Microtubule Binding Kinetics of Membrane-bound Kinesin Predicts High Motor Copy Numbers on Intracellular Cargo

Bidirectional vesicle transport along microtubules is necessary for cell viability and function, particularly in neurons. When multiple motors are attached to a vesicle, the distance a vesicle travels before dissociating is determined by the race between detachment of the bound motors and attachment of the unbound motors. Motor detachment rate constants (k off ) can be measured via single-molecule experiments, but motor reattachment rate constants (k on ) are generally unknown, as they involve diffusion through the bilayer, geometrical considerations of the motor tether length, and the intrinsic microtubule binding rate of the motor. To understand motor attachment dynamics during vesicle transport, we quantified the microtubule accumulation rate of fluorescently-labeled kinesin-1 motors in a 2D system where motors were linked to a supported lipid bilayer. From the first-order accumulation rate at varying motor densities, we extrapolated a k off that matched single-molecule measurements, and measured a two-dimensional k on for membrane-bound kinesin-1 motors binding to the microtubule. This k on is consistent with kinesin-1 being able to reach roughly 20 tubulin subunits when attaching to a microtubule. By incorporating cholesterol to reduce membrane diffusivity, we demonstrate that this k on is not limited by the motor diffusion rate, but instead is determined by the intrinsic motor binding rate. For intracellular vesicle trafficking, this two-dimensional k on predicts that long-range transport of 100 nm diameter vesicles requires 35 kinesin-1 motors, suggesting that teamwork between different motor classes and motor clustering may play significant roles in long-range vesicle transport. Significance Statement Long-distance transport of membrane-coated vesicles involves coordination of multiple motors such that at least one motor is bound to the microtubule at all times. Microtubule attachment of a membrane-bound motor comprises two steps – diffusing through the lipid bilayer to a binding zone near the microtubule, followed by binding. Using a 2D supported lipid bilayer system, we show that membrane diffusion is not the limiting factor for motor attachment. This result suggests that in cells kinesin-1 binding kinetics are not altered by the membrane composition of vesicle cargos. The intrinsically slow binding properties of kinesin-1 suggest that divergent motor binding kinetics and motor clustering regulate long-range vesicle transport.