Control Cdc20 SiRNA Cdc20 Securin 20 2 18 hr in vitro culture Cdc20 is a protein

Question

Control Cdc20 SiRNA Cdc20 Securin 20 2 18 hr in vitro culture Cdc20 is a protein that physically interacts with anaphase promoting complex (APC) to regulate its activity. (A) Western blot analysis of HeLa cells (control), HeLa cells transfected with Cdc20 siRNA, and a Cdc20 mutant HeLa cell line. Blots were stained for Cdc20, Securin, and actin (as a loading control). (B) Embryos from both wild-type control mice and from mice transfected with Cdc20 siRNA were cultured in vitro for 18 hours. Phase-contrast images were captured immediately following the addition of the embryos at the two-cell stage to the culture dish left panel) and 18 hours after incubation (right panel). Asterisks indicate embryos with Cdc20 siRNA and all other embryos are from wild-type control mice. (C) Fluorescence images of embryos cultured in panel B. Embryos were stained using DAPI (DNA marker; blue) and a whole-cell fluorescence marker (to outline the cells; red). (D) Histology sections obtained from two different individuals with breast cancer. Tissue is labelled for nuclei (blue) and securin (brown) 1. What effect does Cdc20 siRNA have on APC activity? Explain 2. Which phase of mitosis is altered by Cdc20 siRNA? 3. Of the two histological images of breast cancer presented in panel D, which image might represent someone with a more aggressive form (e.g. more deadly) of breast cancer? Explain 4. In theory, what effects might the Cdc20 mutant have on cell division?

Explanation / Answer

Lets understand first how Cdc20 play its role in cell cycle. In cell cycle, APC/C (Anaphase promoting complex) is an E3 ubiqitin ligase which mediates onset of metaphase to anaphase transition. There are two substrate trarget of APC/C named CyclinB and Securin. APC target these two proteins for degradation. During the anaphase transitions,there is an event of cohesin degradation (Cohesin is a protein that keep the sister chromaid bound to each other) . This cohesin degradation is mediated by enzyme named separase. During the initiation of metaphase, securin stays attached with the enzyme separase, which results in inactivation of separase enzyme. Cdc20 binds with APC/C results in activation of it, which then ubiquitinate securin results in degradation of securin and activation of separase, therefore helps in onset of anaphase transition during cell cycle.

1) and 2) Cdc 20 SiRNA mediates degradation of Cdc20 mRNA results in no Cdc20 protein, This will results in APC/C deactivation due to the fact that APC/C required Cdc20 in order to exert its effect on cyclin B and securin degradation. Hence cell will be arrested in metaphase.

3) image 1 represent someone with more aggressive form of breast cencer as, brown colour represents the presence of securin that helps in inactivation of enzyme separase. image 1 signify absence of securin protein and without securin, separase enzyme is always active. Therefore, will results in quick transition form metaphase to anaphase and speedup the cell growth.

4) Given data of western blot sugests that , Cdc20 mutant protein is not able to activate APC/C enzyme that has resulted in securin protein in nucleus. securin is still in bound state with separase hence resulted in deactivation of separase enzyme. so we can hypothesis that This Cdc20 mutant is unable to interact with APC/C as this interaction is hampered, cell cycle has been arrested in metaphase.

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