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Milap Thaker - Genetics - Harvard University - Molecular PowerPoint Presentation
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Milap Thaker - Genetics - Harvard University - Molecular

Milap Thaker - Genetics - Harvard University - Molecular

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Milap Thaker - Genetics - Harvard University - Molecular

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  1. DNA Damage Checkpoints Inhibit Mitotic Exit by Two Different Mechanisms Fengshan Liang and Yanchang Wang MOLECULAR AND CELLULAR BIOLOGY, July 2007, p. 5067–5078 Presenter: MilapThaker Proseminar in Molecular and Cellular Biology Harvard University Date 11/29/2007

  2. Key Point 1: Two Different Mechanisms Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  3. Key Point 1: Two Different Mechanisms Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  4. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  5. Relevant context: How to activate Mitotic Exit Network? Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  6. Mitotic Exit Network: Take 2 MilapThaker - Graduate Proseminar - Molecular and Cellular Biology

  7. Alternate Mechanism: The FEAR Pathway Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  8. MEN VS. FEAR: Specific Temporal Differences Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  9. Materials and Methods • Yeast Strains grown in YPD (standard yeast-extract-peptone-dextrose) medium • Arrested in G1 by 5 µg/ml α-factor • After 2 hours, washed 2x with water • Cytology: Cells fixed with 3.7% formaldehyde for 5 min at RT, then washed twice with PBS buffer (phosphate buffered saline) and resuspended in PBS Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  10. DAPI Staining Technique • Cells fixed with 100% MeOH at -20 Celsius for 30 mins. • Cells pelleted, resuspended in DAPI at a final concentration of 2.5µg/ml for 1 min at RT • Cells finally resuspended in 1X PBS • Fluorescence microscopy used to examine GFP and DNA Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  11. Protein Techniques For Immunoprecipitation, cells harvested (50 ml) and protein extracts are prepared with RIPA RIPA = Radioimmunoprecipitation Assay Incubated with Anti-HA (hemagglutinin) antibody Washed in SDS (sodium dodecyl sulfate) protein-loading buffer Boiled 5 mins, resolved with 8% SDS gel

  12. Results 1a KEY ELEMENTS TO NOTE: WILD TYPE HIGH COLONIZATION LANE 2 NO COLONIZATION ALSO, NOTE LANE 6 HIGH COLONIZATION (rad53) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  13. Results 2 – short spindling Fig 1b Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  14. Results 3 – Rebudding Fig 1c Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  15. Re-Incubated Cells Fig 1c Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  16. Results 4 – cdc 14 release Fig 2a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  17. Cdc 14 Localization - Images Fig 2a(2) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  18. Cdc 14 Release Fig 2B Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  19. Rebudding of Cells (2) Fig 3a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  20. Rebudding of Cells (3) Fig 3a(2) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  21. Cdc 14 Release (2) Fig 3B Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  22. Budding, CDC 14 Release (3) Fig 4a,b Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  23. Microcolonies (2) Fig 4C Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  24. Budding, CDC 14 (3) Fig 5a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  25. Budding, Cdc 14 Release (4) Fig 5B Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  26. Microcolonies (3) Fig 5C Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  27. Budding, Cdc14 Release (5) Fig 6a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  28. Budding, Cdc 14 Release (6) Fig 6B Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  29. Colonization across states Fig 6C Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  30. Nuclear Localization, Segregation Fig 7a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  31. Budding, cdc 14 Localization (6) Fig 7b Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  32. Cdc 5-1 mutation vs. Cdc5-1RAD53-21 Fig 7c Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  33. Western Blot, States Figs 8a, 8b Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  34. Master Conclusion Set (1) Fig 8c Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  35. Men & Fear: Proposed Mechanism Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  36. Master Conclusion Set (2) • Rad53 key player in mitotic exit prevention • This is because cdc13-1, rad53021 mutants exhibited cdc14 release • Rad 53 kinase known to phosphorylate Dun1 directly, known activator of Dun1 • Dun1 plays a more direct role in regulating MEN than Rad53 (downstream) • Authors contend possibility that Dun1 kinase phosphorylates Bfa1 and prevents Bfa1/Tem1 dissociation. • Support: Less Bfa1-Tem1 complex in Rad53-21 when DNA damaged Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

  37. Master Conclusion Set (3) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology