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Presentation Transcript
fengshan liang and yanchang wang

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

key point 1 two different mechanisms
Key Point 1: Two Different Mechanisms

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

slide3

Key Point 1: Two Different Mechanisms

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

relevant context how to activate mitotic exit network
Relevant context: How to activate Mitotic Exit Network?

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

mitotic exit network take 2
Mitotic Exit Network: Take 2

MilapThaker - Graduate Proseminar - Molecular and Cellular Biology

alternate mechanism the fear pathway
Alternate Mechanism: The FEAR Pathway

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

men vs fear specific temporal differences
MEN VS. FEAR: Specific Temporal Differences

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

materials and methods
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

dapi staining technique
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

protein techniques

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

results 1a
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

results 2 short spindling
Results 2 – short spindling

Fig 1b

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

results 3 rebudding
Results 3 – Rebudding

Fig 1c

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

re incubated cells
Re-Incubated Cells

Fig 1c

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

results 4 cdc 14 release
Results 4 – cdc 14 release

Fig 2a

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

cdc 14 localization images
Cdc 14 Localization - Images

Fig 2a(2)

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

cdc 14 release
Cdc 14 Release

Fig 2B

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

rebudding of cells 2
Rebudding of Cells (2)

Fig 3a

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

rebudding of cells 3
Rebudding of Cells (3)

Fig 3a(2)

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

cdc 14 release 2
Cdc 14 Release (2)

Fig 3B

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

budding cdc 14 release 3
Budding, CDC 14 Release (3)

Fig 4a,b

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

microcolonies 2
Microcolonies (2)

Fig 4C

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

budding cdc 14 3
Budding, CDC 14 (3)

Fig 5a

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

budding cdc 14 release 4
Budding, Cdc 14 Release (4)

Fig 5B

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

microcolonies 3
Microcolonies (3)

Fig 5C

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

budding cdc14 release 5
Budding, Cdc14 Release (5)

Fig 6a

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

budding cdc 14 release 6
Budding, Cdc 14 Release (6)

Fig 6B

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

colonization across states
Colonization across states

Fig 6C

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

nuclear localization segregation
Nuclear Localization, Segregation

Fig 7a

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

budding cdc 14 localization 6
Budding, cdc 14 Localization (6)

Fig 7b

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

cdc 5 1 mutation vs cdc5 1rad53 21
Cdc 5-1 mutation vs. Cdc5-1RAD53-21

Fig 7c

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

western blot states
Western Blot, States

Figs 8a, 8b

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

master conclusion set 1
Master Conclusion Set (1)

Fig 8c

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

men fear proposed mechanism
Men & Fear: Proposed Mechanism

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

slide36

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

master conclusion set 3
Master Conclusion Set (3)

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology