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Single molecule approach for the study of myosin polymerization

Fig 1: Myosin polymerization induced at the microfluidic chamber level by rapid advection mixing of myosin monomers (40 nM) in high salt with polymerization buffer. a) fluorescent myosin molecules (“seeds”) on an actin decorated surface. b) myosin clusters formed on the surface grow with time. c) The average of traces synchronized with the start of flow more clearly shows a ~10s lag in the fluorescence increase, then a ~ linear increase of fluorescence. The function fitting the data has an exponential decay component due to bleaching and a sigmoidal component, that represents the growth. The sigmoidal type of growth is characteristic for many biopolymers, including actin growth (Ressad,1999), Doubling the concentration of myosin monomer flowed into the chamber leads to growth up to an equivalent of 26 fluorescent molecules, on average.

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.



Nano/Bio Interface Center @ The University of Pennsylvania
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