Nonmuscle myosin 2 (NM2) molecules carry out a wide variety of functions within cells. There are three NM2 heavy chain genes. We are expressing full length NM2 in the baculovirus Sf9 system. We are studying their filament structure and how phosphorylation of both the heavy chain and light chain affects filament formation. We use a single filament motility assay system wherein we can image the movement of fluorescently labeled myosin filaments over actin filaments fixed to the surface. We are examining the copolymerization of NM2A and NM2B form co-polymers in vitro. We are collaborating with the Korn lab at NHLBI to study the effects of heavy chain phosphorylation on filament assembly. With Sarah Hitchcock-DiGregori we have examined the effect of various nonmuscle tropomyosin isoforms on the actin activated ATPase activity and in vitro motility of NM2A, NM2B and NM2C HMM isoforms and find that some TM isoforms activate one or more of the HMMs whereas some do not. Optical trapping studies reveal that NM2A and NM2B are not processive as single molecules. Bipolar filaments of NM2B containing about 30 myosin molecules move processively along actin filaments attached to the surface. Surprisingly, filaments of NM2A do not move processively under these same conditions which may be due to the lower duty ratio of this myosin compared to that of NM2B. NM2A can be co-polymerized with NM2B molecules and these heterotypic filaments move processively provided sufficient NM2B is present. In the presence of 0.5% methylcellulose which mimics the viscosity of the cytoplasm both NM2A and NM2B filaments move processively. We have expressed the S1, HMM and full length constructs for Drosophila nonmuscle myosin 2 and have shown that enzymatically the protein has a low duty ratio with phosphate release being rate limiting and that the enzymatic activity of the HMM and full length proteins require phosphorylation of the regulatory light chain. Electron microscopy shows that the myosin forms bipolar filaments of about 300 nm in length with about 14-16 myosins per filament. We co-expressed these molecules with phospho-mimetic regulatory light chains whereby the the phosphorylatable serine and threonine residues were replaced with negatively charged glutamic acid residues. ATPase activity, in vitro motility and filament formation was studied. Surprisingly, non of the phospho-mimetic mutant light chains replicated the activating effect of true phosphorylation.
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