IMMUNOCHEMICAL STUDIES OF MYOSIN: QUANTITATIVE AND STRUCTURAL ANALYSES (MONOCLONAL ANTIBODIES, NON-MUSCLE ACTOMYOSIN, CELL MOTILITY)

Abstract

Many types of motility in eukaryotic cells appear to share a common molecular basis that involves proteins very similar to the major proteins of muscle, actin and myosin. In non-muscle cells, these proteins are not in stable filament arrays, but form transient contractile structures as needed. Information concerning the myosin polymerization state is essential to understanding how movement is produced and regulated in non-muscle cells.;Monoclonal antibodies were isolated against myosin from the eukaryotic organism Dictyostelium discoideum. Immunoblot analysis using peptide fragments of myosin has shown five classes of epitope specificity amongst the antibodies. A filtration immunoassay has been developed to quantitate total myosin in cells and subcellular fractions. I have determined that myosin is 0.72% of the cell's protein. I have also identified a monoclonal antibody that reacted with monomeric but not filamentous myosin. A competition assay using this antibody was employed to measure the monomeric myosin in a sample. With these assays I have examined myosin in Triton-insoluble cytoskeletons of Dictyostelium amoebae. These contractile structures are greatly enriched in actin and myosin. Isolated cytoskeletons contain 43% of the cell's myosin, all of it in thick filament form; the Triton-soluble myosin appears unpolymerized. Cytoskeletal myosin can be converted to monomeric form under salt conditions known to cause the disassembly of purified myosin thick filaments. Depolymerization of cytoskeletal myosin does not result in its release from the cytoskeleton. Cytoskeletal myosin can be cycled between monomeric and polymeric forms by small changes in ionic and disassembly. Cytoskeletons can undergo contraction only if the myosin is in filamentous form when ATP is added.;The quantitative filtration immunoassay is rapid, sensitive and should be applicable to most proteins for which monoclonal antibodies have been derived. The competition radioimmunoassay to specifically measure monomeric myosin is analogous to the DNase I assay for actin and should be useful in analyzing the assembly state of myosin. The effect of salt concentration suggests that polymerized myosin is a requirement for contraction of cytoskeletons. This is consistent with a sliding filament model of contraction that utilizes bipolar thick filaments.