Release and targeting of polycystin-2-carrying ciliary extracellular vesicles

Extracellular vesicles (EVs) are emerging as a universal means of cell-to-cell communication and hold great potential in diagnostics and regenerative therapies [1Mathieu M. Martin-Jaular L. Lavieu G. Théry C. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication.Nat. Cell Biol. 2019; 21: 9-17Crossref PubMed Scopus (1682) Google Scholar]. An urgent need in the field is a fundamental understanding of physiological mechanisms driving EV generation and function. Ciliary EVs act as signaling devices in Chlamydomonas and Caenorhabditis elegans [2Wood C.R. Huang K. Diener D.R. Rosenbaum J.L. The cilium secretes bioactive ectosomes.Curr. Biol. 2013; 23: 906-911Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 3Wang J. Silva M. Haas L.A. Morsci N.S. Nguyen K.C.Q. Hall D.H. Barr M.M. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication.Curr. Biol. 2014; 24: 519-525Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 4Luxmi R. Kumar D. Mains R.E. King S.M. Eipper B.A. Cilia-based peptidergic signaling.PLoS Biol. 2019; 17e3000566Crossref PubMed Scopus (28) Google Scholar]. Mammalian cilia shed EVs to eliminate unwanted receptors [5Nager A.R. Goldstein J.S. Herranz-Pérez V. Portran D. Ye F. Garcia-Verdugo J.M. Nachury M.V. An actin network dispatches ciliary GPCRs into extracellular vesicles to modulate signaling.Cell. 2017; 168: 252-263.e14Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar] or to retract cilia before entering the cell cycle [6Phua S.C. Chiba S. Suzuki M. Su E. Roberson E.C. Pusapati G.V. Setou M. Rohatgi R. Reiter J.F. Ikegami K. et al.Dynamic remodeling of membrane composition drives cell cycle through primary cilia excision.Cell. 2017; 168: 264-279.e15Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar]. Here, we used our established C. elegans model to study sensory-evoked ciliary EV release and targeting using a fluorescently labeled EV cargo polycystin-2 (PKD-2). In C. elegans and mammals, the autosomal dominant polycystic kidney disease (ADPKD) gene products polycystin-1 and polycystin-2 localize to cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation [7Barr M.M. García L.R. Portman D.S. Sexual dimorphism and sex differences in Caenorhabditis elegans neuronal development and behavior.Genetics. 2018; 208: 909-935Crossref PubMed Scopus (38) Google Scholar]. We find that males deposit PKD-2-carrying EVs onto the vulva of the hermaphrodite during mating. We also show that mechanical stimulation triggers release of PKD-2-carrying EVs from cilia. To our knowledge, this is the first report of mechanoresponsive ciliary EV release and of the directional transfer of ciliary EVs from one animal to another animal. Since the polycystins are evolutionarily conserved ciliary EV cargoes, our findings suggest that similar mechanisms for EV release and targeting may occur in other systems and biological contexts. C. elegans male mating involves stereotyped behavioral steps, including response to hermaphrodite contact, location of the hermaphrodite’s vulva, spicule insertion, and sperm transfer to the hermaphrodite’s uterus [7Barr M.M. García L.R. Portman D.S. Sexual dimorphism and sex differences in Caenorhabditis elegans neuronal development and behavior.Genetics. 2018; 208: 909-935Crossref PubMed Scopus (38) Google Scholar]. To examine male–hermaphrodite EV-mediated interactions during mating, we paired fluorescently labeled transgenic adult males with unlabeled hermaphrodites for 24 hours (Figure 1A). Male sperm transfer was visualized with MitoTracker dye, and ciliary EVs were tracked via the PKD-2::GFP EV cargo protein. In all mated hermaphrodites inseminated with MitoTracker-labeled sperm, we observed deposition of male-derived PKD-2::GFP EVs on the hermaphrodite vulvae (Figure 1B,C). No PKD-2::GFP EVs were found inside the hermaphrodite uterus. Localization of the male-deposited EVs at the hermaphrodite’s vulva is consistent with the position of a male tail during mating and suggests that EVs were released in the time frame between successful location of the vulva and retraction of spicules post-copulation. This time frame represents the closest contact between the male tail and the vulva area of the hermaphrodite, suggesting that the vulva may provide mechanical or chemical cues to stimulate ciliary EV release from the male. Living C. elegans males release EVs when mounted between an agarose-layered slide and a bare glass coverslip [3Wang J. Silva M. Haas L.A. Morsci N.S. Nguyen K.C.Q. Hall D.H. Barr M.M. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication.Curr. Biol. 2014; 24: 519-525Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar], with EVs usually floating close to the surface of the coverslip. To test the hypothesis that the coverslip might mechanically stimulate EV release, we padded the coverslip with a thin layer of agarose gel to reduce mechanical stimulation (see Supplemental Experimental Procedures for details). The use of the agarose-padded coverslip drastically reduced the number of released EVs (Figure 1D,E), indicating that the soft surface did not stimulate PKD-2::GFP EV release. We then proceeded to replace the padded coverslip with a new bare coverslip and observed abundant release of the ciliary EVs (Figure 1D,E). These data suggest that mechanical stimulation from the bare coverslip triggers the PKD-2::GFP EV release. Anatomically, the male tail has ventrally and dorsally positioned rays [8Sulston J.E. Albertson D.G. Thomson J.N. The Caenorhabditis elegans male: postembryonic development of nongonadal structures.Dev. Biol. 1980; 78: 542-576Crossref PubMed Scopus (409) Google Scholar]. Each ray contains a B-type cilium that releases EVs and protrudes through a cuticular pore into the environment (Figure 1D). Our analysis revealed that EVs were more frequently released from ray cilia tips that were adjacent to the bare coverslip (Figure 1D,F). PKD-2::GFP EVs were also released from the hook B-type neuron (HOB) when the ventral side of a male tail faced the bare coverslip (Figure S1 in Supplemental Information). Statistical analysis of the frequencies of EV release events from dorsal and ventral sides indicated that EV release corresponded with the position of the ray pore and cilium against the bare coverslip (Figure 1F, and Table S1 in Supplemental Information). These data support the hypothesis that mechanical stimulation triggers PKD-2::GFP EV release. We propose that this mechanoresponsive nature of PKD-2 EV release in situ may act during male–hermaphrodite mating in vivo. Our finding that the male directly deposited PKD-2::GFP-labeled EVs at the hermaphrodite’s vulva suggests that the EV release is an activity-evoked event rather than a constitutive process. This dynamic implies that the PKD-2 protein should be actively and rapidly transported towards the ciliary tip during appropriate mechanical stimulation, as suggested by our data. An alternative interpretation is that, in addition to mechanical stimulation, EV release might be triggered by the chemical landscape of the bare glass coverslip. We consider the latter scenario unlikely because chemical cues from the glass surface would likely be overridden by the ion-rich phosphate-buffered solution in which worms were mounted during imaging sessions. We predict that, during mating, both mechanical and chemical cues might work in concert to trigger and target male-derived ciliary EVs specifically to the hermaphrodite’s vulva. Our previous studies showed that isolated PKD-2-carrying EVs elicit male tail-chasing behavior and function in animal-to-animal communication [3Wang J. Silva M. Haas L.A. Morsci N.S. Nguyen K.C.Q. Hall D.H. Barr M.M. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication.Curr. Biol. 2014; 24: 519-525Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar,9Silva M. Morsci N. Nguyen K.C.Q. Rizvi A. Rongo C. Hall D.H. Barr M.M. Cell-specific α-tubulin isotype regulates ciliary microtubule ultrastructure, intraflagellar transport, and extracellular vesicle biology.Curr. Biol. 2017; 27: 968-980Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar]. Here, we discovered that male-derived EVs are targeted to the vulva of its mating partner in vivo. However, EV cargo content and the exact function of this targeting cannot be inferred from these studies. Previous transcriptional profiling of these EV-releasing neurons revealed a large variety of adhesive membrane proteins, cellular stress components, and innate immunity modulators, including antimicrobial peptide EV cargo [10Wang J. Kaletsky R. Silva M. Williams A. Haas L.A. Androwski R.J. Landis J.N. Patrick C. Rashid A. Santiago-Martinez D. et al.Cell-specific transcriptional profiling of ciliated sensory neurons reveals regulators of behavior and extracellular vesicle biogenesis.Curr. Biol. 2015; 25: 3232-3238Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar]. Since EVs are reported to carry a variety of signaling molecules and enzymes, the possibilities for their function are numerous. EVs might prevent males from mating with inseminated hermaphrodites, protect inseminated hermaphrodites from microbial invasion, or aid in the mating process by precisely marking the vulva site. Finally, EV release may maintain cilium structure and function, akin to photoreceptor disc shedding in the vertebrate eye. In conclusion, this study provides a new avenue for exploring ciliary EV function in inter-organismal communication and in reproductive biology. Download .pdf (.15 MB) Help with pdf files Figure S1. and Table S1.