706_S2006_PS7 - 7.06 Problem Set #7, 2006 1. Embryonic...

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1 7.06 Problem Set #7, 2006 1. Embryonic epithelial cells grown in rich culture medium appear round and symmetrical. Moving the cells to nutrient-poor medium will cause the cells to differentiate and become polarized. (a) When you move embryonic epithelial cells to nutrient-poor medium in the presence of cytochalasin D, you find that the cells remain round and symmetrical. What can you conclude from these results? (b) You wish to visualize the organization of actin in polarized epithelial cells. How could you visualize actin? (c) Microvilli are cellular projections located on the apical surface of some epithelial cells (such as the intestinal epithelial cells we learned about earlier) that increase surface area. They contain a core of actin filaments arranged in bundles. You suspect that these bundles are maintained by actin-bundling proteins. Actin-bundling proteins are one of the many kinds of actin-binding proteins we learned about in class. In general, how might you isolate and identify actin-binding proteins? (d) Once you have identified actin-binding proteins by way of part (d) , what experiments might you use to see whether it is possible that these proteins are specifically actin-bundling proteins? (e) You are interested in studying the dynamics of actin assembly. You have purified actin that is covalently labeled with a fluorescent dye and a fluorimeter that can be used to measure fluorescence. You have already determined that centrifugation can separate G-actin from F- actin, and that fluorescence will be directly proportional to the amount of actin present in either form. Explain how you would use these tools to study the kinetics of actin assembly.
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2 2. Bart, a stellar undergraduate taking 7.06, decides to pursue his budding fascination with the microtubule cytoskeleton by taking on a new UROP project. He plans on studying microtubule formation and dynamics both in vivo and in vitro . Being the eager beaver that he is, he wakes up at the crack of dawn on Tuesday morning to start his first experiment. Bart adds GTP and purified tubulin to a test tube and observes microtubule polymerization in vitro . He takes a few time points up until 9:30 am (he doesn’t want to be late to 7.06 lecture, after all), and observes the following: amount of polymer time 1 2 (a) Explain Bart’s results at a molecular level – that is, what is happening inside his test tube over time, and what do the stages numbered above represent? (b)
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This note was uploaded on 01/23/2012 for the course BIOLOGY lsm1301 taught by Professor Seow during the Spring '11 term at National University of Singapore.

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706_S2006_PS7 - 7.06 Problem Set #7, 2006 1. Embryonic...

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