ARL13B regulates juxtaposed cilia-cilia elongation in BBSome dependent manner in Caenorhabditis elegans

Abstract

The interaction of cilia with various cellular compartments, such as axons, has emerged as a new form of cellular communication. Cilia often extend in proximity to cilia from neighboring cells. However, the mechanisms driving this process termed juxtaposed cilia-cilia elongation (JCE) remain unclear. We use fluorescence-based visualization to study the mechanisms of coordinated cilia elongation in sensory neurons of Caenorhabditis elegans. Conducting a selective gene-based screening strategy reveals that ARL-13/ARL13B and MKS-5/RPGRIP1L are essential for JCE. We demonstrate that ARL-13 modulates JCE independently of cilia length. Loss of NPHP-2/inversin along with HDAC-6 enhances the cilia misdirection phenotype of arl-13 mutants, while disruption of the BBSome complex, but not microtubule components, partially suppresses the JCE defects in arl-13 mutants. We further show changes in the phospholipid compositions in arl-13 mutants. We suggest that ARL-13 contributes to JCE, in part, through the modulation of the ciliary membrane.

Keywords: cell biology; functional aspects of cell biology; organizational aspects of cell biology.

Graphical abstract

Figure 1

Fluorescence-based visualization of juxtaposed cilia-cilia elongation in C. elegans (A) Shown is a whole worm displaying the positions of ciliated sensory neurons in both the head and tail. The schematic displays cell soma, axon, dendrite, cilia, and basal body (BB) for the ciliated sensory neurons ASE and ASI in the head and PHA and PHB in the tail. Gray arrows in schematic and in vivo fluorescent cilia photos point out the contact positions between both ASE and ASI and PHA and PHB cilia. Fluorescent markers (gcy-5::mCherry and str-3::gfp) are employed to visualize the cilia of ASE and ASI, respectively. The cilia of PHA and PHB were illustrated with CHE-11::mCherry (red) and OCR-2:eGFP (green) fluorescent markers. Scale bar: 2 μm. (B and C) The average lengths of cilia for ASE and ASI and PHA and PHB were measured and depicted in the boxplot (shown in μm). The representative schematic displays the measurements for the ASE and ASI (n for ASE: 21, n for ASI: 18) and PHA and PHB (n for PHA: 30, n for ASI: 30). Distances between the periciliary membrane compartment (PCMC) of ASE and ASI cilia or PHA and PHB were shown with the mean and standard error of the mean (±SEM). The representative measurement for PHA and PHB cilia starts from the PCMC and extends to where two cilia first join, and the subsequent measurement shows beyond the region where two cilia meet. The Wilcoxon two-paired statistical test is utilized to compare the lengths of ASE and ASI and PHA and PHB cilia, where “n” denotes the number of measured cilia (ns indicates non-significance). (D) Shown is the range of PHA and PHB cilia lengths visualized with endogenous IFT-74::GFP. PHA and PHB cilia exhibit variations in length even if they are the same age. In all wild types, the cilia converge at the midpoint and extend to the tip of the cilia, forming the Y-like structure. Scale bar: 2 μm. A stands for anterior, P for posterior, D for dorsal, and V for ventral orientation.

Figure 2

ARL-13 is required for the juxtaposed cilia-cilia elongation (JCE) of PHA and PHB (A) Images depict PHA and PHB cilia in wild-type and 23 indicated single mutants. Fluorescent markers, including single-copy OSM-6::GFP and endogenous single-copy IFT-74::GFP, were used to visualize the PHA and PHB cilia. The cilia length depicted in the images corresponds to the respective cilia shown in the images. The asterisk (∗) indicates the ciliary tip in PHA and PHB cilia. Scale bar: 2 μm. (B) Stack bar charts display the percentage of normal cilia and misdirection cilia phenotype for PHA and PHB cilia in wild-type and indicated single mutants. Fisher’s exact test compares statistical significance between wild-type and indicated mutants (ns = not significant, ∗ = 0.05, ∗∗∗∗ = 0.0001). “n” denotes the number of measured cilia for each mutant. (C) PHA cilia lengths for wild-type and indicated mutants are shown along with the mean. Wilcoxon paired-two statistical test was used for statistical analysis (ns = not significant, ∗ = 0.05, ∗∗ = 0.01, ∗∗∗ = 0.001, ∗∗∗∗ = 1e-04). The number of PHA and PHB cilia counted for each mutant is denoted as “n” for (B) and (C). (D) Images display PHA and PHB cilia for wild-type and arl-13(gk513) mutants. Fluorescent markers CHE-11::mCherry (red) and OCR-2:eGFP (green) were used to visualize the PHA and PHB cilia. The representative schematics are shown next to fluorescent images. Scale bar: 2 μm. (E) Cilia lengths of PHA and PHB, tagged with endogenous single-copy IFT-74::GFP, are displayed for both wild-type and arl-13(gk513) mutants along with the mean. Wilcoxon paired-two test compares cilia lengths (ns = not significant).

Figure 3

The juxtaposed cilia-cilia elongation (JCE) of ASE and ASI requires ARL-13 (A and B) The schematic displays the JCE of several cilia, including ASE (red) and ASI (green) in the head. Shown are images of fluorescent markers, gcy-5::mCherry for ASE and str-3::gfp for ASI cilia in wild-type and arl-13 mutants. Scale bar: 2 μm. (C) The normal and cilia misdirection phenotypes were categorized in wild-type and arl-13(gk513) single mutants and the percentage of counting was plotted. For statistical significance, Fisher’s exact test was employed (∗∗>0.01). The number of analyzed cilia for the wild type (n) and arl-13(gk513) (n) are 39 and 54, respectively. (D) Shown are the cilia lengths of ASE and ASI sensory neurons in both wild-type and arl-13 mutants along with the mean. n represents the number of cilia measured, while “ns” indicates no significant difference. n for WT ASE: 44, n for WT ASI: 46, n for arl-13(gk513) ASE: 45, n for arl-13(gk513) ASI: 45. The asterisk denotes statistical significance (p < 0.05). (E) Shown are images of PHA and PHB cilia labeled with CHE-11::mCherry in arl-13 mutants. The top images display the rescue of arl-13 mutants using the ARL-13::GFP construct. Scale bar: 2 μm. (F) Bar plot displaying the percentage of normal and misdirected phenotypes in arl-13 mutants and arl-13 rescued mutants. n represents the number of cilia analyzed (n for arl-13 (gk513): 53, n for rescue: 37).

Figure 4

Cilia length and JCE are independent of each other (A) Shown are the proportions of phenotypic distribution, categorizing normal and misdirection, in both wild-type and the specified mutants. Data were generated using either srb-6promoter::gfp (on the left side) or endogenously tagged IFT-74::GFP (on the right side). Fisher’s exact statistical test was employed to compare wild-type vs. mutants and arl-13 vs. double mutants (ns = not-significant, ∗∗∗∗ = 0.0001). n represents the number of cilia analyzed for each strain. Refer to the key resources table for more details. (B) Shown are the bar plots displaying the lengths of PHA cilia for both wild-type and stated mutants along with the mean. Wilcoxon paired-two test is employed (ns = not-significant, ∗ = 0.05, ∗∗ = 0.01, ∗∗∗ = 0.001, ∗∗∗∗ = 1e-04). n specifies the analyzed cilia number for each stain.

Figure 5

BB mispositioning accounts for the cilia misdirection phenotype of PHA and PHB cilia in mks-5;nphp-4 and rpi-1;nphp-4 double mutants, but in arl-13 single mutants (A) Shown are the representative confocal images displaying PHA and PHB cilia, marked with endogenous IFT-74::GFP fluorescent protein, from wild-type, arl-13(gk513), mks-5(tm3100), nphp-4(tm925), rpi-1(syb722) single, rpi-1(syb722);nphp-4(tm925), and mks-5(tm3100);nphp-4(tm925) double mutants. The asterisk (∗) points to basal body positions of PHA and PHB. Scale bar: 2 μm. (B) The graph shows the distribution of PHA and PHB cilia phenotype of wild-type and indicated mutants as normal and misdirected. Fisher’s exact statistical test is utilized to compare normal and cilia misdirection phenotypes (ns = not-significant, ∗∗ = 0.01, ∗∗∗∗ = 0.0001). n represents the number of cilia analyzed for each strain. (C, E, and F) The boxplots show PHA cilia length (C), dendrite length (E), and the basal body distance between PHA and PHB cilia along with the mean (F). Wilcoxon paired statistical test is used for comparisons (ns = not-significant, ∗ = 0.05, ∗∗ = 0.01, ∗∗∗ = 0.001, ∗∗∗∗ = 1e-04). The cilia number used is shown in n for (B), (C), (E), and (F) analysis. (D) The illustrations display the whole PHA and PHB sensory neurons, including the cell body, axon, dendrite, basal body (BB), and cilia. Various phenotypes are depicted, including BB mispositioning coupled with cilia misdirection, as well as cilia misdirection without BB mispositioning.

Figure 6

bbs-8 partially rescues the cilia misdirection phenotype in the absence of arl-13 (A) The top image panel shows the PHA and PHB cilia, visualized with srb-6::promoter::gfp in the wild-type and indicated mutants. Scale bar: 2 μm. (B) The stacked bar chart illustrates the percentage distribution of normal and misdirection phenotypic categories for PHA and PHB cilia. Fisher’s exact test was applied for statistical analysis (ns = not significant, ∗∗ = 0.01, ∗∗∗∗ = 0.0001), with “ns” indicating non-significance. n represents the number of cilia analyzed for each strain. (C) PHA cilia lengths are visualized in the graphic in wild-type and indicated mutants along with the mean. The Wilcoxon paired-two test is utilized for comparing the lengths of cilia (ns = not-significant, ∗ = 0.05, ∗∗ = 0.01, ∗∗∗ = 0.001, ∗∗∗∗ = 1e−04). The worm numbers used in (B) and (C) are shown in n for each strain. (D) Shown are the distribution of GFP::PLCδ1-PH (and MKSR-2::tdTomato (a transition zone marker) in wild-type and arl-13 mutants. The representative schematics are shown next to fluorescent images. Scale bar: 2 μm.