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The eukaryotic cell’s microtubule cytoskeleton is a complex 3D filament network. Microtubules cross at a wide variety of separation distances and angles. Prior studies in vivo and in vitro suggest that cargo transport is affected by intersection geometry. However, geometric complexity is not yet widely appreciated as a regulatory factor in its own right, and mechanisms that underlie this mode of regulation are not well understood. We have used our recently reported 3D microtubule manipulation system to build filament crossings de novo in a purified in vitro environment and used them to assay kinesin-1–driven model cargo navigation. We found that 3D microtubule network geometry indeed significantly influences cargo routing, and in particular that it is possible to bias a cargo to pass or switch just by changing either filament spacing or angle. Furthermore, we captured our experimental results in a model which accounts for full 3D geometry, stochastic motion of the cargo and associated motors, as well as motor force production and force-dependent behavior. We used a combination of experimental and theoretical analysis to establish the detailed mechanisms underlying cargo navigation at microtubule crossings.

Transport of cargos along the cytoskeleton is a complex and essential process in all eukaryotic cells. Cargo transport is driven by many different types of molecular motors of the kinesin, dynein, and myosin families, which all have distinct biomechanical properties and can interact with one another in complex ways. Decades of single-motor experiments have illuminated many of the properties of individual motors, but much less work has been done on multiple motor-driven transport. Multiple motor-driven transport adds considerable complexity to cargo routing, both via competition or coordination between different types of motors interacting with the same cargo, and by enabling cargo to simultaneously interact with multiple cytoskeletal filaments. Many individual aspects of this transport system may be regulated by the cell in order to control the final outcomes of cargo routing, but it is not possible in vivo to separate out the individual contributions. In this work, I have applied both optical trapping techniques and temperature dependent studies to highly simplified in vitro systems in order to demonstrate possible mechanisms of regulating cargo routing in cells. I present results from several different projects that show how both microtubule network properties and complex multiple-motor driven motility can significantly impact the route cargos take through the cell. Additionally, I report that the enzymatic properties of several kinesin motors can be regulated via environmental/chemical adjustments, and that this is likely a universal property of molecular motors. Together, these findings support the hypothesis that the complex interactions between molecular motors and the cytoskeleton are regulable by the cell, and this regulation is necessary in order for balanced and consistent transport to occur over the wide range of external conditions experienced by cells.