Kinesin-1 Motors Can Circumvent Permanent Roadblocks by Side-Shifting to Neighboring Protofilaments

Abstract

Obstacles on the surface of microtubules can lead to defective cargo transport, proposed to play a role in neurological diseases such as Alzheimer's. However, little is known about how motor proteins, which follow individual microtubule protofilaments (such as kinesin-1), deal with obstacles on the molecular level. Here, we used rigor-binding mutants of kinesin-1 as roadblocks to permanently obstruct individual microtubule binding sites and studied the movement of individual kinesin-1 motors by single-molecule fluorescence and dark-field scattering microscopy in vitro. In the presence of roadblocks, kinesin-1 often stopped for ∼ 0.4 s before either detaching or continuing to move, whereby the latter circumvention events occurred in >30% after a stopping event. Consequently, and in agreement with numerical simulations, the mean velocity, mean run length, and mean dwell time of the kinesin-1 motors decreased upon increasing the roadblock density. Tracking individual kinesin-1 motors labeled by 40 nm gold particles with 6 nm spatial and 1 ms temporal precision revealed that ∼ 70% of the circumvention events were associated with significant transverse shifts perpendicular to the axis of the microtubule. These side-shifts, which occurred with equal likelihood to the left and right, were accompanied by a range of longitudinal shifts suggesting that roadblock circumvention involves the unbinding and rebinding of the motors. Thus, processive motors, which commonly follow individual protofilaments in the absence of obstacles, appear to possess intrinsic circumvention mechanisms. These mechanisms were potentially optimized by evolution for the motor's specific intracellular tasks and environments.

Heterogeneity in kinesin function

“…In summary, because we observed kinesins which travel with persistently different mean velocities on the same MT (Figure , Movies S2 to S5), and the frequency of velocity groups can be affected by glycerol and temperature as well as pH, we believe that the functional heterogeneity is intrinsic to the motors themselves, not caused by the heterogeneity in MTs or roadblocks on them . Instead, single motors appear to have relatively well defined—but different—mean stepping rates.…”

Kinesin‐2 motors adapt their stepping behavior for processive transport on axonemes and microtubules

“…For example, the direct comparison between kinesin‐1 and kinesin‐2 motors showed that Tau, a MAP known to heavily decorate axonal microtubules in vivo , affected kinesin‐1's but not kinesin‐2's processivity in vitro . Even though kinesin‐1 can also overcome permanent obstacles in vitro , kinesin‐2 appears to do so more efficiently via its higher propensity to switch protofilaments when compared to kinesin‐1 .…”

Interferometric Scattering Microscopy for the Study of Molecular Motors

“…Attachment of nanoparticle labels to kinesin, myosin, and dynein motors has to date been achieved via biotin–streptavidin chemistry (Andrecka et al, 2015; Dunn & Spudich, 2007; Isojima, Iino, Niitani, Noji, & Tomishige, 2016; Mickolajczyk et al, 2015; Schneider, Glaser, Berndt, & Diez, 2013; Schneider, Korten, Walter, & Diez, 2015). One method of introducing biotin into kinesin is to create a cysteine-lite mutant, in which solvent-exposed cysteines are mutated out of the motor domain and a single cysteine is kept to take advantage of sulfhydryl chemistry for attaching a maleimide-functionalized label.…”