Lecture 8

Getting into and out of mitosis

Outline:Uncovering the cyclin/CDK paradigmG2/MExiting M-phase

Paper: Anaphase regulation in budding yeast

2001who won the Nobel prize??

M-phase is induced by a soluble factor

70’s cell fusion experiments

Factor in mitotic cell induces premature mitosisin interphase cell

premature chromosome condensation is a disaster!

the cell cycle must be highly coordinated.

S

M

What is M-phase inducer?

3 lines of experimental approach:yeastXenopus eggs

invertebrate eggs

YEAST

rapidly growing, divides every 90 min

powerful genetics

cell cycle progress easy to follow

Lee Hartwell S. cerevisiae

Paul Nurse S. pombe

Advantages:

Disadvantages:

nuclear envelope does not break down in M-phase

spindle assembly occurs during DNA replication(budding yeast)

little or no chromosome condensation in M-phase

Different from higher eukaryotes

Note on yeast nomenclature

S. cerevisiae (budding yeast)

wild type or dominant CDC28

mutations cdc28-4ts, ∆cdc28

protein Cdc28

S. pombe (fission yeast)

wild type or dominant cdc2+ cdc2D

mutations cdc2ts cdc2-

protein Cdc2

S. pombe = fission yeast

Approach Identify temperature sensitive mutants that block the cell cycle at a specific stage (size)

Fission yeast must reach a critical size to enter mitosis

most arrest at random cell cycle states

cdc mutant “cell division cycle”

asynchronous population

cdc2(ts)loss of function

Cdc2 is the master regulator of cell division

essential, no other mutation can rescue loss of cdc2

cdc2D

gain of function

“wee”

WTalwaysdivide at thesame size

Mitosis-inducing activity of Cdc2 is inhibited by Wee1 andstimulated by Cdc25

cdc2ts

WT

cdc25ts

wee1ts

cdc25ts, wee1ts

cdc2D, wee1ts mitotic catastrophe

Cdc2 - protein kinase

Cdc25 - protein phosphatase

Wee1 - protein kinase

3X wee1+

WT

5X cdc25+

5X cdc2+

can confirminteractions byoverexpression:

Conclusions:

Cdc2 is the master regulator of cell division in fission yeast

Cdc25 is a positive regulator of Cdc2Wee1 is a negative regulator of Cdc2

These genes are highly conserved in all eukaryotes

S. cerevisiae cdc2 homologue = CDC28

human gene also complements~ 65% identical

Invertebrate and Xenopus eggssea urchin, starfish, clam

Advantages:

stripped down cell cycle:

alternating S- and M-phases no G1 or G2 phases

M

Ssynchronous divisions

good for biochemistry

easy to inject

somatic cells

embryonic cells

Eggs divide without growing

SG1 G2 M

3 6 90 12 15 18 21 24

Hours

typical somatic cell cycle

12 embryonic cell cycles

Observation: protein synthesis is required for early embryonic divisions

fertilize eggs synchronous rounds of division

incubate with 35S - methionine

take sample every 10 min

SDS-PAGE and autoradiography

Identification of “cyclins” sea urchin eggs

newly synthesized proteins get labeled

Tim Hunt, Joan Ruderman

mitosis mitosis interphaseinterphase

cyclin A

cyclin B

cyclin Babundance

cell cycle state

I M I M

other proteins

Conclusion

Cyclin synthesis and destruction correlates with progression through the cell cycle

cyclins are potential regulators

Xenopus laevis

1 mm diameter!

diploid haploid

oocyte maturation meiosis fertilization cleavage

diploid

Discovery of MPF

= MaturationPromotingFactor

Masui and Markert1971

MPF = M-phase Promoting Factor

a more general role.....

also present in mitotic somatic cells

KEY EXPERIMENT - Masui, Lohka and Maller

biochemical purification of MPF1 liter of frog eggs 1 g

using oocyte maturation as an assay? no.

monitored nuclear envelope breakdown ofpronuclei in egg extract

MPF has two subunits:p34p46

complex has kinase activity toward histone H1

VERY FAMOUS WESTERN BLOT: 1988

p34 = cdc2

antibody to conserved region of cdc2: “PSTAIRE”

p46 = cyclin B

M-phase promoting factor = MPF =cdk1/cyclinB

1) cdk1 cyclin dependent kinase 1= yeast cdc2• Induces mitosis by phosphorylating specific downstream targets on serine and threonine

protein kinase complex, 2 subunits:

2) cyclin B• regulatory subunit that activates cdk1• abundance oscillates during the cell cycle

cell cycle paradigm: transitions are regulated by cdk/cyclin complexes

In yeast:single cdk,many cyclins

• Cyclin B protein is synthesized continuously• Threshold cyclin B level induces MPF kinase activity • Cyclin B disappears suddenly during anaphase

Experiments to prove that cyclin synthesis drives theembryonic cell cycle

in vitro system

Marc KirschnerAndrew Murray

electric shocksimulates fertilization

cell cycle reconstitution in vitro

high MPFhigh histone H1kinase activity

low MPFlow histone H1kinase activity

cyclin B synthesis can drivethe cell cycle

is cyclin B necessaryto drive the cell cycle?

specifically degradecyclin B mRNA:

cyclin B is both necessary and sufficient

Is cyclin B destruction required to exit mitosis?

YES

add mRNAs to extract:

Other post-translational steps control MPF activation

Why the lag if enough cyclin B has beensynthesized?

fission yeast - Cdc13 (cyclin B) overexpression does not affect cell cycle:

Cdc25 and Wee1 regulate

clams and starfish oocyte maturationpreMPF MPF, no protein synthesis

Drosophila embryolimiting protein is Cdc25, not cyclin B

Other evidence for post-translational regulation

Phosphorylation on different cdk sites can either stimulateor inhibit MPF activity

mutational analysis of cdc2 in yeast:

Tyrosine 15 (Y-15) phosphorylation inhibits Cdc2

Threonine 161 (T-161) phosphorylation activates Cdc2

e.g. mutate tyrosine-15 to phenylalanine wee phenotype

Cdc25Y15 ppase

Wee1Mik1Y15 kinases

MPF activity controlled by:1) cyclin B levels2) phosphorylation of cdk1

Multiple levels of regulation provide input for“checkpoint control”

When MPF level reaches threshold irreversible activation

High MPF levels trigger cyclin B degradation irreversible inactivation

Feedback mechanisms:

Exit from mitosis

Ubiquitin-mediated proteolysis

E1: activates ubiquitinonly 1 gene in yeast

E2: acquires ubiquitin through high energy thioester linkage= ubiquitin-carrier or conjugating enzyme11 genes in yeast

E3: catalyzes attachment of ubiquitin to protein substrate= ubiquitin ligasebinds E2 and substrate, like a scaffoldcan be in large regulatory complexmany versions, provides specificityrate-limiting step!!

Enzymes in ubiquitin pathway

proteosome

EM

E3 ligase for cyclin B = Anaphase Promoting Complex (APC)

8 subunits

identified by fractionating egg extracts, yeast genetics

degrades other proteins besides cyclin B that are required for anaphase and mitotic exit

What are the APC targets?

Xkid

Pds1 - chromosome cohesion regulator

??

mitotic cyclins

temporally specific proteolysis: orderly and irreversible sequence of events

What generates specificity of APC?

Accessory factors: recognize destruction box

Cdc20 - activates APC earlier

targets Pds1, mitotic cyclins

Cdh1 - activates APC later

targets more cyclins, APC subunits

ENTRY INTO INTERPHASE