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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR032858-04A2
Application #
3156428
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1983-07-01
Project End
1993-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Allegheny University of Health Sciences
Department
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19129
Wang, Y; Chantler, P D (1994) Functional analysis of individual brain myosin II isoforms through hybrid formation. FEBS Lett 348:244-8
Li, D; Miller, M; Chantler, P D (1994) Association of a cellular myosin II with anionic phospholipids and the neuronal plasma membrane. Proc Natl Acad Sci U S A 91:853-7
Miller, M; Bower, E; Levitt, P et al. (1992) Myosin II distribution in neurons is consistent with a role in growth cone motility but not synaptic vesicle mobilization. Neuron 8:25-44
Li, D; Chantler, P D (1992) Evidence for a new member of the myosin I family from mammalian brain. J Neurochem 59:1344-51
Sun, W; Chantler, P D (1992) Cloning of the cDNA encoding a neuronal myosin heavy chain from mammalian brain and its differential expression within the central nervous system. J Mol Biol 224:1185-93
Park, H S; Tao, T; Chantler, P D (1991) Proximity relationships between sites on myosin and actin. Resonance energy transfer determination of the following distances, using a hybrid myosin: those between Cys-55 on the Mercenaria regulatory light chain, SH-1 on the Aequipecten myosin heavy chain Biochemistry 30:3189-95
Sun, W D; Chantler, P D (1991) A unique cellular myosin II exhibiting differential expression in the cerebral cortex. Biochem Biophys Res Commun 175:244-9
Chantler, P D; Tao, T; Stafford 3rd, W F (1991) On the relationship between distance information derived from cross-linking and from resonance energy transfer, with specific reference to sites located on myosin heads. Biophys J 59:1242-50
Bower, S M; Chantler, P D (1991) The importance of choice of visualization technique in the use of indirect immunodetection methods: specific reference to the detection of light chain movement on a regulatory myosin. Biotech Histochem 1:37-43