FIG. 1.

Movement of a single fluorescently labelled kinesin molecule along an axoneme. a, Kinesin (uhK560cys) labelled at the C-terminal cysteine residue with Cy3 fluorescent dye was combined with Cy5-labelled axonemes in the presence of ATP. The specimen was illuminated by the evanescent field resulting from total internal reflection of a laser beam at the boundary between the fused silica slide and the aqueous solution⁴. b, Cy5-axoneme. c, uhK560-Cy3. The caret (^) marks a stationary fluorescent spot, and the arrows track a moving fluorescence spot (time in seconds). Other spots visible in individual panels are ones that associated briefly (< 2 s) with the axoneme. Bar, 5 µm.

METHODS. uhK560 with an additional C-terminal peptide containing a reactive cysteine (PSIVHRKCF)⁸ was expressed in Escherichia coli²¹ and purified by phosphocellulose followed by mono-Q chromatography. uhK560-Cy3 (concentration determined from absorbance at 280 nm, A₂₈₀; ref. 22) was labelled to a stoichiometry of 0.1 to 0.5 dyes per polypeptide chain as described⁴. The uhK560-Cy3 preparation was centrifuged at 100,000 r.p.m. for 10 min in a table-top ultracentrifuge to remove any aggregates; additional microtubule-affinity purification¹⁹ was sometimes performed to improve motility. Analysis of low concentrations of uhK560-Cy3 by sucrose gradient velocity sedimentation¹⁶ (5 S) and Superose-6 gel filtration chromatography indicates that it is dimeric. The concentrations used in the sucrose gradient (5 nM) suggest that uhK560-Cy3 remains dimerized under the conditions of the assay. The microtubule-stimulated ATPase²³ properties of uhK560 labelled with Cy3 at a 0.43 molar ratio of dye to polypeptide (k_cat = 22.2 ± 1.0 s⁻¹/site; K_0.5,MT = 1.1 ± 0.2 µM) and at 0.96 stoichiometry (k_cat = 19.2 ± 0.2 s⁻¹/site; K_0.5,MT = 1.6 ± 0.06 µM) were similar to that of the unlabelled protein (k_cat = 25.8 ± 0.8 s⁻¹/site; K_0.5,MT = 0.7 ± 0.08 µM) (measured in the motility buffer: 12 mM PIPES, pH 6.8, 2 mM MgCl₂, 1 mM EGTA). Cy5-labelled axonemes²⁴ were combined with 0.5–1.5 nM uhK560-Cy3 in motility buffer containing 1 mM ATP, 7.5 mg ml⁻¹ BSA, 0.5% mercaptoethanol and an oxygen scavenger system²⁰ at 25 °C. BSA inhibited nonspecific adsorption of kinesin but did not prevent axonemes from attaching to the slide. Details of low-background total internal reflection fluorescence microscopy are described elsewhere⁴. In these experiments, 5.8 ± 1.0 association events per min per µm axoneme (n = 8 axonemes; 5-min observation for each axoneme) were observed when the uhK560-Cy3 concentration was 0.6 nM, whereas only 0.05 ± 0.01 events per min were scored on glass without an axoneme. Kinesin spots moving on an axoneme disappeared at 0.42 ± 0.03 s⁻¹, which was significantly faster than the rate of photobleaching of glass-attached kinesin (0.02 ± 0.0005 s⁻¹).