Set 3 - Problem Set 3 Bi9 Cell Biology Spring 2011...

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Unformatted text preview: Problem Set 3 Bi9 Cell Biology Spring 2011 Instructions: • There are 3 Parts. • Type your answers. • Print and turn in 3 separate sheets. 1 sheet for each part. All answers for each part should not fill more than one page. • You do not have to staple the sheets together, since you will... • Write your FULL NAME on every page. • Problem Set 3 is due on Tuesday April 19th at 5 pm. A box will be available on the first floor of Broad to turn in your work. • Be sure to cite any sources you use besides Molecular Biology of the Cell. If you use websites, include link and date accessed. Problem Set 3 Part I Extracellular Matrix (20 points) You want to study how fibronectin changes conformation in response to cell ­ generated force. Your colleague suggests you use FRET to see how fibrils assemble outside the cell. A. (3 points) List three criteria you should consider when choosing a FRET pair. B. (6 points) Your colleague suggests that you use tetramethylrhodamine dextran and Oregon green 488 carboxylic acid to label opposite ends of fibronectin. Using the website below, explain which would be the donor fluorophore and which would be the acceptor fluorophore. How do you know? http://www.invitrogen.com/site/us/en/home/support/Research ­Tools/Fluorescence ­SpectraViewer.html C. (6 points) Guanidinium hydrochloride (GuHCl) is used to denature (unfold) proteins. As a control, you put your labeled fibronectin in varying GuHCl concentrations and measure the intensity at the donor and acceptor emission wavelengths. Draw a graph of acceptor emission intensity/donor emission intensity versus [GuHCl]. D. (5 points) Once you have labeled your fibronectin, you incubate it with fibroblasts and look at them after one hour and after four hours. Below are two images of a single cell taken at 1 hour and 4 hours after incubation. Based on your answers in B, explain what you are seeing in the images below. * scale bars are 10µm Part II Intracellular Compartments and Protein Sorting (20 points) A (4 points) Signal sequences for import into the ER are surprisingly diverse. Below are some sample signal sequences. Highlight two features common to all of the sequences below. Give a 1 ­2 sentence explanation of why each feature is common to signal sequences.  ­ BiP  ­ MKLSLVAAMLLLLSAARA  ­ EGF ­receptor MRPAGTAGAALLALLAALCPRA  ­ Prolactin MNIKGSPWGSLLLLLVSNLLLCQSVAP  ­ Glucagon MKSIYFVAGLFVMLVQG Your colleague is studying the role of the ER in Peptide Z is secretion. He decides to try to reconstitute the translation machinery in vitro from heterologous components. First he isolates ribosomes from rabbit reticulocytes, prepares microsomes from dog pancreas, and prepares mRNA transcripts of Peptide Z. Then he creates an assay buffer (Buffer A) that contains HEPES (pH 7.3), MgCl2, DTT, ATP, GTP, creatine phosphate, creatine phosphokinase, 14C labeled amino acids, nucleotides, proteins required for translation, and the rabbit reticulocyte ribosomes. To conduct the experiment, he incubates rough microsomes in the presence and absence of peptide Z mRNA in Buffer A at 37ºC. At the end of the incubation, your colleague uses SDS ­PAGE to assay his samples in addition to a sample of Peptide Z secreted from rabbit reticulocytes. He dries the gel and visualizes it by autoradiography. His results are shown below: B.(2 points) What makes dog pancreas cells a good model to study secretion in the ER as opposed to another cell type such as hepatocytes? (two sentences) C.(4 points) Which are the control lanes? What are they controls for?For each lane, give a one sentence summary of why it is included in the gel. D. (3 points) According to your experimental conditions, the arrows on the gel image should indicate Peptide Z. Why do you think the Peptide Z band shows up at different molecular weights under different conditions? Do not worry about the slight difference in MW of peptide Z between lanes 3 and 4. The presence of membranes in lane 4 has a modest effect on how the SDS ­PAGE runs and the slight upshift in mobility in lane 4 is not consequential. E. (3 points) Describe a model that corroborates these data. F. (4 points) Can you think of an experiment that you might do to further test the hypothesis that the two distinct forms of peptide Z observed in this experiment (e.g. lane 2 vs. lane 4) reside in different compartments? Part III Vesicle Trafficking (20 points) A. (5 points) There are multiple sources of specificity in vesicle transport. Please explain TWO of them and comment on how they work, when they exert their effects relative to budding, coating, uncoating, docking, and fusion. Why do you think there are multiple mechanisms that contribute to specificity? (Your answer shouldn’t be more than 3 lines for each of the sources) B. (5 points) The fusion of two membrane structures is very unlikely to happen without SNAREs. According to what we discussed v ­SNAREs and t ­SNAREs bind to each other to form a very strong complex that facilitates membrane fusion. Although the exact mechanism is not known, one hypothesis about how they work has been put forward which was discussed in lecture. According to this model, what is the major advantage that SNAREs provide in facilitating membrane fusion? (Explain in less than 5 lines). C. (10 points) You want to determine if SNARES are sufficient for membrane fusion. Design an experiment that will let you investigate your hypothesis. Explain what controls would you need to include. What are the potential pitfalls? (Answer should be less than 10 lines) ...
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