There are four alternatively spliced isoforms of NMHC II-B and II-C, each with inserts in loop 1 or loop 2 in the myosin head domain. The function of the nonmuscle myosin II-C isoforms remains unclear. We are expressing the different full length isoforms of nonmuscle myosin II-C (II-C0, NMHC II-C1, and NMHC-II-C2 and NMHC II-C1,C2) in vitro using the baculovirus expression system in order to characterize the full length NM II-C isoforms. We are also expressing heavy meromyosin version of the various NM II-C isoforms using the baculovirus system. Recently we have generated NM II-C-GFP transgenic mice, to localize expression of NM II-C in mice in vivo.In this case GFP was introduced into the 3 prime end of the endogenous gene encoding for NMHC II-C. These experiments will help us understand the function of NM II-C in both mice and humans. We are also using using the full length baculovirus expressed proteins to study myosin filament formation. In separate experiments, It has been shown in vitro that myosin II adopts a compact folded conformation (10S) when unphosphorylated, or inactive. This conformation has been indirectly shown to exist for smooth muscle myosin in human airway smooth muscle cells. Our results show that there exist two distinct pools of NM II in COS-7 and 3T3 cells. We hypothesize that one pool contains 10S, or soluble, NM II and the other contains filamentous (6S), or insoluble, NM II. Here, we propose the use of FRET to directly image the 10S conformation of NM II in COS-7 cells. Through the strategic placement of two FRET probes on NM II, we plan to detect the 10S conformation when the FRET probes are in close proximity of one another. FRET signal will be absent when the cell is stimulated for form NM II filaments, thereby increasing the 6S pool at the expense of the 10S pool.
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