Prof. R. V. Skibbens. Cell Cycle, Cell Division and Cancer

Transcription

1 Prof. R. V. Skibbens November 18, 2010 BIOS 10: BioScience in the 21 st Century Cell Cycle, Cell Division and Cancer

2 4 Stages of the cell cycle: Genome segregation

3 Interphase DNA Microtubules

4 Microtubules as Railways Microtubule motors Dynein toward cell center Kinesin toward cell periphery

5 Interphase A single centrosome, a single microtubule array Microtubules are polarized Minus end anchored Plus end distal

6 Now that s a fish of a different color Railway is polarized Dynein move toward center Kinesin move toward periphery

7 Proportions? What are the relative contributions to Interphase... S-phase is easy...

8 Vertebrate cells analyses H 3- thymidine BrDU Relative contributions to Interphase... S-phase

9 Vertebrate cells analyses H 3- thymidine BrDU Bromo-deoxyuridine used in place of thymidine Relative contributions to Interphase... S-phase

10 Proportions? What are the relative contributions to Interphase... G1-phase also easy...

11 DNA content indicates G1 portion of cell cycle Flow cytometry - Laser-based inquiry of DNA

12 M-phase the easiest of ALL

13 Chromosomes condensed during Mitosis note TWO microtubule arrays...

14 Simple scopes - mitosis

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16 Centrosomes Microtubules Pulling Forces Kinetochores - protein complex assembled onto centromere DNA Microtubule capturing cpu complex

17 G2 is whatever is left...

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19 To understand aneuploidy have to understand dregulation of cell cycle

20 Directionality of the cell cycle S-phase promoting factor triggers DNA replication in G1 cell G1 cell is competent to enter S

21 Directionality - clocks go in only one direction No going in reverse (S-phase cell does not induce G2 cell to re-enter S-phase) G2 cell is Inhibited from Re-entering S or Only G1 cells Are competent to enter S You can t rush S

22 Directionality - clocks go in only one direction G2 cell can not induce G1 cell to replicate DNA G2 cell has lost the S-phase promoting factor Molecular mechanism For losing and gaining an Activity...

23 The Nobel Prize in Physiology or Medicine 2001 The Nobel Prize in Physiology or Medicine 2001 "for their discoveries of key regulators of the cell cycle" Leland H. Hartwell R. Timothy Hunt Sir Paul M. Nurse 1/3 of the prize 1/3 of the prize 1/3 of the prize USA United Kingdom United Kingdom Fred Hutchinson Cancer Research Imperial Cancer Imperial Cancer Center Research Fund Research Fund Seattle, WA, USA London, UK London, UK b b b budding Clam, urchin fission

24 Budding yeast (brewers yeast or bakers yeast)

25 Cell morphology and DNA content provide detailed information regarding cell cycle distribution Flow cytometry

26 Random mutagenesis allows for identification of Random mutagenesis allows for identification of genes required for specific cell phase activities

27 CDCs - Cell Division Cycle mutants ua What gene, when mutated, locks cells in mitosis (G1 or S or.. )?

28 Master Regulator of the Cell Cycle: Cyclin-dependent Kinase (CDK) Cell cycle progression requires CDK activity Control event timing sequence Respond to cues Partners in crime: Cyclin binds/activates kinase subunit

29 Master Regulator of the Cell Cycle: Cyclin-dependent Kinase (CDK) Cell cycle progression requires CDK activity Control event timing sequence Respond to cues Budding yeast - only 1 CDK = Cdc28 Partners in crime: Cyclin binds/activates kinase subunit Fission yeast only 1 CDK = Cdc2 Hmm... but regulate G1, S, G2, M

30 Cyclin-dependent Kinase (CDK) Nuclear envelope breakdown Condense chromosomes (Lamins) (Histone 1) Microtubule re-organization (MAPs) Cell cycle progression requires CDK activity Different cyclins - different substrates Transcription of DNA replication factors (Polymerase, RFCs) Initiate DNA replication (ORC)

31 Cyclin-CDK complexes: budding vertebrate G1 Cdc28 CDK4, CDK6 CLN3 Different flavors of cyclin specify CDK activities i i G1/S Cdc28 CDK2 through the cell cycle CLN1,2 S Cdc28 CDK2 CLB5,6 M Cdc28 CDK1 CLB1-4

32 Cyclin-CDK complexes: budding Different catalytic CDK kinase proteins vertebrate G1 Cdc28 CDK4, CDK6 CLN3 G1/S Cdc28 CDK2 CLN1,2 S Cdc28 CDK2 CLB5,6 M Cdc28 CDK1 CLB1-4

33 Cyclin-CDK complexes: budding Different catalytic CDK kinase proteins vertebrate G1 Cdc28 CDK4, CDK6 CLN3 Cyclin D1-3 G1/S Cdc28 CDK2 CLN1,2 S Cdc28 CDK2 Cyclin E CLB5,6 Cyclin A M Cdc28 CDK1 CLB1-4 Cyclin B... And Different cyclins

34 Cyclin-dependent Kinase (CDK) Nuclear envelope breakdown Condense chromosomes (Lamins) (Histone 1) Microtubule re-organization (MAPs) Cell cycle progression requires CDK activity Different cyclins - different substrates Unidirectionality - cyclin expression and degradation d IN TURN Transcription of DNA replication factors (Polymerase, RFCs) Initiate DNA replication (ORC)

35 CDK regulation Cyclin degradation d Mitotic cyclin degradation = Anaphase Promoting Complex or APC G1 and S-phase cyclin degradation SCF (Skp1-Cullin-F box complex)

36 Observing cell cycles through the microscope Timing - clock Not all cell divisions are equal!

37 Changes in cell cycles: developmental regulation

38 Cues and Changing the clock Nuclear:cell volume Ratio maintained Which phase changes?

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40 Low CDK G1 cells can enter Quiescence (G 0 0) G 0 Differentiated cells or Cells that control the clock via external cues Understanding cancer often involves understanding how cells Reenter the cell cycle in the absence of cues!