New Methods for Molecular Motor and Cell Motility Research

Abstract

Several novel methods for high-speed, high resolution microscopy have enabled new insights into function of molecular motors in vitro and in live cells. Several groups have developed methods to track the position and orientation of bi-functional organic fluorophores or quantum rods bound to specific motor subunits. We have now improved the time resolution by time-multiplexing excitation polarization every 100 μs and resolving structural changes by the consequent sudden changes in photon collection rates. In addition, we developed a multi-channel change-point method to detect structural states within the noisy signals and estimate rotations to within a few photons. These improvements enabled new insights into the walking mechanism of myosin V: we directly observe 10-20 ms periods of high mobility while myosin V detached heads search for actin. The gradual left-handed path of myosin V is produced by intermittent large side-ways motions among long stretches of straight walking where the lever arms mostly tilt in a plane very close to that containing the actin filament. For in vivo studies, detecting forces and motions of intracellular cargos have been compromised, until recently, by uncertainties about calibrating optical traps in cells. In the last year, four groups published novel methods for improving trap calibration in living cells. In our method, the thermal fluctuations of a phagocytosed bead are analyzed together with its motions upon sinusoidal excitation over a series of frequencies, giving trap sensitivity and local sub-diffusive viscoelastic parameters of the cytoplasm. This method is insensitive to active biological processes in the cell, as the forced response is used to resolve the elastic responses of the trap and the environment. Forces on these cargos indicate several kinesin and dynein motors operating near force balance. Supported by NIH grants P01GM087253 and R01GM086352.