Recently, you identified a new protein in human cells, Xuf7. To elucidate the fu
ID: 266289 • Letter: R
Question
Recently, you identified a new protein in human cells, Xuf7. To elucidate the function of this protein, you have undertaken studies in which Xuf7 either has been depleted from cells, or its levels have been increased by overexpression of exogenous Xuf7. The consequences on progression through mitosis were then assessed. Based on your data (shown below) and your knowledge of the cell cycle, answer the following questions.
(a) In control cells, why do you see a decrease in Cyclin B levels over time?
(b) When control cells are treated with nocodazole, why do Cyclin B levels NOT decrease?
(c) What are the consequences of depleting Xuf7 from nocodazole-treated cells? What defect do you think depletion of Xuf7 is causing?
Time in minutes (0 indicates mitotic entry) 0 10 20 30 40 50 60 70 1. control anti- 2. control + nocz Cyclin B Western blots 3. Xuf 7-depleted 3. Xuf7-depleted + noczExplanation / Answer
1) Cyclin B levels rise during mitosis phase of the cell cycle. Binding of CdK1 to cyclin B, which are then activated during mitosis phase. The CDk1-cyclin B complex phosphorylates key proteins causing reorganization of the cell and separation of sister chromatids of the mitotic spindle.
As the cell exits the mitosis phase, cyclin B levels should fall. Mitosis in cell last for an hour. Hence, there is a gradual decline in cyclin B protein 30 min post entry into mitosis (O minutes) in control cells. The expression of cyclin B in control cells declines completely by one hour (60 minutes). By this time, Mitosis is completed. Degradation of cyclin B by proteosome-dependent mechanism is required for exit from mitosis after the completion of microtubule attachment to kinetochores. Anaphase-promoting complex (APC) is involved in this degradation. Degradation of cyclin B is required for onset of anaphase. It also inactivates M-phase promoting factor to exit mitosis.
2) When control cells are treated with nocodazole, microtubules depolymerization is induced, that results in loss of microtubules. Cyclin B is associated with unattached kinetochores during prometaphase where it contributed to microtubule attachment to kinetochores. After the kinetochore-microtubule attachment is completed, cyclin B is transported from each kinetichore. As there is loss of microtubules in nocodazole treated cells, microtubules will not be attached to kinetochores in prometaphase cells. A false signal is created to prometaphase cells that there is insufficient amount of cyclin B to proceed through mitosis. Hence, the cells will keep synthesizing cyclin B even after 60-70 minutes. Nocodazole prevents the entry of prometaphase cells into anaphase.
3) Depletion of Xuf7 to control cells decreases cyclin B protein faster. Hence, the cells will exit mitosis faster that in control conditions. Mitosis is completed by 30 min in the Xuf7 depleted cells as compared to 1 hour in control cells. Depletion of Xuf7 from nocodozole treated cells also causes down regulation of cyclin B in the cells during mitosis and possible exit from Mitosis faster. Mitosis is completed by 30 min in Xuf7 depleted cells in presence and absence of nocodazole.
Thus, Xuf7 depletion is possibly blocking the action of nocodozole and preventing microtubule depolymerization and subsequent loss of microtubules. Further, it enhances the binding of microtubules to kinetochore. Xuf7 depletion causes degradation of cyclin B to allow cells to enter anaphase faster. Low cyclin B levels will hasten inactivation of M-phase promoting factor. Faster exit from mitosis will result in enhanced proliferation and tumorigenesis.
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