Figure 3. DYF-11 is a component of the intraflagellar (IFT) transport machinery. (A) Kymograph analyses show the bi-phasic anterograde movement of DYF-11::GFP in amphid cilia. Middle segment (MS) motion was determined to be 0.74±0.08 um/sec, while the distal segment (DS) velocity was 1.15±0.21 um/sec. The second and fourth panels are actual kymographs, whereas the third and fifth panels are representative traces of moving particles in the indicated middle or distal segments; X axes denote time whereas Y axes denote distance covered by fluorescently-labeled particles. In the still fluorescence microscopy image taken from Movie S1 (left panel), solid arrowheads denote the positions of representative transition zones, while hollow arrowheads denote unexpected dendritic endings. (B) Human MIP-T3 tagged with a V5 epitope (V5-MIP-T3) localizes to centrosomes in IMCD3 kidney cells that have not yet ciliated, and to the axoneme when cilia are present. Centrosomes/basal bodies are stained with an antibody against gamma-tubulin (red), V5-MIP-T3 is marked with an antibody against the V5 epitope (green), and the nucleus is stained with DAPI (blue). Arrowheads and arrows point to centrosomes/basal bodies, and brackets denote ciliary axonemes. Scale bar, 5 um. (C) Similar to other well characterized IFT components, DYF-11::GFP accumulates in the cilia of
che-11 mutants, which displays retrograde transport defects. Arrowheads represent the positions of transition zones, brackets show ciliary axonemes, and stars denote accumulations (compare the localization of DYF-11::GFP in head amphid cilia of wild-type (A) and
che-11 mutant (C) animals; see also Figure 4A for normal DYF-11::GFP localization). Scale bar, 5 um. Schematics represent anterograde transport in wild-type animals (no accumulations) and
che-11 mutants (accumulations at the tip of cilia, shown as stars). K, Kinesin-II; O, OSM-3-kinesin; A, IFT subcomplex A; B, IFT subcomplex B; S, BBS protein complex. TZ, transition zones.