The objective of our research is to understand the mechanism of the assembly of contractile structures in non-muscle and developing muscle cells. A combination of modern techniques, including microinjection, fluorescent analogue cytochemistry, digital image processing, and laser photobleaching will be used to obtain information directly from living cells. We will first microinject fluorescently labeled actin nucleation sites, and determine if they are capable of activating actin polymerization. The results will indicate whether the high level of unpolymerized actin in living cells is maintained by deactivating actin subunits, or by capping filament ends. We will next treat cells with phosphoinositides or protein kinase C activators, to determine their possible roles in regulating actin polymerization. We will also identify endogenous sites that are capable of incorporating actin subunits, by microinjecting fluorescent actin subunits and extracting cells after a short period of incubation. Active sites will be determined by comparing the distribution of injected actin with that of total filamentous actin using ratio imaging techniques. The ability and pattern of incorporation into leading lamellipodia, contractile rings, and developing myofibrils will be analyzed. Furthermore, the incorporation into posterior versus anterior regions of polarized cells will be compared, in order to detect a possible flux or cycling of the actin-containing cortex. Finally, we will study the elongation of stress fibers and myofibrils, by labeling them with fluorescent probes, and using laser microbeams to mark reference points near the ends of elongating fibrils. The movement of the spot, relative to the terminus of the fibril, will indicate where and how the elongation of filament bundles takes place. Together, these experiments will provide important information on the mechanisms of the regulation of actin polymerization and of the assembly of actin-containing structures in living cells.
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