Lecture 8 Getting into and out of mitosis Outline: Uncovering the cyclin/CDK paradigm G2/M Exiting...
Transcript of Lecture 8 Getting into and out of mitosis Outline: Uncovering the cyclin/CDK paradigm G2/M Exiting...
![Page 1: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/1.jpg)
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??
![Page 2: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/2.jpg)
![Page 3: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/3.jpg)
M-phase is induced by a soluble factor
70’s cell fusion experiments
Factor in mitotic cell induces premature mitosisin interphase cell
![Page 4: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/4.jpg)
premature chromosome condensation is a disaster!
the cell cycle must be highly coordinated.
S
M
![Page 5: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/5.jpg)
What is M-phase inducer?
3 lines of experimental approach:yeastXenopus eggs
invertebrate eggs
![Page 6: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/6.jpg)
YEAST
rapidly growing, divides every 90 min
powerful genetics
cell cycle progress easy to follow
Lee Hartwell S. cerevisiae
Paul Nurse S. pombe
Advantages:
![Page 7: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/7.jpg)
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
![Page 8: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/8.jpg)
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
![Page 9: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/9.jpg)
S. pombe = fission yeast
![Page 10: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/10.jpg)
![Page 11: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/11.jpg)
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
![Page 12: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/12.jpg)
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
![Page 13: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/13.jpg)
Mitosis-inducing activity of Cdc2 is inhibited by Wee1 andstimulated by Cdc25
cdc2ts
WT
cdc25ts
wee1ts
cdc25ts, wee1ts
cdc2D, wee1ts mitotic catastrophe
![Page 14: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/14.jpg)
Cdc2 - protein kinase
Cdc25 - protein phosphatase
Wee1 - protein kinase
3X wee1+
WT
5X cdc25+
5X cdc2+
can confirminteractions byoverexpression:
![Page 15: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/15.jpg)
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
![Page 16: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/16.jpg)
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
![Page 17: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/17.jpg)
somatic cells
embryonic cells
Eggs divide without growing
![Page 18: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/18.jpg)
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
![Page 19: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/19.jpg)
![Page 20: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/20.jpg)
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
![Page 21: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/21.jpg)
mitosis mitosis interphaseinterphase
cyclin A
cyclin B
cyclin Babundance
cell cycle state
I M I M
other proteins
![Page 22: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/22.jpg)
Conclusion
Cyclin synthesis and destruction correlates with progression through the cell cycle
cyclins are potential regulators
![Page 23: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/23.jpg)
Xenopus laevis
![Page 24: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/24.jpg)
1 mm diameter!
diploid haploid
oocyte maturation meiosis fertilization cleavage
diploid
![Page 25: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/25.jpg)
Discovery of MPF
= MaturationPromotingFactor
Masui and Markert1971
![Page 26: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/26.jpg)
MPF = M-phase Promoting Factor
a more general role.....
also present in mitotic somatic cells
![Page 27: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/27.jpg)
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
![Page 28: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/28.jpg)
VERY FAMOUS WESTERN BLOT: 1988
p34 = cdc2
antibody to conserved region of cdc2: “PSTAIRE”
p46 = cyclin B
![Page 29: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/29.jpg)
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
![Page 30: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/30.jpg)
cell cycle paradigm: transitions are regulated by cdk/cyclin complexes
In yeast:single cdk,many cyclins
![Page 31: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/31.jpg)
• Cyclin B protein is synthesized continuously• Threshold cyclin B level induces MPF kinase activity • Cyclin B disappears suddenly during anaphase
![Page 32: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/32.jpg)
Experiments to prove that cyclin synthesis drives theembryonic cell cycle
in vitro system
Marc KirschnerAndrew Murray
electric shocksimulates fertilization
![Page 33: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/33.jpg)
cell cycle reconstitution in vitro
high MPFhigh histone H1kinase activity
low MPFlow histone H1kinase activity
![Page 34: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/34.jpg)
cyclin B synthesis can drivethe cell cycle
is cyclin B necessaryto drive the cell cycle?
![Page 35: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/35.jpg)
specifically degradecyclin B mRNA:
cyclin B is both necessary and sufficient
![Page 36: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/36.jpg)
Is cyclin B destruction required to exit mitosis?
YES
add mRNAs to extract:
![Page 37: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/37.jpg)
Other post-translational steps control MPF activation
Why the lag if enough cyclin B has beensynthesized?
![Page 38: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/38.jpg)
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
![Page 39: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/39.jpg)
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
![Page 40: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/40.jpg)
Cdc25Y15 ppase
Wee1Mik1Y15 kinases
![Page 41: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/41.jpg)
MPF activity controlled by:1) cyclin B levels2) phosphorylation of cdk1
Multiple levels of regulation provide input for“checkpoint control”
![Page 42: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/42.jpg)
When MPF level reaches threshold irreversible activation
High MPF levels trigger cyclin B degradation irreversible inactivation
Feedback mechanisms:
![Page 43: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/43.jpg)
Exit from mitosis
Ubiquitin-mediated proteolysis
![Page 44: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/44.jpg)
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
![Page 45: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/45.jpg)
proteosome
![Page 46: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/46.jpg)
EM
![Page 47: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/47.jpg)
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
![Page 48: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/48.jpg)
What are the APC targets?
Xkid
Pds1 - chromosome cohesion regulator
??
mitotic cyclins
temporally specific proteolysis: orderly and irreversible sequence of events
![Page 49: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/49.jpg)
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
![Page 50: 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.](https://reader036.fdocuments.in/reader036/viewer/2022062717/56649e545503460f94b4b7eb/html5/thumbnails/50.jpg)