Calmodulin is a calcium-binding protein found in all eukaryotic cells; it appears to be responsible for mediating the regulatory effects of calcium on numerous biological processes. The protein has been highly conserved, so its structure and functions can be conveniently examined in lower eukaryotes as well as vertebrate cells. This project would characterize calmodulin from the eukaryotic microorganism Dictyostelium discoideum and explore its roles, particularly in the regulation of motility. Earlier work has revealed that this protein, although functionally similar to vertebrate calmodulins, has several amino acid differences, including the absence of trimethylated lysine residue. Determination of the primary structure will be completed, and the consequences of the amino acid differences will be explored. Interaction with Ca++, troponin I, and vertebrate enzymes will be examined in this regard. Monoclonal antibodies will be raised against Dictyostelium calmodulin; the site (or sites) toward which these antibodies are directed will be determined, and mutants producing calmodulin altered at this site will be sought. Proteins that interact with Dictyostelium calmodulin will be identified and characterized, with emphasis on proteins that may be involved in regulating the structural states or interaction of myosin and actin. The ability of calmodulin to modulate myosin force production through a light chain kinase or other means will be tested directly. Antibodies against Dictyostelium calmodulin, myosin, and selected calmodulin-binding proteins will be used to visualize the localization of these proteins in mitotic and chemotaxing cells; for some of these proteins, monoclonal antibodies will be raised to facilitate purification and to map functional domains. The gene(s) encoding Dictyostelium calmodulin will be isolated and sequenced, as an initial step toward a long range goal of analyzing the function of the protein by genetically modifying it.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029723-07
Application #
3277363
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1981-07-01
Project End
1988-07-31
Budget Start
1987-07-01
Budget End
1988-07-31
Support Year
7
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Zhu, Q; Hulen, D; Liu, T et al. (1997) The cluA- mutant of Dictyostelium identifies a novel class of proteins required for dispersion of mitochondria. Proc Natl Acad Sci U S A 94:7308-13
Zhu, Q; Liu, T; Clarke, M (1993) Calmodulin and the contractile vacuole complex in mitotic cells of Dictyostelium discoideum. J Cell Sci 104 ( Pt 4):1119-27
Fok, A K; Clarke, M; Ma, L et al. (1993) Vacuolar H(+)-ATPase of Dictyostelium discoideum. A monoclonal antibody study. J Cell Sci 106 ( Pt 4):1103-13
Heuser, J; Zhu, Q; Clarke, M (1993) Proton pumps populate the contractile vacuoles of Dictyostelium amoebae. J Cell Biol 121:1311-27
Liu, T; Williams, J G; Clarke, M (1992) Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the completion of cytokinesis. Mol Biol Cell 3:1403-13
Zhu, Q; Clarke, M (1992) Association of calmodulin and an unconventional myosin with the contractile vacuole complex of Dictyostelium discoideum. J Cell Biol 118:347-58
Hulen, D; Baron, A; Salisbury, J et al. (1991) Production and specificity of monoclonal antibodies against calmodulin from Dictyostelium discoideum. Cell Motil Cytoskeleton 18:113-22
Rathi, A; Kayman, S C; Clarke, M (1991) Induction of gene expression in Dictyostelium by prestarvation factor, a factor secreted by growing cells. Dev Genet 12:82-7
Kayman, S C; Birchman, R; Clarke, M (1988) motA1552, a mutation of Dictyostelium discoideum having pleiotropic effects on motility and discoidin I regulation. Genetics 118:425-36
Clarke, M; Yang, J; Kayman, S C (1988) Analysis of the prestarvation response in growing cells of Dictyostelium discoideum. Dev Genet 9:315-26

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