The mitotic kinesin-14 KlpA contains a context-dependent directionality switch

Abstract

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.

Figure 1. Surface-immobilized KlpA molecules exhibit minus end-directed motility to glide microtubules.

(a) Schematic diagrams of the full-length KlpA and the recombinant GFP-KlpA. The full-length KlpA consists of three consecutive coiled coils (CC1, aa 153–249; CC2, aa 250–297; and CC3, aa 298–416), a neck (aa 417–421) and a catalytic microtubule-binding motor domain (aa 422–756). GFP-KlpA contains an N-terminal polyhistidine-tag (not shown). (b) Coomassie-stained SDS–polyacrylamide gel electrophoresis (SDS–PAGE) of purified recombinant GFP-KlpA. (c) Schematic diagram of the microtubule-gliding assay. Movement of microtubules driven by surface-immobilized GFP-KlpA molecules was visualized by TIRF microscopy. Microtubules were fluorescently labelled with tetramethylrhodamine (TMR), and polarity-marked with a dim minus end and a bright plus end. (d) Representative TIRF microscopy images of polarity-marked microtubules moving on the coverslip surface with the bright plus ends leading (yellow arrowheads). (e) Histogram showing the microtubule-gliding velocity distribution of GFP-KlpA. Red line indicates a Gaussian fit to the velocity histogram. Scale bar, 5 μm.

Figure 2. KlpA moves processively towards the plus end on single microtubules.

(a) Schematic diagram of the in vitro KlpA motility assay. Microtubules were fluorescently labelled with Hilyte 647, and polarity-marked with a dim minus end and a bright plus end. (b) Example kymograph showing GFP-KlpA molecules (green), at relatively high protein input levels, form a plus end-directed flux and accumulate there on a surface-immobilized polarity-marked microtubule (red). Yellow arrow indicates GFP-KlpA accumulation at the microtubule plus end, and white arrow indicates minus end-directed movement of a GFP-KlpA aggregate. (c) Example kymographs showing that individual GFP-KlpA molecules (green) move towards the plus end on single polarity-marked microtubules (red) in a processive manner. (d) Velocity histogram of individual GFP-KlpA molecules on single microtubules. Red line indicates a Gaussian fit to the velocity histogram. (e) Run-length histogram of individual GFP-KlpA molecules on single microtubules. Red line indicates a single exponential fit to the run-length histogram. Scale bars, 1 min (vertical) and 5 μm (horizontal).

Figure 3. Plus end-directed processive motility of KlpA on single microtubules requires its N-terminal nonmotor microtubule-binding tail.

(a) Schematic diagrams of the full-length KlpA and the recombinant GFP-KlpA-Δtail. GFP-KlpA-Δtail contains a polyhistidine-tag (not shown) and a GFP at the N terminus, and residues 303–770 of KlpA. (b) Representative TIRF microscopy images of GFP-KlpA-Δtail driving polarity-marked microtubules (red) to glide with the bright plus ends leading (white arrowheads). (c) Velocity histogram of microtubule-gliding by GFP-KlpA-Δtail. Red line indicates a Gaussian fit to the velocity histogram. (d) Example kymograph showing that individual GFP-KlpA-Δtail molecules (yellow) exhibit nonprocessive movement on a single polarity-marked microtubule with a bright plus end. White arrow indicates minus end-directed movement of a rare GFP-KlpA-Δtail aggregate. Scale bars, 1 min (vertical) and 5 μm (horizontal).

Figure 4. KlpA exhibits opposite directional preference inside and outside the microtubule overlaps.

(a) Schematic diagram of the microtubule-sliding assay showing that KlpA contains context-dependent opposite directional preference. Track and cargo microtubules were fluorescently labelled with Hilyte 647 and TMR, respectively, and polarity-marked with a dim minus end and a bright plus end. (b) Example kymographs of GFP-KlpA motility inside and outside the antiparallel microtubule overlap. Yellow arrow indicates GFP-KlpA accumulation at the microtubule plus end outside the antiparallel microtubule overlap. White arrow indicates minus end-directed movement of GFP-KlpA inside the antiparallel microtubule overlap. (c) Example kymographs of GFP-KlpA motility inside and outside the parallel microtubule overlap. Yellow arrow indicates GFP-KlpA accumulation at the microtubule plus end outside the parallel microtubule overlap. White arrow indicates GFP-KlpA accumulation at the microtubule minus end inside the parallel microtubule overlap. Scale bars, 30 s (vertical) and 5 μm (horizontal).