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