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
Cells only come from pre-existing cells
cytokinesis
mitosis
Growth and division must be carefully regulated
Unregulated cell growth = cancer
The eukaryotic cell cycle is partitioned into four “phases”
Division occurs in “M-phase:”
“mitosis” and “cytokinesis” (<1
hr)
4C
4C
2C
(DNA replicated,
diploid chr #)
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)…
2C
4C
ECB 18-2
2C
(unreplicated DNA,
diploid chr #)
G1+S+G2=“Interphase”
Division = “M-phase”
A “typical” cell cycle for animal
cells is 24-48 hrs long, but
varies…
Cell cycle times vary
Can determine phase of cell cycle from DNA content
Number of cells
Cells in G1
Adapted from MBoC figures
17-5 and 17-6
Where are cells in G1, S, G2 and
M on plot?
Which phase has most cells in it?
Lasts longest?
Cells in
G2/M
Cells in
S
1
2
DNA content (arbitrary units)
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)
See HWK 618-619
– 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
1. Fission yeast “cell division cycle (cdc)” mutants define a
master regulator (tigger) of the G2/M transition
“Wild-type” fission yeast
WT
cdc2- (loss of function)
“cdc”
WEE2 = cdc2D
(gain of function)
Mutant
“wee”
Phenotype
wee1- (loss of function)
wee
cdc13-
(loss of function)
cdc
cdc25- (loss of function)
cdc
wee1 cdc25
G2
cdc2
cdc13
Genetic pathway
M
2. Frogs: unfertilized eggs contain an M-phase Promoting Factor
Transfer M-phase cytoplasm
to interphase oocyte…
ECB 18-9
Control expt;
Transfer interphase cytoplasm to
interphase cell - no effect
Nucleus
Egg in
“M-phase”
Oocyte in
“interphase”
Oocyte “matures”
(enters M-phase)…
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
ECB figure 18-5
MPF activity cycles during the cell division cycle
M-phase
Interphase
MPF activity
MPF peaks in
M-phase
Time
ECB 18-10
M-phase
Interphase
Peak MPF induces
M-phase
3. Surf clams and sea urchins: the abundance of
“cyclin” proteins varies with the cell cycle
Continuously label fertilized eggs with 35S-methionine
Analyze incorporation into proteins by SDS-PAGE
Cyclin A
Cyclin B
Ribonucleotide reductase
(control)
M-phase
“Cyclin” abundance varies
with cell cycle:
MPF peaks in
M-phase
Cyclin B mRNA induces
M-phase when injected
into Xenopus oocytes
MPF activity
continuously synthesized…
degraded at end of Mphase
Interphase
Cyclin synthesis
Time
ECB 18-6
M-phase
Interphase
Peak MPF induces
M-phase
Cyclin degraded
Three models of the eukaryotic cell cycle
wee1 cdc25
G2
cdc2
M
cdc13
Cdc2 gene product is a master
regulator of the G2-M transition
MPF regulates entry into M-phase
Bringing it all together
Cyclin B mRNA (clam) induces Mphase in frog oocytes
cdc13 encodes a yeast cyclin
MPF consists of frog cdc2 homolog
and cyclin B
Abundance of “cyclins” in clam
eggs varies with the cell cycle
Cell cycle control: from models to molecules
Inhibitory
kinase
CLB
(cdc13)
wee1
CLB
cdc2
Inactive
(weakly active)
Active MPF
(CDK1)
P
CLB
CLB
(cdc13)
(cdc13)
cdc2 P
cdc2 P
cdc2
CDK1
Inactive
P
(cdc13)
Remove
inhibitory
phosphate
P
cdc25
Activating
kinase
Positive
feedback
ECB 18-11 and 18-12
“MPF” contains two components:
cdc25
(inactive)
cdc2 gene product = catalytic subunit of protein kinase
cyclin B (CLB = cdc13): regulatory subunit activates kinase
MPF = “Cyclin-dependent kinase (CDK1)”
MPF (CDK1) activity is also regulated by phosphorylation
wee 1 is inhibitory kinase
cdc25 is activating phosphatase
“Switching on” CDK1 (MPF) drives cell into M-phase
Phosphorylate
M-phase
substrates
Histones
Lamins
MAPs
etc
MPF triggers its own inactivation
“anaphase promoting complex (APC)”; targets cyclin B for degradation
Cyclin B accumulation
activates MPF
Metaphase (mid-M)
High cyclin B
MPF (CDK1) active
Prophase (early-M)
Activation of CDK1 by
cyclin and cdc25
MPF activates APC
CLB
APC inactivates MPF by
degrading cyclin B
(cdc13)
cdc2 P
A cytoplasmic oscillator
Accumulation
of cyclin B
APC
APC
Inactive
CLB
Active
(cdc13)
CLB
cdc2 P
(cdc13)
Polyubiquitin
Interphase
APC is turned off
cdc2
Telophase (late-M)
Low cyclin B
MPF inactive
Cyclin B degraded by
proteosome
Anaphase
Review:
M-phase
Interphase
M-phase
Interphase
ECB 18-6
MPF activity
MPF peaks in
M-phase
Cyclin synthesis
Cyclin degraded
Time
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
Multiple CDKs regulate progression through the cell cycle
Trigger M-phase
M-phase
cyclins (B)
M-phase cyclin
degraded…
Active M-phase CDK (MPF)
Done M-phase
P
M
G1-CDKs; drive cells
through G1 (won’t discuss)
G2
M-phase
CDK
(CDK1)
S-phase
cyclins
degraded…
G1
S-phase
CDKs
S-phase cyclins and
CDKs regulate DNA
replication
S-phase cyclins
S
P
Active S-phase CDKs
ECB 18-13
At least 6 different
CDKs and multiple
cyclins in mammals
Trigger S-phase
Degradation of Sphase 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
ECB 18-14
Origin is ready to fire
Active S-Cdk
1- phosphorylates ORC causing origin to fire = replication
2-phosphorylates Cdc6 leading to ubiquitination and degradation
Cdc6 not made until next G1 - prevents origin from double firing
Completion of critical cellular processes is monitored
at cell cycle “check points”
ECB 18-17
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
The DNA damage checkpoint:
p53 induced expression of an S-phase CDK inhibitor
p53
(inactive)
DNA damage activates p53
p53 (active)
Active p53 acts as a
transcription factor to turn
on genes, including p21
RNA pol
DNA
p21 gene
Transcription
p21 protein inhibits G1/S
phase CDKs, blocking entry
into S-phase
Translation
p21
ECB 18-15
Cell arrests in G1 until
damage repaired, or
undergoes apoptosis
(programmed cell death)
P
Active S-phase CDK
P
P21 binds and
inactivates S-phase CDK
If checkpoint is activated
Exit cell cycle
(temporary or permanent)
neurons
most plant cells
Or undergo apoptosis (in a minute)
Zones of division and growth in plant roots
Arabidopsis thaliana
Only a fraction of cells still actively dividing
Zone of differentiation - cells cease
growing and terminally differentiate
Zone of cell elongation - growth
but not division; Cells in G0
Meristem - zone of active cell
division cells remain in cell cycle
Regulation of each zone is not well understood in plants but involves hormones
In animals:
mitogens stimulate cell proliferation (block checkpoints)
growth factors stimulate cell growth (stimulate biosynthesis, inhibit
degradation)
Apoptosis: A tale of tadpole tails and mouse paws
what do they have in common?
ECB figure 18-19
Tadpole tails are
resorbed during
metamorphosis
ECB figure 18-18
Paws develop from “paddles”
Both processes involve “programmed cell death (apoptosis)”
ECB - “programmed cell death is a commonplace, normal, and benign event. It is
the inappropriate proliferation and survival of cells that presents real dangers”
Apoptosis is visibly distinct from necrosis
ECB 18-20
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 condense
Corpses of apoptotic cells are often engulfed by their neighbors or specialized
phagocytic cells
Apoptosis is mediated by a “caspase cascade”
Death
protein
Survival
factor
Caspase
(inactive)
Inactive
Active
“Caspases” are proteases; inactive precursors
activated by proteolysis
Presence of suicide signals and/or withdrawal of
needed survival factor activates first caspase in
cascade
Initial caspase proteolytically activates
downstream caspases
…which activate additional caspases, and so on
Activated caspases degrade nuclear
and cytoplasmic proteins (lamins,
cytoskeletal proteins, etc)…
Activated endonucleases cut
chromosomal DNA…
ECB 18-21
Caspase cascade must be carefully regulated
Bcl-2 family of proteins are death proteins
Form pores in outer mitochondrial membrane
releasing cytochrome c (respiratory chain)
Cytochrome c binds adaptor and complex activates first procaspase