Synthetic Biology Approaches to Dissecting Multiple Motor Functions in Living Cells

Abstract

Dissecting how the coordination of cytoskeletal motor proteins, (kinesin, dynein and myosin) has become increasingly important to characterizing various transport processes in eukaryotes. Most subcellular commodities (ribonucleoprotein, particles, organelles, vesicles etc.) are driven by “teams” of motor proteins that are composed of multiple copies of the same motors or even mixtures of different motors that move in opposite directions, with different velocities, or along different types of filament tracks. Consequently, numerous intracellular transport processes and regulatory mechanisms depend fundamentally on how these collections of similar and dissimilar motors either cooperate or compete with one another. Understanding these behaviors are important to elucidating various disease pathologies stemming from motor and other transport-related protein mutations since their impact on cargo transport will ultimately depend on the extent to which they perturb the composite dynamics of motor systems that also contain wild type motors. Our work has focused on developing genetically engineered cells that can be used to characterize endogenous cargo transport and trafficking response to variation in motor number, type and ratio and cargo size. While integrating multiple genetic control elements, these cells also exploit drug- and light-dependent protein dimerization switches to allow competitions between different types of wild-type and mutant motors to be adaptively regulated. We are also developing micro-fabrication techniques to pattern these cells into defined shapes as a way to control the organization of their cytoskeleton and facilitate direct comparisons of transport responses within the cell population. Given these unique handles, these cells provide a powerful platform to examine fundamental mechanisms of intracellular transport regulation and assess the impact of disease-related motor mutations.