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

Abstract

Extracellular vesicles (EVs) are emerging as a universal means of cell-to-cell communication and hold great potential in diagnostics and regenerative therapies [1]. 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 C. elegans [2-4]. Mammalian cilia shed EVs to eliminate unwanted receptors [5] or to retract cilia before entering the cell cycle [6]. Here we used our established C. elegans model to study sensoryevoked 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 [7]. 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 nature of the ciliary EV release and of ciliary EV directional transfer 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 [7]. 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, whereas 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. Location 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 timeframe between successful location of the vulva and retraction of spicules post-copulation. This timeframe 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 [3], with EVs usually floating close to the surface of the coverslip. To

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

“…C. elegans EV-releasing cilia are specialized structurally and functionally. The cilia of C. elegans EV-releasing neurons (EVNs) protrude to the environment from cuticular pores 25–27 and shed EVs in response to mechanical pressure 28 . EVN ciliary membrane homeostasis of PKD-2 is modulated by a phosphoinositide 5-phosphatase 29 and caveolin 30 .…”

Ectosome uptake by glia sculptsCaenorhabditis eleganssensory cilia

“…Previous work showed that only a subset of ciliary proteins enters the C. elegans male EVs (21,25). To explore which cilia proteins can enter basal ectosomes to be exported to the embedding glial cells, we fluorescently tagged several ciliary membrane proteins endogenously expressed in AFD, ASER and ASK neurons and known to localise to their cilia.…”

“…In C. elegans, the polycystin-2 ortholog PKD-2 localises to the cilia of a subset of male ciliated sensory neurons (20). These male ciliated neurons were shown to release PKD-2-containing EVs into the external environment, where they contribute to interindividual communication (17,21). Electron microscopy of the male cephalic sensilla revealed EVs accumulating in the lumen surroundings of the cilia base of male cephalic neurons (CEM neurons), hinting for a release at the ciliary base (17).…”

The tubulin code specializes neuronal cilia for extracellular vesicle release

“…Depending on the cellular context, EVs can contain beneficial or pathogenic cargos that, for example, promote wound healing or neurodegeneration (Raposo & Stoorvogel, 2013). Traditionally considered receivers of signals, cilia can also release signals in the form of extracellular vesicles (EVs), and the green alga Chlamydomonas reinharditii and C. elegans have offered insights into the contents, function, biogenesis, and release of ciliary EVs (Akella et al., 2020; Cao et al., 2015; Luxmi et al., 2019; Maguire et al., 2015; O'Hagan et al., 2017; Silva et al., 2017; Wang et al., 2014, 2015; Wood et al., 2013); Wang et al., 2020).…”

“…EVs isolated from C. elegans cultures when applied on wild‐type males induce a male mating behavior termed “tail‐chasing” (Silva et al., 2017; Wang et al., 2014). During mating, males deposit PKD‐2::GFP‐carrying EVs onto the vulva of the hermaphrodite, indicating that male‐derived EVs are targeted to specific locations on the hermaphrodite's body during the mating process (Wang et al., 2020).…”