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Mitosis and Meiosis Paul Shapiro, Room 222 Pharmacy Hall pshapiro@rx.umaryland Office: 6-8522

MMCB601. Mitosis and Meiosis Paul Shapiro, Room 222 Pharmacy Hall pshapiro@rx.umaryland.edu Office: 6-8522 Lab (Rm 269): 6-2980. Objectives: A. Define major mitotic structural changes. B. Define major mitotic signaling events. C. Current mitosis paper discussion. The Cell Cycle:.

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Mitosis and Meiosis Paul Shapiro, Room 222 Pharmacy Hall pshapiro@rx.umaryland Office: 6-8522

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  1. MMCB601 Mitosis and Meiosis Paul Shapiro, Room 222 Pharmacy Hall pshapiro@rx.umaryland.edu Office: 6-8522 Lab (Rm 269): 6-2980 Objectives: A. Define major mitotic structural changes. B. Define major mitotic signaling events. C. Current mitosis paper discussion.

  2. The Cell Cycle: History: Pre20th century- Mitotic and interphase cells. 1953-Cell cycle phases identified. 1971-Masui and Markert-Identified maturation promoting factor (MPF) “Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes.” J Exp Zool. (1971) 177(2):129-45. 1974-Hartwell et al., Identified cell division cycle (cdc) proteins in yeast. “Genetic control of the cell division cycle in yeast.” Science (1974) 246:46. 1983-Evans et al. Identified cyclin proteins. “Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division.” Cell (1983) 33(2):389-96. 1989 -Minshull et al. Role of protein synthesis, phosphorylation and degradation. “The role of cyclin synthesis, modification, and destruction in the control of cell division.” J. Cell Science (1989) 12:77-97. 1992-Norbury and Nurse. Identified cyclin dependent kinases (cdks). “Animal cell cycles and their control.” Ann. Rev. Biochem. (1992)61:441-470.

  3. Mitosis: Prophase Metaphase Anaphase Telophase Entry: *Chromosome condensation *Spindle pole formation *Nuclear envelope breakdown *Other structural changes Exit: *Chromosome congression *Microtubule attachment *Chromosome segregation *Nuclear envelope reassembly *Other structural reassembly

  4. Stages of mitosis: Start  4 meters of DNA in 46 chromosomes per G2 cell.

  5. The Microtubule Organizing Center: A. Animal cells: centrosome B. Yeast: spindle pole body. Major Components: 1) Centriole pair surrounded by pericentriolar material. 2) g-tubulin - acts as tether for a and b tubulin and microtubule nucleation (+ and - ends). 3) Motor proteins: Dynein and kinesin. (+ accessory proteins)

  6. Centrosomes and Centrioles: 1) Duplication at G1/S boundary is distinct from maturation during G2/M transitions. A) Control of duplication: Protein kinases Mps1p (S. cerevisiae)- (M. Winey-Univ. of Colorado) Cyclin E and A/cdk2 - (T. Stearns-Stanford) B) Centrosome maturation: growth in size during G2/M. -Recruitment of many cytoplasmic proteins. -Nucleation of microtubules. Centrosome proteins: Maybe ~50, including signaling proteins, chaperones, motor proteins, structural proteins. (Hsp 90, Lange et al., 2000, EMBO J. Vol. 19: p1252)

  7. Centrosome maturation: role of phosphorylation. A. Regulation by protein kinases: Polo-like kinases (Plk). -Human homologue to Drosophila polo gene product. -Plk active during M-phase and may regulate entry and exit. B. Plk regulation of G2/M transitions: Microinjection studies showed profound effects on centrosome maturation and bipolar spindle formation. *Differences between normal diploid cells and tumor cells suggested the presence of a centrosome checkpoint. C. Plk is regulated by upstream kinases: D. Potential target proteins: -Centrosome? -Cdc25 -Anaphase promoting complex (APC) (3 proteins: Cdc16,27, and Tsg24) (E. Nigg-Univ. of Geneva/Max Planck) (J. Maller-Univ. of Colorado)

  8. Nuclear Envelope Breakdown (NEB) during mitosis: A. Nuclear envelope versus nuclear membrane: Envelope consists of: Nuclear pore complex Outer membrane Inner membrane Lamina: -(A/C and B-type lamins) -lamina-associated polypeptides (LAPs) -lamin B receptor (LBR) B. Rapid NEB at the end of prophase. C. NEB regulated by phosphorylation of lamins and other lamina proteins: Not simply due to increases in cdc2/cyclin B activity. Other lamin kinases: PKC, Arg-Ser (RS) kinase, PKA D. Reassembly of nuclear envelope following dephosphorylation: Protein Phosphatase 1 (PP1) targeting of lamin B involved.

  9. NEB: Regulation may involve functional nuclear pores and lamina. A. Lamin B and p56 (inner membrane protein and putative lamin B receptor) interactions weakened by hyperphosphorylation. B. Intact nuclear pore complexes may be required for hyperphosphorylation. C. Similarly, nuclei disassembly does not occur without lamina. D. Pores likely provide access to lamin kinases. (+) mitotic extracts From Collas (1998) J. Cell Science, Vol.111:1293.

  10. Organelle disassembly during mitosis: Golgi fragmentation Warren (Yale): Cdc2 mediated GM130 phosphorylation and p115 interactions. Malhotra (UCSD): MEK1 activity.

  11. Entry into mitosis: activation of cdc2: A. Cdc2 activity regulated by phosphorylation and association with cyclin B. 1) Phosphorylation on Thr 161, Thr 14, and Tyr 15. Inhibitory phosphorylations: -Wee1 / Mik1 target Tyr 15. -Myt1 targets Thr 14 and Tyr 15. 2) Successful G2: active Cdc25 and cdc2 Thr 14 and Tyr 15 dephosphorylation. (H. Piwnica-Worms, Wash. Univ.) (P. Russell-Scripps) 3) Cyclin B phosphorylation and nuclear translocation. Kinases involved - cdc2 and others? 4 known phosph’n sites- Serines at position 94, 96, 101, 113. (S. Kornbluth - Duke Univ.)

  12. Centromere / Kinetochore: From: Rieder and Salmon (1998) Trends in Cell biology, Vol 8: p310.

  13. Poleward movement at kinetochores: 1) ~2 mm per minute. 2) Oscillation between poleward shortening (MT depolymerization) and growing away from pole (MT polymerization). 3) Movement mediated by motor proteins. 4) Unresolved questions: Why switch between states? What controls switching? (R. McIntosh-Univ. of Colorado) Figure from: Rieder and Salmon (1998) Trends in Cell biology, Vol 8: p310.

  14. Spindle Assembly Checkpoint: (also called mitotic or kinetochore-attachment checkpoint) Function: Inhibit progression into anaphase in order to prevent chromosome segregation errors. Questions: 1) What senses proper MT attachment or tension? 2) How does one unattached chromosome inhibit anaphase in the attached chromosomes? (G. Gorbsky-Univ. of VA) (A. Murray-UCSF) (B. Nicklas-Duke) Figure from: W. Wells (1996) Trends in Cell biology, Vol 6: p228.

  15. Spindle checkpoint proteins: MAD (mitotic arrest-deficient) BUB (budding uninhibited by benzimidazole) MAD1/2/3 - All interact with anaphase promoting complex proteins (at least in 2-hybrid screens) and are thought to inhibit. MAD1-phosphoprotein (by Mps1?) (pre-meta kinetochores). MAD2-cointeracts with MAD1 (pre-meta kinetochores). MAD3-unknown function. BUB1/2/3 - BUB1 - S/T kinase (pre-meta kinetochores). BUB2 - unknown function. BUB3 - associates with BUB1, BUB1 substrate (pre-meta kinetochores).

  16. Mitotic Exit: Anaphase Promoting Complex (APC) / Cyclosome. 1) Composed of at least 8 subunits in vertebrates. 2) Catalyzes the transfer (ligation) of ubiquitins to a N-terminus 9 amino acid destruction box (D-box) sequence on cyclins and other proteins. (RXALGXIXN) E1: ubiquitin-activating enzyme. E2: ubiquitin carrier. E3: ubiquitin protein ligase. 3) Ubiquitinated proteins targeted for degradation. 4) APC activity regulated, in part, by phosphorylation. (Kinases involved and mechanisms not well defined) 5) Protein-protein interactions involved in APC activation. (ATP-independent)

  17. Regulators of APC activity during metaphase: WD40 repeat proteins - Humans; hCDC20 (p55CDC) and hCDH1 S. Cer. (Cdc20p, Cdh1p/Hct1p) D. mel. (fizzy, fzy and fizzy-related, fzr) CDC20 and CDH1 bind APC and activate cyclin ubiquitination activity. CDC20 regulates APC in mitosis and confers a strict D-box dependence on APC, CDH1 is not strictly dependent on D-box. CDC20 protein levels increase in G2/M, whereas, CDH1 levels are relatively constant throughout the cell cycle. CDH1 may facilitate G1 degradation of proteins lacking D-box (eg. Plk).

  18. Model for cell cycle regulation of APC: From Fang et al., Mol. Cell (1998) Vol.2:163 (M. Kirschner-Harvard) Degradation Cyclin B/Cdc2 Other kinases? Mitotic Checkpoint CDC20 CDH1 CDC20 and P CDC20/CDH1 APC-P CDH1 APC APC APC-P S/G2 Metaphase Early Anaphase Late Mitosis/G1

  19. Meiosis: Goal: reduce number of chromosomes by half (haploid) so that when egg and sperm unite the normal diploid number is restored. Reproductive cells (gametes): 2 rounds of division that are separated by interphase in the absence of DNA replication. Model system: Oocytes from Xenopus laevis. Stages of oocyte development: Oogenesis, Maturation, and Embryogenesis. Signaling proteins and oocyte developmental regulators: -Mechanisms of cdc2 activation. -Progesterone receptor signaling. -cAMP/protein kinase A signaling. -Mos/MEK1/p42 MAP kinase pathway.

  20. Stages of oocyte development: Figures takenfrom: Ferrell (1999) Bioesssays, 21:833-842. Oogenesis: Chromosome replication, condensation, homologous pairing of chromosomes, genetic exchange. Oocyte remains in G2-like state until progesterone induces maturation.

  21. Oocyte maturation: Progesterone-induced entry into meiosis I and germinal vesicle (nucleus) breakdown or GVBD. P GVBD F Summary of mechanisms: 1) Progest. Inhibits cAMP / PKA 2) A lag time allows for protein synthesis (Mos). 3) Mos activates p42 MAP kinase and Plx1 pathways. 4) Cdc25 activation and Myt1 inhibition promote cdc2 activation.

  22. Oocyte maturation: signaling pathways.

  23. Post-fertilization: p42 MAPK activity maintains metaphase arrest until fertilization. p42 MAPK role in subsequent mitotic phases not clear. Cdc2 p42 MAPK

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