Preparation and characterization of recombinant C. elegans kinesin-II and OSM-3. (A) SDS gels of Sf9 cell high speed supernatant (left), Talon column eluate (middle), and Sephacryl S-300 purified kinesin-II (right). (B) SDS gels of purified kinesin-II and OSM-3. (C and D) Double reciprocal plots of kinesin-II– (C) and OSM-3 (D)–driven MT motility versus [Mg-ATP] in standard MT gliding assays. (E) MT gliding velocity driven by kinesin-II (circles), OSM-3 (squares), and OSM-3–G444E (triangles) under standard assay conditions but varying concentrations of K₂-Pipes. Error bars represent the standard deviations. (F and G) On sucrose density gradients (F) and gel filtration columns (G), the KLP-11, KAP-1, and KLP-20 subunits elute as a monodisperse heterotrimeric complex (S value = 9.8; Rs = 7.1 nm; and native molecular mass = 287 kD) in a KLP-11/KLP-20/KAP-1 molar stoichiometry of 1.0:1.17:0.89 (protein standard peak positions are also indicated).

MT gliding rate versus mole fraction for mixtures of OSM-3 and kinesin-II showing alternating action and mechanical competition model fits. (A and B) Velocity histograms of gliding rates in competitive motility assays as a function of the percentage of wild-type OSM-3 (A) and OSM-3–G444E mutant (B) versus kinesin-II. Between 0 and 100% OSM-3, gliding rates intermediate between those produced by each motor alone are observed. (C and D) Gliding assay plotted versus mole fraction of wild-type (WT) OSM-3. Experimental data (black dots) with standard deviations (error bars) are shown with best fits for the alternating action (red line; C) and the mechanical competition (blue line; D) models. The parameters are as follows: v_kinesin-II = 0.34 μm/s and v_osm-3 = 1.09 μm/s (C); and , , and γ = 0.98 ≈ 1 (D). (E and F) Gliding assay velocities plotted versus mole fraction of OSM-3–G444E. Experimental data (black dots) with standard deviations are shown with the best fit for the alternating action (red line; E) and the mechanical competition (blue line; F) models. The parameters are as follows: v_kinesin-II = 0.46 μm/s and v_osm-3 = 0.99 μm/s (E); and , , and γ = 0.7 (F).

Model showing BBS proteins antagonizing mechanical competition between kinesin-II and OSM-3 to maintain IFT particle integrity. (A) Demonstrates distinct phenotypes predicted by the two models in bbs-;motor double mutants. The alternating action model predicts that in bbs-;klp-11 double mutants, IFT-A cannot be moved by either kinesin-II or OSM-3–kinesin and will not enter cilia, so IFT-A will form aggregates in the endings of truncated cilia, mimicking the phenotype of IFT-A mutants. On the other hand, in bbs-;osm-3 double mutants, IFT-B cannot be moved by kinesin-II or OSM-3, and ciliary length will decrease. In contrast, the mechanical competition model predicts that in either bbs-;klp-11 or bbs-;osm-3 double mutants, there will be no mechanical competition between the two motors or no drag exerted through IFT particles, so even in the absence of BBS proteins, IFT particles can be maintained in a single complex, and A and B subcomplexes will display identical transport profiles. (B) Summary of the results of transport assays that test the predictions (Fig. 4). In wild type (WT), BBS proteins maintain IFT particle integrity by antagonizing the mechanical competition between kinesin-II and OSM-3. In bbs-7/-8 single mutants, mechanical competition between kinesin-II and OSM-3 is not counterbalanced by BBS proteins, so IFT particles dissociate into subcomplexes A and B. In bbs-7/-8;kinesin-II or bbs-7/-8;osm-3 double mutants, no mechanical competition is generated, so IFT particles do not dissociate but are moved by kinesin-II or OSM-3 alone.

Transport assays of IFT particle subcomplexes A and B in bbs-7 single and bbs-7 or bbs-8; kinesin-2 double mutants. Micrographs of the distribution of IFT-A (CHE-11∷GFP) and -B (CHE-2∷GFP and OSM-6∷GFP) subcomplexes along sensory cilia (CHE-11∷GFP in A, B, E, G, I, and K; CHE-2∷GFP in C, D, F, H, J, and L; and OSM-6∷GFP in M–O). Kymographs and corresponding graphs in E–O (right) show the diagonal lines that represent trajectories of movement along the initial (M and M′) and distal segments (D and D′). Arrowheads point to initial-distal segment junctions. In wild-type (wt) animals (A and C), CHE-11∷GFP and CHE-2∷GFP move identically along initial and distal segments. In bbs-7 mutants (B and D), IFT-A and -B dissociate, CHE-11∷GFP only moves within the initial segment, and CHE-2∷GFP moves along both the initial and distal segments. In klp-11;bbs-7 or bbs-8 double mutants, CHE-11∷GFP, CHE-2∷GFP, and OSM-6∷GFP move at OSM-3–kinesin's fast velocity along the initial and distal segment (E, F, I, J, and M). In osm-3; bbs-7 or bbs-8 double mutants, CHE-11∷GFP, CHE-2∷GFP, and OSM-6∷GFP move at kinesin-II's slow rate in the remaining initial segment (G, H, K, L, and N). OSM-6∷GFP moves at OSM-3's fast rate in klp-11 mutants (O). Unlike in bbs single mutants, IFT particles are stable and do not dissociate into IFT-A and -B in bbs-7 or bbs-8; kinesin-2 double mutants.