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Gary kerr dundee uni seminar feb 2013
- 1. Meiotic nuclear divisions in budding yeast require PP2ACdc55- mediated antagonism of Net1 phosphorylation by Cdk Gary Kerr University of Dundee 13th February 2013
- 2. Mitosis Parent Cell Parent Cell DNA Replicates DNA Replicates 2 Daughter Cells 4 Daughter Cells Meiosis I Meiosis II
- 3. Metaphase Cohesin Anaphase Cleavage of Scc1/ Rad21 Mitosis in budding yeast
- 4. 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
- 5. Meiosis in budding yeast
- 6. 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
- 7. 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.
- 8. • 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
- 9. Is PP2ACdc55 required for meiotic nuclear divisions? cdc55-mn strains fail to divide their nuclei and form monads 1n 2n 4n CDC55 PCLB2
- 10. Is PP2ACdc55 required for pre-meiotic DNA replication? PP2ACdc55 is not required for pre-meiotic DNA replication
- 11. 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+
- 12. Is PP2ACdc55 required for Synaptonemal Complex (SC) assembly and disassembly? PP2ACdc55 is not required for SC assembly or disassembly Cells(%)Cells(%) Sourav Sarkar
- 13. 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 %Cells
- 14. 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
- 15. Is PP2ACdc55 required for Spindle Pole Body (SPB) separation during meiosis? PP2ACdc55 is required for SPB separation during meiosis 4 SPB’s 1 SPB 2 SPB’s SPC42-GFP
- 16. FEAR network regulates CDK activity during mitosis and meiosis Cdc14 is a phosphatase that decreases CDK activity a) Degradation of B-Cyclins b) Dephosphorylation of CDK substrates Metaphase Anaphase Cdc14 Net1 P P PCdk PP2ACdc55 Cdc14 Early Anaphase Release (FEAR) CDK Cdc14 Net1 Esp1
- 17. 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?
- 18. 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
- 19. 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
- 20. 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-nucleates Binucleates Mononucleates
- 21. Conclusion PP2ACdc55 is required to prevent premature exit from meiosis I
- 22. Does PP2ACdc55 have a role in meiotic chromosome segregation?
- 23. A screen was performed to isolate a role for PP2ACdc55 in monopolar attachment 987 transformants Two 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
- 24. • 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 spo11Δ spo12Δ mam1Δ spo11Δ spo12Δ spo11Δ spo12 Δ cdc55Δ WT FG4 FG7 FG8 FG9 FG10 FG12 FG14 HSV allele Mutations FG4 K405N FG7 L520S FG8 F64S FG9 Q12P, C60Y, N144T FG10 V132E FG12 I28F,A481E FG14 I190V, S252P FG15 L27P, N66S
- 25. cdc55-MP has no apparent effect on meiotic chromosome segregation during wild-type meiosis Does cdc55-MP affect chromosome segregation during wild-type meiosis?
- 26. • 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
- 27. Acknowledgements University of Warwick Dr Prakash Arumugam Dr Sourav Sarkar Miss Kate Tibbles Professor Jonathan Millar All members of M116 Strains & Reagents Professor Raymond Deshaies Professor Kim Nasmyth Professor Angelika Amon Dr Kyung Lee Cancer Research UK – London Dr Mark Petronczki Advisory Committee Dr Kevin Moffat Dr Graham Ladds Dr Lorenzo Frigerio School of Life Sciences Mr Paul Goode Technical & Admin Support Staff Prep & Media Prep Staff
Editor's Notes
- In order to understand meiosis, we must first understand meiosis Mitosis is the production of somatic cells and occurs in all embryonic tissue and most adult tissue to produce genetically identical daughter cells Meiosis, on the other hand, is the production of gametes and occurs only in gonadal tissue. Through recombination and formation of chiasmata, gametes produced during meiosis are genetically unique Mitosis is an equational division where the daughter cells have the same number of chromosomes as the parent cell (they are 2n). This is achived by one round of DNA replication followed by one nuclear division Meiosis is a reductional division from diploid cell to haploid cell. This is achieved by one round of DNA replication followed by two consecutive nuclear divisions (meiosis I and meiosis II) So, how is the formation of haploid and diploid cells achieved?...
- So, mitosis is characterised by one round of DNA replication followed by two rounds of nuclear division to produce two identical daughter cells Since errors at either DNA replication or nuclear division will be deleterious, cells have evolved elaborate mechanisms to ensure accurate replication of its genome and faithful segregation of chromosomes to daughter cells Immediately after DNA replication the sister chromatids are held together by a protein complex called cohesin (cohesin comprises 4 subunits illustrated in the slide) – Cohesin forms a ring around the sister chromatids holding them together Each chromosome has a centromeric DNA sequence on which a multi-protein complex known as the kinetochore assembles (illustrated in green) The kinetochore is a specialised structure that binds the growing ends of microtubules that emanate from the microtubule organising centre When sister kinetochores are captured by microtubules emanating from the MTOCs at opposite spindle poles, they are said to be bi-oriented Bi-orientation of sister kinetochores results in a tug-of-war where a pulling force is generated by the bi-polar spindle and counteracted by cohesin between sister chromatids The resulting tension stabilises bi-oriented sister kinetochores At metaphase when all sister kinetochores are bi-oriented, cells destroy cohesin by activating separase (Esp1) which cleaves Scc1 (cohesin subunit). This triggers the transition into anaphase where sister chromatids move towards opposite poles of the cell There are some key differences between mitosis and meiosis and we’ll look at those now
- The first key difference between meiosis and mitosis is that during meiosis I, reciprocal recombination and formation of chiasmata occurs (illustrated on figure) Secondly, centromeric cohesin is protected from separase cleavage during meiosis I and only non-centromeric cohesin is cleaved Thirdly, monopolar attachment instead of bipolar attachment of sister kinetochores during meiosis I. This results in the co-segregation of sister centromeres to the same spindle pole Finally, DNA replication is inhibited between meiosis I and meiosis II divisions: this allows for the production of haploid cells during meiosis We will now look at the process of meiosis in detail…
- During metaphase, separase is inhibited by securin At the metaphase to anaphase transition, the anaphase promoting complex involving Cdc20 destroys securin thus releasing an active form of separase and this separase can cleave non-centromeric cohesin during meiosis I During meiosis II, sister chromatids bi-orient and move towards opposite poles and this is promoted by the destruction of centromeric cohesin at the metaphase II – anaphase II transition So why are we studying meiosis in budding yeast?...
- Why do we study meiosis? Meiosis results in the formation of gametes and in humans. Errors in meiotic chromosome segregation can lead to aneuploidy. In humans, aneuploidy is a leading cause of spontaneous abortion (estimated 1/7 pregnancies result in miscarriage) Although most aneuploidies are non-viable, viable aneuploidies include trisomies 13, 18 and 21) The karyotype shows a male with Trisomy 21 – Down syndrome DS is associated with profound learning difficulty and associated clinical features Sex chromosome aneuplodies can result in disease such as Turners and Kleinfelters syndromes
- Budding yeast is a genetically tractable organism Wide range of genetic & biochemical techniques available Can be studied in the haploid or diploid form Meiosis is well characterised in S. cerevisiae Meiosis produces ascus (4 haploid ascospores) Many fundamental processes are conserved from humans to yeast
- Cdc55 is a regulatory subunit of PP2A PP2A comprises 3 subunits: A scaffold (A) subunit – Tpd3 (invariant) A Regulatory (B) subunit – Rts1, Rts3 or Cdc55 (variable and exchangeable) -A Catalytic (C) subunit – Pph21 or Pph22 PP2ACdc55 is a highly conserved serine-threonine phosphatase (Human counterpart is B55α) implicated in various processes in budding yeast like Cellular morphogenesis, Protein translation, Spindle Checkpoint and Mitotic exit Until now, the meiotic function of PP2ACdc55 has not been characterised in any organism
- It has previously been reported that cdc55Δ cells are too sick for meiotic analysis. We therefore generated a meiotic null allele of CDC55 where its promoter was replaced with that of the mitosis-specific CLB2 Western analysis shows that Cdc55 is expressed mitotically in cdc55-mn cells, but is not expressed during meiosis To test if PP2ACdc55 is required for meiosis, we induced CDC55 and cdc55-mn strains to sporulate >60% of CDC55 cells went through 2 rounds of nuclear division and formed tetrads after 10h in SPM, whereas cdc55-mn strains remained largely mononucleate (around 97%) cdc55-mn strains fail to divide their nuclei and form monads, suggesting that Cdc55 is required for cellular meiotic progression
- Flow cytometry shows that both CDC55 and cdc55-mn strains accumulate a 4C peak following transfer into SPM WT cells – initiated pre-meiotic DNA replication after 3h cdc55-mn cells – initiated pre-meiotic DNA replication after 1h This indicates that PP2ACdc55 is not required for pre-meiotic DNA replication
- Failure to progress through meiotic nuclear divisions could be caused by an inability to degrade securin and/or cleave cohesin. To test this possibility, we followed the levels of securin (Pds1) and meiosis-specific cohesin subunit Rec8 in sporulating CDC55 and cdc55-mn cells by immunofluorescence and immunoblotting. In WT, Pds1 and Rec8 levels accumulate and reach maximum around 5 hours and then levels start to decline. This is confirmed by western blotting START – is timing of replication initiation (as indicated by flow cytometry) These data suggest that meiotic cell cycle events appear to proceed normally in the absence of PP2ACdc55 activity but occur in the absence of any nuclear division
- Since SC assembly in budding yeast depends on recombination, we asked whether PP2ACdc55 is required for SC assembly or disassembly by staining chromosome spreads prepared from sporulating CDC55 and cdc55-mn cells using antibodies for the central SC component Zip1 and for Rec8 Mutants that are defective in SC assembly in budding yeast accumulate Zip1 aggregates referred to as Polycomplexes. However in both WT and cdc55-mn cells, we detected full SC formation after 4h in SPM and its disappearance after 8h suggesting that PP2ACdc55 is not required for SC assembly and disassembly.
- Pachytene checkpoint and SAC are 2 known surveillance mechanisms that block/delay meiotic progression in response to errors in recombination and KT-MT attachment, respectively. In order to identify if the failure of cdc55-mn cells to undergo meiotic nuclear divisions due to activation of pachytene checkpoint or spindle assembly checkpoints, we performed genetic assays Activation of pachytene checkpoint is dependent on Rad24 and presence of DSBs Deletion of SPO11 (an endonuclease that creates DSBs on DNA) and RAD24 did not suppress the nuclear division defect of cdc55-mn cells Deletion of MAD2 (which is necessary for SAC) did not rescue the nuclear division defect of cdc55-mn cells. These data demonstrate that failure of cdc55-mn cells to undergo nuclear divisions is not due to activation of pachytene checkpoint or SAC.
- Since a bipolar spindle is required for nuclear division, we tested whether PP2ACdc55 is required for formation of metaphase I spindles by immunofluorescence using anti-tubulin antibodies. WT cells go through 2 rounds of nuclear division and assembled metaphase I, anaphase I and meiosis II spindles whereas cdc55-mn cells formed very short spindles or no spindles and did not divide their nuclei. Therefore, PP2ACdc55 is required for bipolar spindle assembly during meiosis
- The failure of cdc55-mn cells to form a bi-polar spindle could be caused by a defect in SPB separation. To assay SPB separation, we tagged Spc42 with GFP and counted the number of GFP dots in sporulating CDC55 and cdc55-mn cells. In WT, >70% of cells went through 2 rounds of SPB separation and showed 4 SPB’s. In cdc55-mn, 95% of cells contained a single GFP dot after 10h in SPM, suggesting that PP2ACdc55 is required for SPB separation during meiosis
- CDK activity is required for SPB separation and formation of a bipolar spindle during mitosis CDK activity is regulated by the FEAR network during meiosis and mitosis During metaphase, Cdc14 remains in the nucleolus and is released during anaphase Cdc14 is an inhibitor of CDK activity During metaphase, Net1 binds Cdc14 and thus prevents it from leaving the nucleolus Metaphase into Anaphase transition is facilitated by both the FEAR and MEN pathways during mitosis During mitosis, FEAR is not essential as MEN also promotes Cdc14 release; however, FEAR is essential for exit of meiosis I Cdc14 then inhibits CDK’s resulting in cell exit of mitosis Cdc14 is a phosphatase that decreases CDK activity by Degradation of B-Cyclins b) Dephosphorylation of CDK substrates PP2ACdc55 has an antagonistic role in keeping Net1 under-phosphorylated during metaphase and thus keeping Cdc14 in the nucleolus
- In order to test why the release of Cdc14 from the nucleolus is transient, we asked if PP2ACdc55 is required for preventing premature Cdc14 release from the nucleolus During early anaphase, destruction of mitotic cyclins by APC/Cdc20 is thought to decrease Cdk activity to an extent that is unable to sustain Net1 phosphorylation and thereby causing relocation of Cdc14 to the nucleolus. If FEAR operated in a similar manner during meiosis, then one would predict a robust nuclolar release of Cdc14 in cdc55-mn cells in the absence of APC/Cdc20 activity. To test this, we synchronised CDC55 and cdc55-mn cells in metaphase I by depletion of Cdc20 and monitored Cdc14 localization. In WT cells, Cdc14 was nucleolar as measured by co-localization with Net1. However, in cdc55-mn cells, Cdc14 was progressively released from the nucleolus. Nucleolar release of Cdc14 initiated after 5h and by 8h, more than 60% of cells had Cdc14 distributed all over the nucleus. Western analysis shows that Net1 from cdc55-mn cells arrested in metaphase I is up-shifted, suggesting that it is hyper-phosphorylated. PP2ACdc55 is therefore required for preventing premature release of Cdc14 from the nucleolus during meiosis
- Since Cdc14 was released in cdc55-mn cells arrested in metaphase I, we tested whether Cdc14 release sufficient for blocking meiotic nuclear divisions and spindle assembly. To mimic premature release of Cdc14, we expressed TAB6, a dominant mutant allele of Cdc14 that binds poorly to Net1. Using the GAL4-ER system, we constructed a strain that expressed TAB6 in the presence of estradiol. Induction of TAB6 expression in cells after 4, 5 and 6h into sporulation affected tetrad formation Addition of estradiol to the strain that lacked the pGAL1-TAB6 allele had little or no effect. These results indicate that premature release of Cdc14 is sufficient for blocking meiotic spindle assembly and nuclear divisions.
- Phosphorylation of Net1 at 6 Cdk consensus sites is required for Cdc14 release during early anaphase. We therefore constructed diploid strains that contained either CDC55 or cdc55-mn allele in combination with either NET1 or net1-6Cdk allele. We induced the 4 strains to undergo meiosis and analyzed the kinetics of nuclear division. Wild type 60% of NET1 CDC55 cells went through 2 rounds of nuclear division and formed tetrads NET1 cdc55mn These cells failed to undergo any nuclear division and arrested as mononucleates net1-6Cdk CDC55 These cells formed 50% tetrads, a high proportion of cells (18%) formed dyads, very similar to WT Double mutant Crucially, around 40% of net1-6Cdk cdc55-mn cells went through 2 rounds of nuclear division and formed tetrads/triads This indicates that 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.
- It has previously been shown that cdc55Δ cells are too sick for performing any meiotic analysis. In order to test if the inability of cdc55Δ cells to sporulate is due to premature FEAR activation, we tested the ability of the net1-6Cdk allele to suppress the sporulation defect of cdc55Δ strains. As previously reported, NET1 cdc55Δ failed to sporulate. Remarkably, net1-6Cdk cdc55Δ cells formed a high proportion of dyads and tetrads This suggests that the major function of PP2ACdc55 during meiosis is to control the timing of FEAR activation by opposing Net1 phosphorylation by Cdk.
- In conclusion, PP2ACdc55 is required to prevent premature exit from meiosis I Meiotic nuclear divisions in budding yeast require PP2ACdc55-mediated antagonism of Net1 phosphorylation by Cdk. Unlike mitosis, premature activation of FEAR during meiosis blocks spindle assembly and nuclear divisions. This work emphasises the importance of FEAR in regulating the meiotic cell cycle.
- Currently, we are investigating if negative regulation of FEAR is the only function of PP2ACdc55 during meiosis. A paper in 2003 showed that Cdc55 was a candidate gene for involvement in monopolar attachment We therefore testing if PP2ACdc55 has a role play in chromosome segregation during meiosis. We have found that spore viability is very low (around 52%) in the net1-6Cdk cdc55-mn double mutant, compared to around 90% in WT and net1-6Cdk alone. In addition, we have identified a subtle defect in chromosome segregation in anaphase I cells where there is an increased proportion of cells in the double mutant where sister centromeres split either equationally or reductionally with split sisters The previous study linking PP2ACdc55 with a possible role in monopolar attachment found that cdc55Δ cells were too sick to work with. We therefore devised a method to isolate mis-sense mutations in Cdc55
- A screen was performed to isolate a role for PP2ACdc55 in monopolar attachment Cdc55 was subject to PCR-mediated mutagenesis From the screen, two classifications of mutants were generated The first classification of mutants were those that were suppressors of spo12Δ dyad phenotype. These mutants prematurely released Cdc14 from the nucleolus. This is consistent with my previous findings that PP2ACdc55 has a role in negatively regulating FEAR during meiosis. In addition to the FEAR mutants, we identified a second class of mutants that suppress the low spore viability of spo11Δspo12Δ strains. SPO11 – required for DSB formation SPO12 – part of FEAR network Monopolin mutants have been shown to be suppressors of the spo11Δspo12Δ low spore viability This is suggestive of an additional role of PP2ACdc55 during meiosis in chromosome segregation Therefore, the negative regulation of FEAR role of PP2ACdc55 is genetically separable from its role in meiotic chromosome segregation I will address this role using the mis-sense mutations identified in this screen