Description

A subset of sensory neurons in C. elegans contains compartmentalized sensory structures termed cilia at their distal dendritic ends (Ward et al. 1975; Perkins et al. 1986; Doroquez et al. 2014). Cilia present on different sensory neuron types are specialized both in morphology and function, and are generated and maintained via shared and cell-specific molecules and mechanisms (Perkins et al. 1986; Evans et al. 2006; Mukhopadhyay et al. 2007; Mukhopadhyay et al. 2008; Morsci and Barr 2011; Doroquez et al. 2014; Silva et al. 2017). The bilaterally symmetric pair of URX oxygen-sensing neurons in the C. elegans head (Figure 1A) is thought to be non-ciliated (Ward et al. 1975; Doroquez et al. 2014) but nevertheless exhibits intriguing morphological similarities with ciliated sensory neurons. URX dendrites extend to the nose where they terminate in large bulb-like complex structures (Ward et al. 1975; Doroquez et al. 2014; Cebul et al. 2020) (Figure 1A). These structures concentrate oxygen-sensing signaling molecules (Gross et al. 2014; Mclachlan et al. 2018) suggesting that similar to cilia, these structures are specialized for sensory functions. Microtubule growth events similar to those observed in ciliated sensory neurons were also reported at the distal dendritic regions of URX, implying the presence of a microtubule organizer such as a remodeled basal body (Harterink et al. 2018). Moreover, a subset of ciliary genes is expressed in URX (Kunitomo et al. 2005; Harterink et al. 2018; Mclachlan et al. 2018). We tested the hypothesis that URX dendrites contain cilia at their distal ends.

We first examined the localization of proteins associated with different ciliary compartments in URX. The basal body component DYF-19/FBF1 (Wei et al. 2013) was enriched at single puncta at the distal dendritic ends of URX (Figure 1A). A similar localization pattern was previously reported for the centrosome-associated protein gamma-tubulin (Harterink et al. 2018). The MKS-5::tagRFP (Williams et al. 2011) and NPHP-4::GFP (Jauregui and Barr 2005; Williams et al. 2011) transition zone fusion proteins were also localized to single puncta at the URX dendritic tips, distal to the region of DYF-19 localization (Figure 1B). In addition, ARL-13::GFP, a well-characterized ciliary membrane marker (Cevik et al. 2010; Li et al. 2010) was restricted to a region distal to the DYF-19::tagRFP and NPHP-4::GFP puncta (Figure 1C). The mKate-tagged oxygen-sensing soluble guanylyl cyclase GCY-35 was also highly enriched at the distal dendritic ends of URX in a domain that was defined proximally by the GIP-2 component of gamma-TuRC expressed from its endogenous locus (Figure 1C) (Wang et al. 2015; Harterink et al. 2018). We noted that overexpression of ARL-13 and GCY-35 resulted in an expansion of the presumptive cilium-like structure in URX; overexpression of ARL-13 has previously been reported to alter cilia morphology in other cell types (Hori et al. 2008; Larkins et al. 2011). Together, these observations imply the presence of a cilium-like structure at the distal dendritic ends of URX that houses signaling molecules, and that is delineated proximally by a transition zone and basal body (summarized in Figure 1D). We speculate that the short length of this structure may have precluded its identification in previous ultrastructural studies (Ward et al. 1975; Perkins et al. 1986; Doroquez et al. 2014).

We next investigated whether intraflagellar transport (IFT) necessary for cilia generation and maintenance (Rosenbaum and Wittman, 2014) could be detected in this cilium-like organelle in URX. Although the KAP-1 kinesin-2 motor component, OSM-3 homodimeric motor, and XBX-1 dynein light chain subunit were localized to the very distal ends of the URX dendrites, we were unable to detect movement of these proteins under standard conditions (Figure 1E). Similar to the localization of these proteins, the IFT core component OSM-6::GFP was also present in a small domain distal to that occupied by the DYF-19::tagRFP puncta (Figure 1E). However, we observed anterograde movement of OSM-6::GFP in the URX dendritic ends at an average speed of 0.61 ± 0.20 µm/sec (Figure 1F), consistent with kinesin-2-mediated transport. We were unable to detect and/or quantify retrograde movement of OSM-6::GFP. Unlike in other characterized sensory cilia which typically exhibit robust IFT, episodes of OSM-6::GFP anterograde movement in the presumptive URX cilia-like structure were infrequent and could be observed in only ~20% of examined neurons under specific conditions (see Methods).

We asked whether IFT proteins are necessary for the structure and/or function of the URX cilium-like structure. Although we were unable to definitively determine whether the very short cilium-like structure was further truncated in osm-6 mutants, we found that ARL-13::tagRFP was mislocalized to the URX dendrite in all examined osm-6 mutants (Figure 1G). Interestingly, ~40% of URX neurons in osm-6 mutants also exhibited multiple branches emanating from their distal dendritic ends; these branches contained ARL-13::GFP (Figure 1G). Similar, albeit less extensive, branches (also referred to as ‘posterior projections’) have been reported from the distal ends of a subset of ciliated sensory neuron dendrites in animals mutant for IFT genes in C. elegans (Lewis and Hodgkin 1977; Perkins et al. 1986; Fujiwara et al. 1999; Murayama et al. 2005; Kunitomo and Iino 2008; Maurya et al. 2019).

URX responds to a 7%-21% rise in oxygen with a tonic increase in intracellular calcium levels (Zimmer et al. 2009; Busch et al. 2012). We found that only 45% (20/44) of URX neurons in osm-6(p811) mutants exhibited oxygen-evoked responses as assessed via changes in YC2.60 fluorescence, as compared to 100% of neurons (21/21) in wild-type animals. Moreover, response amplitudes were significantly decreased in responding URX neurons in osm-6 mutants (Figure 1H). We conclude that a subset of IFT molecules is necessary for the morphological and functional integrity of the cilia-like compartment in URX.

The sensory compartments at the distal ends of URX dendrites exhibit several features characteristic of cilia. Basal body, transition zone, IFT, and ciliary membrane and membrane-associated markers, including sensory signaling molecules, are localized in domains whose relative organization at the URX dendritic ends is similar to those present in bona fide sensory cilia. Moreover, OSM-6 appears to undergo IFT in a subset of URX cilia-like structures and is required for correct neuronal morphology, ciliary protein localization and sensory functions. However, we did not observe movement of any additional IFT proteins including motors. It is possible that motor movement in the short URX cilium is difficult to detect due to technical considerations. Alternatively, URX may employ cell-specific mechanisms including cell-specific motors to build its cilia. Finally, environmental manipulations have been shown to alter neuronal and cilia morphology including URX dendritic morphology in C. elegans (Albert and Riddle 1983; Mukhopadhyay et al. 2008; Procko et al. 2011; Schroeder et al. 2013; Cohn et al. 2019). Thus, trafficking in URX cilia may be modulated in a context-specific manner, perhaps to regulate cilium length or protein composition in response to specific cues. In the future, it will be interesting to correlate IFT in URX cilia with neuronal responses under defined external and internal conditions.