Description

C. elegans EGL-36 displays extensive similarity to human KCNC proteins, the conserved potassium channel subfamily KV3 (the Shaw subfamily). This potassium channel subfamily contains four genes KCNC1, KCNC2, KCNC3, and KCNC4 encoding KV3.1 (Shaker), KV3.2 (Shab), KV3.3 (Shaw), and KV3.4 (Shal), respectively. Previous studies have demonstrated that C. elegans EGL-36 is expressed in the egg-laying muscles, ventral cord neurons, and the subset of ciliated sensory neurons including ADEL, ADER, PHB through its subcellular localization in the ciliated sensory neurons remains unknown (Elkes et al. 1997; Johnstone et al. 1997). To observe the subcellular localization of EGL-36 in the ciliated sensory neurons in C. elegans, we tagged green fluorescent protein (GFP) at the C-termini of EGL-36 with a cilia-specific promoter. We observed overexpressed EGL-36::GFP in the subsets of sensory neurons in the head and tail including PHB sensory neurons in the tail but not in the PHA sensory neuron, which is in line with previous findings (Elkes et al. 1997; Johnstone et al. 1997) (Figure 1A). Confocal laser scanning microscopy shows that EGL-36::GFP is predominantly located at the base of the cilium. The EGL-36::GFP signal can be found in the neuronal cell body, with weak dendrite staining, where they may function in the dendritic excitability (Figure 1A).

Cilia have several sub-compartments including the basal body, the transition zone, the middle segment, and the ciliary tip. We further examined the exact localization of GFP-tagged EGL-36 relative to these compartments. To this end, we used the transition zone marker MKS-6/CC2D2A and the cilia marker IFT-140 (intraflagellar transport 140), and co-expressed EGL-36::GFP with either of these markers (Qin et al.. 2001; Williams et al.. 2011; Prevo et al. 2015 Mijalkovic et al. 2018). Our microscopy analysis revealed that some of EGL-36::GFP signal was, however, found to overlay with the proximal zone of IFT signal (marked with IFT-140::mCherry), while the majority of EGL-36:: GFP signal was predominantly localized in a compartment, proximal to the ciliary base (Figure 1A). This strong EGL-36 staining likely corresponds to the periciliary membrane compartment (PCMC) (Kaplan et al. 2012). There is no overlay of EGL-36::GFP signal with MKS-6::mCherry (Figure 1B and C).

We next went on characterizing potential ciliary functions of EGL-36 in C. elegans, we therefore obtained two mutants for egl-36 including egl-36(n728) and egl-36(n728n398). egl-36(n728) mutant contains a missense variant, conversion of glutamic acid at the 142^nd position of EGL-36 to lysine (p.E142K). EGL-36(p.E142K) represents a gain of function variant of egl-36, which displays defects in egg-laying in C. elegans. On the other hand, proline to serine conversion (p.P439S) in egl-36 suppresses the egg-laying defect of p.E142K variant (Elkes et. al., 1997; Johnstone et. al., 1997). egl-36(n728n398)] mutant contains both p.E142K and p.P439S variants. First, we performed the DiI-filling assay, which has been used to test ciliary structural defects in C. elegans. The lipophilic fluorescent dye is not taken up by C. elegans with abnormal cilia structures, while wild types always stain their ciliated sensory neurons with this fluorescent dye (Herman and Hedgecock 1990). The dye analysis coupled with microscopy imaging revealed that similar to wild type, both egl-36 mutants fill up their ciliated sensory neurons with the lipophilic fluorescent dye, suggesting that egl-36 mutants have no gross cilia abnormalities in C. elegans (Figure 1E). We next examined the cilia morphologies of phasmid (tail) sensory neurons in egl-36(n728) mutant. We generated transgenic strains expressing OSM-6/IFT52::GFP in egl-36(n728) mutants, and our microscopy analysis revealed that PHB (tail) displayed no abnormality in cilia morphology in egl-36(n728) mutants (Figure 1F).