Optical Control of Fast and Processive Engineered Myosins 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 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 individual motors tagged with fluorescent protein arrays [3]. For non-processive plus-end directed myosins, the most responsive constructs are approximately stopped in the dark and reach several microns/second in the presence of blue light. We also report on speed modulation in minus-end directed motors. For processive myosins, we demonstrate controllable bidirectional transport, and observe subcellular localization at filopodial tips under optical stimulation. An extended set of optogenetic motors will contribute to an expanded toolkit for programmable control of nanoscale transport and force generation. [1] P. van Bergeijk, et al. (2015) Nature 518, 7537. [2] M. Nakamura et al. (2014) Nat. Nanotechnol 9, 693. [3] R.P. Ghosh et al. (2017) BioRxiv https://doi.org/10.1101/159004.