Regulation Of Smooth and Nonmuscle Myosin

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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 NM2proteins and fragments of these myosins 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 lab of Laci Nytray at Eotvos Lorand University in Budapest 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. 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, In addition, we have obtained high resolution structures of both NM2A and NM2B bound to actin in the presence and absence of ADP using cryo-electron microscopy. In collaboration with the Nyitray lab we showed that GFP-capped myosin tail fragments formed bipolar filaments similar to those formed by full length myosin. S100a is a small calcium binding protein that is upregulated in metastatic cells. This protein has previously been shown to bind to the distal tail of NM2A with high affinity. Its affinity for NM2B is 3 orders of magnitude less. S100a addition to preformed NM2A/NM2B co-filaments results in the extraction of NM2A from these filaments. Thus in a cell, NM2A could effectively sequester NM2A in the presence of calcium while leaving NM2B in filamentous form.