Optical Control of Fast and Processive Engineered Myosins: Optimization and Characterization in Vitro and in Living Cells

Abstract

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 light-activated 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 potential 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, single-molecule tracking of processive complexes, and live cell imaging of motors tagged with fluorescent protein arrays [3]. For non-processive plus-end directed myosins, we measure up to 4X speed increases in the presence of blue light, reaching velocities of 4-5 microns/s. For processive myosins, we demonstrate controllable bidirectional processive transport, and report on cellular localization under optical stimulation. [1] P. van Bergeijk, et al. (2015) Nature 518 (7537) “Optogenetic control of organelle transport and positioning” [2] M. Nakamura et al. (2014) Nat. Nanotechnol 9, 693 “Remote control of myosin and kinesin motors using light-activated gearshifting” [3] R.P. Ghosh et al. (2017) “A Fluorogenic Array Tag for Temporally Unlimited Single Molecule Tracking”, BioRxiv doi: 10.1101/159004.