Kinesin-14s are commonly known as nonprocessive minus end-directed microtubule motors that function mainly for mitotic spindle assembly. Here we show using total internal reflection fluorescence microscopy that KlpA—a kinesin-14 from Aspergillus nidulans—is a context-dependent bidirectional motor. KlpA exhibits plus end-directed processive motility on single microtubules, but reverts to canonical minus end-directed motility when anchored on the surface in microtubule-gliding experiments or interacting with a pair of microtubules in microtubule-sliding experiments. Plus end-directed processive motility of KlpA on single microtubules depends on its N-terminal nonmotor microtubule-binding tail, as KlpA without the tail is nonprocessive and minus end-directed. We suggest that the tail is a de facto directionality switch for KlpA motility: when the tail binds to the same microtubule as the motor domain, KlpA is a plus end-directed processive motor; in contrast, when the tail detaches from the microtubule to which the motor domain binds, KlpA becomes minus end-directed.

Kinesins are commonly homodimers with two identical heavy chains (protomers) and play indispensable roles in many intracellular processes. Engineered heterodimeric kinesins with two distinct protomers are important tools for dissecting coordination and regulation of naturally occurring kinesin homodimers. Here, we report a chemical-biology-based approach that generates kinesin heterodimers by combining genetic incorporation of reactive noncanonical amino acids and small-molecule-based cross-linking. We verified using yeast kinesin-8/Kip3 as a model system that our method yields kinesin heterodimers of desired properties without introducing unintended motility disruption. To demonstrate the utility of our method, we engineered a crippled Kip3 heterodimer that contains both a wild-type-like protomer and a catalytically inactive one, and our results revealed that the resulting heterodimer moves on the microtubule with a significant reduction in velocity but not processivity. Due to its versatility, we expect that our method can be broadly adopted to create novel heterodimers for other kinesins and will thus greatly expand the studies on kinesin mechanisms.

Kinesins are microtubule-based motor proteins that convert chemical energy from ATP hydrolysis into mechanical work for a variety of essential intracellular processes. Kinesin-14s (i.e. kinesins with a C-terminal motor domain) are commonly considered to be nonprocessive minus end-directed motors that mainly function for mitotic spindle assembly and maintenance. Here, we show that KlpA – a mitotic kinesin-14 motor from the filamentous fungus Aspergillus nidulans – contains a context-dependent directionality switch. KlpA exhibits canonical minus end-directed motility inside microtubule bundles, but on individual microtubules it unexpectedly moves processively toward the plus ends. Removal of the N-terminal nonmotor microtubule-binding domain renders KlpA diffusive on individual microtubules but does not abolish its minus end-directed motility to collectively glide microtubules, suggesting that the nonmotor microtubule-binding domain likely acts as a switch for controlling the direction of KlpA motility. Collectively, these findings provide important insights into the mechanism and regulation of KlpA functions inside the mitotic spindle.

Eukaryotic chromosome segregation requires the kinetochore, a megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP-A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its dedicated centromere-specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNAbinding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP-A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.