Lecture 28: Cell Cycle

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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
      • 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
      • 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
    • 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
      • 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
    • 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
      • 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


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