Single molecule approach for the study of myosin polymerization
PIs: Alina Popescu, Henry Shuman
The mechanism and kinetics of myosin filament growth are studied using time resolved Total Internal Reflection Fluorescence Microscopy (TIRFM) and rapid mixing in a novel microfluidics chamber. The microfluidic mixing chamber, which provides an observation port for high resolution optical imaging, was developed in a collaboration between the groups of Haim Bau and Henry Shuman. We found that myosin filament formation is monomer concentration dependent, has a lag phase on a time scale of seconds, followed by a fast growth phase, characteristic for the nucleation-elongation assembly of many biopolymers. Myosin molecules at 3 nM, in equilibrium with single filaments, bound to the filaments at the rate of 0.25 ± 0.08 s-1 mm-1 which was two orders of magnitude larger then the rate monomers bound to the adjacent surface. Quantitation of the step increases of filament fluorescence suggests that elongation of single striated muscle myosin thick filaments occurs by addition of myosin dimers, confirming earlier predictions (Davis, 1988). Preliminary tests with high salt rapid mixing suggest that freshly formed myosin filaments rapidly depolymerize but filaments stored for >1 day depolymerize more slowly.