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. 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. By copolymerizing full length NM2B with a Halo-capped tail fragment of NM2B, we show that between 5 and 8 motor domains per half filament are required for processive movement. 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 can also image the movement of unlabeled NM2 filaments using iSCAT microscopy. We have reexamined the activity of an N93K mutant of NM2A which we previously published was inactive with regards to ATPase activity and in vitro motility. Recent work suggests that if properly expressed in Sf9 cells, the protein has substantial ATPase activity. We believe that the reason for the previous determination of inactivity may be related to problems in properly folding the molecule and this may explain some of the disease phenotypes in humans bearing this mutation. In collaboration with others, we have determined the 2.25 Angstrom structure of a motor domain of NM2C and have shown that there is an allosteric communications pathway that operates from the distal end of the motor domain to the active site via a positively charged residue present at the interface between the N-terminal subdomain, the converter and the lever arm. 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. In collaboration with the Hammer lab (NHLBI) we showed that myosin-18 does not directly bind GOLPH3 as suggested by a prior publication.
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