Module 37: Cell Cycle Overview (PDF)

MODULE 37 Slide 2: Cell Cycle Overview At any given time, only a small percentage of cells in a tissue or cell culture are typically observed in mitosis. This is because mitosis is just one phase of the cell cycle, and it’s relatively short compared to the other phases. Cell Cycle Phases: Interph...

MODULE 37 Slide 2: Cell Cycle Overview At any given time, only a small percentage of cells in a tissue or cell culture are typically observed in mitosis. This is because mitosis is just one phase of the cell cycle, and it’s relatively short compared to the other phases. Cell Cycle Phases: Interphase: G1 Phase (Gap 1): Cell growth and preparation for DNA synthesis. S Phase (Synthesis): DNA replication; centrosomes duplicate. G2 Phase (Gap 2): Final preparations for mitosis, with continued growth and protein synthesis. M Phase (Mitosis): Prophase: Chromosomes condense, nuclear envelope disassembles. Prometaphase: Mitotic spindle attaches to kinetochores. Metaphase: Chromosomes align at the metaphase plate. Anaphase: Sister chromatids separate and move to opposite poles. Telophase and Cytokinesis: Chromosomes decondense, nuclear envelope re-forms, and the cell divides. Slide 3: Cell Cycle Analysis - Flow Cytometry DNA per Cell (X) vs. Numbers of Cells (Y) In this flow cytometry profile, the G₁ phase peak (left peak) is the greatest peak. This indicates that the majority of cells are in the G₁ phase at any given time, with a single set (1x) of DNA. This is typical because cells spend the most time in the G₁ phase, where they grow and carry out regular functions before preparing for DNA replication. To summarize: Greatest peak: G₁ phase (left peak) – most cells are here. Smaller peaks: S phase (middle region) and G₂/M phase (right peak) – fewer cells are in these phases since they are shorter parts of the cell cycle. Pi Intensity (X) vs. Counts (Y) The tallest peak (G₀/G₁) shows most cells are in this resting or growth phase. The S phase region has fewer cells, as DNA synthesis is a shorter part of the cell cycle. The G₂/M peak is relatively small, as these phases are also brief. The Sub-G1 region may suggest some cell death or apoptosis in the sample. MODULE 37 Slide 4: Determining Cell Cycle Duration - Radioactive Thymidine Labeling Radioactive Thymidine Labeling A brief, ½ hour pulse labels the cells in S phase (i.e., during DNA synthesis) The time until the label shows up in mitotic cells equals the length of G2 phase One mitotic peak to another equals the length of one cell cycle PERCENTAGE OF CELLS = TIME OF CELL CYCLE PHASES From the labeling, the length of the cell cycle and G2 phase are calculated; measuring the percentage of cells in S and M phases allows for the calculation of the phase length of each For example, if the cell cycle is 24 hours, and 25% of cells are in S phase, then S phase is 6 hours Once you know the length of the cell cycle, S, G2, and M phases, the remainder of the cycle duration is the length of G1 phase Slide 5: Cell Fusion Experiments Cell Fusion Evidence for the Role of Cytoplasmic Chemical Signals in Cell Cycle Regulation SPF (S Phase Promoting Factor): S can induce S in G1 but not G2 (SPF) MPF (Mitosis Promoting Factor): M can induce M in G1, S, or G2 (MPF) Slide 6: Cell Cycle Regulation in Yeast Cyclin concentrations: highest in late G1 and late G2CDK Binding with Cyclin: CDK initially binds with a mitotic cyclin to form an inactive Mitosis Promoting Factor (MPF) complex. MODULE 37 This image depicts the activation process of Cyclin-Dependent Kinase (CDK) in yeast, specifically through the roles of Wee1, CAK (CDK-activating kinase), and Cdc25. Here’s a breakdown of the steps: 1. CDK Binding with Cyclin: CDK initially binds with a mitotic cyclin to form an inactive Mitosis Promoting Factor (MPF) complex. 2. Wee1 Phosphorylation: Wee1, a kinase, phosphorylates CDK at the Y15 residue, keeping the MPF complex inactive despite the presence of cyclin. 3. CAK Phosphorylation: CAK phosphorylates CDK at T161, resulting in a double-phosphorylated, but still inactive, MPF complex due to the inhibitory phosphorylation on Y15. 4. Cdc25 Activation: Cdc25, a phosphatase, removes the inhibitory phosphate from Y15, leaving the activating phosphate on T161. This activates the MPF complex. 5. Active MPF: The MPF complex is now active, enabling it to bind to substrates, which promotes progression through the cell cycle. Slide 8: Cyclins in the Mammalian Cell Cycle Cyclins accumulate and are degraded in a regulated manner to ensure proper cycle progression. Slide 9: Cyclin Levels and Cell Cycle Progression G₁/S cyclins: essential for the control of the cell cycle at the G₁/S transition Cyclin A / CDK2 – active in the S phase Cyclin D / CDK4, Cyclin D / CDK6, and Cyclin E / CDK2 – regulate the transition from the G₁ phase to the S phase. G₂/M cyclins – essential for the control of the cell cycle at the G₂/M transition (mitosis). G₂/M cyclins accumulate steadily during G₂ and are abruptly destroyed as cells exit from mitosis (at the end of the M-phase). Cyclin B / CDK1 – regulates progression from G₂ to M phase Slide 10: Summary of Cell Cycle Progression The cell cycle consists of interphase (I) and the M phase, with each phase further divided into significant and distinct events. Flow cytometry, radioactive labeling, and cell fusion experiments are methods to determine cell cycle parameters. Cell cycle complexes are made up of cyclins and cyclin-dependent kinases (CDKs). The progression of the cell cycle is driven by phosphorylation and dephosphorylation patterns in these complexes (e.g., Wee1, CAK, and CDC25 in yeast). The mammalian cell cycle is more intricate, featuring a greater variety of cyclins and CDKs than in yeast.