To extract useful work from biological motor proteins, it is necessary to orient microtubules traveling over kinesin-coated surfaces properly. Toward this goal, we have used microfabrication to construct 1.5-µm-deep channels in SU-8 photoresist patterned on glass. Although motor proteins bind to all surfaces, these channels localize motility exclusively to the glass surface, and the photoresist creates steep walls that direct microtubule movement. This technique provides a general approach for lithographically patterning enzyme activity.

Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple kinesins coordinate their activity during cargo transport are poorly understood. To understand these multi-motor coordination mechanisms, defined pairs of kinesin-1 and kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than kinesin-1 at the single-molecule level, addition of kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the kinesin-2 microtubule reattachment rate was shown to be 4-fold faster than that of kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of gold-nanoparticle-labeled cargo with 1 ms and 2 nm precision revealed that kinesin-2 motors detach and rebind to the microtubule much more frequently than do kinesin-1. Finally, cargo transported by kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multi-motor transport and suggest a coordinated transport model in which kinesin-1 motors step effectively against loads while kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.

Kinesins are biological motors that transport cargo unidirectionally along microtubule tracks. These motors are attractive candidates for carrying out biomolecular separations, directed assembly of nanoparticles, or for powering nano- or microscale devices. However, a prerequisite for harnessing kinesins is properly aligning the microtubule tracks that they walk along. We describe a method for constructing an array of aligned microtubules on a two-dimensional surface. The process involves immobilizing short microtubule seeds, polymerizing long microtubules uniquely from one end, and then attaching the elongated filaments to the surface. To quantitate the extent of microtubule alignment, we analyzed microtubule orientations from four different arrays and found a standard deviation of 12.8°, which is comparable to the alignment of oriented microtubule arrays observed in migrating fibroblasts. By producing a field of aligned microtubules, this array provides a launching point for employing kinesins for directed assembly or nanoscale force generation.

Dynein–dynactin–BicD2 (DDB) is highly processive, but also shows transient pausing and diffusion, which we analyzed using iSCAT microscopy. Blocking dynactin p150 results in more diffusion of isolated DDB and a plus-end shift of kinesin-1–DDB complexes. Thus, we conclude that p150 is an allosteric activator of dynein in the DDB complex.