1400
0.01
0.02
0.03
0.04
0.05
0.06
0 20 40 60 80 100 120 1400
0.0010.0020.0030.0040.0050.0060.007
Time (minutes)
Con
cent
ration
(M
)
Time (minutes)
Con
cent
ration
(M
)
Cdk1-Cln1,2cyt Cdk1-Clb5,6nuc
Time (minutes)Time (minutes)
OE-CLN3
cln3
WT
OE-FAR1
far1WT WT
OE-CLN3
cln3
WT
far1
OE-FAR1
Cdk1-Clb5,6nuc
Con
cent
ration
(M
)
Con
cent
ration
(M
)
A B
C DCdk1-Cln1,2cyt
Model Validation by altering the Gene Dosage
17
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
SIMPLER ???
“ … modeling is a very concise
way of thinking about a system
and generating hypothesis … ”
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
(Barberis M.*, Beck C.* et al., Mol. BioSyst., 2011, 7: 2804-2812)
Stochastic Model
Sic1Clb5 nuc
SIC1 mRNA
Clb5 cyt
Model of Sic1 Expression and Degradation
18
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
(Barberis M.*, Beck C.* et al., Mol. BioSyst., 2011, 7: 2804-2812)
Mean Sic1
SD Sic1 CV Sic1
Q
Initial SIC1 mRNA
SIC1 mRNA transcri
ption
SIC1 mRNA degradatio
n
Transcriptional Noise at the G1/S Transition
Q =CV Sic1
CV SIC1 mRNACV Sic1 =
SD Sic1
mean Sic1
CV, Q = noise
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
(Barberis M.*, Beck C.* et al., Mol. BioSyst., 2011, 7: 2804-2812)
Initial SIC1 mRNA = 2 Initial SIC1 mRNA = 10
Mean Sic1
SD Sic1 CV Sic1
Q
Low SIC1 mRNA ensures Robust G1/S TimingInitial SIC1 mRNA = 6
Initial SIC1 mRNA 2 6 10
Max mean Sic1 320 molecules 350 molecules 380 molecules
Max noise Q 250 275 320
Max Timing noise Q 390 min 450 min 500 min
19
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
How many SIC1 mRNA Molecules?
(Barberis M. et al., Mol. BioSyst., 2011, 7: 2804-2812)
+
MS2 GFP (x3)
x12 loopsSIC1 ORF
3’ UTR
• Majority of cells show 0 or 1 SIC1 mRNAs
• Observed range between 0 and 10 mRNAs
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
• Cdk1/Cyclins and Sic1 activities show alternate oscillations
Oscillations of Cdk1/Cyclins and Sic1
(Zachariae W. & Nasmyth K., Genes Dev., 1999, 13: 2039-2058)
Cdk1-Clb5,6
Cdk1-Clb3,4Cdk1-Clb1,2
20
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Which Mechanisms control Cdk1/Clb Waves ?
Humboldt University BerlinInstitute for Biology
Max Planck Institutefor Molecular Genetics
(Morgan D.O., The Cell Cycle: Principles of Control, 2007, pp. 210)
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Cyclin-DependentKinase Inhibitor (CKI)
Sic1
InactiveComplexes
Cdk1 (Cdc28)
Cyclins
Cyclin-DependentKinase (CDK)
Clb5, Clb6Clb3, Clb4Clb1, Clb2
ActiveComplexes
ODE Model of Cdk1/Clb Activity
21
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Potential TranscriptionalRegulation of Clb Cyclins
CLB5,6
CLB3,4
CLB1,2
Clb6
Clb4Clb3
Clb5
Clb2Clb1
MBF
Fkh2
C
DB
TF
A
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Sic1
Cdk1-Clb5,6
Cdk1-Clb1,2
Cdk1-Clb3,4
Clb5,6
Clb1,2
Clb3,4
6
1
8
10
C
A
B
9
7
D
26
C1
C2
C3
Cdk1-Clb5,6-Sic1 Sic1
2
3Sic1
C1 C2 C3
5
4
Cdk1-Clb3,4-Sic1
15
16Sic1
C1 C2 C3
18
17
Cdk1-Clb1,2-Sic112
11
Sic1
C1 C2 C3
14
13
Ensemble Modeling of Sic1 Involvement
22
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Ensemble Modeling to study Sic1 Involvement
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
0 10 20 30 40 50 600
0.3
0.6
0.9
1.2
Time (minutes)
Clb3,4tot
Clb5,6tot
Clb1,2tot
Con
cent
ratio
n(a
.u.) • Global Sensitivity Analysis:
is the time delay independent on the parameter choice ?
The timing of Clb cyclinsappearance is Sic1-dependent
Sic1 potentially regulates the Appearance of Clb Cyclins
(Barberis M. et al., Biotechnol. Adv., 2012, 30: 108-130)
23
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
G1 S G2 M
Cell cycle progression
Cyc
lin
leve
ls
Clb5,6 Clb3,4 Clb1,2
t5,6 t3,4 t1,2
(t3,4- t5,6)
(t1,2- t5,6)
(t1,2- t3,4)
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
0 2 4 6 8
0
5
10
15
20
0 2 4 6 8
0
5
10
15
20
t1,2 t5,6 (min) [Version 3]
t 1,2
t 5
,6(m
in)
[V
ers
ion
1]
t 1,2
t 3
,4(m
in)
[V
ers
ion
1]
t1,2 t3,4 (min) [Version 3]
The Time Delay is Sic1-dependent
tClb1,2 tClb5,6 tClb1,2 tClb3,4
r = 0.497
r = 0.0026
• The timing of appearance of Clb cyclins is due to the binding of Sic1 to all Cdk1/Clb complexes
(Barberis M. et al., Biotechnol. Adv., 2012, 30: 108-130)
24
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Structural Modeling of Sic1-Cdk1/Clb InteractionSic1-KID
Clb5
Cdk1
• Interfaces have steric features that allowthe formation of a stable ternary complex
(Barberis M. et al., Biochem. J., 2005, 387: 639-647;
Barberis M. et al., Biochem. Biophys. Res. Commun., 2005, 336: 1040-1048)
Leu 224
Val 225
(Barberis M., Adv. Exp. Med. Biol., 2012, 736: 135-167;
Barberis M., FEBS J., 2012, 279: 3386-3410)
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Sic1 interacts with all Clb Cyclins
(Barberis M. et al., Biotechnol. Adv., 2012, 30: 108-130)
25
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
(courtesy of Andreas Herrmann, Humboldt University Berlin)
Förster Resonance Energy Transfer (FRET)
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Fluorescence Lifetime Imaging Microscopy (FLIM)
(courtesy of Andreas Herrmann, Humboldt University Berlin, Germany)
26
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
mYFPmCer• m, A206K mutation
preventing any potential dimerization of fluorescent proteins
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Transient Sic1-Clb Interactions in Living Cells
(Schreiber G.*, Barberis M.* et al., FASEB J., 2012, 26: 546-554)
27
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
error bar = SEM (Standard Error of the Measurement)
Efficiency of Energy Transfer E (%)
D
DAE
1
• τDA, average lifetime of the donor measured in presence of the acceptor
• τD, average lifetime of the donor expressed alone
(Schreiber G.*, Barberis M.* et al., FASEB J., 2012, 26: 546-554)
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
0 10 20 30 40 50 600
0.3
0.6
0.9
1.2
Time (minutes)
Clb3,4tot
Clb5,6tot
Con
cent
ratio
n(a
.u.)
Does Sic1 regulate Timing and Oscillatory Behavior of Clb Cyclins?
Clb1,2tot
SIC1
0 10 20 30 40 50 600
0.1
0.2
0.3
Time (minutes)
Clb1,2tot Clb3,4tot
Clb5,6totCon
cent
ratio
n(a
.u.)
sic1
(Barberis M. et al., Biotechnol. Adv., 2012, 30: 108-130)
28
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
• Wild type strain shows Clb periodicity, which is delayed but still present in the SIC1-0P strain
Clb2
Clb3 Clb5 Sic1
0 30 60 90 100 110 120 130 140 150 160 170 180
SIC1
Time (min)
180
160
140
120
100
90
60
30
0
58/46
46/53
14/81
34/67
65/29
74/9
97/4
99/3
100/0
1C/Bud
SIC1
Sic1 may be Responsible for Clb Timing during Cell Cycle Progression
Clb2
Clb3
Clb5
sic1Δ
1C/Bud
42/74
49/73
50/58
58/47
63/38
67/25
70/14
76/14
80/13
sic1Δ• In the sic1Δ strain, timing of Clb appearance is lost, although cells proceed into S phase
G1 S G2 M
(Barberis M. et al., Biotechnol. Adv., 2012, 30: 108-130)
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Hcm1 is a Transcriptional Activator of S Phase
(Pramila T. et al., Genes Dev., 2006, 20: 2266-2278)
29
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
The Sir2 Histone Deacetylase
• Sir2 is a NAD-dependent histone deacetylaseinvolved in silencing telomeres and mating locus, maintaining genome integrity and blocking inappropriate gene expression
• sir2Δ shortens life span, whereas SIR2 extra copy extend lifespan
(Guarente L., Genes Dev., 2000, 14: 1021-1026)
Calorie excess Calorie restriction
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Sir2 interacts and influences Hcm1 Localization
(Rodriguez-Colman M.J. et al., J. Biol. Chem., 2010, 285: 37092-37101)
Hcm1-HA
Is Sir2 involved in the regulation of Fkh1/2 for the timing of mitotic events?
30
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
(Rodriguez-Colman M.J. et al., J. Biol. Chem., 2010, 285: 37092-37101)
Sir2 interacts with Fkh1 and Fkh2
(Linke C., …, Barberis M.* and
Krobitsch S.*, under revision)
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Sir2 interacts with Fkh Transcription Factors
SBI Seminar, Dublin Matteo Barberis 3.12.2012
31
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
(Linke C ., …, Barberis M.* and Krobitsch S.*, under revision)
Sir2 inhibits Cell Growth in a Different Extent
SDII SDIVpBTM + pACT
pBTM-Fkh1 + pACT
pBTM-Fkh1 + pACT-Sir2
pBTM-Fkh2 + pACT
pBTM + pACT-Sir2
pBTM-Fkh2 + pACT-Sir2
pBTM-Hcm1 + pACT
pBTM-Hcm1 + pACT-Sir2
Sir2 inhibition: Fkh1 > Fkh2 > Hcm1
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Sir2 binds to CLB2 Promoter
Sir2 binds to CLB2 promoter in G1 and M phases
0
1
2
3
4
5
6
ACT1 CLB2
Exponential phase
G1 phase
S phase
G2/M phase
Fold
chan
ge
(Linke C ., …, Barberis M.* and Krobitsch S.*, under revision)
32
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Sir2-VC+ VN-Fkh1Ndd1-VC+ VN-Fkh2
(Linke C ., …, Barberis M.* and Krobitsch S.*, under revision)
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Transmitted light
Venus
FACS
Transmitted light
Venus
FACS
50100 4020 30
60 70 80 90 100 110
Sir2_VC +VN_Fkh1
33
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Sir2 binds to CLB2 Promoter via Fkh1/Fkh2
Fold
chan
ge
0
1
2
3
4
5
ACT1 CLB2
Exponential phase
Stationary phase
2 mM H2O2
0
1
2
3
4
5
ACT1 CLB2
Fold
chan
ge
wild type
fkh1
fkh2
(Linke C., …, Barberis M.* and
Krobitsch S.*, under revision)
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Sir2 Nuclear Localization requires Fkh1/Fkh2
(Linke C ., …, Barberis M.* and Krobitsch S.*, under revision)
34
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Activation and Repression in Clb Regulation
Clb2 Sir2Ndd1
Fkh1
Fkh2Physical Interaction
Transcriptional activation
Transcriptional repression
Sir2 Hcm1
Fkh1
Fkh2
(Linke C ., …, Barberis M.* and Krobitsch S.*, under revision)
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Conclusion
Timing of Cdk1/Clb activation is achieved by coupling multiple mechanisms:
(i) Sic1-dependent Clb inhibition
(ii) Sir2-dependent inhibition via Fkh1/Fkh2
35
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
StructuralBiochemical
Computational
Microscopy
PPIs Modeling
Modeling
DNAPIs
SYSTEMS
BIOLOGY
Assays
(Barberis M., FEBS J., 2012, 279: 3386-3410)
SBI Seminar, Dublin Matteo Barberis 3.12.2012
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
36
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
From Ph.D. Student to Assistant Professor
Prof. Edda Klipp Prof. Hans WesterhoffProf. Lilia Alberghina
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Claudia Beck (Bachelor)
Aouefa Amoussouvi (Ph.D.)Christian Linke (Ph.D., Post-doc)
Alberto González-Novo (Post-doc)
Gabriele Schreiber (Post-doc)
Christine Klaus (Technician)Silvia Scolari (Ph.D.)
Adriana Supady (Master)
Miquel Á. Adrover (Ph.D.)
Thomas Spiesser (Master, Ph.D.)Christian Diener (Ph.D.)
37
ICYSB, Göteborg Matteo Barberis
Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM
Thank You!
Questions welcome.
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