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.