Myosin monomer density and exchange in synthetic thick filaments investigated using fluorescence microscopy with single molecule sensitivity

The number of myosin molecules in synthetic thick filaments, prepared by dialysis at 0.12 M NaCl and pH 5.0, was estimated to be between 400 and 800 molecules per micrometre under conditions appropriate for in vitro motility assays. This estimate was based on a number count of Cy3-labelled myosin molecules incorporated into filaments at a nominal ratio of 1:1000. At this dilution, single fluorescent spots were resolved corresponding to individual labelled myosins. The spots usually bleached with a one- or two-step profile but, in around 30% of the cases: fluctuations were observed indicating that additional photophysical or photochemical events had occurred. Myosin molecules were shown to exchange between filaments in suspension on a time-scale of several hours at 4 degrees C, but the reaction was only 75% complete after 48 h, suggesting a non-exchangeable core. However, myosin exchange does not appear to be the predominant source of the fluctuations in fluorescence intensity in the single molecule assays.