Biol110-08-Methods-Problem Set Key#1

Biol110-08-Methods-Problem Set Key#1 - Biol110 Cell Biology...

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Biol110 Cell Biology Spring 2008 Problem Set Key #1 ______________________________________________________________ Question #1 A novel compartment in human cells was discovered, which appears when cells are exposed to a chemical (e.g. ALLN) that inhibits the function of a cytoplasmic protease termed the proteasome. The new compartment was termed “the aggresome” and is always seen in close apposition to the nuclear envelope. How would you determine if the aggresome is a membrane-bound compartment? Describe at least two different methods (one microscopic and the other more biochemical) that one could use to test that theory. I would use microscopic techniques as well as biochemical techniques: Microscopic: First, I would use a lipophilic fluorophore such as DiOC 6 to stain cellular membranes and would look under the fluorescence microscope for a fluorescent ‘boundary’ around the aggresome. Unfortunately, this would not distinguish between a single membrane surrounding the aggresome from small membrane-vesicles ‘coating’ the aggresome (in the latter case, the proteins within the aggresome would not be membrane enclosed/protected). To examine the aggresome compartment boundaries in greater detail, I would process the cells for transmission electron microscopy (fix, dehydrate, embed in resin, section and stain the membranes with osmium tetroxide or potassium permanganate to add contrast). I would look under the microscope for the presence of the characteristic ‘railroad track’ appearance of membranes surrounding the aggresome. Serial section EM followed by three-dimensional reconstitution of the image would tell me if membrane surrounds the entire aggresome. Biochemical: I would add a fluorescently-labeled antibody that recognize a protein in the aggregate to cells whose plasma membrane has been perforated gently with digitonin. If the fluorescent antibodies concentrate on the surface of the aggresome (visible under the microscope) it would mean that its contents are exposed and not fully surrounded/protected by a membrane (note that antibody molecules cannot cross intact biological membranes). A control using immune-detection in Figure: CFTR molecules accumulate in aggresomes. ( A ) Time course of F508 accumulation. HEK cells stably expressing low
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This note was uploaded on 06/23/2008 for the course BIO 110 taught by Professor Rexach during the Spring '08 term at UCSC.

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Biol110-08-Methods-Problem Set Key#1 - Biol110 Cell Biology...

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