Microtubule Motor Traffic Jams

Abstract

In the cell, microtubules spread throughout the cytoplasm, creating an intracellular highway system, with plus ends pointing towards the periphery. Kinesin-1 uses energy derived from ATP hydrolysis to walk towards microtubule plus ends to deliver cargoes, such as organelles or mRNA to the cell periphery. Multiple motors often work together to transport a single cargo. Transport properties of both single kinesin motors and single cargoes carried by multiple motors have been characterized. However, these studies are typically done in dilute conditions that do not accurately represent the cellular environment, where crowding or motor exchange on cargoes can occur. To address crowding and motor exchange, we use quantum dots, known to spontaneously bind kinesin-1, as cargo in our experiments and high concentrations of kinesin-1 to mimic crowded conditions on microtubules. Our system allows cargoes to self-assemble with exchanging kinesin-1 motors as the cargo is transported. Using Total Internal Reflection Fluorescence (TIRF) Microscopy, we tracked individual cargoes over a wide range of kinesin-1 concentrations to mimic varying degrees of crowded conditions. We found that while the velocity of cargoes decreased as conditions became more crowded, the run length and total association time of cargoes increased. We observed that cargoes paused more frequently in crowded conditions. Interestingly, we also observed cargo reversal events during runs, which were more likely to occur in crowded conditions. We believe these reversals occur when multiple motors are bound to a single cargo. If the leading motor is stretched and under strain, if it detaches, the cargo will rock backwards. Using coarse-grained Brownian dynamics simulations combined with model convolution microscopy, we can recapitulate velocity reduction, increased pausing, and reversals in silico.