Optical Control of Fast and Processive Engineered Myosins In Vitro and in Living Cells

Abstract

The interaction between actin and myosin II plays an important role in a variety of cellular functions. In particular, myosin II is involved in muscle contraction, which is attributed to the sliding of thin filament actin past the thick myosin II filaments. Past studies on the structure of myosin have linked severe pathologies to defects in myosin, making it important to understand the mechanism of the system. In this study, we focus our analysis on the powerstroke of the myosin II cross bridge cycle, which is the active process of muscle contraction. To do this, we use an algorithm called Milestoning which partitions the dynamics into a sequence of trajectories between ''milestones'' along the reaction pathway. The structure of myosin II bound to actin in the rigor state was used as a starting point, and a structure for the bound pre-powerstroke state was developed using existing published structures for the unbound pre-powerstroke state as well as experimental data gathered about the movement of myosin II during the powerstroke. With both the beginning and final states of the powerstroke, we can interpolate between these structures to build intermediate states along the reaction pathway. A total of 97 all-atom structures along the pathway of the powerstroke were developed to serve as guides and ''anchors'' along the reaction pathway. Milestoning short trajectories between cells defined by the anchors will allow for the computation thermodynamics and kinetics of the myosin II powerstroke. This work will lead to a significant improvement in our understanding of the complete powerstroke mechanism, which will in turn facilitate future research on the effects of structural defects in myosin II on powerstroke function and muscle contraction. 1280-Pos Optical Control of Fast and Processive Engineered Myosins In Vitro and inLiving Cells Spatiotemporal control of cytoskeletal transport can provide new possibilities for dissecting cellular processes or for constructing complex artificial devices. Optogenetic approaches have been used for both controlled recruitment of motors to cellular cargos [1] and direct modulation of motor speed and direction [2]. Here we have worked to create optimized and diversified engineered myosin motors with velocities that can be optically controlled using dynamic changes in lever arm geometry. Previous designs for lightactivated gearshifting [2] were non-processive, and suffered from either low velocities (< 10 nm/s) or modest degrees of velocity modulation (15%) in response to light. These limitations preclude many applications in cell biology, devices, and reconstituted systems. We have now engineered (i) non-processive myosin motors that combine large optical modulation depths with high velocities and (ii) processive myosin motors with optically controllable directionality. We have characterized a series of optimized constructs using in vitro motility assays of propelled actin filaments, singlemolecule tracking of processive complexes, and live-cell imaging of individual motors tagged wi...