Synthetic Tools for Delineating Multiple Motor Functions in Living Cells

Abstract

Characterizing the collective dynamics of cytoskeletal motors has become increasingly important to resolving transport regulatory mechanisms and the impact of mutated motor molecules in disease. Yet, many analyses multiple motor behaviors, especially those in living cells, have been limited by inabilities to control and / or characterize the number of motors responsible for cargo motion, their organization on cargos, as well as the mechanical barriers motors face during intracellular transport. Here, we describe an approach to engineer COS7 cells that provides genetic-level control over the densities and ratios of different motors on vesicular cargos (peroxisomes), as well as the loads imposed experienced by motors via the regulation of cargo size. Such control is facilitated by the construction of multi-component gene regulatory constructs and opens new opportunities to evaluate the collective responses of different types of motor molecules systematically in living cells. Assays comparing the collective behaviors of kinesin-1 and myosinVa motors - via analyses of the responses of peroxisome velocities, run lengths, and position noise to motor density and cargo size - indicate that multiple myosinVa motors can cooperate more productively that groups of kinesins. Overall, these results support predictions from in vitro experiments and theoretical analyses suggesting that the susceptibilities of motor velocity and filament detachment rates to forces are primary determinants of how effectively teams of motors can cooperate under the applied load imposed by the COS7 cell cytoplasm. The role of such behaviors in mechanisms regulating cargo motion will be discussed.