Endocytosis genes facilitate protein and membrane transport in C. elegans sensory cilia

Abstract

Background: Multiple intracellular transport pathways drive the formation, maintenance, and function of cilia, a compartmentalized organelle associated with motility, chemo-/mechano-/photosensation, and developmental signaling. These pathways include cilium-based intraflagellar transport (IFT) and poorly understood membrane trafficking events. Defects in ciliary transport contribute to the etiology of human ciliary disease such as Bardet-Biedl syndrome (BBS). In this study, we employ the genetically tractable nematode Caenorhabditis elegans to investigate whether endocytosis genes function in cilium formation and/or the transport of ciliary membrane or ciliary proteins.

Results: Here we show that localization of the clathrin light chain, AP-2 clathrin adaptor, dynamin, and RAB-5 endocytic proteins overlaps with a morphologically discrete periciliary membrane compartment associated with sensory cilia. In addition, ciliary transmembrane proteins such as G protein-coupled receptors concentrate at periciliary membranes. Disruption of endocytic gene function causes expansion of ciliary and/or periciliary membranes as well as defects in the ciliary targeting and/or transport dynamics of ciliary transmembrane and IFT proteins. Finally, genetic analyses reveal that the ciliary membrane expansions in dynamin and AP-2 mutants require bbs-8 and rab-8 function and that sensory signaling and endocytic genes may function in a common pathway to regulate ciliary membrane volume.

Conclusions: These data implicate C. elegans endocytosis proteins localized at the ciliary base in regulating ciliary and periciliary membrane volume and suggest that membrane retrieval from these compartments is counterbalanced by BBS-8 and RAB-8-mediated membrane delivery.

Figure 1. Localisations of C. elegans endocytosis-associated proteins overlap with a distinct compartment at the base of sensory cilia

(A) Co-localisation of indicated GFP-tagged endocytic proteins and mCherry-tagged CHE-13/IFT57 in multiple amphid or phasmid cilia. DYN-1 localisation is shown in the AWB olfactory cilium. GFP-tagged fusion genes were expressed under gene promoters active in ciliated cells, namely che-12 (CLIC-1, CHE-13) [43], arl-13 (RAB-5) [29], rab-3 (DPY-23) [23] and str-1 (DYN-1b) [27]. The str-1p::dyn-1b::gfp and rab-3p::dpy-23::gfp constructs are functional (Figure 2D, E, and Figure S2A, and ref [23]); functional assessment using phenotypic rescue could not be determined for gfp::rab-5 and clic-1::gfp. Arrows; pools of accumulated endocytosis-associated protein near the ciliary base. Arrowheads; pools of CHE-13::mCherry at the ciliary base. cil; cilia. den; dendrite. Scale bars; 2 μm. (B) Fluorescence intensity of CHE-13::mCherry relative to CLIC-1::GFP or GFP::RAB-5 indicating endocytosis-associated protein signals (green arrows) partly overlap with the IFT pool (red arrows). Data shown from one experiment. White arrows indicate starting position of measurements shown below each panel. Graphs aligned with fluorescence images. a.u.; arbitrary units. Scale bars; 2 μm. (C) PHA/B neuronal cilia in WT animals expressing the transcriptional PHA/B marker, srb-6p::gfp. cil; cilia. TZ; transition zone. den; dendrite. Scale bar; 1 μm (D–E) Low magnification (D; scale bar 500 nm) and high magnification (E; scale bar 200 nm) TEM images of longitudinal sections through ciliated amphid channel neurons. In D, cilia outlined in blue, TZ in green, far distal dendrite in red and more proximal dendrite in yellow. Belt junctions indicated in D. Black arrow in E denotes possible coated vesicle; other vesicles are unlabelled. (F) Schematic showing relative localisation of IFT and endocytic proteins within cilia and in the far distal dendrite pocket, which we call the periciliary membrane compartment (PCMC). TZ; transition zone. (G) Chlamydomonas clathrin heavy chain (CHC) colocalises with the centriolar basal body marker ε-tubulin [44] at the flagellar base. Scale bar; 5 μm.

Figure 2. Disrupting endocytic gene function expands ciliary and PCMC membranes

(A, B) Quantification of cilium lengths in indicated genetic backgrounds. Cilia visualised using osm-6/IFT52::gfp (PHA/B cilia), che-13/IFT57::mCherry (PHA/B cilia; used in animals overexpressing N-terminal gfp-tagged rab-5 variants), str-1p::gfp (AWB cilia) and gcy-5p::gfp (ASER cilia) reporters. >20 cilia analysed in B. Error bars; standard error of the mean. * p<0.001 compared with WT (one-way ANOVA followed by Dunnett’s posthoc test). (C) Representative images of AWB cilium morphology categories. Asterisks denote base of cilia. Arrows denote expanded membrane or ectopic ciliary branches. Brackets indicate fans in WT cilia. Scale bars; 3 μm. (D) AWB cilia morphology phenotypes by category in the indicated genetic backgrounds. p<0.001 (cross-tabs and Chi-square test; compared with *WT or #dyn-1(ky51)). (E) Box and whisker distribution plots of AWB ciliary fan area measurements. Boxes; quartiles. Whiskers; 5^th and 95^th percentiles. * p<0.001 (non-parametric Mann-Whitney U test; comparison with WT values). # p<0.001 (non-parametric Mann-Whitney U test; comparison with dyn-1(ky51) values). (F–I) PCMC is enlarged in AP-2 disrupted worms.Shown in F are fluorescence images (scale bars; 3 μm) from WT and dpy-23(e840) worms expressing srb-6p::gfp (PHA/B) or gcy-5p::gfp (ASER) markers. Distribution of PCMC areas are shown as box and whisker plots in G. Shown in I are TEM images (scale bars; 300 nm) of longitudinal sections of amphid channel cilia from the indicated strains. Corresponding box and whisker plot distributions of PCMC areas from electron micrographs are shown in H. * p<0.001 (non-parametric Mann Whitney U test; compared with WT). Arrows in F denote PCMC, showing enlargement in dpy-23 mutants. cil; cilium. TZ; transition zone. den; dendrite. n; number of cilia/PCMCs analysed.

Figure 3. Genetic relationship between sensory signaling, rab-8, bbs-8 and endocytic genes in regulating ciliary and PCMC membrane volume in AWB neurons

(A, B) Representative images and box and whisker plot distribution of AWB cilium fan areas (A) and PCMC areas in PHA/PHB (B) in the indicated genotypes expressing str-1p::gfp or srb-6p::gfp transgenes, respectively. For (A), p<0.005 (Mann-Whitney U test; compared with * WT, # dpy-23(e840), or % dyn-1(ky51)). For (B), p<0.005 compared between indicated (*) genotypes (Mann-Whitney U test). Arrowheads in A; ciliary fans. Arrowheads in B; PCMC. Scale bars; 3 μm. cil; cilium. den; dendrite. n; number of cilia analysed.

Figure 4. Differential requirements for AP-2 complexes in targeting transmembrane and IFT proteins to cilia

(A, B) Representative fluorescence images of cilia from animals expressing GFP-tagged proteins in the indicated neurons. c; ciliary axoneme, d; dendrite, Arrowhead; periciliary membrane. Arrows; punctate accumulations. scale bars; 2 μm. (C) Quantification of localisation phenotypes. * p<0.001 (cross-tabs and Chi-square test; compared with WT). # p<0.001 (cross-tabs and Chi-square test; compared with dpy-23(e840)). (D, E) Quantification of SRBC-66::GFP signal intensities in the PCMC and cilium of ASK neurons. Data acquired from images taken at identical exposure settings. All data normalized against WT values and data in E also adjusted for area. Each dataset comprises 39 measurements. a.u.; arbitrary units. Error bars; SEM. * p<0.001 (t-test; two-tailed).

Figure 5. Sensory signaling may act in the same pathway as endocytic genes to modulate cilia membrane volume

(A) Analysis of PCMC areas in the indicated genotype expressing str-1p::gfp. All p-values <0.005 (Mann-Whitney U test) (compared with * WT, # dpy-23(e840), $ str-1p::rab-5(wt) or % str-1p::rab-5(S33N)). (B) Model of C. elegans ciliary and PCMC membrane homeostasis. In WT worms, RAB-8 and BBS-8 facilitate addition of membrane to PCMC and ciliary regions, whereas endocytic AP-2, DYN-1 and RAB-5 facilitate retrieval of membrane, resulting in steady-state membrane volume at the PCMC and/or within the cilium. Membrane retrieval/addition is proposed to occur at the PCMC membrane. In endocytosis gene-disrupted worms, membrane retrieval is reduced relative to membrane delivery, thus membrane accumulates within cilia and the PCMC. This membrane accumulation/retrieval defect can be reversed by simultaneously disrupting membrane delivery via rab-8 and bbs-8 directed processes. Sensory signaling may act either upstream or downstream of endocytic genes.