Actin microfilaments have been implicated to be the essential force-generating machinery for animal cell locomotion, tumor cell metastasis, endocytosis and exocytosis, cytokinesis, muscle contraction, organelle and granule movement, cell attachment, and maintenance of cell shape. The long-term goal of this research project is to understand the structural and functional significance of microfilaments in cell motility and the maintenance of cell shape. Our approach is to focus on tropomyosin isoforms and caldesmon, so-called actin-binding proteins, which are believed to govern the temporal and spatial distribution of microfilaments and then to control the function of microfilamentes in nonmuscle vertebrate cells. Several monoclonal antibodies against tropomysin isoforms and caldesmon have been prepared and characterized. More antibodies will be prepared. These will be used to study the intracellular distribution by immunofluorescence and immunoelectron microscopy, and to probe the physiological roles of these two regulatory proteins by microinjection of antibodies into living cells. We propose to purify individual tropomyosin isoforms from human and chicken cells, and caldesmon from human platelets. The in vitro interactions of these two proteins with actin as well as other actin-binding proteins will be examined by actin-binding assay and myosin ATPase assay. The effect of monoclonal antibodies on these interactions will also be examined in vitro. This information is also very useful in interpreting the results from microinjection experiments. In order to study the control of tropomyosin isoform expression in normal and transformed cells, we will continue to isolate and characterize cDNA expression clones encoding tropomyosin isoforms human nonmuscle cells by immunological screening with out collection of polyclonal and monoclonal antibodies. These cloned cDNAs will be used to screen the genomic library and to study the DNA encoded by these cDNA clones will also be used as tropomyosin isoform- specific antigens for immunization of mice and for preparation of isoform-specific monoclonal antibodies. These studies will lead us to understand how the multiple isoforms of tropomyosin play a fine regulatory role in stabilizing and organizing microfilaments, and in cell motility.
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