Sensory cilia act as a specialized venue for regulated extracellular vesicle biogenesis and signaling

Abstract

Ciliary extracellular vesicle (EV) shedding is evolutionarily conserved. In Chlamydomonas and C. elegans, ciliary EVs act as signaling devices.^1-3 In cultured mammalian cells, ciliary EVs regulate ciliary disposal but also receptor abundance and signaling, ciliary length, and ciliary membrane dynamics.^4-7 Mammalian cilia produce EVs from the tip and along the ciliary membrane.^8,9 This study aimed to determine the functional significance of shedding at distinct locations and to explore ciliary EV biogenesis mechanisms. Using Airyscan super-resolution imaging in living C. elegans animals, we find that neuronal sensory cilia shed TRP polycystin-2 channel PKD-2::GFP-carrying EVs from two distinct sites: the ciliary tip and the ciliary base. Ciliary tip shedding requires distal ciliary enrichment of PKD-2 by the myristoylated coiled-coil protein CIL-7. Kinesin-3 KLP-6 and intraflagellar transport (IFT) kinesin-2 motors are also required for ciliary tip EV shedding. A big unanswered question in the EV field is how cells sort EV cargo. Here, we show that two EV cargoes- CIL-7 and PKD-2-localized and trafficked differently along cilia and were sorted to different environmentally released EVs. In response to mating partners, C. elegans males modulate EV cargo composition by increasing the ratio of PKD-2 to CIL-7 EVs. Overall, our study indicates that the cilium and its trafficking machinery act as a specialized venue for regulated EV biogenesis and signaling.

Keywords: C. elegans; cilia; ectosomes; exosomes; extracellular vesicles; kinesin-2; kinesin-3; myristoylation; polycystin; super-resolution microscopy.

Figure 1:. PKD-2 EVs are shed at two sites: the ciliary tip and base of the cephalic male CEM cilium.

A-D PKD-2:GFP EVs captured at the moment of shedding from the tip (in A and B) or from the base (in C and D) of the CEM cilium. The axoneme is labeled with β-tubulin TBB-4::tdTomato, transition zone is labeled with NPHP-1::dsRed. White arrowheads indicate environmental EVs outside the animal, white arrows point to the transition zone. Dashed white lines outline base EVs in the process of shedding, solid white lines indicate PCM, periciliary membrane this is situated below the transition zone. E, Schematic cartoon showing the relationship between PKD-2 levels in ciliary tip EVs, the ciliary membrane and the region containing the PCM and ciliary base EVs. F-G, Correlation plots showing that fluorescence intensity of the tip PKD-2::GFP EVs positively correlates with that of the ciliary shaft (F), whereas fluorescence intensity of the base PKD-2::GFP EVs inversely correlates with the presence of PKD-2::GFP at the ciliary shaft (G). Spearman test, 35 data pairs. r = 0.47, p = 0.004 for (f), r = − 0.91, p < 0.0001 for (G). Each circle indicates relative fluorescence intensity of the compartment, i. e. the environmental EVs, the ciliary shaft or the ciliary base for each cilium. Scale bars are 1 μm. See also Table S1, Video S1–2.

Figure 2.. PKD-2 release from the tip of CEM cilium requires its distal ciliary enrichment mediated by CIL-7,but CIL-7 EV biogenesis is independent of PKD-2.

A-B, Depth coded 3D projections of PKD-2::GFP distribution in the CEM cilia in wild type (strain name PT621) and the cil-7 mutant (strain name PT2681). In the wild type, PKD-2 is distributed evenly along the cilium including ciliary base, shaft, and tip. The cil-7 mutant cilium lacks PKD-2::GFP at the tip and at the distal region, whereas considerable accumulations of PKD-2::GFP are observed at the ciliary shaft and the ciliary base. C, Measured PKD-2::GFP relative fluorescence intensity distribution along ciliary shaft (Ci) and ciliary base of wild type and the cil-7 mutant cilia. Each circle indicates the relative fluorescent intensity of the compartment for the cilium. The cil-7 mutant demonstrates heavily skewed distribution of PKD-2 with significant accumulations at the ciliary base. ***p<0.001 by Kruskal-Wallis test with Dunn’s multiple comparison, n=11 wild type and 11 cil-7 mutant cilia. D-E, Close-up image of PKD-2::GFP distribution along the cil-7 mutant cilium. Dashed line with arrowhead indicates direction of fluorescent intensity profiling depicted in E. F, The PKD-2 absent zone in the cil-7 mutant reaches up to 1 μm, which is significantly larger than in most wild-type cilia. ***p<0.001 by Mann-Whitney test, n=31 wild type and 42 cil-7 mutant cilia. G, Representative image of the wild-type CEM cilium showing PKD-2::GFP enrichment at the ciliary tip. Newly formed string of tip EVs is labeled as EVs and indicated by white line; it extends from the PKD-2 enriched ciliary tip to above focal planes. Green and magenta arrows point to the corresponding positions of PKD-2 and CIL-7 enrichment points shown in H. H, Fluorescent intensity profiling of the cilium from panel G shows that PKD-2 and CIL-7 are enriched at distinct locations positioned at 256 nm apart from each other. I, Distances measured between PKD-2 and CIL-7 enrichment points at the distal parts of wild-type cilia, n=11. All the plots show median values with 95% confidence intervals. J-K, Representative images of C. elegans adult male head releasing CIL-7::mNeonGreen EVs in wild type and the pkd-2 mutant. CIL-7::mNeonGreen transgenic animals were generated by CRISPR (Strain name for WT is PT3602, for pkd-2 mutant is PT3621). In the wild type (J) and the pkd-2 mutant (K) CIL-7 EVs are released by male-specific CEM cilia (outlined and indicated by figure bracket) and the sex shared IL2 cilia (arrows) in the head. Ciliary localization and EV biogenesis look similar in wild-type and pkd-2 mutant males (n=5 animals for wild type, n=9 animals for the pkd-2 mutant). Circles indicate environmentally released EVs. Scale bar, 5 μm. See also Figure S1–4, Video S3.

Figure 3.. PKD-2 and CIL-7 require kinesin motors for ciliary tip localization.

A-C, Representative images of L4 male CEM cilia of wild type (A), kinesin-3 klp-6 mutant (B), and kinesin-2 osm-3 klp-11 double mutant (C). Top to bottom: PKD-2, CIL-7, merged channel, and cartoon summarizing the observed PKD-2 and CIL-7 localization. A, In the wild type, PKD-2::GFP puncta are observed at the ciliary membrane and are enriched at the ciliary tip. The CIL-7 is mostly enriched at the ciliary tip at places of active EV biogenesis. B, In the kinesin-3 klp-6 mutant, PKD-2 and CIL-7 fail to reach the ciliary tip and are ectopically enriched at the ciliary shaft. Note that PKD-2::GFP is organized into small protrusions adjacent to CIL-7 enriched areas. C, In the kinesin-2 osm-3 klp-11 double mutant, PKD-2 fails to enter stunted cilia and accumulates considerably at the ciliary base. In contrast, CIL-7 locates to the cilium and abnormally accumulates at the ciliary base. The CIL-7 enrichment at the base corresponds to the formation of PKD-2 enriched protrusions, similar to phenotype observed at the ciliary shaft of the klp-6 mutant (B). D, Quantification of the length of PKD-2 and CIL-7 along the cilia of wild type, klp-6, and osm-3 klp-11 animals. CIL-7, but not PKD-2, traverses a significantly shorter distance in the klp-6 mutant compared to wild type. PKD-2 localization is significantly affected in the osm-3 klp-11 double mutant, but not in the klp-6 mutant. E, Quantification of number of EVs labeled by PKD-2 and CIL-7 in late L4 male tail molting cuticle. Each data point represents total EV numbers in one animal. klp-6 and osm-3 klp-11 mutants are defective in both PKD-2 and CIL-7 EV release. Median values with 95% confidence intervals are indicated. *p < 0.05, ** p<0.01 by Kruskal-Wallis test with Dunn’s multiple comparison, n=8, 8, and 10 for wild type, klp-6, and osm-3 klp-11 in D, n=22, 15 and 18 for wild type, klp-6 mutant, and osm-3 klp-11 double mutant in E. Scale bars are 1 μm.

Figure 4.. C. elegans males dynamically modify content of environmentally released EVs.

A, Scheme of experimental design for PKD-2 and CIL-7 EVs quantification in the presence or absence of mating partners. Males were isolated from a mixed population at the larval stage L4 and were imaged as adult virgin males alongside adult males picked from the mixed population with adult hermaphrodites. B, The ratio of PKD-2 to CIL-7 EV numbers is significantly increased in males cultured in mixed population as compared to isolated virgin males. **P<0.01 by Mann Whitney test, n=19 for isolated and 31 for mixed population conditions. C, Male head PKD-2 and CIL-7 EVs quantification in the presence or absence of mating partners. The increased ratio of PKD-2 to CIL-7 EVs is not simply due to an increase in the number of EVs released by the CEMs when males are exposed to hermaphrodite mating partners. Furthermore, the IL2 neurons produce a negligible amount of EVs that do not contribute substantially to the entire pool of CIL-7 EVs. This table shows that the number of CIL-7 EVs released by males exposed to mating partners decreased when compared to virgin males. See also Figure S2, 4.