Intracellular Myosin Motor Protein Motion Using Laser Scanning Confocal Microscopy

Abstract

We focus on live cell single molecule imaging and tracking with particular focus on measuring single myosin V steps along actin filaments within live cells. The goal of the work is to acquire information that will offer new insights into the mechanism of these motors' processivity. Conventionally, single molecule Myosin studies have been accomplished through total internal reflection fluorescence microscopy (TIRF).1,2 However, TIRF methods have very limited applicability to live cell in vivo studies. As such, we apply confocal laser scanning microcopy (CLSM) to imaging and tracking of Myosin V. CLSM also offers the capability to provide full 3-dimensional tracking of biomolecular motors. Our experiments have confirmed step size results from previous TIRF experiments. In terms of instrumentation, we overcome the nominally slow scanning speed of CLSM by the use of a fiber scanning technique that allows us to scan with image acquisition times of less than 100 ms. We make use of home-made streptavidin-functionalized quantum dots in conjunction with this tracking technique to increase the quantum efficiency and stability of the fluorophores. The QD fabrication and sample preparation techniques will also be presented.1. A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization,” Science 300 (5628), 2061-2065 (2003).2. T. Sakamoto, A. Yildez, P. R. Selvin, and J. R. Sellers, “Step-size is determined by neck length in myosin V,” Biochemistry 44 (49), 16203-16210 (2005).