DNA tensiometer reveals catch-bond detachment kinetics of kinesin-1, -2, and -3

Abstract

Abstract

Bidirectional cargo transport by kinesin and dynein is essential for cell viability and defects are linked to neurodegenerative diseases. Computational modeling suggests that the load-dependent off-rate is the strongest determinant of which motor 'wins' a kinesin-dynein tug-of-war, and optical tweezer experiments find family-dependent differences in the sensitivity of detachment to load, with kinesin-3 > kinesin-2 > kinesin-1. However, in reconstituted kinesin-dynein pairs vitro, all three kinesin families compete nearly equally well against dynein. Modeling and experiments have confirmed that vertical forces inherent to the large trapping beads enhance kinesin-1 dissociation rates. In vivo, vertical forces are expected to range from negligible to dominant, depending on cargo and microtubule geometries. To investigate the detachment and reattachment kinetics of kinesin-1, 2 and 3 motors against loads oriented parallel to the microtubule, we created a DNA tensiometer comprising a DNA entropic spring attached to the microtubule on one end and a motor on the other. Kinesin dissociation rates at stall were slower than detachment rates during unloaded runs, and the complex reattachment kinetics were consistent with a weakly-bound 'slip' state preceding detachment. Kinesin-3 behaviors under load suggested that long KIF1A run lengths result from the concatenation of multiple short runs connected by diffusive episodes. Stochastic simulations were able to recapitulate the load-dependent detachment and reattachment kinetics for all three motors and provide direct comparison of key transition rates between families. These results provide insight into how kinesin-1, -2 and -3 families transport cargo in complex cellular geometries and compete against dynein during bidirectional transport.