fission yeast

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Transcript fission yeast

Lecture 8
Getting into and out of mitosis
2001
who won the
Nobel prize??
Outline:
Uncovering the cyclin/CDK paradigm
G2/M
Exiting M-phase
Paper: Anaphase regulation in budding yeast
M-phase is induced by a soluble factor
70’s cell fusion experiments
Factor in mitotic cell
induces premature mitosis
in interphase cell
M
premature
chromosome
condensation
is a disaster!
S
 the cell cycle must be highly coordinated.
What is M-phase inducer?
3 lines of experimental approach:
yeast
Xenopus eggs
invertebrate eggs
YEAST
Advantages:
rapidly growing, divides every 90 min
powerful genetics
cell cycle progress easy to follow
Lee Hartwell  S. cerevisiae
Paul Nurse
 S. pombe
Disadvantages:
Different from higher eukaryotes
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
Note on yeast nomenclature
S. cerevisiae (budding yeast)
wild type or dominant
mutations
protein
CDC28
cdc28-4ts, ∆cdc28
Cdc28
S. pombe (fission yeast)
wild type or dominant
mutations
protein
cdc2+ cdc2D
cdc2ts cdc2Cdc2
S. pombe = fission yeast
Fission yeast must reach a critical size to enter mitosis
Approach
 Identify temperature sensitive mutants that
block the cell cycle at a specific stage (size)
asynchronous population
most arrest at random
cell cycle states
cdc mutant
“cell division cycle”
Cdc2 is the master regulator of cell division
WT
always
divide at the
same size
cdc2(ts)
loss of
function
cdc2D
gain of
function
“wee”
 essential, no other mutation can rescue loss of cdc2
Mitosis-inducing activity of Cdc2 is inhibited by Wee1 and
stimulated by Cdc25
WT
cdc2ts
cdc25ts
wee1ts
cdc25ts, wee1ts
cdc2D, wee1ts
mitotic catastrophe
Cdc2 - protein kinase
Wee1 - protein kinase
Cdc25 - protein phosphatase
WT
5X cdc2+
3X wee1+
5X cdc25+
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:
M
 alternating S- and M-phases
no G1 or G2 phases
synchronous divisions
easy to inject
good for biochemistry
S
Eggs divide without growing
somatic cells
embryonic cells
12 embryonic cell cycles
typical somatic cell cycle
G1
0
3
S
6
9
12
G2
15
18
M
21
Hours
Observation: protein synthesis is required
for early embryonic divisions
24
Identification of “cyclins”
 sea urchin eggs
fertilize eggs  synchronous rounds of division
incubate with 35S - methionine
take sample every 10 min
SDS-PAGE and autoradiography
 newly synthesized proteins get labeled
Tim Hunt, Joan Ruderman
mitosis
interphase
mitosis
interphase
cyclin A
cyclin B
other proteins
cyclin B
abundance
I
M
I
M
cell cycle state
Conclusion
Cyclin synthesis and destruction correlates
with progression through the cell cycle
 cyclins are potential regulators
Xenopus laevis
oocyte maturation
diploid
meiosis
fertilization
haploid
1 mm diameter!
cleavage
diploid
Discovery of
MPF
=
Maturation
Promoting
Factor
Masui and Markert
1971
a more general role.....
MPF = M-phase Promoting Factor
also present in mitotic somatic cells
KEY EXPERIMENT - Masui, Lohka and Maller
biochemical purification of MPF
1 liter of frog eggs  1 mg
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
antibody to conserved region of cdc2: “PSTAIRE”
p34 = cdc2
p46 = cyclin B
M-phase promoting factor = MPF =cdk1/cyclinB
protein kinase complex, 2 subunits:
1) cdk1 cyclin dependent kinase 1
= yeast cdc2
• Induces mitosis by phosphorylating specific
downstream targets on serine and threonine
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
electric shock
simulates fertilization
in vitro system
Marc Kirschner
Andrew Murray
cell cycle reconstitution in vitro
low MPF
low histone H1
kinase activity
high MPF
high 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?
add mRNAs to extract:
YES
Other post-translational steps control MPF activation
Why the lag if enough cyclin B has been
synthesized?
Other evidence for post-translational regulation
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
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
Wee1
Mik1
Y15 kinases
Cdc25
Y15 ppase
MPF activity controlled by:
1) cyclin B levels
2) phosphorylation of cdk1
Multiple levels of regulation provide input for
“checkpoint control”
Feedback mechanisms:
When MPF level reaches threshold
 irreversible activation
High MPF levels trigger cyclin B degradation
 irreversible inactivation
Exit from mitosis
Ubiquitin-mediated proteolysis
Enzymes in ubiquitin pathway
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!!
proteosome
EM
E3 ligase for cyclin B
= Anaphase Promoting Complex (APC)
identified by fractionating egg extracts, yeast genetics
8 subunits
 degrades other proteins besides cyclin B that are
required for anaphase and mitotic exit
What are the APC targets?
mitotic cyclins
Pds1 - chromosome cohesion regulator
Xkid
??
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