Simultaneous Tracking of Multiple Myosins in Sub-Diffraction Scale Based on Spectral Division

Abstract

In the past couple of decades, single molecule measurement techniques have tremendously unveiled dynamic behavior and function of individual motor protein molecules. In many living systems, however, multiple molecules work with sort of cooperativity to increase the energy efficiency and the robustness. To understand the physicochemical mechanism of the motor protein systems, the attention is now shifting to upper hierarchy; from individual elements to cooperative motion of multiple protein molecules. We have proposed a novel technique for simultaneous nanometric tracking of dynamic behavior of multiple motor proteins along a one-dimensional rail protein filament. By simple combination of a total internal reflection fluorescence microscope with an imaging spectrometer, multiple motor proteins labeled with quantum dots of different colors can be simultaneously observed with the millisecond time resolution. Even two or more adjacent motor proteins working within nanometric scale, which is much smaller than spatial resolution of optical microscopy, can be separately tracked. Our microscopy system has successfully tracked one-dimensional processive movement of multiple myosins (myosin VI) with a few nanometers precision, and realized observation of how they were chasing each other back and forth on an actin filament. We also applied the microscopy to dynamic observation of two heads of a single myosin (myosin V), and showed the hand-over-hand walking manner, which is consistent with previous works. Furthermore, we extended the technique to two dimensional tracking and applied it to tracking of multiple myosin V on two-dimensional plane. We believe that this would be a useful tool to investigate cooperative movement of motor protein molecules in nanoscale.