C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication

Center for C. elegans Anatomy, Albert Einstein College ofMedicine, 1410 Pelham Parkway, Bronx, NY 10461, USASummaryCells release extracellular vesicles (ECVs) that play impor-tant roles in intercellular communication and may mediatea broad range of physiological and pathological processes[1–4]. Many fundamental aspects of ECV biogenesis andsignaling have yet to be determined, with ECV detectionbeing a challenge and obstacle due to the small size(100 nm) of the ECVs. We developed an in vivo system tovisualize the dynamic release of GFP-labeled ECVs. Weshow here that speciﬁc Caenorhabdidits elegans ciliatedsensory neurons shed and release ECVs containing GFP-tagged polycystins LOV-1 and PKD-2. These ECVs are alsoabundant in the lumen surrounding the cilium. Electrontomography and genetic analysis indicate that ECV biogen-esis occurs via budding from the plasma membrane at theciliary base and not via fusion of multivesicular bodies.Intraﬂagellar transport and kinesin-3 KLP-6 are required forenvironmental release of PKD-2::GFP-containing ECVs.ECVs isolated from wild-type animals induce male tail-chasing behavior, while ECVs isolated from klp-6 animalsand lacking PKD-2::GFP do not. We conclude that environ-mentallyreleasedECVsplayaroleinanimalcommunicationand mating-related behaviors.ResultsCiliated Sensory Neurons Shed and ReleasePolycystin-Containing Extracellular VesiclesCaenorhabdidits elegans ciliated sensory neurons monitorinternal and external conditions. The hermaphrodite has 60ciliated sensory neurons, whereas the male possesses anadditional 52 [5, 6]. Six inner labial type 2 (IL2) and 21male-speciﬁc B type sensory neurons are unique in that theirsensory cilia protrude into the environment via a cuticularpore [5–7]. The C. elegans polycystins LOV-1 and PKD-2are expressed exclusively in 21 male-speciﬁc B type sensoryneurons that include four cephalic male (CEM) neurons inthe head and hook B type (HOB) and bilateral ray B type(RnB) neurons (where n = 1w9, but not 6) in the tail (Fig-ure 1)[8, 9].GFP-tagged LOV-1 and PKD-2 extracellular vesicles (ECVs)are released from the tip of the nose, where CEM cilia areexposed, and from the tips of the male tail rays, where theRnB cilia are exposed in late larval L4 and in adult males (Fig-ures1A–1D).PKD-2::GFP-labeledECVsareshedandreleasedbylateL4malesandtrappedintheL4moltedcuticle(MovieS1available online). Another cilia-enriched protein, coexpressedwith polycystin-1(CWP-1) [10], is abundantly shed andreleased by male-speciﬁc B type sensory neurons (Figures1E and 1F) and by IL2 neurons in both hermaphrodites andmales throughout development (data not shown). We canobserve GFP-tagged ECV release from individual RnB ciliatedneurons (see inset of Figures 1B, 1D, and 1F). Inner labialsensilla, male cephalic sensilla, male ray sensilla, and themale hook sensillum are similar in that each contain twociliated dendrites, with the tips of the IL2, CEM, RnB, andHOB cilia completely penetrating the cuticle [5] and releasingECVs (Figure 1; Table 1).pkd-2 is required for male mating behavior, so we askedwhether PKD-2::GFP-containing ECVs are produced in ahermaphrodite-dependent manner. Adult males shed andrelease PKD-2::GFP ECVs whether cultured as single males(virgin) or in mixed populations (mated), suggesting that ECVproduction is constitutive in these conditions (Figure 2A).ECVs were harvested by washing off worms from cultureplates, pelleting the worms, and removing the supernatent.ECVs in the supernatent were puriﬁed by repeated centri-fugations and ﬁltering, as described in the SupplementalExperimental Procedures. Movie S1 shows isolated PKD-2::GFP-containing vesicles. Negative staining of the ECVpreparation reveals vesicle sizes averaging 91.7 6 92.5 nm(average 6 SD; n = 329). By staining with a monoclonalLOV-1 antibody and using ultra-small (<0.8 nm) gold-labeledsecondaryantibodydetectionfollowedbysilverenhancement[1], we observe endogenous LOV-1-containing ECVs (Figures1G–1J)thatare111.2631.1nmindiameter(n=210;seeTableS1 for ECV size comparison). These results conﬁrm thatC. elegans ECVs contain endogenous LOV-1 and that ECVsheddingisnotaconsequenceofoverexpressedGFP-taggedproteins.To test for cargo speciﬁcity of the shed vesicles, weexamined GFP-tagged reporters of known ciliary compo-nents (Table 1). We did not observe environmental releaseof GFP-tagged b-tubulin TBB-4, IFT-A polypeptide IFT140/CHE-11, IFT-B polypeptide IFT52/OSM-6, motors (kinesin-II,kinesin-2, and kinesin-3 KLP-6), or soluble GFP fromB type, IL2, or any other ciliated sensory neurons. Therefore,in contrast to the polycystins LOV-1 and PKD-2, ciliumstructural components, intraﬂagellar transport (IFT) poly-peptides, and ciliary motors are not ECV cargo. Likewise, aGFP-labeled G protein-coupled receptor (GPCR) ODR-10::GFP that is expressed in amphid wing A (AWA) neuronsis not shed.Lysosome-associated membrane protein 1 (LAMP1) is amarker of both exosomes and microvesicles (types of ECVs)[11, 12]. LMP-1::GFP is shed and released from male B typeciliated neurons, but not from other ciliated sensory neurons.Hence, ECV shedding and release is selective, constitutive,and abundant in IL2 and male-speciﬁc B type ciliated sensoryneurons and is not simply a consequence of breakage fromthe cilium.