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