TABLE 1.

Motility of monomeric and dimeric kinesin

Microtubule gliding		Single molecule fluorescence
Kinesin	Velocity (nm s−1)	Velocity (nm s−1)	Travel distance (nm)	Association events (events per min per µm MT)
dK340	7 ± 2	–	–	0.08 ± 0.02
dK350	120 ± 10	–	–	0.22 ± 0.07
dK365	110 ± 10	–	–	0.30 ± 0.04
dK440	570 ± 70	340 ± 90	400 ± 40	2.48 ± 0.40

Microtubule translocation induced by monomeric and dimeric Drosophila kinesin proteins (number of amino acids indicated) in multiple motor gliding or single fluorescent molecule motility assays. Velocities are the means and s.d. of > 20 measurements (microtubules in the case of the gliding assay, and axonemes in the case of the single molecule fluorescence assay), and the association events are the means and s.d. from > 3 axonemes (5–20 min of observation per axoneme (a minimum of 10 events were scored)). The dK440 travel distance and error were determined as shown in Fig. 3. Dashes signify that no movement was detected. dK340cys, dK350cys, dK365cys, and dK440cys were generated by polymerase chain reaction using the Drosophila kinesin gene, expressed in bacteria and purified by phosphocellulose followed by mono-Q chromatography. Hydrodynamic studies indicated that, consistent with previous work¹²,¹⁴,¹⁵, dK440 is a dimer and that dK365, dK350 and dK340 are monomers. Sucrose gradient velocity sedimentation indicates that dK440-Cy3 (5 S) remains a dimer at 3 nM. For microtubule gliding assays, the kinesin reactive cysteine was modified with long-chain biotin–maleimide⁸, streptavidin was added in an 8:1 ratio, and the kinesin–biotin–streptavidin complexes were isolated by microtubule affinity¹⁸,¹⁹. For the motility assay, 0.5 mg ml⁻¹ biotinylated BSA was introduced into a microscopic flow chamber. After washing, streptavidin-modified kinesin motor (1–5 µM) was introduced in motility buffer with 3 mg ml⁻¹ casein. Next, rhodamine-labelled microtubules¹⁸ (~10 µg ml⁻¹) were introduced in the above buffer with 1 mM ATP and an oxygen scavenger system²⁰ and examined by fluorescence microscopy. For the single molecule fluorescence assay, Drosophila kinesin proteins were labelled with Cy3 and assayed for motility as described for Fig. 1.