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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

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lectures 21 and 22 the regulation and mechanics of cell division
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
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

4C 2C

2C 4C

The eukaryotic cell cycle is partitioned into four “phases”

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…

4C

(DNA replicated,

diploid chr #)

2C

(unreplicated DNA,

diploid chr #)

ECB 18-2

can determine phase of cell cycle from dna content

Adapted from MBoC figures

17-5 and 17-6

Number of cells

1

2

DNA content (arbitrary units)

Can determine phase of cell cycle from DNA content

Where are cells in G1, S, G2 and M on plot?

Cells in G1

Which phase has most cells in it?

Lasts longest?

Cells in G2/M

Cells in S

ECB 18-2

transition from one phase to another is triggered
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
Regulating the eukaryotic cell cycle: studies in four model organisms

See HWK 618-619

  • 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
slide8

wee1

cdc25

Phenotype

Mutant

cdc13

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)

“wee”

wee1-(loss of function)

wee

G2

M

cdc2

cdc13-(loss of function)

cdc

cdc25-(loss of function)

cdc

Genetic pathway

2 frogs unfertilized eggs contain an m phase promoting factor

ECB figure 18-5

Transfer M-phase cytoplasm to interphase oocyte…

2. Frogs: unfertilized eggs contain an M-phase Promoting Factor

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

mpf activity cycles during the cell division cycle

M-phase

M-phase

Interphase

Interphase

MPF peaks in M-phase

MPF activity

Time

MPF activity cycles during the cell division cycle

Peak MPF induces M-phase

ECB 18-10

3 surf clams and sea urchins the abundance of cyclin proteins varies with the cell cycle

Cyclin A

Cyclin B

Ribonucleotide reductase (control)

M-phase

M-phase

Interphase

Interphase

MPF peaks in M-phase

MPF activity

Cyclin degraded

Cyclin synthesis

Time

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” abundance varies with cell cycle:
    • continuously synthesized…
    • degraded at end of M-phase
  • Cyclin B mRNA induces M-phase when injected into Xenopus oocytes

Peak MPF induces M-phase

ECB 18-6

three models of the eukaryotic cell cycle

wee1

cdc25

G2

M

cdc2

cdc13

Cdc2 gene product is a master regulator of the G2-M transition

MPF regulates entry into M-phase

Abundance of “cyclins” in clam eggs varies with the cell cycle

Three models of the eukaryotic 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

wee1

P

CLB

(cdc13)

CLB

(cdc13)

CLB

(cdc13)

CLB

(cdc13)

cdc2

cdc2

cdc2

cdc2

P

cdc25

P

cdc25

(inactive)

Cell cycle control: from models to molecules

Inhibitory

kinase

Remove inhibitory phosphate

Inactive

(weakly active)

Active MPF (CDK1)

P

P

CDK1

Inactive

Phosphorylate

M-phase substrates

Histones

Lamins

MAPs

etc

Activating

kinase

Positive feedback

ECB 18-11 and 18-12

  • “MPF” contains two components:
    • cdc2gene 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

mpf triggers its own inactivation anaphase promoting complex apc targets cyclin b for degradation

CLB

(cdc13)

CLB

(cdc13)

CLB

(cdc13)

cdc2

cdc2

cdc2

P

P

Accumulation of cyclin B

APC

Inactive

APC

Active

Polyubiquitin

MPF triggers its own inactivation“anaphase promoting complex (APC)”; targets cyclin B for degradation

Cyclin B accumulation activates MPF

MPF activates APC

APC inactivates MPF by degrading cyclin B

A cytoplasmic oscillator

  • Metaphase (mid-M)
    • High cyclin B
    • MPF (CDK1) active
  • Prophase (early-M)
    • Activation of CDK1 by cyclin and cdc25
  • Interphase
    • APC is turned off
  • Telophase (late-M)
    • Low cyclin B
    • MPF inactive
    • Cyclin B degraded by proteosome
  • Anaphase
review

M-phase

M-phase

Interphase

Interphase

MPF activity

MPF peaks in M-phase

Cyclin degraded

Cyclin synthesis

Time

Review:

ECB 18-6

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 cyclin degraded…

Active M-phase CDK (MPF)

M-phase cyclins (B)

M

G2

P

S-phase CDKs

M-phase CDK

(CDK1)

P

G1

S

S-phase cyclins degraded…

S-phase cyclins

Active S-phase CDKs

Trigger S-phase

At least 6 different CDKs and multiple cyclins in mammals

Done M-phase

Multiple CDKs regulate progression through the cell cycle

G1-CDKs; drive cells through G1 (won’t discuss)

S-phase cyclins and CDKs regulate DNA replication

Degradation of S-phase cyclins promotes exit from S-phase into G2

ECB 18-13

s cdk regulates dna replication
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

ECB 18-14

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
Completion of critical cellular processes is monitored at cell cycle “check points”

ECB 18-17

  • Is DNA undamaged?
  • Is DNA replicated?
  • Is cell big enough?
  • Yes? Enter M phase
  • Have all chromosomes
  • attached to spindle?
  • Yes? Proceed to anaphase
  • Is the cell big enough?
  • Is the environment favorable?
  • Is DNA undamaged?
  • Yes? Enter S phase

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

RNA pol

p53 (active)

p21 gene

Translation

Transcription

p21

P

P

The DNA damage checkpoint: p53 induced expression of an S-phase CDK inhibitor

p53

(inactive)

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)

DNA

ECB 18-15

P21 binds and

inactivates S-phase CDK

Active S-phase CDK

if checkpoint is activated
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
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”

Apoptosis: A tale of tadpole tails and mouse pawswhat do they have in common?

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

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 visibly distinct from necrosis

ECB 18-20

apoptosis is mediated by a caspase cascade

Death protein

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

Survival factor

Apoptosis is mediated by a “caspase cascade”

Caspase

(inactive)

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
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