Gary kerr dundee uni seminar feb 2013
Transcript of Gary kerr dundee uni seminar feb 2013
Meiotic nuclear divisions in budding yeast require PP2ACdc55-mediated antagonism of Net1 phosphorylation by Cdk
Gary KerrUniversity of Dundee13th February 2013
Mitosis
Parent Cell Parent Cell
DNA ReplicatesDNA Replicates
2 Daughter Cells4 Daughter Cells
Meiosis I
Meiosis II
Metaphase
Cohesin
Anaphase
Cleavage of Scc1/ Rad21
Mitosis in budding yeast
Key differences between mitosis & meiosis
1. Reciprocal recombination & formation of chiasmata during meiosis I
2. Centromeric cohesin protected from separase cleavage during meiosis I
3. Monopolar attachment of sister kinetochores during meiosis I
4. DNA replication inhibited between meiosis I and meiosis II
F
chiasmata
monopolar attachment
Protection of centromeric cohesin
Meiosis in budding yeast
Why study meiosis?
• Aneuploidy leading cause of spontaneous abortion
• Viable human aneuploidies:• Patau, Edward & Down syndromes
(trisomies 13, 18 and 21)
• Profound learning difficulties and associated clinical features
• Sex chromosome aneuploidies: Turners (XO) and Kleinfelters (XXY) syndromes
Why use budding yeast to study meiosis?
Genetically tractable organism;
Wide range of genetic & biochemical techniques available;
Can be studied in haploid or diploid;
Meiosis produces ascus;
Meiosis well characterised in budding yeast;
Many fundamental processes conserved from yeast to humans.
• PP2ACdc55 is a highly conserved serine-threonine phosphatase
• Implicated in various processes in budding yeast like:– Cellular morphogenesis– Protein translation– Spindle checkpoint– Mitotic exit
• The meiotic function of PP2ACdc55 has not been characterised in any organism
Catalytic (C) subunit(Pph21 or Pph22)
Regulatory (B) subunit (Rts1, Rts3 or Cdc55)
Scaffold (A) subunit (Tpd3)
Protein Phosphatase PP2ACdc55
Is PP2ACdc55 required for meiotic nuclear divisions?
cdc55-mn strains fail to divide their nuclei and form monads
1n
2n
4n
CDC55PCLB2
Is PP2ACdc55 required for pre-meiotic DNA replication?
PP2ACdc55 is not required for pre-meiotic DNA replication
Is cdc55-mn inability to progress through meiosis caused by an inability to degrade securin and/or cleave cohesin?
Degradation of securin and cohesin appears to proceed normally in the absence of PP2ACdc55 activity but occur in the absence of any nuclear division
Rec8+
2n Pds1+
1n Pds1+
Is PP2ACdc55 required for Synaptonemal Complex (SC) assembly and disassembly?
PP2ACdc55 is not required for SC assembly or disassembly
Cel
ls (%
)C
ells
(%)
Sourav Sarkar
Is the failure of cdc55-mn strains to undergo meiotic nuclear divisions due to activation of pachytene or spindle assembly
checkpoints?
The failure of cdc55-mn strains to undergo nuclear divisions is not due to activation of the pachytene checkpoint or spindle assembly checkpoint
% C
ells
Is PP2ACdc55 required for bipolar spindle assembly during meiosis?
PP2ACdc55 is required for bipolar spindle assembly during meiosis
Metaphase I spindles
Meiosis II spindles
Anaphase I spindles
Is PP2ACdc55 required for Spindle Pole Body (SPB) separation during meiosis?
PP2ACdc55 is required for SPB separation during meiosis
4 SPB’s1 SPB
2 SPB’s
SPC42-GFP
FEAR network regulates CDK activity during mitosis and meiosis
Cdc14 is a phosphatase that decreases CDK activitya) Degradation of B-Cyclins b) Dephosphorylation of CDK substrates
Metaphase Anaphase
Cdc14
Net1
PP
PCdk
PP2ACdc55
Cdc14 Early Anaphase Release (FEAR)
CDK
Cdc14
Net1
Esp1
PP2ACdc55 is required for preventing premature release of Cdc14 from the nucleolus during meiosis
Is PP2ACdc55 required for preventing premature Cdc14 release from the nucleolus?
Is premature release of Cdc14 from the nucleolus sufficient for inhibiting meiotic nuclear divisions?
Premature release of Cdc14 from the nucleolus is sufficient for blocking meiotic nuclear divisions
Tab6-Cdc14
Net1
Cdc14
Net1
Kate Tibbles
Is the inability of cdc55-mn cells to separate their nuclei is due to premature release of Cdc14?
The nuclear division defect of cdc55-mn cells is caused by untimely phosphorylation of Net1 by Cdk and consequent release of Cdc14 from the nucleolus
1n
2n
4n
Is the inability of cdc55Δ cells to sporulate due to premature FEAR activation?
The major function of PP2ACdc55 during meiosis is to control the timing of FEAR activation by opposing Net1 phosphorylation by Cdk
Tri/Tetra-nucleatesBinucleatesMononucleates
Conclusion
PP2ACdc55 is required to prevent premature exit from meiosis I
Does PP2ACdc55 have a role in meiotic chromosome segregation?
A screen was performed to isolate a role for PP2ACdc55 in monopolar attachment
987 transformantsTwo classifications of mutants
Suppressors of spo12Δ dyad phenotype
Release Cdc14 prematurely from the
nucleolus
(Role in FEAR)
Suppress low spore viability of spo11Δspo12Δ
Feature of monopolin mutant
(Role in monopolar attachment?)
CDC55
FEAR role of PP2ACdc55 genetically separable from role in meiotic chromosome segregation
• Plasmids from the screen were isolated and re-introduced into the parent spo11Δ spo12 Δ cdc55 Δ strain
• A second screen identified suppressors of the low-spore viability phenotype• To maximise the effect of the mutations, FG4 and FG9 mutations were
combined to create a cdc55-MP allele
A screen produced mutations that suppressed spo11Δ spo12Δ low-spore viability phenotype
spo11Δspo12Δmam1Δ
spo11Δspo12Δ
spo11Δspo12 Δcdc55Δ
WT FG4 FG7 FG8 FG9 FG10 FG12 FG14HSV allele Mutations
FG4 K405N
FG7 L520S
FG8 F64S
FG9 Q12P, C60Y, N144T
FG10 V132E
FG12 I28F, A481E
FG14 I190V, S252P
FG15 L27P, N66S
cdc55-MP has no apparent effect on meiotic chromosome segregation during wild-type meiosis
Does cdc55-MP affect chromosome segregation during wild-type meiosis?
• High-spore viability mutations have an effect on reductional segregation of chromosomes during meiosis
• But no detectable effect on wild-type meiosis
• We do not yet know the precise function of PP2ACdc55 that is affected by cdc55-MP
• How does cdc55-MP effect monopolar attachment in achiasmate cells?
• What effect does cdc55-MP have on release of Csm1/Lrs4 release and association with kinetochores?
Summary
AcknowledgementsUniversity of WarwickDr Prakash ArumugamDr Sourav SarkarMiss Kate TibblesProfessor Jonathan MillarAll members of M116
Strains & ReagentsProfessor Raymond DeshaiesProfessor Kim NasmythProfessor Angelika AmonDr Kyung Lee
Cancer Research UK – LondonDr Mark Petronczki
Advisory CommitteeDr Kevin MoffatDr Graham LaddsDr Lorenzo Frigerio
School of Life SciencesMr Paul GoodeTechnical & Admin Support StaffPrep & Media Prep Staff