DNA tensiometer reveals catch-bond detachment kinetics of kinesin-1, -2 and -3
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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 that the load-dependent detachment sensitivity of transport kinesins is 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.
Significance Statement
Kinesin and dynein motor proteins transport intracellular cargo bidirectionally along microtubule tracks, with the speed and directionality of transport involving competition between motor teams. We created a ‘DNA tensiometer’ that uses DNA as a spring to measure kinesin performance against loads oriented parallel to the microtubule. We find that dissociation rates for all three families of transport kinesins slow down with imposed loads. Dyneins are also thought to possess this ‘catch-bond’ behavior, meaning that both motors will hang on tightly during a tug-of-war. Building on previous work that showed combined vertical and horizontal loads cause faster detachment rates under load, we conclude that the effectiveness of kinesins during bidirectional transport depends strongly on the geometry of their cargo.
