Post on 28-Dec-2015
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Lectures 21 and 22: The regulation and mechanics of cell division
• Today - cell cycle (regulation of cell division)
– Cell proliferation
– The eukaryotic cell cycle
– Measuring the cell cycle
– Models of the cell cycle: from fungi to frogs
– The cell cycle is regulated by cyclin-dependent kinases
• Next time - mechanisms of cell division
A cell cycle is one round of growth and division
mitosis
cytokinesis
Growth and division must be carefully regulatedUnregulated cell growth = cancer
Cells only come from pre-existing cells
Division occurs in “M-phase:” “mitosis” and “cytokinesis” (<1 hr)
Most cell growth occurs during “G1” (6-20+ hrs; duplicate organelles, double in size)
DNA replication occurs during “S-phase” (4-10+ hrs)…
“G2” prepares cells for division (1-6+ hrs)…
G1+S+G2=“Interphase”
Division = “M-phase”
A “typical” cell cycle for animal cells is 24-48 hrs long, but varies…
The eukaryotic cell cycle is partitioned into four “phases”
ECB 18-2
2C(unreplicated DNA,diploid chr #)
4C(DNA replicated,diploid chr #)
4C 2C
2C 4C
Cell cycle times vary
Adapted from MBoC figures17-5 and 17-6
DNA content (arbitrary units)1 2
Num
ber
of
cells
Cells in G1
Cells in G2/M
Cells in S
Can determine phase of cell cycle from DNA content
Where are cells in G1, S, G2 and M on plot?
Which phase has most cells in it?Lasts longest?
ECB 18-2
Transition from one phase to another is triggered
We will take a historical perspective to ‘triggers’
Regulating the eukaryotic cell cycle: studies in four model organisms
• Marine invertebrates:– Surf clam (Spisula)
– Sea urchins and starfish
• Frog eggs and embryos:– Rana pipiens (Northern leopard frog)
– Xenopus laevis (African clawed frog)
• Cultured cells– HeLa (Human cervical carcinoma)
• Yeast cell division cycle (“cdc”) mutants:– Saccharomyces cerevisiae “budding” yeast
– Schizosaccharamyces pombe “fission” yeast
See HWK 618-619
1. Fission yeast “cell division cycle (cdc)” mutants define a master regulator (tigger) of the G2/M transition
cdc2- (loss of function)
WEE2 = cdc2D
(gain of function)
“cdc”
“wee”
cdc13- (loss of function) cdc
cdc25- (loss of function) cdc
wee1- (loss of function) wee
“Wild-type” fission yeast WT
Mutant Phenotype
cdc2
cdc25
cdc13
wee1
G2 M
Genetic pathway
Nucleus
Egg in“M-phase”
Oocyte in“interphase”
Transfer M-phase cytoplasm to interphase
oocyte…
Oocyte “matures” (enters M-phase)…
ECB figure 18-5
2. Frogs: unfertilized eggs contain an M-phase Promoting Factor
Transfer of cytoplasm from egg to oocyte induces M-phase: “M-phase promoting factor (MPF)”
Not restricted to egg cytoplasm - Any M-phase cytoplasm will induce M-phase
Control expt;Transfer interphase cytoplasm to interphase cell - no effect
ECB 18-9
MPF activity cycles during the cell division cycle
Time
MP
F a
ctiv
ity
MPF peaks in M-phase
Interphase M-phaseM-phase Interphase
Peak MPF induces M-phase
ECB 18-10
Time
MP
F a
ctiv
ity
MPF peaks in M-phase
3. Surf clams and sea urchins: the abundance of “cyclin” proteins varies with the cell cycle
“Cyclin” abundance varies with cell cycle:
continuously synthesized…
degraded at end of M-phase
Cyclin B mRNA induces M-phase when injected into Xenopus oocytes
Continuously label fertilized eggs with 35S-methionine
Analyze incorporation into proteins by SDS-PAGE
ECB 18-6
Ribonucleotide reductase (control)
Cyclin A
Cyclin B
Interphase M-phaseM-phase Interphase
Peak MPF induces M-phase
Cyclin synthesis Cyclin degraded
Cdc2 gene product is a master regulator of the G2-M transition
cdc2
cdc25
cdc13
wee1
G2 M
Three models of the eukaryotic cell cycle
MPF regulates entry into M-phase
Abundance of “cyclins” in clam eggs varies with the cell cycle
Bringing it all together
Cyclin B mRNA (clam) induces M-phase in frog oocytes
cdc13 encodes a yeast cyclin
MPF consists of frog cdc2 homolog and cyclin B
Cell cycle control: from models to molecules
Inactive(weakly active)
Active MPF (CDK1)
“MPF” contains two components:cdc2 gene product = catalytic subunit of protein kinase
cyclin B (CLB = cdc13): regulatory subunit activates kinase
MPF = “Cyclin-dependent kinase (CDK1)”
Remove inhibitory phosphate
ECB 18-11 and 18-12
PhosphorylateM-phase substrates Histones Lamins MAPs etc
cdc2
CLB(cdc13)
cdc2
CLB(cdc13)P
P cdc2
CLB(cdc13)
Pcdc2
CLB(cdc13)
P cdc25
cdc25(inactive)
wee1P
Positive feedback
CDK1Inactive
Inhibitory kinase
Activating kinase
MPF (CDK1) activity is also regulated by phosphorylationwee 1 is inhibitory kinase
cdc25 is activating phosphatase
“Switching on” CDK1 (MPF) drives cell into M-phase
MPF triggers its own inactivation “anaphase promoting complex (APC)”; targets cyclin B for
degradation
Polyubiquitin
InterphaseAPC is turned off
cdc2
CLB(cdc13)
P
cdc2
cdc2
CLB(cdc13)
P
APCInactive
APCActive
Cyclin B degraded by proteosome
Anaphase
Accumulation of cyclin B
CLB(cdc13)
Metaphase (mid-M)High cyclin BMPF (CDK1) active
Telophase (late-M)Low cyclin BMPF inactive
Prophase (early-M)Activation of CDK1 by cyclin and cdc25
Cyclin B accumulation activates MPF
MPF activates APC
APC inactivates MPF by degrading cyclin B
A cytoplasmic oscillator
Review:
Time
MP
F a
ctiv
ity
MPF peaks in M-phase
Interphase M-phaseM-phase Interphase
Cyclin synthesis Cyclin degraded
Accumulation of cyclin B above threshold activates MPF (CDK1) and promotes entry into M-phase
Activation of APC by MPF promotes cyclin destruction, MPF inactivation, and exit from M-phase
ECB 18-6
Multiple CDKs regulate progression through the cell cycle
M
G2
S
G1
ECB 18-13
S-phase cyclins
At least 6 different CDKs and multiple cyclins in mammals
Done M-phase
S-phase CDKs
P
Active S-phase CDKs
Trigger M-phase
S-phase cyclins degraded…
P
Active M-phase CDK (MPF)M-phase cyclin degraded…
Trigger S-phase
M-phase CDK
(CDK1)
M-phase cyclins (B)
S-phase cyclins and CDKs regulate DNA replication
G1-CDKs; drive cells through G1 (won’t discuss)
Degradation of S-phase cyclins promotes exit from S-phase into G2
S-Cdk regulates DNA replication
Origin recognition complex - protein scaffolding for assembly of other proteins
Cdc6 increases in G1; binds ORC and induces binding of other proteins forming pre-replicative complex
Origin is ready to fire
Active S-Cdk 1- phosphorylates ORC causing origin to fire = replication2-phosphorylates Cdc6 leading to ubiquitination and degradation
Cdc6 not made until next G1 - prevents origin from double firing
ECB 18-14
Completion of critical cellular processes is monitored at cell cycle “check points”
Is the cell big enough?Is the environment favorable?Is DNA undamaged?Yes? Enter S phase
Is DNA undamaged?Is DNA replicated?Is cell big enough?Yes? Enter M phase
Have all chromosomes attached to spindle?Yes? Proceed to anaphase
Of these, the G1/S checkpoint for damaged DNA is best understood
ECB 18-17
RNA pol
The DNA damage checkpoint: p53 induced expression of an S-phase CDK
inhibitor
DNA damage activates p53
Active p53 acts as a transcription factor to turn on genes, including p21
p21 protein inhibits G1/S phase CDKs, blocking entry into S-phase
Cell arrests in G1 until damage repaired, or undergoes apoptosis (programmed cell death)
ECB 18-15
PP
p53(inactive)
P21 binds andinactivates S-phase CDKActive S-phase CDK
p53 (active)
Translation
Transcription
p21
DNA
p21 gene
If checkpoint is activated
Or undergo apoptosis (in a minute)
neuronsmost plant cells
Exit cell cycle (temporary or permanent)
Zones of division and growth in plant roots
Meristem - zone of active cell division cells remain in cell cycle
Zone of cell elongation - growth but not division; Cells in G0
Zone of differentiation - cells cease growing and terminally differentiate
Regulation of each zone is not well understood in plants but involves hormonesIn animals:
mitogens stimulate cell proliferation (block checkpoints)growth factors stimulate cell growth (stimulate biosynthesis, inhibit degradation)
Arabidopsis thaliana
Only a fraction of cells still actively dividing
Apoptosis: A tale of tadpole tails and mouse pawswhat do they have in common?
Both processes involve “programmed cell death (apoptosis)”
Tadpole tails are resorbed during metamorphosis
ECB figure 18-19
Paws develop from “paddles”
ECB figure 18-18
ECB - “programmed cell death is a commonplace, normal, and benign event. It is the inappropriate proliferation and survival of cells that presents real dangers”
Necrosis (cell death following injury) often results in lysis, spilling the contents into the surrounding space and causing inflamation
During apoptosis (“programmed cell death”), cells remain intact and condenseCorpses of apoptotic cells are often engulfed by their neighbors or specialized phagocytic cells
Apoptosis is visibly distinct from necrosis
ECB 18-20
Apoptosis is mediated by a “caspase cascade”“Caspases” are proteases; inactive precursors activated by proteolysis
Presence of suicide signals and/or withdrawal of needed survival factor activates first caspase in cascade
Death protein
Survival factor
Inactive
Activated caspases degrade nuclear and cytoplasmic proteins (lamins, cytoskeletal proteins, etc)…
Activated endonucleases cut chromosomal DNA…
Active
Caspase(inactive)
ECB 18-21
Initial caspase proteolytically activates downstream caspases
…which activate additional caspases, and so on
Caspase cascade must be carefully regulated
Bcl-2 family of proteins are death proteinsForm pores in outer mitochondrial membrane releasing cytochrome c (respiratory chain)
Cytochrome c binds adaptor and complex activates first procaspase