Download Lecture 19 Cell Cycle 4.pptx

What we hope you learnt
Phosphorylation & degradation run the cell cycle engine
Today’s specials
Mitosis is an example of parallel paths ! self assembly
Positive & negative feedback make a limit cycle
G proteins (GTPases) run & regulate much of biology
Most cycles have more cyclins and Cdks
DNA and motors collaborate to assemble the spindle
The engine integrates a variety of inputs
There is general theory for complex biological processes
DNA must replicate once and only once
Discriminating between models for growth/division coordination
Replication is regulated by controlling origin firing
Microbial cells grown when they can (mostly!)
Animal cells need to be told to grow and divide
Cancer results from two sorts of mutations
Proto-oncogenes are activated to become oncogenes
Tumor suppressor genes are inactivated
Two routes to spindle assembly
A three step model for chromosome-driven spindle assembly
Chromosomes nucleate microtubules
by locally activating nucleation factors
Chromokinesins move micrortubules so
their + ends are nearest to the chromosomes; bipolarity visible
Dynein clusters the minus ends of microtubules to form two focused spindle poles
Centrosome-driven
Chromosome-driven
GTP hydrolysis regulates the activity of many proteins
GTP
GTP hydrolysis regulates the activity of many proteins
Guanine nucleotide
exchange factor (GEF)
GDP
GTP
GDP
Free
GTP
GDP
GTP
On microtubules
GDP
Tubulin
G Protein
P
P
GTPase activating
protein (GAP)
Importin !!!Ran-GDP
Importin !!!R
Chromosomes use Ran-GTP to nucleate microtubules
Invention recycling: Ran drives nuclear import
CYTOPLASM
Rcc1
(Ran GEF)
Ran-GDP
Ran-GTP
Spindle Assembly
Factor (SAF)
Ran-GDP + Importin ß
Importin ß: Spindle Assembly Factor
Cargo Protein + Importin ß
Importin ß:Ran-GTP
Ran GAP
Importin ß: Cargo Protein
Ran-GDP + Importin ß
+
Spindle Assembly Factor (SAF)
Microtubule nucleation
Spindle assembly
Ran-GTP
Cargo Protein
Importin ß:Ran-GTP
NUCLEUS
4 Slides from Dan Needleman
Physics undergrad
Physics PhD: polymer bundling
Postdoc: w Mitchison @ HMS
Behaviors of microtubules in spindles
Nucleation Polymerization and
Depolymerization
Interactions
and Forces
Polymerization
Following single microtubules in spindles
Prof in SEAS
tubulin
oligomers
Goal: understand spindle assembly
Phenomenology: follow all aspects of MT behavior
Result: a theory for spindle assembly
Depolymerization
stable
nucleus
Adapted From Kollman, Merdes, Mourey,
Adapted from Walczak, Cai, Khodjakov, Nature Reviews MCB, 11, 2010
Agard, Nature Review MCB, 12, 2011
What experiments reveal
Nucleation Polymerization and
Depolymerization
Nucleators,
activated by
chromosomes,
diffuse and bind
microtubules
Spatially
regulated
Semiquantitative
understanding
Not spatially
Regulated
General behaviors
well characterized
and explained by
simple theory
Interactions
And forces
Local interactions
Mutual alignment
and sliding
Contractile
Adapted from
Howard, Hyman
Nature 422, 2003
Nucleators localized by a positive feedback loop
Chromosomal
signaling
pathway (Ran)
Activates
Nucleators
Generates
Microtubules
Localizes
Without Microtubules
With Microtubules
Coarse-grained
theoretical
explanation
active
nucleator
inactive
nucleator
…or this sort of theory,…
Not this sort of theory,…
Anaphase promoting
complex (APC)
…but this sort of theory!
With Appropriate Symmetries, Conservation Laws, etc. . .
Nematic Order
Polar Order
Concentration
Momentum
No coordination !size variation
Try this rule:
cells split in middle ± 8% then double in size
Number of cells
Stresses
Divison 1
Division 3
Division 5
Division 7
Division 9
10000
Microtubule Polarity
Momentum
Division with 8% mean error in symmetry
12000
Microtubule Orientation
8000
6000
4000
2000
Microtubule Density
with Jan Brugues
0
0
10
20
Cell Size
30
40
50
Coordinating growth & division
The original model
Two phases of the cell cycle
Variable: birth to Start
Constant: Start to division
No Start until size threshold reached
Comparing coordination models
The constant volume addition model
The new model
Attempt to add constant volume between Starts
Variable: Start to Start
A titration scheme for start
R is an inhibitor: stable, inherited in a constant amount
Eg: DNA
Start cannot be induced until all inhibitor bound by activator
mRNA
Protein
For budding yeast and bacteria:
Data fits constant ∆volume model
For fission yeast
Data doesn’t fit constant ∆volume model
A
Start
+
R
A R
Threshold size: unstable activator
Threshold size: unstable activator
Activator amount proportional to volume
Separation of time scales: t0.5,A << tD
OK to ignore volume increase while considering [A]
Constant volume: periodically unstable activator
Constant volume: periodically unstable activator
Destroyed after each Start, stable otherwise
Activator amount proportional to volume increase
d[A]
= kS,A
dt
dAAMOUNT
= kS,AV
dt
d[A]
= kS,A − kdeg,A [A]SS = 0
dt
kdeg,A [A]SS = kS,A
[A]SS =
kS,A
kdeg,A
t
AAMOUNT = ∫ kS,AV dt
0
AAMOUNT = [A]SS V
Budding yeast: grow if you can
Ras is conserved, its targets are not
Budding Yeast:
Ras:GTP ! Adenyl cyclase (Cdc35)
Fission yeast:
Ras:GTP ! Byr2 (kinase, pheromone response)
Ras:GTP ! GEFCDC42 (actin cytoskeleton)
Mammals
Ras:GTP ! >6 different effectors
Is it always grow & divide if you can?
Cells face a choice as nutrient levels fall
Conservative:
Stop,hoard nutrients, prepare to starve
Go for it
Try one more division,hope nutrients reappear soon
Experimental evolution yields go for it mutants
Animal cells need growth & division instructions
Most animal cells neither grow nor divide
Typically, division requires growth
Protein signals stimulate growth & division
Reduced O2 ! more Epo ! more RBC
Clotting platelets release PDGF ! growth & divn
(Platelet-derived growth factor)
Growth control via increased ribosome production
x
Growth factors detected by receptor tyrosine kinases
Tor = key regulatory kinase
Cancer is a multistep process
Steps to a benign tumor
Abnormally controlled cell growth
Abnormally controlled cell division
Escape from programmed cell death (apoptosis)
Induce new blood vessel formation (angiogenesis)
Steps to malignant tumor (cancer)
Enter & exit blood vessels
Form tumors at new sites (metastasis)
Two sorts of cancer mutations
Proto-oncogene ! Oncogene
Ras mutations are prominent in cancer
Guanine nucleotide
exchange factor (GEF)
Mutations increase activity or reduce regulation
Genes normally promote cell growth & division
Example: Ras
Inactivation of tumor suppressor genes
Mutations destroy or reduce activity
Genes normally restrain cell growth & division
Examples: Retinoblastoma protein, p53
Losing a tumor suppressor gene causes retinoblastoma
GTP
Guanine nucleotide
exchange factor (GEF)
GDP
GTP
GTP
GTP
GDP
GDP
Ras
Ras
G12V
P
GTPase activating
protein (GAP)
Ras: proto-oncogene
GDP
Ras-G12V: oncogene
Rb inhibits S phase cyclin production