An important challenge in cell biology is to describe the force-generating mechanisms for such essential eukaryotic processes as exocytosis, endocytosis, and overall cell motility. In recent years, a single-headed, nonfilamentous form of myosin, called myosin I, has emerged as a likely motor to support actin-dependent membrane motility. Until now, myosin I had been purified and partially characterized only from protozoans and avian intestinal brush border. We have now succeeded in isolating mammalian forms of the enzyme from bovine adrenal medulla, adrenal cortex, and brain. This proposal focuses on two major questions regarding mammalian myosin I. The first is the regulation of the enzyme's catalytic activity and its interaction with actin and membranes and the second is its role in neuroendocrine cell function. To study the regulation of myosin I we will clone it and identify and characterize domains on the molecule which interact with F-actin, membranes, and calmodulin. The role of Ca2+ and myosin I phosphorylation in regulating these functional interactions will be investigated, both in vitro and in cultured adrenal chromaffin cells. To address the question of the physiological function of myosin I, we will first localize the protein in resting and stimulated chromaffin cells and in PC12 cells before and after they have been induced to differentiate into neuron-like cells by nerve growth factor. Subcellular fractionation will reveal whether myosin I is selectively targeted to specific organelles. If so, the basis for this selectivity will be investigated. Finally, we will introduce antibodies into cultured chromaffin cells. The effect of these antibodies on the exocytosis/endocytosis cycle and on catecholamine release will be examined. This proposal will provide a structural and functional characterization of the mammalian myosin I enzyme and will lay the foundation for future studies on the role of myosin I as an intracellular motor.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038567-07
Application #
2179416
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1988-07-01
Project End
1997-03-31
Budget Start
1995-04-01
Budget End
1997-03-31
Support Year
7
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Barylko, B; Binns, D D; Albanesi, J P (2001) Activation of dynamin GTPase activity by phosphoinositides and SH3 domain-containing proteins. Methods Enzymol 329:486-96
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Earnest, S; Khokhlatchev, A; Albanesi, J P et al. (1996) Phosphorylation of dynamin by ERK2 inhibits the dynamin-microtubule interaction. FEBS Lett 396:62-6
Reizes, O; Barylko, B; Li, C et al. (1994) Domain structure of a mammalian myosin I beta. Proc Natl Acad Sci U S A 91:6349-53
Wagner, M C; Barylko, B; Albanesi, J P (1992) Tissue distribution and subcellular localization of mammalian myosin I. J Cell Biol 119:163-70
Barylko, B; Wagner, M C; Reizes, O et al. (1992) Purification and characterization of a mammalian myosin I. Proc Natl Acad Sci U S A 89:490-4
Hildebrandt, E; Albanesi, J P (1991) Identification of a membrane-bound, glycol-stimulated phospholipase A2 located in the secretory granules of the adrenal medulla. Biochemistry 30:464-72
Yoo, S H; Albanesi, J P (1991) High capacity, low affinity Ca2+ binding of chromogranin A. Relationship between the pH-induced conformational change and Ca2+ binding property. J Biol Chem 266:7740-5
Yoo, S H; Albanesi, J P; Jameson, D M (1990) Fluorescence studies of nucleotide interactions with bovine adrenal chromogranin A. Biochim Biophys Acta 1040:66-70
Yoo, S H; Albanesi, J P (1990) Inositol 1,4,5-trisphosphate-triggered Ca2+ release from bovine adrenal medullary secretory vesicles. J Biol Chem 265:13446-8

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