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The anterograde IFT motor, hetero-trimeric kinesin-II7, consists of two hetero-dimerized kinesin-related motor subunits and one accessory subunit (KAP)8. To observe kinesin-II-driven IFT within chemosensory cilia, we used transgenic lines of C. elegans expressing GFP fused to the kinesin-II KAP and to a presumptive cargo molecule, OSM-6, a component of IFT rafts that has an essential role in chemosensory ciliary function5,9.

With a fluorescence microscope, we observed that KAP::GFP and OSM-6::GFP polypeptides accumulate in the region of the transition zone at the base of the sensory cilia. This is consistent with previous immunofluorescence data on IFT motors and raft polypeptides in other systems5 (Fig. 1). We observed small fluorescent dots corresponding to the kinesin-II KAP and the OSM-6 cargo emerging from these regions and moving out towards the distal tip of the sensory cilia. Both the motor and its presumptive cargo moved anterogradely at identical rates (0.65±0.11 μm s-1 (n =50) for the KAP compared with 0.65±0.10 μm s-1 (n =50) for OSM-6), which is similar to the velocity of microtubule motility driven by purified heterotrimeric kinesin-II in a motility assay7. In contrast, the sensory ciliary transmembrane receptor ODR-10 moved at a faster rate (1.59±0.28 μm s-1 (n =10), confirming that the identical velocities displayed by KAP::GFP and OSM-6::GFP are not an artefact of the recording technique.

Figure 1: Visualization of intraflagellar transport (IFT).
figure 1

a, Schematic diagram of Caenorhabditis elegans chemosensory cilia. Top, a differential interference contrast light micrograph of the head of an adult worm orientated to the left. The boxed region shows the position of an amphid channel containing sensory cilia. This region is represented in the diagram (bottom), which shows a close-up of sensory cilia orientated with their distal endings facing left, and contacting the external environment through openings in the cuticle. Green indicates fluorescent kinesin-II or OSM-6, which accumulate at the transition zone at the base of the cilia (orange arrowhead) and move (black arrow) as dots (orange arrow) to the distal endings of the cilia. b, Fluorescence micrographs of sensory cilia in GFP transgenic worms as represented by the boxed region and the diagram in a. Fluorescent kinesin-II motors and fluorescent OSM-6 subunits of IFT rafts accumulate at the base of the transition zones where they appear as large dots (arrowheads). Arrows indicate position of dots of fluorescent kinesin-II and fluorescent OSM-6 as they travel to the distal tip of the sensory cilium (left, as in a). A movie of this process can be seen at http://www.mcb.ucdavis.edu/faculty-labs/scholey/. Details of the methods are available on request from J. M. S.

This direct viewing of the intracellular transport of a motor and its cargo in vivo provides strong support for the hypothesis that heterotrimeric kinesin-II is the motor protein that drives anterograde IFT5. In chemosensory neurons of C. elegans, it is likely that kinesin-II-driven IFT delivers structural components of sensory ciliary axonemes and components of the sensory signalling machinery that are concentrated in these cilia.

Genetic studies have identified 25 genes, including osm-6, that are essential for cili-ary function in this system10, and the ability to view IFT in organisms carrying mutations in these genes will make it possible to determine which of the corresponding gene products are linked to the kinesin-II transport pathway. In a broader context, our approach should allow the direct observation of motor and cargo molecules participating in IFT in a broad range of cilia and flagella.