Regulation Of Smooth and Nonmuscle Myosin

National Heart, Lung, And Blood Institute

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Abstract

Nonmuscle myosin 2 (NM2) molecules carry out a wide variety of functions within cells. There are three NM2 heavy chain genes in mammals. We are expressing full length NM2 proteins and fragments of these myosins in the baculovirus Sf9 system. The find that NM2A moves actin filaments the fastest and NM2C, the slowest of the three paralogs. We are studying their structure both at the level of the single molecule folded autoinhibited state as well as the structure of myosin filaments. We are particularly interested in how phosphorylation of both the heavy chain and light chain affects filament formation and activity. 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 in vitro and wish to understand the dynamics of 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. In collaboration with others, 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 and have crystallized NM2A in the presence of ATP analogs. This will give us the ability to observe high resolution structures of myosin in all four cross-bridge states. We also have solved the structure of the folded, autoinhibited form of both smooth muscle myosin and nonmuscle myosin 2B at less than 4 Angstrom resolution. This has allowed us to determine the structural basis for this 10S structure and to speculate on why phosphorylation activates the myosin. A collaborating lab identified an alternatively spliced variant of NM2A containing 21 additional amino acids in loop 2. We expressed this myosin in Sf9 cells and have examined the effect of this splicing on enzymatic activity, motility and filament formation. Preliminary data suggest that the spliced form is much less active. We are using micropatterning in conjunction with TIRF microsocopy to create higher order actin structures to examine their interaction with NM2 paralogs. One of these involves creating parallel stripes of various separation on a microscope coverslip which can be coated with formin, an actin nucleator. Upon adding G-actin the formin nucleates polymerization of actin in which the barbed in is bound to the stripe. In the middle of the patterns between stripes there is an antiparallel array of actin filaments. When NM2B filaments are added to this system they bind to the actin filaments and begin to generate tension which aligns the actin filament. The bipolar myosin filaments near the center area between stripes encounter actin filaments of opposite polarity which causes the myosin filaments to effectively stall where they are likely generating isometric tension on the actin filaments within the stripes. In these cases the myosin filaments remain bound to the actin filaments for a long time, likely because the kinetic cycle has been slowed by force dependent processes. We are using a myosin that had a FRET-based force sensor embedded into the S2 region of the myosin. Preliminary data show that as the actin filaments become tensed a portion of the myosin molecules in a filament become force bearing. We are using fluorescently-labeled myosin molecules to examine the exchange of myosin regulatory light chain using TIRF microscopy. Preliminary data show that the RLC exchanges in a slow time scale between heavy chains of different myosins. We are also using TIRF microscopy to examine the exchange of myosins between filaments. Early findings show that filaments composed of NM2A exchange more rapidly than those of NM2B.