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Part IV. Cbemotaxis
Purt
zv.
C bemotaxis
The next layer of cell biological complexity, chemotaxis, naturally follows the section on cell motility. Part IV begins with a chapter by Konijn and Van Haastert, who describe how to measure chemotaxis. Several different assays are described, as well as methods for giving localized pulses of chemoattractants, time-lapse cinematography, and other procedures pertinent to the study of the chemotactic response. Devreotes et al. next describe the cyclic AMP signaling system and methods to purify and study the cAMP receptor. Their chapter includes further information on growth and development of cells, how to monitor spontaneous oscillations, several chemotaxis assays, ways of measuring intracellular and secreted CAMP, and how to assay for adenylate cyclase. They go on to describe binding assays for cAMP and its analogs, receptor photoaffinity labeling, receptor modification, and purification procedures for the cAMP receptor. Their chapter concludes with a section on characterization of transmembrane signaling mutants. In Chapter 18, Berlot describes methods for examining changes in myosin phosphorylation during the chemotactic response. Her chapter includes conditions for labeling cells in vivo with [32P]orthophosphate, conditions that eliminate phosphatase and protease activities in extracts, and methods to isolate phosphorylated protein rapidly by immunoprecipitation. The last chapter in Part IV is by Fukui et af.,who describe detailed procedures for an agaroverlay technique for immunofluorescence. This technique provides higher resolution than others that have been employed, so that individual myosin thick filaments and their dynamics during the chemotactic response have been able to be measured.
28 I
zv.
C bemotaxis
The next layer of cell biological complexity, chemotaxis, naturally follows the section on cell motility. Part IV begins with a chapter by Konijn and Van Haastert, who describe how to measure chemotaxis. Several different assays are described, as well as methods for giving localized pulses of chemoattractants, time-lapse cinematography, and other procedures pertinent to the study of the chemotactic response. Devreotes et al. next describe the cyclic AMP signaling system and methods to purify and study the cAMP receptor. Their chapter includes further information on growth and development of cells, how to monitor spontaneous oscillations, several chemotaxis assays, ways of measuring intracellular and secreted CAMP, and how to assay for adenylate cyclase. They go on to describe binding assays for cAMP and its analogs, receptor photoaffinity labeling, receptor modification, and purification procedures for the cAMP receptor. Their chapter concludes with a section on characterization of transmembrane signaling mutants. In Chapter 18, Berlot describes methods for examining changes in myosin phosphorylation during the chemotactic response. Her chapter includes conditions for labeling cells in vivo with [32P]orthophosphate, conditions that eliminate phosphatase and protease activities in extracts, and methods to isolate phosphorylated protein rapidly by immunoprecipitation. The last chapter in Part IV is by Fukui et af.,who describe detailed procedures for an agaroverlay technique for immunofluorescence. This technique provides higher resolution than others that have been employed, so that individual myosin thick filaments and their dynamics during the chemotactic response have been able to be measured.
28 I