MOLECULAR AND CELLULAR BIOLOGY ,  July 2007, p. 5067–5078 Presenter: Milap Thaker Proseminar in Molecular and Cellular Bio...
Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Key Point 1: Two Different Mechanisms
Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Key Point 1: Two  Different  Mechanisms
Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Relevant context:  How to activate Mitotic Exit Network?
Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Mitotic Exit Network: Take 2
Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Alternate Mechanism: The FEAR Pathway
Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology MEN  VS. FEAR: Specific Temporal Differences
Materials and Methods <ul><li>Yeast Strains grown in YPD (standard yeast-extract-peptone-dextrose) medium </li></ul><ul><l...
DAPI Staining Technique <ul><li>Cells fixed with 100% MeOH at -20 Celsius for 30 mins. </li></ul><ul><li>Cells pelleted, r...
<ul><li>For Immunoprecipitation, cells harvested (50 ml) and protein extracts are prepared with RIPA </li></ul><ul><li>RIP...
KEY ELEMENTS TO NOTE: WILD TYPE HIGH COLONIZATION LANE 2 NO COLONIZATION ALSO, NOTE LANE 6 HIGH COLONIZATION (rad53) Milap...
Results 2 – short spindling Fig 1b Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Results 3 – Rebudding Fig 1c Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Re-Incubated Cells Fig 1c Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Results 4 – cdc 14 release Fig 2a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Cdc 14 Localization - Images Fig 2a(2) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Cdc 14 Release Fig 2B Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Rebudding of Cells (2) Fig 3a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Rebudding of Cells (3) Fig 3a(2) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Cdc 14 Release (2) Fig 3B Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Budding, CDC 14 Release (3) Fig 4a,b Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Microcolonies (2) Fig 4C Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Fig 5a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Budding, CDC 14 (3)
Fig 5B Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Budding, Cdc 14 Release (4)
Fig 5C Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Microcolonies (3)
Budding, Cdc14 Release (5) Fig 6a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Fig 6B Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Budding, Cdc 14 Release (6)
Colonization across states Fig 6C Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Fig 7a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Nuclear Localization, Segregation
Budding, cdc 14 Localization (6) Fig 7b Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Cdc 5-1 mutation vs. Cdc5-1RAD53-21 Fig 7c Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
Figs 8a, 8b Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Western Blot, States
Fig 8c Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Master Conclusion Set (1)
Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Men & Fear: Proposed Mechanism
<ul><li>Rad53 key player in mitotic exit  prevention </li></ul><ul><ul><li>This is because cdc13-1, rad53021 mutants exhib...
Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Master Conclusion Set (3)
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Milap Thaker - Biology Powerpoint: Harvard University - DNA Damage Checkpoints

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  • Specific areas of note: Cdc13-1 has high probability of short spindles Also, note that cdc13-1 rad 53 mutants look “most healthy” of mix
  • Milap Thaker - Biology Powerpoint: Harvard University - DNA Damage Checkpoints

    1. 1. MOLECULAR AND CELLULAR BIOLOGY , July 2007, p. 5067–5078 Presenter: Milap Thaker Proseminar in Molecular and Cellular Biology Harvard University Date 11/29/2007 Fengshan Liang and Yanchang Wang
    2. 2. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Key Point 1: Two Different Mechanisms
    3. 3. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Key Point 1: Two Different Mechanisms
    4. 4. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    5. 5. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Relevant context: How to activate Mitotic Exit Network?
    6. 6. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Mitotic Exit Network: Take 2
    7. 7. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Alternate Mechanism: The FEAR Pathway
    8. 8. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology MEN VS. FEAR: Specific Temporal Differences
    9. 9. Materials and Methods <ul><li>Yeast Strains grown in YPD (standard yeast-extract-peptone-dextrose) medium </li></ul><ul><li>Arrested in G1 by 5 µg/ml α -factor </li></ul><ul><li>After 2 hours, washed 2x with water </li></ul><ul><li>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 </li></ul>Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    10. 10. DAPI Staining Technique <ul><li>Cells fixed with 100% MeOH at -20 Celsius for 30 mins. </li></ul><ul><li>Cells pelleted, resuspended in DAPI at a final concentration of 2.5µg/ml for 1 min at RT </li></ul><ul><li>Cells finally resuspended in 1X PBS </li></ul><ul><li>Fluorescence microscopy used to examine GFP and DNA </li></ul>Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    11. 11. <ul><li>For Immunoprecipitation, cells harvested (50 ml) and protein extracts are prepared with RIPA </li></ul><ul><li>RIPA = Radioimmunoprecipitation Assay </li></ul><ul><li>Incubated with Anti-HA (hemagglutinin) antibody </li></ul><ul><li>Washed in SDS (sodium dodecyl sulfate) protein-loading buffer </li></ul><ul><li>Boiled 5 mins, resolved with 8% SDS gel </li></ul>Protein Techniques
    12. 12. 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. 13. Results 2 – short spindling Fig 1b Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    14. 14. Results 3 – Rebudding Fig 1c Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    15. 15. Re-Incubated Cells Fig 1c Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    16. 16. Results 4 – cdc 14 release Fig 2a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    17. 17. Cdc 14 Localization - Images Fig 2a(2) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    18. 18. Cdc 14 Release Fig 2B Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    19. 19. Rebudding of Cells (2) Fig 3a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    20. 20. Rebudding of Cells (3) Fig 3a(2) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    21. 21. Cdc 14 Release (2) Fig 3B Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    22. 22. Budding, CDC 14 Release (3) Fig 4a,b Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    23. 23. Microcolonies (2) Fig 4C Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    24. 24. Fig 5a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Budding, CDC 14 (3)
    25. 25. Fig 5B Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Budding, Cdc 14 Release (4)
    26. 26. Fig 5C Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Microcolonies (3)
    27. 27. Budding, Cdc14 Release (5) Fig 6a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    28. 28. Fig 6B Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Budding, Cdc 14 Release (6)
    29. 29. Colonization across states Fig 6C Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    30. 30. Fig 7a Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Nuclear Localization, Segregation
    31. 31. Budding, cdc 14 Localization (6) Fig 7b Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    32. 32. Cdc 5-1 mutation vs. Cdc5-1RAD53-21 Fig 7c Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology
    33. 33. Figs 8a, 8b Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Western Blot, States
    34. 34. Fig 8c Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Master Conclusion Set (1)
    35. 35. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Men & Fear: Proposed Mechanism
    36. 36. <ul><li>Rad53 key player in mitotic exit prevention </li></ul><ul><ul><li>This is because cdc13-1, rad53021 mutants exhibited cdc14 release </li></ul></ul><ul><ul><li>Rad 53 kinase known to phosphorylate Dun1 directly, known activator of Dun1 </li></ul></ul><ul><ul><li>Dun1 plays a more direct role in regulating MEN than Rad53 (downstream) </li></ul></ul><ul><ul><li>Authors contend possibility that Dun1 kinase phosphorylates Bfa1 and prevents Bfa1/Tem1 dissociation. </li></ul></ul><ul><ul><li>Support: Less Bfa1-Tem1 complex in Rad53-21 when DNA damaged </li></ul></ul>Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Master Conclusion Set (2)
    37. 37. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology Master Conclusion Set (3)

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