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. Following are various experimental approaches designed to characterize actin assembly and the effects of actin-binding proteins on actin assembly.
a. The graph in part (a) of the figure 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 actin filaments of a predefined length to rabbit actin maintained at the concentrations labeled A, B, and C in the figure. Diagram the appearance of the filaments after a 10-minute incubation at each of the indicated actin concentrations, if the original filaments are depicted as follows:
Make sure to mark the location of the original (+) and (—) ends of the filament on your diagrams.
b. A novel actin-binding protein (X) is overexpressed in certain highly malignant cancers. You wish to determine if protein X caps actin filaments at the (+) or (—) end. You incubate an excess of protein X with various concentrations of G-actin under conditions that induce polymerization. Control samples are incubated in the absence of protein X. The results are shown in part (b) of the figure. How can you conclude from these data that protein X binds to the (+) end of actin filaments? Design an experiment, using myosin S1 fragments and electron microscopy, to corroborate the conclusion that protein X binds to the (+) end. What results would you expect if this conclusion is correct?
c. An in vitro system was developed to study actin assembly and disassembly in nonmuscle cells. In this study, tissue culture cells were incubated for several hours with [35S]me-thionine so that all the actin monomers in each filament were labeled. Actin filaments were then collected by differential centrifugation and put into a buffer containing one of three c E
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