SUMMARY Diverse cytoskeletal motors perform essential cellular functions including spindle assembly, nuclear positioning, and polarized transport of mRNA, proteins, and membranous cargos along microtubules and actin filaments. Engineering biomolecular motors with tunable and dynamically controllable properties can provide (1) rigorous tests of models relating molecular structures to mechanical functions, (2) novel tools for selective perturbation of mechanical processes inside living cells, and (3) optimized components for complex tasks such as molecular sorting and directed assembly in vitro. This project seeks to develop and characterize a comprehensive set of modified cytoskeletal motors with defined properties — including speed, direction, and force generation — than can be controlled using external cues such as light. A modular protein engineering approach will be applied to both actin-based and microtubule-based transport. During successive design cycles, chimeric motors will be constructed based on structural models, and then functionally characterized using gliding filament assays, single fluorophore imaging, gold nanoparticle tracking, and optical trapping. Complementary structural characterization using cryoelectron microscopy will be used to compare the experimental conformations of filament-bound motors to the original structural designs, and to yield new insights into class-specific structure-function relationships. Finally, pilot studies will be conducted to test the function of engineered motors inside living cells. The specific aims of this project are (1) to create diverse myosin motors that exploit dynamic changes in lever arm structure in order to shift gears — speed up, slow down, or change directions — when exposed to blue light; (2) to develop diverse microtubule-based motors with artificial lever arms, including light-activated gearshifts, by exploiting a mechanistic analogy between myosins and class-14 kinesins, and (3) to create processive multimeric assemblies of controllable engineered myosins and kinesins, and characterize their force-generating properties. If successful, this work will dramatically expand the potential applications of engineered molecular motors, and provide unprecedented control over nanoscale motion. Genetically encoded light- responsive motors will expand the optogenetics toolkit, complementing precise perturbations of ion channels and intracellular signaling with spatiotemporal control of cytoskeletal transport and contractility. Optogenetic control of bidirectional transport will enable dynamic relocalization of biomolecules and organelles; highly processive and controllable motors will have potential applications in gene and drug delivery; and controllable motors may be used to sort, shuttle, and concentrate analytes in microfabricated diagnostic devices.