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 (koff) can be measured via single-molecule experiments, but motor reattachment rate constants (kon) 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 the attachment dynamics of motors bound to fluid lipid bilayers, we quantified the microtubule accumulation rate of fluorescently labeled kinesin-1 motors in a 2-dimensional (2D) system where motors were linked to a supported lipid bilayer. From the first-order accumulation rate at varying motor densities, we extrapolated akoffthat matched single-molecule measurements and measured a 2Dkonfor membrane-bound kinesin-1 motors binding to the microtubule. Thiskonis 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 thiskonis not limited by the motor diffusion rate, but instead is determined by the intrinsic motor binding rate. For intracellular vesicle trafficking, this 2Dkonpredicts 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.

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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 (koff) can be measured via single-molecule experiments, but motor reattachment rate constants (kon) 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 koff that matched single-molecule measurements, and measured a two-dimensional kon for membrane-bound kinesin-1 motors binding to the microtubule. This kon 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 kon 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 kon 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 StatementLong-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 membranebound 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.

Microtubules (MTs) and their associated proteins are essential for many cellular processes, including maintenance of cellular structure, cell motility, cell division, and intracellular transport. Kinesin superfamily (KIFs) proteins are molecular motors that directionally transport organelles and cargos along MTs. As the first-discovered kinesins, the Kinesin-1 superfamily (KIF5s) is a group of highly processive motor proteins. Despite KIF5B's importance in cellular health, atomic level insight to the structure, dynamics, and microtubule interface are lacking. Magic Angle Spinning (MAS) NMR spectroscopy is well suited for structure and dynamics characterization of KIF5B motor domain in complex with MTs. We present an investigation into the atomic-resolution structure of KIF5B motor domain in complex with polymeric MTs by MAS NMR. We applied multidimensional (2D and 3D) homo-and heteronuclear experiments in U-13 C, 15 N-kinesin/MT complex for resonance assignments. All homo-and heteronuclear correlation spectra exhibited high resolution and revealed that more than 80% of the residues are present in the spectra. Chemical shift predictions were performed by ShiftX2 using the X-ray structure of KIF5B bound to a/b tubulin dimer. Based on the 2D/3D spectra, together with SHIFTX2 predictions, characteristic spin system assignments were identified and one third of residue specific assignments have been achieved. In addition, we applied homonuclear experiments in [1,6-Glu-13 C]-[U-15 N]-kinesin/MT complex and 1 H detection experiments under fast MAS in U-2 H, 13 C, 15 N-kinesin/MT complex to reduce spectral complexity and enhance resolution.