XBX-1 encodes a dynein light intermediate chain required for retrograde intraflagellar transport and cilia assembly in Caenorhabditis elegans

Abstract

Intraflagellar transport (IFT) is a process required for flagella and cilia assembly that describes the dynein and kinesin mediated movement of particles along axonemes that consists of an A and a B complex, defects in which disrupt retrograde and anterograde transport, respectively. Herein, we describe a novel Caenorhabditis elegans gene, xbx-1, that is required for retrograde IFT and shares homology with a mammalian dynein light intermediate chain (D2LIC). xbx-1 expression in ciliated sensory neurons is regulated by the transcription factor DAF-19, as demonstrated previously for genes encoding IFT complex B proteins. XBX-1 localizes to the base of the cilia and undergoes anterograde and retrograde movement along the axoneme. Disruption of xbx-1 results in cilia defects and causes behavioral abnormalities observed in other cilia mutants. Analysis of cilia in xbx-1 mutants reveals that they are shortened and have a bulb like structure in which IFT proteins accumulate. The role of XBX-1 in IFT was further confirmed by analyzing the effect that other IFT mutations have on XBX-1 localization and movement. In contrast to other IFT proteins, retrograde XBX-1 movement was detected in complex A mutants. Our results suggest that the DLIC protein XBX-1 functions together with the CHE-3 dynein in retrograde IFT, downstream of the complex A proteins.

Figure 1.

DAF-19 regulation of xbx-1 expression. (A) Northern blot analysis of xbx-1 expression in daf-19(+) and daf-19(m86) worms. xbx-1 expression is reduced in daf-19(m86) worms. The DAF-19 independent gene snb-1 was used as a loading control. (B and C) In vivo analysis of xbx-1 promoter driven GFP expression in (B) daf-19(+) and (C) daf-19(m86) mutant worms. xbx-1 is expressed in the ciliated sensory neurons of the wild-type worms. xbx-1 expression is significantly reduced in the absence of DAF-19. Anterior of the worm is directed toward the left. (D) Genomic organization of the xbx-1 gene (F02D8.3), which maps on linkage group (LG) V. Numbered boxes, exons; black boxes, exons with significant sequence similarities to DLIC proteins from other species (cf. Grissom et al., 2002, and Perrone et al., 2003). The region of the gene deleted in the xbx-1(ok279) mutant allele is shown above the schematic.

Figure 2.

Restoration of cilia in xbx-1 mutants by transgenic rescue. All panels are bright field images overlaid with fluorescent images to show dye-filling (red) and XBX-1:: YFP localization (yellow). (A) Six pairs of amphid neurons of wild-type N2 worms fill with the fluorescent dye DiD. (B) In contrast, xbx-1(ok279) mutant worms fail to take up the dye. (C) Transgenic expression of xbx-1::yfp in xbx-1(ok279) mutant worms restores the ability to uptake fluorescent dye (red). The XBX-1::YFP protein is detected at the transition zones and within cilia on the ends of the amphid neurons (yellow, arrowhead). Anterior is toward the left in all panels.

Figure 3.

Movement of XBX-1::YFP along the cilia axoneme in phasmid neurons of a wild-type hermaphrodite. Using time-lapse fluorescence image analysis, XBX-1::YFP movement was observed in both (A) anterograde and (B) retrograde directions similar to that seen for other IFT proteins (Signor et al., 1999a; Haycraft et al., 2001; Qin et al., 2001). The transition zone is marked (*). The arrowhead indicates the initial position of the particle at time zero (t = 0). The arrows indicate the same particle at subsequent times (t = 0.5 s, and t = 1 s). The distal tips of the phasmid cilia are directed toward the left. Three sequential frames from Movie 1 are shown for each.

Figure 4.

Analysis of cilia morphology in wild-type and IFT mutants using fluorescence-tagged IFT proteins. (A) OSM-5::GFP in wild-type worms shows full-length cilia with OSM-5::GFP localized at the transition zones and within cilia. (B) Complex B mutants (osm-5 (m184)) exhibit severely truncated cilia axonemes as visualized by localization of OSM-6::YFP to the base of the truncated cilia. (C) Because of loss of retrograde IFT movement in che-3 mutants, the cilia are severely shortened and exhibit a significant accumulation of OSM-5::GFP along the enlarged cilia axonemes. (D) The cilia in complex A mutants (che-11(e1810)) as determined by using OSM-5:: GFP are similar to those seen in the che-3 mutants. The cilia are truncated and show significant accumulation of OSM-5::GFP along the swollen cilia axonemes. (E) Analysis of the cilia morphology in xbx-1 mutants using OSM-5::GFP. Similar to that seen in both che-3 and complex A mutants, the cilia of xbx-1 mutants were truncated and OSM-5::GFP concentrated along the enlarged axonemes. (F) Schematic diagram depicting the cilia morphology as determined using the IFT::G/YFP fusion proteins. Arrows pointing right indicate retrograde movement while arrows pointing left indicate anterograde movement. Two lines drawn through the arrows indicate defects in transport. Images A–E are 2-D confocal projections from the amphid region of the worm. In all panels, arrowheads point to the cilia axonemes of one bundle of amphid neurons. The distal end of the cilium is directed toward the left in all panels.

Figure 5.

Localization of IFT proteins in xbx-1 mutants. To evaluate the effect that the xbx-1(ok279) mutation has on the localization of IFT proteins, we crossed transgenic lines expressing either complex B or complex A IFT proteins fused to either GFP or YFP into the xbx-1 mutant background. The loss of XBX-1 results in accumulation of both complex A and B proteins along the axoneme as shown using (A) OSM-5::GFP (complex B), (B) OSM-6::YFP (complex B), and (C) CHE-11::GFP (complex A). Arrowheads indicate the swollen cilia axonemes. Anterior of the worm and the distal tips of the cilia are directed toward the left. All panels are 2-D confocal projections.

Figure 6.

The effect of IFT mutations on XBX-1 localization. Localization of XBX-1::YFP was analyzed by moving the XBX-1::YFP transgene into the indicated IFT mutant backgrounds. (A) XBX-1::YFP localizes to the base of cilia and within cilia in wild-type N2 worms. (B) XBX-1::YFP localizes to the transition zone at the base of cilia in the osm-5 complex B mutant, with little if any fluorescence detected along the cilia axoneme. (C) XBX-1::YFP is localized to the base of cilia in the che-11 complex A mutant. Unlike other IFT proteins analyzed in complex A mutants, there is no significant accumulation of XBX-1::YFP within cilia (see Figure 4D). (D) XBX-1::YFP accumulates in large bulb-like endings in the che-3 IFT dynein mutant. This is similar to that reported for other IFT proteins (see Figure 4C). Anterior of the worm and distal tip of the cilium are toward the left. Arrowheads indicate the position of the cilia axonemes on one bundle of amphid neurons.

Figure 7.

Movement of XBX-1::YFP along the cilia axonemes on one pair of phasmid neurons of a che-11 complex A mutant. Using time-lapse fluorescence image analysis, XBX-1::YFP movement was observed in the (A) anterograde direction along the stunted cilia axoneme of the che-11 mutants. In contrast to other IFT proteins, XBX-1::YFP transport was also detected in the (B) retrograde direction. This continued retrograde trafficking of XBX-1 in che-11 mutants is consistent with the failure of XBX-1 to accumulate in the cilia axoneme. Images were captured at a rate of two frames per second. The distal tips of the phasmid cilia are directed toward the left. The transition zone is marked (*). The arrowhead indicates the initial IFT particle at time zero (t = 0). The arrows indicate the same particle at the subsequent times indicated (t = 0.5 s, and t = 1 s). Three frames representative of the movement seen in Movie 2 are shown for each direction.