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