Experimental and Computational Investigations into Cooperative Cargo Transport by Mixtures of Kinesins from Different Families

Abstract

Transport of intracellular cargo often involves multiple motor types, either having opposite directionality such as during bidirectional transport of vesicles, or having the same directionality but different speeds. While significant progress has been made in characterizing motors at the single-molecule level, predicting their ensemble behavior is still challenging. To uncover the force-dependent properties of diverse kinesins and to understand how diverse kinesins attached to the same cargo coordinate their movement, we carried out microtubule gliding assays using pairwise mixtures of motors from the kinesin-1, 2, 3, 5 and 7 families. To match their processivities and ensure identical binding to the glass substrate, the motors were fused to the dimerization domain and coil-1 of kinesin-1, and the neck linkers were adjusted to have a uniform length of 14 amino acids. Uniform motor densities were used and microtubule-gliding speeds were measured as the ratio of fast motors varied from 0 to 1. Depending on the motor pair, velocity versus motor fraction curves varied from convex up to nearly linear to convex down. These findings were recapitulated using a coarse-grained computational model of gliding assays. The simulations incorporate force dependent velocities and dissociation rates from the literature along with mechanical interactions between motors bound to the same microtubule. The simulations also suggest that the motor compliance plays a minimal role in the observed gliding speed compared to observations in quantum dots. The gliding assays combined with the modeling allows us to test hypotheses regarding the characteristics of diverse kinesins under predominantly axial load, avoiding the large normal forces inherent in optical tweezer experiments.