706_S2006_PS7key - 7.06 Problem Set#7 2006 1 Embryonic...

Info icon This preview shows pages 1–3. Sign up to view the full content.

View Full Document Right Arrow Icon
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.
Image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
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 form. Explain how you would use these tools to study the kinetics of actin assembly.
Image of page 2
Image of page 3
This is the end of the preview. Sign up to access the rest of the document.

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern