CPM Seminar

Probing Mechanisms of Myosin Molecular Motors Using Single Molecule Techniques

Mary Elting

Department of Applied Physics
Stanford University

Members of the myosin family of molecular motors perform a variety of essential functions in the cell, including powering muscle contraction and cell division, transporting cargo, and serving as anchors and tension generators. Myosins accomplish these tasks by transforming chemical energy from ATP hydrolysis into mechanical work, as they either move along their actin filament tracks, or power the movement of the actin filaments. Most of my work is on one member of the myosin family of motor proteins, myosin VI, which exhibits the ability to take multiple steps along actin filaments, termed ‘processivity’.

In the first section of my talk, I will describe our analysis of myosin VI processivity via structurally engineered mutant constructs, which we examined using single molecule fluorescence. Myosin VI is both structurally and functionally unusual among myosins. In order to probe our understanding of its mechanism, we replaced its lever arm with a variety of engineered artificial lever arms, and tested whether it responded as we would expect. As part of this work, I also developed a quantitative model of processivity of myosin VI, which I used to analyze the expected effects of decreasing intramolecular communication between the heads of a processive myosin. This model has implications not only for myosin VI, but for other two headed processive motors, including processive myosins, kinesins, and dyneins. In this work, I found that processivity is markedly robust to decreased inter-head communication.

In order to further characterize intramolecular communication in processive myosins V and VI, I hoped to directly visualize ATP molecules binding and releasing from myosin molecules as they walked along actin filaments. This required the development of techniques to allow the resolution of single fluorescent molecules at higher concentrations of fluorophore than has previously been possible. I approached this challenge using two technological approaches: linear zero mode waveguides (ZMW, Pacific Biosciences) and convex lens induced confinement (CLIC, Dr. Sabrina Leslie). While the direct visualization of nucleotide gating remains a challenge, I will discuss my progress toward applying these techniques to better understanding myosin~Rs processive mechanism.