l'horloge circadienne, le cycle cellulaire et leurs ... · are involved that impose...
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L'horloge circadienne, le cycle cellulaire et leurs interactions
?
?
Madalena CHAVES
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What do all these living organisms have in common?
Cyanobacteria in Baltic sea
Neurospora crassa
Arabidopsis thaliana
Mus musculus
Drosophila melanogaster Synechococcus elongatus
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Cell cycle
● Cell cycle is the process by which cells growth and divide
celldivision
cellgrowth
DNAreplication
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Circadian rhythms● Anticipate environment cycles (day/night) and prepare living organism
for upcoming events (eg., expressing required genes)
● Generate an AUTONOMOUS molecular clock: cycle goes on independently of other cellular processes
● Temperature compensation: cycle always adjusts to 24h
MelatoninTo promote sleep
CortisolLipid degradation,
Glucose production(energy)
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2017 Nobel Prize
Drosophila Melanogaster→ mouche du vinaigre
● J. Hall and M. Rosbash (1984) first to identify gene “period”, responsible for circadian rhythm in drosophila
● Hypothesis: protein PER contributes to eventually repress its own gene, thus creating a negative feedback loop
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Negative feedback loop
PERprotein
PermRNA
OFF OFF
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Negative feedback loop
PERprotein
PermRNA
ON OFFOFF OFF
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Negative feedback loop
PERprotein
PermRNA
ON OFF
ON ON
OFF OFF
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Negative feedback loop
PERprotein
PermRNA
ON OFF
ON ON
OFF OFF
OFF ON
Generates an oscillatory
Cycle !
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Negative feedback loop
PERprotein
PermRNA
PER proteinaccumulates
during the night
Zehring,..., Hall and Rosbash, Cell 1984
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A basic negative feedback loopis not enough
● How to guarantee that oscillations are sustained ?
→ increase the number of steps:
PERper PER-ph
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A basic negative feedback loopis not enough
● How to guarantee that oscillations are sustained ?
→ increase the number of steps:
● How to generate a 24 hour cycle ?
PERper PER-ph
TIM
[P:T]
nucleus
Other proteins are involvedthat impose “waiting” times
“timeless” (TIM) and “doubletime”,discovered byM. Young 1994, 98
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Circadian clock in Drosophila
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Similar clock mechanisms across organisms
Drosophila melanogaster
Arabidopsis thaliana
Neurosporacrassa
Mammalian clock
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Autonomous clock: cyanobacteria
“In vitro” system: only proteins in a test tube; no genes, no transcription or translation involved !
Oscillations are due to an ordered cycle of phosphorylations and dephosphorylations of a protein, Kai C
Kondo lab, 2005-2007, Rust et al. 2007
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Kai C -T Kai C -ST
Kai A
Kai C Kai C -S
Modeling cyanobacteria clock
x A = k Ah-(xS ,θS)−γA x A
xT = kT h+ (xU ,θU )h+(x A ,θA)−γT xT
˙xTS = kTSh+(xT ,θT )h
+(x A ,θA )−γTS xTS
xS = kSh+(xTS ,θTS)h
-(x A ,θA2)−γS xS
xU=x total−xT−xTS−xS
Need to calibrate model that is find the values of parameters:
k A , kT , kTS , kS , γA ,γT , γTS ,γS , ...
Chaves & Preto, Chaos 2013
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Model calibration
Kai C -T Kai C -ST
Kai A
Kai C Kai C -S
Kai C -T Kai C -ST
Kai A
Kai C Kai C -S
Experiment 1Dephosphorylation
Experiment 2Phosphorylation
Data sets: Rust et al, Science 2007
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Kai C -T Kai C -ST
Kai A
Kai C Kai C -S
Model validation
Values of parameters were obtained from two distinct sets of experiments, each for a “partial” model.
Putting all parameters together yields the correct oscillatory solution!
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Kai C -T Kai C -ST
Kai A
Kai C Kai C -S
Data vs. model comparison
Values of parameters were obtained from two distinct sets of experiments, each for a “partial” model.
Putting all parameters together yields the correct oscillatory solution!
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Closed circles:Basin of attraction of x*
Open circles:Basin of attraction of periodic orbit
PREDICTION: there exist critical points in the periodic orbit, where the system my be perturbed and the circadian cycle arrested – such as when adding a large amount of Kai A.
(θS ,θA, 2)
Shaded:InvariantRegion (PWA)
Model analysis
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Model predictions: response to light
Chaves & Preto, Chaos 2013
Phase response to 5h pulses of ADP, applied at different instants during the circadian cycle.This experiment mimics clock entrainment by light – which varies the ratio ATP/(ATP+ADP).
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Cell cycle and circadian rhythmsHow do they interact?
??
Bmal1
● The general belief (cf. cyanobacteria): autonomous molecular clock
● Very few possible links are known from clock to cell cycle (Bmal1 → Wee1)
● Almost no knowledge on how cell cycle affects clock (at mitosis gene expression shuts down)
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Independent? Coordinated?Gating (cell division allowed only at certain clock phases)
Cell cycle and circadian rhythmsHow do they interact?
No gating in mammals
Observed in Cyanobacteria
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Mammalian clock and cell cycle experiments
Red: cell cycle, G1 phase cells are growing
Blue: cell cycle, G2/S phase cells are getting ready to divide
Green: clock, high phaseright after division
Feuillet et al, PNAS 2014 (Delaunay Lab) (Cells: mouse fibroblasts)
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Mammalian clock and cell cycle experiments
– cell cycle period decreases
– clock decreases by same amount
– 1:1 lock
Feuillet et al, PNAS 2014 (Delaunay Lab) (Cells: mouse fibroblasts)
Change the cell cycle period ?
Apply different concentrations of growth hormone
cell cycle clock
cell cycle clock
Evidence in favour of:
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Mammalian clock and cell cycle experiments Feuillet et al, PNAS 2014 (Delaunay Lab) (Cells: mouse fibroblasts)
– synchronized cells
– two subpopulations (20% fbs):
1:1 lock
3:2 (cc:clock)
cell cycle clock
cell cycle clockChange the clockperiod ?
Apply a drug thatsynchronizes cells clock,dexamethasone
Evidence in favour of:
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How to identify the coupling mechanism ?
MammalianClock +
Cell cycleToo complex!
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How to identify the coupling mechanism ?→ SYNTHETIC BIOLOGY APPROACH
MammalianClock +
Cell cycleToo complex!
Synthetic Oscillators,
Biological bricks
DNA Proteins
Activations,Inhibitions
collaboration F. Delaunay Lab
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MammalianClock +
Cell cycleToo complex!
Synthetic Oscillators,
Biological bricks
MathematicalModels
Too large!
Model reduction&
Interconnections
How to identify the coupling mechanism ?→ DYNAMICAL ANALYSIS & PREDICTIONS
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MPFCyclinB
APC/cdc20
Reduced cell cycle model
Growth factor (%FBS)
Per
iod
(h)
Model predictions(on growth factor)
Experimental data(fetal bovine serum) (F. Delaunay lab)
S. Almeida et al, IFAC 2017
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MA
R
B1
B2C2=ν2
M2
δ22+M 2
C1=ν1
B12
δ12+B1
2
Interconnecting cell cycle and clock
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• Each oscillator has a different “individual” period
• When coupled, the two oscillators synchronize 1:1 (or stop oscillating)
• Question 1: range of periods over which the two oscillators synchronize?
• Question 2: is an oscillator either a “controller” or a “follower” ?
ie., is the final period closer to the clock or cell cycle individual periods?
• Characterize dependence of these observations on the coupling strength
Interconnecting cell cycle and clock
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In summary
Cell cycle and circadian rhythms: Are they interconnected? And how? Many questions ! A current and lively topic
Synthetic biology approach: minimal circuit design with desired properties
Our tools: model reduction, mathematical analysis, and control
Merci !