Our overall objective is to provide a better understanding of the molecular mechanisms underlying cellular movements. Specifically, it is proposed to extend our understanding of the structure, function and organization of actin, a major contractile element, using an immunochemical approach. This will entail the development of monoclonal and polyclonal antibodies against specific amino acid sequences found in skeletal muscle actin using synthetic peptides as immunogens. The first peptides to be synthesized will be chosen on the basis of their postulated involvement of certain amino acid residues in actin functions such as polymerization, myosin activation, or binding to accessory elements. The relative hydrophilicity of the peptide sequence will also be taken into account since this parameter is a good indicator of antigenicity and surface orientation in globular proteins. In later experiments, additional peptides will be prepared to hydrophilic sequences spaced throughout the actin molecule. Currently available monoclonal and polyclonal antibodies will also be used in these studies. These antibodies will be tested as to their ability to bind to native, denatured, monomeric and filamentous actin and the spatial relationships of the peptides in native actin will be assessed by determining the degree of overlap, if any, in the native molecule. It is also proposed to analyze the relationship between each antibody binding site and functional sites in the actin by determining the effects of these antibodies on actin polymerization, binding/activation of myosin and its proteolytic fragments, and binding of accessory proteins. Conversely, the ability of these proteins to interfere with antibody binding will be determined. This immunochemical approach should provide data to support or negate current hypotheses regarding the three dimensional organization of actin and should provide new and important information as to those regions of the molecule that are essential to its many vital functions of this contractile element. Moreover, the physiological relevance of this information will be tested by assaying the effects of the antibodies on living cells. Ultimately this approach, in combination with crystallographic, biochemical, and ultrastructural studies, should provide detailed insight into the molecular mechanisms underlying cell movements in processes such as division, migration, and contraction.
| Sawtell, N M; Hartman, A L; Lessard, J L (1990) Conserved tissue-restricted expression of HUC 1-1 actin phenotype among eumetazoan organisms. J Exp Zool 256:54-62 |
| Hartman, A L; Sawtell, N M; Lessard, J L (1989) Expression of actin isoforms in developing rat intestinal epithelium. J Histochem Cytochem 37:1225-33 |
| Sawtell, N M; Hartman, A L; Lessard, J L (1988) Unique isoactins in the brush border of rat intestinal epithelial cells. Cell Motil Cytoskeleton 11:318-25 |
