C-terminal PRD restricts ARL-13 to the doublet segment of the cilium

In contrast to most small GTPases, which are ~20 kD and composed of only a GTPase domain, Arl13B has an atypical long C terminus that contains a coiled-coil helix and a PRD (Fig. 1 A). This unique structural pattern is conserved in all Arl13B protein family members from the green algae C. reinhardtii to mammals. In zebrafish, ciliary targeting of Arl13B requires both the N and C termini (Duldulao et al., 2009). To characterize the putative cilia targeting signal sequence of C. elegans ARL-13, we generated transgenic animals that express different GFP-tagged ARL-13 truncations. Interestingly, both the ARL-13 NT truncation (which retains the GTPase domain plus the coiled-coiled helix) and the ARL-13 PRD truncation (which possesses only the PRD domain) were targeted to cilia (Fig. 1 C), indicating redundant ciliary targeting signals in the N and C termini of the worm ARL-13 protein. Nonetheless, ARL-13 PRD::GFP showed restricted middle segment localization, whereas ARL-13 NT::GFP was present in both middle and distal segments. This result suggests that the PRD domain is critical for restriction of ARL-13 to the doublet segment. Although full-length ARL-13 protein was completely absent from the cell body, we observed significant GFP signals in the cytoplasmic region for ARL-13 NT::GFP and ARL-13 PRD::GFP (Fig. 1 C), which suggests that both the N and C termini are required for efficient ciliary targeting of ARL-13.

ARL-13 is required for amphid and phasmid ciliogenesis

Two deletion alleles are available for the arl-13 gene, gk513 and tm2322. The gk513 allele removes a 1.1-kb DNA fragment, including the promoter and first exon (Fig. 1 A). The tm2322 allele, an in-frame deletion that removes exons 7–9, encodes a truncated protein with an intact GTPase domain (Fig. 1 A). Both arl-13(gk513) and arl-13(tm2322) animals are healthy and show no gross anatomical abnormalities. We used gk513 as a reference allele. Because of the unique expression pattern of the ARL-13 protein (Fig. 1 B), we focused our experiments on cilia.

In C. elegans, cilia integrity can be assessed by a simple and efficient dye-filling assay. In living worms, amphid and phasmid neurons take up lipophilic fluorescent dye through sensory cilia that are exposed to the outside environment (Hedgecock et al., 1985). Worm mutants with abnormal cilia cannot take up dye and are termed dye-filling defective (Dyf; Perkins et al., 1986). Under the fluorescence dissecting microscope, arl-13(gk513) amphids showed normal dye uptake, whereas ~40% of phasmids were Dyf (Fig. 2 A). Although no Dyf phenotype was observed in amphids, our transmission electron microscopy (TEM) experiments revealed that some arl-13(gk513) amphid cilia were malformed (see Fig. 4). The Dyf phenotype of arl-13 mutants could be fully rescued by introducing an ARL-13::GFP transgene (Fig. 2 A). Furthermore, overexpression of ARL-13 NT::GFP and ARL-13 PRD::GFP both caused Dyf in wild-type animals, which indicates the importance of the GTPase and PRD domains in ARL-13 ciliary function (Fig. 2 A).

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Figure 2.

arl-13 mutants have deformed cilia and disrupted IFT. (A) Dye-filling assay was used to examine ciliogenesis. Phasmid cilia of arl-13 mutants showed a clear Dyf phenotype. Introduction of a copy of wild-type (WT) ARL-13::GFP but not ARL-13^R83Q::GFP, which carries a human pathogenic mutation, fully rescued the Dyf phenotype in arl-13 mutants. Overexpression of ARL-13 NT, ARL-13 PRQ, or ARL-13^R83Q::GFP had mild dominant-negative effects on ciliogenesis. At least 100 animals were scored per genotype (~400 individual phasmid cilia were observed). (B) The ciliary localization of ARL-13^R83Q::GFP was normal. (C) Phasmid cilia expressing OSM-6::GFP. Note the bulblike accumulations in arl-13 mutants. (D) Phasmid cilia expressing various ciliary markers. In arl-13 mutants, the IFT-A component CHE-11::GFP and the kinesin-II motor KAP-1::GFP showed no accumulation along cilia, whereas IFT-B components (CHE-13::GFP and OSM-5::GFP), the IFT-B–associated motor OSM-3, and BBS proteins (BBS-7::GFP and BBS-8::GFP) aggregated in similar bulblike structures. (E) GFP-tagged CHE-11, KAP-1, OSM-6, and OSM-3 were expressed in wild-type and arl-13 worms, and IFT transport event ratios within middle segments of phasmid cilia were quantified. Note that the curves of the IFT-A component CHE-11 and kinesin-II subunit KAP-1 significantly shifted to the left for arl-13 mutants compared with wild-type worms, suggesting that significant amounts of IFT-A and the associated kinesin-II move at a slower rate in the mutants. In contrast, a right shift of the curves for the IFT-B component OSM-6 and the OSM-3 motor was observed, indicating that IFT-B and the associated OSM-3 motor tend to move at a faster rate in ARL-13–deficient cilia. In all figures, solid arrows indicate the TZs, dashed arrows point to the approximate middle to distal segment transition points, and arrowheads mark the distal ends. Statistical analysis was performed using a two-tailed paired Student’s t test. *, P < 0.01. Error bars indicate SEM. Bars, 5 µm.

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Figure 4.

ARL-3 depletion corrects the abnormal ultrastructure of arl-13 amphid cilia. TEM analyses of amphid cilia in wild-type (A, E, I, M, and Q), arl-13 (B, F, J, N, and R–T), arl-3; arl-13 (C, G, K, and O), and ARL-3 DA; arl-13 (D, H, L, and P) animals are shown. The reduced cilia number (B and F) in arl-13 single mutants could be restored to nearly the wild-type level in arl-3; arl-13 double mutants (C and G). Overexpression of DA ARL-3 exacerbated the ciliogenesis defect of arl-13 mutants (D, H, and L). TZs are characterized by the typical y links that connect the axoneme with the ciliary membrane (M–P, white arrows). Note that fewer cilia were seen in the TZ area in ARL-3 DA(Xs); arl-13 animals (L), which indicate a more severe ciliogenesis defect. In all mutants, doublets were occasionally misplaced to the ciliary lumen (T, arrowhead). (R) An abnormal membrane structure that was occasionally found in arl-13 mutants is shown. (S) A cilium in an arl-13 mutant that exhibited an increased number of microtubule doublets surrounding the outer edge, most having a B-tubule seam break, is shown. This may represent a fused cilium. More than eight amphids were sectioned for each genotype. Black arrows indicate the abnormal membrane structure, and black arrowheads mark the misplaced doublet. Bars: (A–L) 0.5 µm; (M–T) 100 nm.

We also generated a mutant ARL-13^R83Q that resembles the R79Q mutation identified in human JS patients. The human ARL13B^R79Q mutant retains half of the GTP-binding efficiency of the wild-type protein (Cantagrel et al., 2008). In C. elegans, ARL-13^R83Q showed a wild-type ciliary localization pattern (Fig. 2 B). However, as expected, the mutant could only partially rescue the arl-13(gk513) Dyf phenotype (Fig. 2 A). Overexpression of ARL-13^R83Q also caused Dyf in wild-type animals. Collectively, our data suggest a conserved role for the ARL-13 protein and its GTPase activity in ciliogenesis.

arl-13 mutants show various cilia deformations and compromised IFT

We used ciliary markers to examine cilia morphology in arl-13 mutants. To minimize differences in expression levels of the markers, we introduced each gfp transgene from wild-type strains into arl-13 mutants by genetic crossing. Expression of OSM-6::GFP, an IFT-B marker, revealed that ~40% phasmid cilia are truncated, and the cilia often show abnormal bulblike structures in the middle segments (Fig. 2 C). This ratio is comparable with the Dyf ratio of arl-13(gk513) phasmid cilia.

Further analyses revealed that IFT-B proteins (OSM-5, OSM-6, and CHE-13), the IFT-B–associated OSM-3 motor, and IFT regulators (BBS-7 and BBS-8) all accumulated in bulblike structures in middle segments, whereas the IFT-A protein CHE-11 and its associated motor KAP-1 did not exhibit bulblike accumulation along the cilium (Fig. 2 D). All observed bulblike structures were located proximal to the middle to distal transition point (Fig. 2 D, dashed arrows), which might indicate a defective transition of IFT-B subcomplexes (including associated BBS proteins and the OSM-3 motor) from middle segments to distal segments in arl-13 mutants. In transition zones (TZs; Fig. 2 D, solid arrows), all IFT markers showed similar enrichment in both arl-13 and wild-type worms.

Next, we examined IFT motility in arl-13 mutants. In wild-type animals, kinesin-II and OSM-3 cooperate in moving the same IFT particle along the middle segment at 0.7 µm/s. Then, the OSM-3 kinesin alone moves the IFT particle along the distal segment at the faster rate of 1.3 µm/s. Cytoplasmic dynein mediates retrograde movement of the IFT particle along the whole axoneme at a rate of 1.1 µm/s (Signor et al., 1999; Snow et al., 2004; Ou et al., 2005). In bbs-7 and bbs-8 mutants, kinesin-II and OSM-3 separate in the middle segment, which results in IFT-A moving with kinesin-II at a slower rate of 0.5 µm/s and IFT-B moving with OSM-3 at a faster rate of 1.3 µm/s (Ou et al., 2005).

In arl-13 mutants, anterograde IFT movement along distal singlets and retrograde IFT movement occurred at characteristic wild-type rates (Table S1). Because IFT components accumulated abnormally in middle segments, we performed detailed analyses of IFT movement in these regions. The recorded IFT velocities in phasmid cilia were distributed in a normal curve that resembled the previous observation made in amphid cilia (Fig. 2 E; Snow et al., 2004). In arl-13 mutants, the velocity curves of IFT-A and the IFT-A–associated motor kinesin-II shifted to the left (slower) side, whereas those of IFT-B and the IFT-B–associated motor OSM-3 shifted to the right (faster) side (Fig. 2 E).

To determine the molecular basis of the abnormal IFT movement in arl-13 mutants, we subtracted the transport event ratio of wild-type animals from that of arl-13 mutants at each velocity step. Analyses of the distribution curves revealed that in arl-13 cilia, IFT-A and kinesin-II moved at a rate of ~0.5 µm/s, whereas IFT-B and OSM-3 moved at ~1.3 µm/s (see Fig. 5). For all IFT reporters used in our analyses, fewer IFT particles were found to move at the normal rate of ~0.7 µm/s in arl-13 mutants than in wild-type animals. These observations suggest that in ARL-13–deficient cilia, the IFT complex tends to dissociate, and the separated subcomplexes are transported by either kinesin-II or OSM-3 alone (see Fig. 7). The dissociation of IFT-A and IFT-B may account for the ciliogenesis defects and accumulation of IFT-B subcomplexes in arl-13 mutants.

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Figure 5.

ARL-13 and ARL-3 influence the integrity of the IFT macromolecular complex. The IFT transport event ratios of GFP-tagged IFT proteins of wild-type (WT) animals were subtracted from those of arl-3(tm1703), arl-13(gk513), and arl-3; arl-13 mutant animals. Positive value, negative value, and zero (y axis) represent the ratio of more, fewer, or an equal number of particles moving at a given specific velocity step (x axis) in mutants compared with wild-type animals. The simple spline curves in each figure were generated using SigmaPlot software (version 8; Systat Software, Inc.). For all mutants, the curves fit into a bimodal Gaussian distribution, with fewer particles moving at the normal rate of ~0.7 µm/s (transport by kinesin-II and OSM-3 together) and more moving at either ~0.5 (kinesin-II alone) or ~1.3 µm/s (OSM-3 alone), depending on the IFT markers used in the analysis. IFT-A and IFT-B tended to dissociate and move with their own motors in arl-13 mutants, whereas IFT-B tended to dissociate from OSM-3 and move together with IFT-A–kinesin-II in arl-3 and arl-3; arl-13 double mutants. Error bars indicate SEM.

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Figure 7.

Schematic model of ARL involvement in the regulation of IFT stability. (A) In wild-type (wt) cilia, ARL-13 and ARL-3 stabilize the IFT complex. The holding force between IFT-A and IFT-B is counterbalanced by the dragging force generated by fast-moving OSM-3 motors and slow-moving kinesin-II motors. OSM-3 and kinesin-II coordinate the movement of IFT particles at an intermediate rate. (B) Because of the attenuated association between IFT-A and IFT-B in arl-13 mutants, the holding force is smaller than the dragging force. Some IFT-A and IFT-B are dissociated and moved by their own motors at slow or fast rates. (C and D) In both arl-3 single (C) and arl-3; arl-13 double (D) mutants, the weakened connection between OSM-3 and the IFT-B subcomplex reduces the dragging force that OSM-3 exerts on the rest of the IFT complex, and thereby indirectly restores the binding efficiency of IFT-A and IFT-B.

Depletion of ARL-3 partially rescues deformations in ARL-13–deficient cilia

Incomplete dissociation of the IFT complex in arl-13 cilia indicates the involvement of redundant players in this process. arl-3 and arl-6, the C. elegans homologues of human Arl3 and Arl6/BBS3, are promising candidates because they are expressed exclusively in ciliated cells. Similar to ARL-13, ARL-3::GFP does not show detectable movement in cilia (Fig. S2 A), axons, or dendrites (not depicted). In contrast, ARL-6 exhibits typical IFT motility in cilia (Fan et al., 2004). Although ARL-3 was reported to be expressed exclusively in ciliated neurons, with little ciliary localization (Blacque et al., 2005), analysis of our GFP-tagged ARL-3, whose expression was driven by its own 2.5-kb promoter, revealed that a significant amount of ARL-3::GFP localized to cilia (both middle and distal segments; Fig. 3 B). We further analyzed arl-3(tm1703) mutants, which carry a genomic deletion that encompasses nearly all of the arl-3 gene (Fig. 3 A). To our surprise, not only were arl-3(tm1703) animals superficially wild type, they also had no detectable sign of cilia malformation by the dye-filling assay and IFT markers (Fig. 3, C and D).

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Figure 3.

ARL-3 depletion rescues cilia deformations in arl-13 mutants. (A) ARL-3 is a typical small GTPase. The tm1703 allele removes most of the coding region, resulting in a null mutant. (B) In the adult worm, ARL-3::GFP localized to the cilium. Bars, 10 µm. (C) Depletion of ARL-3 partially rescued the phasmid cilia Dyf in arl-13 mutants. However, overexpression of the DA ARL-3^Q72L mutant in an arl-13 background caused a much more severe Dyf. (D) The morphology of phasmid cilia in arl-3 mutants appeared normal. Bar, 5 µm. (E) ARL-3 depletion significantly reduced the occurrence of the bulblike structures observed in arl-13 mutants. At least 100 animals were scored per genotype (~400 individual phasmid cilia were observed). (F) Middle segment length was also partially restored in arl-3; arl-13 double mutants. At least 50 individual phasmid cilia were observed for each genotype. Solid arrows indicate the TZ, dashed arrows point to the approximate middle to distal segment transition points, and arrowheads mark the distal ends. WT, wild type. Statistical analysis was performed using a two-tailed paired Student’s t test. *, P < 0.001. Error bars indicate SEM.

To address the possibility that arl-3 negatively regulates ciliogenesis, we generated a transgenic strain that overexpresses ARL-3^Q72L, a constitutive GTP-binding (dominant active [DA]) mutant. In accordance with our prediction, DA ARL-3^Q72L overexpression caused Dyf in wild-type phasmid cilia (Fig. 3 C). We characterized the genetic interaction between arl-3 and arl-13. As shown in Fig. 3 C, arl-3; arl-13 double mutants showed less phasmid Dyf, suggesting that depletion of ARL-3 can partly rescue cilia deformations in ARL-13–deficient cilia. This result confirms that ARL-3 negatively regulates ciliogenesis. Consistently, overexpression of DA ARL-3 in arl-13 had an additive effect in causing much more severe Dyf (Fig. 3 C). Further analysis indicated that the abnormal bulblike accumulations of IFT-B components, BBS proteins, and the OSM-3 motor in arl-13 mutants were significantly rescued by the introduction of the arl-3–null allele (Fig. 3 E).

In middle segments of wild-type animals, IFT-A and IFT-B subcomplexes are transported together by kinesin-II and OSM-3 at the intermediate rate of 0.7 µm/s. According to our measurements, the length of the phasmid cilia along which IFT particles move at 0.7 µm/s is ~2.2 µm (Fig. 3 F). In arl-13 mutants, this length decreases to ~1.5 µm. As expected, the length of middle segment can be partly rescued to ~2.0 µm in arl-3; arl-13 double mutants (Fig. 3 F).

Ultrastructural defects in cilia of arl-13 and arl-3; arl-13 mutants

To investigate cilia deformations at the ultrastructural level, we used TEM to examine amphid cilia in various mutants (Fig. 4). Wild-type amphids contained 10 cilia bundled together (Fig. 4 A), whereas arl-13 mutants had only six to eight distal segments, likely as a result of premature truncation of the distal segments of some cilia (n = 8; Fig. 4 B). The remaining distal segments contained normal microtubule singlets. Some truncations in the distal segments continued further, usually resulting in one to two missing middle segments in the worms we sectioned (Fig. 4 F). In contrast to the normal pattern in middle segments of nine peripheral doublets adjacent to the membrane, misplaced doublets were frequently seen in the ciliary lumen of arl-13 mutants (Fig. 4, F and T, arrowheads), a phenomenon indicative of a disrupted axoneme architecture or microtubule: membrane connection.

Several other cilia deformations were observed in arl-13 mutants. For example, an abnormal membrane distortion (seen in two of eight amphids of arl-13 mutants; Fig. 4, F and R, black arrows) in middle segments may correlate with the bulblike structure observed in Fig. 2 C. Occasionally, we observed an increased number of outer doublets (14 rather than 9 doublets; Fig. 4 S) in a cilium, with most showing B-tubule seam breaks, and some doublets were seen in the ciliary lumen. This structure represents a fused cilium with misplaced doublets in the middle of the ciliary lumen. The C. elegans TZ typically contains a circular array of doublet microtubules with y links between the axoneme and ciliary membrane (Fig. 4, M–P, white arrows; Perkins et al., 1986). The number and morphology of amphid cilia in the TZ area of arl-13 mutants were comparable with those of wild-type animals (Fig. 4, I, J, M, and N).

Consistent with our finding that ARL-3 depletion can rescue various ciliary deformations in arl-13 mutants, the distal segments of arl-3; arl-13 mutants were restored to 9–10 cilia, comparable with those of wild-type worms (Fig. 4 C). The abnormal membrane distortion (Fig. 4, F and R, arrows) and uncommon ciliary fusion with broken B-tubules were not detected in any examined arl-3; arl-13 amphid cilium (n = 8), indicating a rescue effect. In accordance with the result that ARL-3 depletion could not completely rescue ciliogenesis defects in arl-13 mutants, middle segments of arl-3; arl-13 mutants possessed misplaced doublets in the ciliary lumen (Fig. 4 G, arrowhead).

However, overexpression of the ARL-3^Q72L DA mutant caused a much more severe ciliogenesis defect in arl-13 animals. Only three to six middle and distal segments existed in the amphid cilia of ARL-3^Q72L(Xs); arl-13 (Fig. 4, D and H). Unexpectedly, overexpression of ARL-3^Q72L DA also reduced the number of cilia in the TZ area (Fig. 4 L). Checking IFT markers in ARL-3^Q72L(Xs); arl-13 mutants (Fig. S4 B) and examining TEM sections deeper into the dendrite region did not reveal mispositioned TZs or cilia. Thus, we conclude that some cilia are completely missing in ARL-3^Q72L(Xs); arl-13 mutants. On the basis of these results and the observation that a significant amount of ARL-3 protein is cytosolic (Fig. 3 B), we propose that ARL-3 plays a role in a very early stage of ciliogenesis, possibly before TZ formation. Collectively, the TEM data complement our previous findings and support a model in which ARL-3 and ARL-13 act in opposite directions to regulate ciliogenesis.

ARL-3 depletion restores IFT-A–IFT-B binding in arl-13 mutants

To gain a mechanistic insight into how ARL-3 depletion rescues ciliogenesis defects in arl-13 mutants, we performed detailed IFT analysis on arl-3 single and arl-3; arl-13 double mutants. arl-13 mutants showed a destabilized IFT complex (Fig. 2 E). To our surprise, a significant proportion of the IFT-A particles and kinesin-II motor moved at the slower rate of 0.5 µm/s in arl-3; arl-13 mutants, as they did in arl-13 mutants (Fig. 5, A and B). Moreover, the IFT-B component OSM-6 also tended to move at the slower 0.5 µm/s rate in arl-3; arl-13 double mutants (Fig. 5 C). Interestingly, in contrast to all other IFT components tested, the OSM-3 motor tended to move at a faster rate of 1.3 µm/s in arl-3; arl-13 mutants (Fig. 5 D). Thus, our data reveal that in arl-3; arl-13 mutants, IFT-A and IFT-B subcomplexes move together and OSM-3 tends to dissociate from the IFT complex and move alone in the middle segments (see Fig. 7).

ARL-3 depletion does not rescue arl-13 ciliogenesis defects by restoring the integrity of the entire IFT particle. Rather, the absence of ARL-3 destabilizes the association between IFT-B and the OSM-3 motor, which indirectly restores binding between IFT-A and IFT-B subcomplexes. A similar scenario exists in bbs mutants. In bbs single mutants, IFT-A and IFT-B subcomplexes separate, but in bbs; osm-3 double mutants, the subcomplexes move together by kinesin-II alone because of the absence of a dragging force generated by the faster OSM-3 motor (Pan et al., 2006). To support this conclusion, we examined arl-3 mutants and found that more IFT-A and IFT-B moved together at a slower rate, whereas more OSM-3 moved at a faster rate (Fig. 5). In all mutants examined, the anterograde movement in distal segments and the retrograde movement were unchanged (Table S1). Notably, although the IFT integrity in arl-3; arl-13 is reminiscent of that in arl-3, the former still show Dyf phenotype. This finding indicates that compromised IFT integrity is not the sole explanation for the ciliogenesis defects in arl-13 mutants.

Effectors of ARL-3 and ARL-13

Although IFT integrity in arl-3; arl-13 mutants was restored to a similar state as that in arl-3 mutants (Fig. 5; and Fig. 7, C and D), arl-3; arl-13 mutants still display mild ciliogenesis defects. One hypothesis to explain this observation is that the effectors of ARL-13 may have two distinct functions: one related to regulating IFT integrity and the other a non-IFT–related function, such as membrane–microtubule architecture. This hypothesis is supported by the observation with TEM that arl-3; arl-13 mutants possess misplaced doublets. ARL-3 depletion indirectly restores IFT but is not able to correct other non-IFT–related ciliogenesis defects; therefore, we did not see a complete rescue effect for the arl-3; arl-13 double mutants.

The cellular effectors of ARL-3 and ARL-13 are poorly understood. However, experiments on ARL mutants with altered GTP-binding capacity indicate that normal ciliogenesis requires the GTPase activities of both ARL-3 and ARL-13 (Fig. 2 A and Fig. 3 C). Identification of the putative GTPase exchange factor and GTPase-activating protein that bind to ARL-3 and ARL-13 will be critical to fully dissect their roles in cilia biogenesis and IFT regulation. Remarkably, the proposed roles of ARL-13 and ARL-3 in IFT regulation resemble the roles proposed for the BBSome and DYF-1 in this system (Ou et al., 2005). It would be informative to study how ARLs, BBS proteins, and DYF-1 converge in the same pathway to regulate ciliogenesis and IFT.

The C-terminal PRD of ARL-13 is indispensable for its specific targeting to the doublet segment of the cilium. The PRD and its major interacting domain, SH3 (Src homology 3), have been shown to be involved in various protein–protein interactions. Interestingly, a PRD is found in many microtubule-associated proteins such as MAP2, τ, and MAP4 and is required for the activities of microtubule-associated proteins in microtubule binding and assembly (West et al., 1991; Kanai et al., 1992; Ferralli et al., 1994; Tokuraku et al., 1999). Additionally, the PRD of the GTPase dynamin regulates its effector binding and microtubule association (Gout et al., 1993; Hamao et al., 2009). In our experiments, overexpression of the ARL-13 PRD domain induced a dominant-negative effect on ciliogenesis (Fig. 2 A). Overexpression of the ARL-13 NT truncation, which lacks the PRD domain but contains the entire GTPase domain, could also cause Dyf in the phasmid cilia of wild-type animals. Similarly, the arl-13(tm2322) allele is an in-frame deletion and encodes a truncated ARL-13 protein that lacks the PRD domain but retains the intact GTPase domain (Fig. 1 A). Both arl-13(gk513) and arl-13(tm2322) animals show comparable ciliogenesis defects (unpublished data). Remarkably, contrary to our observation that none of the IFT reporters interfere with ciliogenesis in arl-13(gk513) mutants, IFT transgene overexpression can enhance the ciliogenesis defect of arl-13(tm2322) mutant cilia (Cevik et al., 2010). These findings provide support for the view that the PRD domain of ARL-13 is critical for both ciliary targeting and cilia formation, and loss of the PRD domain could result in a truncated ARL-13 protein with deregulated GTPase activity. Thus, characterization of the ciliary proteins that bind the ARL-13 PRD domain is of great interest.

What are HDAC6’s substrates in ciliogenesis?

We demonstrate in this study that ARL-3 acts through the deacetylase HDAC6 to regulate cilia formation in C. elegans. HDAC6 deacetylates not only histones (Grozinger et al., 1999) but also α-tubulin (Hubbert et al., 2002), Hsp90 (Kovacs et al., 2005), and cortactin (Zhang et al., 2007). Inhibition of HDAC6 enzymatic activity causes increased transport of the kinesin-1 cargo complex along microtubules in neuronal cells (Reed et al., 2006). Reminiscent of our findings, activation of HDAC6 can promote cilia disassembly, and loss of HDAC6 activity selectively stabilizes cilia in human retinal epithelial cells (Pugacheva et al., 2007).

Currently, HDAC6 is thought to regulate microtubule-dependent processes by deacetylating α-tubulin. However, this model is inaccurate for the C. elegans ciliogenesis pathway because MEC-12, the only α-tubulin capable of being acetylated, is not expressed in ciliated amphids and phasmids (Fukushige et al., 1999). Another HDAC6 substrate, Hsp90, was shown to associate with β-tubulin and may be involved in stabilizing tubulin polymerization in cilia (Takaki et al., 2007). However, worms with a null allele of daf-21, the C. elegans homologue of Hsp90, possess normal cilia in our analysis (unpublished data). These data rule out α-tubulin and Hsp90 as candidate HDAC6 substrates during ciliogenesis in C. elegans. On the basis of our model, we propose that HDAC6 deacetylates an unknown adaptor protein in IFT-B–OSM-3 subcomplexes.

Dye-filling assay

The stock DiI (2 mg/ml in dimethyl formamide; Invitrogen) was diluted 1:200 in M9. Worms were incubated in diluted dye for 1 h at room temperature. After incubation, the animals were washed at least three times with M9 and observed using a fluorescence microscope (M2Bio; Carl Zeiss, Inc.).

Microscopy

Animals were raised at 20°C and imaged using standard C. elegans slide mounts and a Plan Apochromat 60× 1.49 NA oil objective (Nikon) on an imaging microscope (TE 2000-U; Nikon).

IFT measurement

We performed all IFT analyses in phasmids for easier observation. IFT motility was observed using a Plan Apochromat 100× 1.49 NA oil total internal reflection fluorescence objective (Nikon). In wild-type worms, the IFT velocities in phasmid cilia were identical to those reported in amphid cilia (Table S1). Motility stacks were recorded using a charge-coupled device camera (QuantEM 512SC; Roper Industries), and kymographs were produced using MetaMorph software (MDS Analytical Technologies). Worms were anesthetized in a drop of M9 containing 10 mM levamisole, transferred to an agarose mount slide, and imaged immediately.

TEM

For TEM, worms were fixed in 2.5% glutaraldehyde in cacodylate buffer on ice. Heads were cut off, moved to fresh fixative, and held overnight at 4°C. Animals were rinsed in buffer and stained with 1% osmium tetroxide in cacodylate buffer for 1 h at 4°C. After embedding small groups of worms in agarose, the specimens were dehydrated and embedded in Embed812 resin according to the general procedures described previously (Hall, 1995). Thin sections were collected on a diamond knife and poststained before viewing on an electron microscope (JEM-1400; JEOL).

We thank the Caenorhabditis Genetics Center, the Japanese Bioresource Project, and Drs. Jonathan Scholey, Michel Leroux, and Maureen Barr for strains. We also thank Drs. Maureen Barr, Peter Harris, Vicente Torres, Nicholas LaRusso, Jeffery Salisbury, and Ms. Natalia Morsci for helpful discussions.

This research was funded by the Polycystic Kidney Disease (PKD) Foundation Young Investigator Award (grant 04YI09a to J. Hu). J. Hu is also supported by a FULK Career Development award, the Zell PKD Disease Research Fund, and the Upjohn PKD Research Fund from Mayo Clinic.