Figure 2. The extracellular vesicle (EV) marker TSP-6-wrmScarlet localizes to the cilia region, is loaded into EVs, and exported to surrounding glial cells.

(A) TSP-6 and TSP-7 were C-terminally tagged with wrmScarlet. Expression patterns based on molecular cell profiling show that tsp-6 and tsp-7 genes are enriched in ciliated or non-ciliated neurons, respectively. (B) Scheme of ciliary domains: ciliary tip, distal segment (DS), middle segment (MS), transition zone (TZ), and the periciliary membrane compartment (PCMC) located at the base of the cilia in contact with the distal dendrite. (C) Expression of TSP-6-wrmScarlet in ASER driven by gcy-5 promoter. TSP-6-wrmScarlet-carrying EVs exported from ASER are observed within the cytoplasm of AMsh. Left panels show a magnification of ASER cilium region, EVs are released in the cilia pore anterior to the ASER cilia tip (green arrowhead). Right panels show EVs within AMsh cell body (magenta arrowheads). Scale bar: 20 μm middle panel and 5 μm insets. (C′) TSP-6-wrmScarlet is enriched in ASER cilium: representative confocal projection showing TSP-6-wrmScarlet enrichment in PCMC and cilium. (C′′) Fluorescence quantification in animals expressing TSP-6-wrmScarlet in ASER neuron. Brown–Forsythe ANOVA, multiple comparisons corrected by Dunnett´s test. (D) Expression of TSP-6-wrmScarlet under the sra-6 promoter (driving expression in ASH and ASI neurons). TSP-6-wrmScarlet is enriched in both ASH and ASI cilia. Left panel shows TSP-6-wrmScarlet-carrying EVs released by ASI and/or ASH in the cilia pore (green arrowhead), EVs released by ASH/ASI were also seen within the cytoplasm of AMsh surrounding ASI/ASH cilia (magenta arrowheads). Few vesicles were also observed in AMso (blue dashed outline, middle panel). Right panel shows AMsh soma with multiple EVs (magenta arrowhead). Scale bar: 20 μm middle panel and 5 μm insets. (E) Expression of TSP-6-wrmScarlet in AFD neurons driven by gcy-8 promoter. TSP-6-wrmScarlet is enriched in AFD microvilli and PCMC. Left panel shows TSP-6-wrmScarlet-carrying EVs within the cytoplasm of AMsh that surrounds AFD terminals (magenta arrowheads). Right panel shows AMsh soma with multiple EVs (magenta arrowhead). Scale bar: 20 μm middle panel and 5 μm insets. (F) Co-expression of TSP-6-wrmScarlet and cytoplasmic mEGFP in IL2 neurons (driven by klp-6 promoter). mEGFP can be observed within EVs that are environmentally released (green arrowhead) while TSP-6-wrmScarlet is observed on EVs located within the cytoplasm of ILsh and ILso glial cells (magenta arrowheads). Theoretical position of ILsh and ILso was outlined (magenta dashed circles), IL2 neurons position was drawn with green filled outlines. Scale bar: 20 μm top panels and 5 μm insets.

Figure 2—figure supplement 1. TSP-7-wrmScarlet also localizes to the cilia region, is loaded into extracellular vesicles (EVs), and exported to surrounding glial cells.

(A) Cell-specific expression of TSP-7-wrmScarlet in ASER: the fusion protein localizes to the cilia and is exported to EVs observed within the AMsh cytoplasm. Left panel shows EVs captured by AMsh glia in the surroundings of ASER cilia region (magenta arrowheads). Right panel shows TSP-7-wrmScarlet-carying EVs in the AMsh cell body (magenta arrowheads). Scale bar: 20 μm middle image and 5 μm insets. (B) TSP-7-wrmScarlet expressed driven by the ocr-2 promoter (expression in ADF, ADL, ASH, and AWA). TSP-7-wrmScarlet expression overlaps with the XBX-1-mEGFP marker in the cilium and PCMC of ADF, ADL, ASH, and AWA. Scale bar: 5 μm. (B′) In these animals, TSP-7-wrmScarlet-carrying EVs were also exported to AMsh cytoplasm (magenta arrowheads), XBX-1-mEGFP was not observed in these EVs. Theoretical position of AMsh was outlined (blue dashed line). Scale bar: 20 μm. (C, C′) Co-expression of TSP-7-wrmScarlet and cytoplasmic mEGFP in IL2 neurons: (C) mEGFP expression locates in all ciliary compartments while TSP-7-wrmScarlet is heavily enriched in IL2 PCMC. (C′) TSP-7-wrmScarlet carrying EVs are seen within the cytoplasm of ILsh and ILso (magenta arrowheads, ILsh and ILso are outlined in magenta dashed lines). On some occasions, mEGFP and/or TSP-7-wrmScarlet-carrying EVs were also released by cilia tip into the environment (Video 3). (D) Cytoplasmic mCherry expression in IL2 neurons shows EVs that have already been environmentally released (green arrowheads). Scale bar: 5 μm in (C), 20 μm in (C′), 5 μm in (D).

Figure 2—figure supplement 2. TSP-7-wrmScarlet localizes to AFD nerve receptive endings (NREs), is loaded into extracellular vesicles (EVs), and exported to embedding AMsh glia.

(A) Cell-specific expression of TSP-7-wrmScarlet in AFD shows TSP-7-wrmScarlet enrichment in the microvilli and periciliary membrane compartment (PCMC) of AFD. Non-saturated images show a TSP-7-wrmScarlet vesicular compartment within PCMC of AFD neuron, possibly corresponding to a membrane recycling compartment. TSP-7-wrmScarlet-carrying EVs are released and captured within the cytoplasm of AMsh in the vicinity of AFD microvilli (magenta arrowheads). Image was digitally saturated by increasing the TSP-7 channel brightness in order to display the fluorescence of released vesicles. Scale bar: 5 μm. (B) Longitudinal acquisitions across developmental stages from the first larval stage to day 1 adult show the amount of TSP-7-wrmScarlet fluorescent EVs observed within AMsh cell body progressively build up as the animal ages. Scale bar: 20 μm in top panels, 5 μm in bottom panels.