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Unformatted text preview: 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? The assembly of F-actin is required for the establishment of cell polarity. (b) You wish to visualize the organization of actin in polarized epithelial cells. How could you visualize actin? You could perform immunofluorescence using anti-actin antibodies or you could label the actin using fluorescently labeled phalloidin. If you wanted to examine the localization of actin in live cells, you could transfect your cells with a DNA construct that expresses a translational actin-GFP fusion and visualize actin in real time by fluorescence microscopy. (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? You could use an affinity chromatography approach where actin is conjugated to the column matrix. You could prepare a cell lysate from your cell type of interest and run the lysate over your column. After washing, the proteins bound to the matrix actin could be eluted using excess purified actin. The eluate could then be run on an SDS-PAGE gel and then the gel could be stained. The identity of the protein bands on the gel would then be determined by mass spectrometry. (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? You could look to see if these proteins are found in the microvilli. However this would only show a correlation, and would not prove that your proteins are actually causing the bundling of microfilaments. To show this, you could add your actin-binding proteins to in vitro polymerization assays, and look to see how they affect the formation of microfilaments in vitro. You could also isolate mutations in the genes encoding these proteins, and see if mutant cells do not bundle their actin properly in the microvilli. 2 (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...
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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.
- Spring '11