Cell Cycle Stages (00. MODULE 37 P.PDF)

Slide 3: The cell cycle consists of the interphase (I) and M phase. Interphase is subdivided into the G1 phase (when the cell grows), S phase (when the cell duplicates DNA and centromere), and G2 phase (when the cell grows again). The M phase consists of mitosis and cytokinesis. Typically, a small p...

Slide 3: The cell cycle consists of the interphase (I) and M phase. Interphase is subdivided into the G1 phase (when the cell grows), S phase (when the cell duplicates DNA and centromere), and G2 phase (when the cell grows again). The M phase consists of mitosis and cytokinesis. Typically, a small percentage of cells in a tissue or cell culture is in mitosis at any given time. We will discuss mitosis in more detail in a future presentation; note that it is subdivided into prophase, prometaphase, metaphase, anaphase, and telophase, followed by cytokinesis. Each phase of mitosis has events associated with it (please, study the information on the slide and make sure that you know these events). Cells that are not proliferating and are “outside the cell cycle” (i.e., in the resting phase, quiescent cells) are said to be in G0. 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 4: One way of measuring the cell cycle is via flow cytometry. DNA is stained and the amount of DNA per cell is tracked (on the X-axis) vs. the number of cells (on the Y-axis). DNA content of 1 is indicative of phase G1. DNA content increases from 1 to 2 in the S phase (this is the DNA replication phase). DNA content of 2 is detected in the G2/M phase. Sub-G1, with a DNA content of less than 1 is when cells undergo apoptosis/necrosis. Slide 5: Radioactive thymidine labeling is another method for determining the phases of the cell cycle and how long each phase is. Thymidine is a DNA base; it is taken up into the cells and incorporated into the DNA during the S phase (during DNA synthesis). A brief, ½ hour pulse of labeling is applied. The time until the radioactive label shows up (is detected) in all mitotic cells equals the length of phase G2. G2 is the gap of time (i.e., the phase) separating S from M; therefore, the time it takes from the initial labeling pulse to the time when all mitotic cells are labeled is G2. The length of a cycle is measured as the length between one mitotic peak to another. Going from one M phase to the next M phase is one full cell cycle. This cycle length differs between cell types. Many human cancer cells in culture have a cell cycle of approximately 24 hours. How do we measure the length of the S and M phases? Please, remember that: THE PERCENTAGE OF CELLS IN A CYCLE PHASE = LENGTH OF THIS CELL CYCLE PHASE The longer the phase of the cell cycle is, the greater is the probability of finding cells in that phase. From a labeling experiment, one can find the total cell cycle time, as well as G2. One can also measure the percentage of cells in S and M phases, and then calculate how long each phase is. For example, if the cell cycle is 24 hours, and 25 % of the cells are in the S phase, then S is 6 hours long. Once you know the length of the cell cycle, S, G2, and M phases, the remainder of the time is G1 [i.e., cell cycle length – (S + M + G2) = G1] Slide 6: Does a Mitosis Promoting Factor (MPF) exist? What about an S phase-promoting factor (SPF)? Both factors exist. Cell fusion experiments have demonstrated that fusing an M- phase cell with a second cell in another phase of the cell cycle “pushes” the second cell into the M phase. Thus, an M-phase cell induces the M phase in G1-, S-, or G2-phase cells. However, an S-phase cell can induce the S phase in G1-phase but not in the G2-phase cell. Why? The cell cycle moves forward, not backward. The G2-phase cell cannot re-duplicate its DNA (the cell has already gone through the S phase). Slide 7: Consider yeast, a simple eukaryotic organism. The Mitosis Promoting Factor (MPF) is a cdc2 kinase complexed with mitotic cyclins. MPF levels are highest in late G2 and MPF pushes the cell into the M phase (mitosis). The SPF is the same kinase complexed with G1 cyclins. SPF levels are highest in late G1, and SPF pushes the cell into the S phase. Slide 8: While complex formation is necessary for cell cycle progression, it is not sufficient. Let us consider the G2 to M transition in yeast. The complex of cdc2 kinase and mitotic cyclins is required, but this complex might be inactive. Two kinases play a role in the G2 to M transition: Wee1 and CAK. Wee1 phosphorylates the cdc2 kinase at tyrosine 15 (Y15), and this is an inactivating modification. CAK phosphorylates the cdc2 kinase at threonine161 (T161), and this is an activating modification. When both are phosphorylated at the same time, the Y15 is dominant, and the complex is inactive, the cell is in G2 and growing. When it is time to enter the M phase, a phosphatase Cdc25 removes the phosphorylation at Y15, allowing the active complex to promote mitosis. These interactions were discovered with yeast mutants. Specifically, temperature-sensitive yeast mutants were used. At one temperature, such mutant yeast cells are normal’ whereas, at another temperature, the mutation is manifested (through a phenotype). Here are some of the analyzed mutants: A Wee1 mutant cannot add the phosphor group to Y15, and the cell enters the M phase too quickly. Such cell does not grow enough in G2, and the daughter cells are small (“wee” means “small”). A Cdc25 mutant cannot remove the phosphor group from Y15, and the cell is “stuck” in G2; the cell becomes big. A CAK mutant is likely similar to a Cdc25 mutant (think why). Slide 9: The mammalian cell cycle is similar to this of yeast. However, in mammalian cells, there are more types of cyclin-dependent kinases (Cdks) and cyclins, with different sets of complexes at each phase. These complexes move the cell forward and prevent it from going backward. For example, an S-phase cell + a G2-phase cell fusion cannot make the G2 cell go back to the S phase, as the factors in the G2 cell prevent this. Slide 10: There are two main groups of cyclins: The G1/S cyclins that are essential for the control of the cell cycle at the G1/S transition --- Cyclin A / CDK2 is active in the S phase. --- Cyclin D / CDK4, Cyclin D / CDK6, and Cyclin E / CDK2 regulate the transition from G1 to S phase. G2/M cyclins that are essential for the control of the cell cycle at the G2/M transition (mitosis). G2/M cyclins accumulate steadily during the G2 phase and are abruptly destroyed as cells exit from mitosis (at the end of the M-phase). --- Cyclin B / CDK1 regulates the progression from G2 to M phase Slide 12: Fundamental points. The cell cycle is composed of the interphase and M phase. Each of these is subdivided into distinct components of events. The cell cycle parameters can be determined through flow cytometry, radioactive labeling, and cell fusion experiments. The cell cycle complexes are composed of cyclins and cyclin-dependent kinases (CDKs). The phosphorylation and dephosphorylation of these complexes drive the cell cycle forward (e.g., Wee1, CAK, and CDC25 in yeast) The mammalian cell cycle is more complex than this of yeast, with more cyclins and CDKs.