Lecture 28: Cell Cycle
- Created by: annie.edge
- Created on: 05-04-16 20:48
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- Cell Cycle (Lec. 28)
- Experiments: How do we measure the cell cycle?
- 1. How to determine if cells are in G0 or actively cycling?
- Ki-67 protein is expressed when cycling
- % of Ki-67 marked cells gives Ki-67 index, or % of actively cycling cells
- Ki-67 protein is expressed when cycling
- 2. How to measure if in G1, S, or G2?
- Measure DNA content labelled with red fluorescent, propidium iodide
- G1 phase: 2N, G2 phase: 4N, S phase: in between
- Measure with a flow cytometer
- Measure DNA content labelled with red fluorescent, propidium iodide
- 3. How long the cell cycle takes to complete?
- Roughly-- can count # of cells every 24 hours
- Assumes no cells die/ all actively cycling
- Accurately: use FUCCI
- Counts proteins Cdt1 (RFP, active in G1) and Geminin (GFP, in S/G2/M)
- Observe color change on video
- Counts proteins Cdt1 (RFP, active in G1) and Geminin (GFP, in S/G2/M)
- Roughly-- can count # of cells every 24 hours
- 1. How to determine if cells are in G0 or actively cycling?
- Checkpoints: How do cells know when to begin M-phase?
- DNA damage checkpoint (Start Mitosis)
- Cyclin B protein accumulates from S thru M, forms a Cyclin-Cdk1 complex in late G2
- 2nd layer of regulation: Cdc25 phosphatase activates 2 sites on complexed Cdk1
- If double strand breaks, cascade of Chk1/2 kinases activate the degradation of Cdc25
- Prevents dephosphorylation of Cdk1 complex --> G2/M arrest
- Cyclin B protein accumulates from S thru M, forms a Cyclin-Cdk1 complex in late G2
- Spindle assembly checkpoint (Start Dividing)
- APC/C activates/ degrades securin, which allows separase to to cleave cohesin
- APC/C is activated if chromosomes are not attached/ MT tension is incorrect
- DNA damage checkpoint (Start Mitosis)
- Mitosis
- Prophase: condense DNA into chromosomes
- Metaphase: line up @ metaphase plate (spindle assembly forms)
- Anaphase: sister chromatids split apart
- Centromere-kinetochore complex: motor protein attached to MT attached to kinetochore
- Inner/outer kinetochore; anaphase-promoting complex (APC/C) is on the kinetochore complex
- Cohesin proteins bind sister chromatids together
- Separase enzyme cleaves cohesin during anaphase
- Securin protein inhibits separase until APC/C signal degrades it
- Centromere-kinetochore complex: motor protein attached to MT attached to kinetochore
- Telophase: cells pulled to each end, and membranes re-form
- Interphase (G1, S-phase [DNA synthesis], G2) + Mitosis
- Cells can leave the cycle in G0
- Experiments: How do we measure the cell cycle?
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