Lecture 8 Getting into and out of mitosis Outline: Uncovering the cyclin/CDK paradigm G2/M Exiting M-phase Paper: Anaphase regulation in budding yeast 2001 who won the Nobel prize??

M-phase is induced by a soluble factor 70’s cell fusion experiments Factor in mitotic cell induces premature mitosis in 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: yeast Xenopus 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 dominantCDC28 mutationscdc28-4 ts, ∆cdc28 proteinCdc28 S. pombe (fission yeast) wild type or dominantcdc2 + cdc2 D mutationscdc2 ts cdc2 - proteinCdc2

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 cdc2 D gain of function “wee” WT always divide at the same size

Mitosis-inducing activity of Cdc2 is inhibited by Wee1 and stimulated by Cdc25 cdc2 ts WT cdc25 ts wee1 ts cdc25 ts, wee1 ts cdc2 D, wee1 ts mitotic catastrophe

Cdc2 - protein kinase Cdc25 - protein phosphatase Wee1 - protein kinase 3X wee1 + WT5X cdc25 + 5X cdc2 + can confirm interactions by overexpression:

Conclusions: Cdc2 is the master regulator of cell division in fission yeast Cdc25 is a positive regulator of Cdc2 Wee1 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 eggs sea urchin, starfish, clam Advantages: stripped down cell cycle:  alternating S- and M-phases no G1 or G2 phases M S synchronous divisions good for biochemistry easy to inject

somatic cells embryonic cells Eggs divide without growing

SG1G2M 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 35 S - 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 interphase cyclin A cyclin B abundance cell cycle state IMIM other proteins

Conclusion Cyclin synthesis and destruction correlates with progression through the cell cycle  cyclins are potential regulators

Xenopus laevis

1 mm diameter! diploidhaploid oocyte maturationmeiosisfertilizationcleavage diploid

Discovery of MPF = Maturation Promoting Factor Masui and Markert 1971

MPF = M-phase Promoting Factor a more general role..... also present in mitotic somatic cells

KEY EXPERIMENT - Masui, Lohka and Maller biochemical purification of MPF 1 liter of frog eggs  1  g using oocyte maturation as an assay? no.  monitored nuclear envelope breakdown of pronuclei in egg extract MPF has two subunits: p34 p46 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 the embryonic cell cycle in vitro system Marc Kirschner Andrew Murray electric shock simulates fertilization

cell cycle reconstitution in vitro high MPF high histone H1 kinase activity low MPF low histone H1 kinase activity

cyclin B synthesis can drive the cell cycle is cyclin B necessary to drive the cell cycle?

specifically degrade cyclin 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 been synthesized?

fission yeast - Cdc13 (cyclin B) overexpression does not affect cell cycle: Cdc25 and Wee1 regulate clams and starfish oocyte maturation preMPF  MPF, no protein synthesis Drosophila embryo limiting protein is Cdc25, not cyclin B Other evidence for post-translational regulation

Phosphorylation on different cdk sites can either stimulate or 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

Cdc25 Y15 ppase Wee1 Mik1 Y15 kinases

MPF activity controlled by: 1) cyclin B levels 2) 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 ubiquitin only 1 gene in yeast E2: acquires ubiquitin through high energy thioester linkage = ubiquitin-carrier or conjugating enzyme 11 genes in yeast E3: catalyzes attachment of ubiquitin to protein substrate = ubiquitin ligase binds E2 and substrate, like a scaffold can be in large regulatory complex many versions, provides specificity rate-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