MYOSIN LINKED REGULATION IN MUSCLE AND NON-MUSCLE CELLS

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This proposal is aimed at furthering an understanding of the mechanism and function of myosin-linked regulation, both in muscle and non-muscle cells. In regulatory myosins, the interaction between actin and myosin is controlled solely by events occurring on the myosin molecule alone, either in response to direct calcium binding or phosphorylation. We will take advantage of the unique and versatile properties of scallop striated adductor myosin in order to incorporate foreign light-chains, both regulatory and essential, into the myosin molecule. These light-chains will be modified at differing locations in their primary sequence. Our experiments will monitor intra-head conformational changes by fluorescence resonance energy transfer (FRET). These events will be manifest as changes in distance and/or orientation between probes placed on each of the two light-chain types and between probes on either light-chain and the active site, or other locations on both actin and myosin. Changes in the degree of transfer will be sought in response to changing conditions designed to mimic the physiological states of 'rest', 'rigor' or the 'active state.' The complementary technique of protein crosslinking will also be used, when appropriate. We also intend to perform a comparative study of two regulatory myosins which exhibit both light-chain and heavy-chain phosphorylation and to observe the relationship of such regulation to the specific functions of myosin in situ. By using molluscan catch adductor myosin we will further our understanding of the regulation of catch contraction. By using myosin isolated from a neuronally-derived cell line, we will ultimately be able to evaluate the role of myosin in neurotransmitter release during synaptic transmission. An antibody to this myosin will enable us to probe the location and function of myosin within the developing central nervous system. A detailed understanding of these mechanisms is of fundamental importance.