MICB688L/MOCB639 Advanced Cell Biology Exam II May 10, 2001 Name: 1. Briefly describe the four major classes of cell surface receptors and their modes of action (immediate downstream only) (8) 2. Please list names of three secondary messengers, and briefly describe how they are generated (immediate upstream) and their modes of action (immediate down stream). (9) 1
3. Briefly describe two experimental approaches used to map the order of events in a signaling pathway, such as to identify the adaptor proteins that link EGF receptor to Ras protein. (10) 4. Both of β 2 -adrenergic receptor and EGF receptor can lead to the activation of MAP kinase pathway. In order to understand the relationship between the signaling pathways of these two receptors, Maudsley and colleagues did a simple experiment. The result of this experiment is showed in Figure 1. What can you conclude from this set of data? (6) ERK1/2 a component of MAP kinase pathway NS - no stimulation Iso a ligand of β 2 -adrenergic receptor Propranolol an antagonist of β 2 -adrenergic receptor Tyrophostin AG1478 EGF receptor-specific tyrosine kinase inhibitor PP2 Src-family kinase inhibitor 2
5. Please identify the similarities (at least four) and differences (at least four) between microfilaments and microtubules. (8) 6. Individuals who are genetically defective in dynein demonstrate a characteristic cough, often have chronic bronchitis, and are sterile. Please briefly discuss the molecular basis for these clinical symptoms. (8) 3
7. Understanding of actin filaments has been greatly facilitated by the ability of scientists to purify actin and actin-binding proteins, and the ability to assemble actin filaments in vitro. Below are two experimental approaches designed to characterize actin assembly and the effects of actin-binding proteins on this assembly. a. (4.5) Figure 2A depicts the actin polymerization rate at the plus (+) and minus (-) ends of rabbit actin as a function of actin concentration. Assume that you could add microfilaments of a predefined length to rabbit actin maintained at the concentrations labeled A, B and C in this figure. Diagram the appearance of the filaments after a 10-min incubation at each of the indicated actin concentrations, if the original filaments are depicted as follows: Original filament: + - Make sure to mark the location of the original (+) and (-) ends of the filament on your diagrams. Figure 2A b. (4.5) An in vitro system was developed to study actin assembly and disassembly in non-muscle cells. In this study, tissue culture cells were incubated for several hours with [ 35 S]methionine so that all the actin monomers in each filament were labeled. Actin filaments were then collected by different centrifugation and put into a buffer containing one of three different cytosolic extracts (A, B, or C). The amounts of soluble actin in each sample were monitored over time (Figure 2B). What do these data indicate about the effects of A, B, and C on the assembly and disassembly of actin filaments? Figure 2B 4
8. Name two major approaches used by Verhey and his colleagues to demonstrate JIP scaffolding proteins as cargo of kinesin, (2) and briefly describe the major point that each of these two approaches demonstrates. (4) What is the significance of their discovery? (3) 9. Name three critical regulatory mechanisms for cell cycle regulation. (4.5) Give an example for each of these mechanisms. (4.5) 5
10. Please use a hand-drawing figure to illustrate the mitotic apparatus of animal cells, and name each major part of the mitotic apparatus. (8) 11. What features of yeasts make this an excellent model system for studying cell cycle regulation? (2) You have mutagenized a culture of the budding yeast S. cerevisiae and have isolated three different clones (A, B, and C). Two of the clones (A and B) exhibit temperature-sensitive growth. After shifting the cultures to the non-permissive temperature, you exam cells from each clone under a light microscope. Their appearance is depicted in Figure 3. Which of these clones is a wild type, a cdc mutant, and non-cdc mutant? (3) Give a one-sentence explanation. (3) Figure 3 6
12. The most commonly mutated tumor suppressor gene associated with human cancer is p53. To study the function of p53 in cell cycle, Murray and Hunter analyzed effect of p53 mutation on cell cycle. Cultured human cells were incubated with fluorescent nucleotides, and then exposed to radiation that induces DNA damage. The distribution of cells in the different phases of cell cycle was analyzed using flow cytometry. Figure 4 shows you the results. Please briefly describe what each histogram tells you, and what can you conclude from this set of data? (8) Figure 4 7