Ciliary intrinsic mechanisms regulate dynamic ciliary extracellular vesicle release from sensory neurons

Abstract

Extracellular vesicles (EVs) are submicron membranous structures and key mediators of intercellular communication.^1,2 Recent research has highlighted roles for cilia-derived EVs in signal transduction, underscoring their importance as bioactive extracellular organelles containing conserved ciliary signaling proteins.^3,4 Members of the transient receptor potential (TRP) channel polycystin-2 (PKD-2) family are found in ciliary EVs of the green algae Chlamydomonas and the nematode Caenorhabditis elegans^5,6 and in EVs in the mouse embryonic node and isolated from human urine.^7,8 In C. elegans, PKD-2 is expressed in male-specific EV-releasing sensory neurons, which extend ciliary tips to ciliary pore and directly release EVs into the environment.^6,9 Males release EVs in a mechanically stimulated manner, regulate EV cargo content in response to mating partners, and deposit PKD-2::GFP-labeled EVs on the vulval cuticle of hermaphrodites during mating.^9,10 Combined, our findings suggest that ciliary EV release is a dynamic process. Herein, we identify mechanisms controlling dynamic EV shedding using time-lapse imaging. Cilia can sustain the release of PKD-2-labeled EVs for 2 h. This extended release doesn't require neuronal transmission. Instead, ciliary intrinsic mechanisms regulate PKD-2 ciliary membrane replenishment and dynamic EV release. The kinesin-3 motor kinesin-like protein 6 (KLP-6) is necessary for initial and extended EV release, while the transition zone protein NPHP-4 is required only for sustained EV release. The dynamic replenishment of PKD-2 at the ciliary tip is key to sustained EV release. Our study provides a comprehensive portrait of real-time ciliary EV release and mechanisms supporting cilia as proficient EV release platforms.

Keywords: C. elegans; KLP-6; NPHP-4; PKD-2; cilia; extracellular vesicles; kinesin-3; male; neuron; polycystin; transition zone.

Figure 1.. Extended release of PKD-2::GFP-labeled extracellular vesicles (EVs) by C. elegans male sensory neurons.

(A) Representative images capturing the head and tail regions of C. elegans males at 0, 1, and 2 hours following mounting on a glass slide. Blue lines indicate the outline of the male head or tail. Orange lines indicate the outline of the EV clouds released by the head and the tail. Orange arrowheads point to EVs that are released in a string-like pattern. (B) Quantification of EV counts from the head and tail regions at 0, 1, and 2 hours. The scatter plot with lines indicates the mean ± SEM. Each data point represents the total EV count released by an individual C. elegans male, either from its head (left) or tail (right). n = 16 and the data were scored over 4 days. Statistical analysis was performed using one-way ANOVA with Bonferroni correction. ns denotes not significant (p ≥ 0.05), and * denotes p < 0.05. (C) Individual trajectories depicting the EV release pattern from the head and tail regions of C. elegans males. Each trajectory represents EV counts from a single animal. The EV count at new time points includes newly released EVs in addition to previously released EVs that remain visible post-photobleaching. Photobleaching explains the decline in EV counts among certain animals at the 2-hour mark, where newly released EVs are fewer than the photobleached older EVs (photobleached twice). See also Figure S1, Video S1 - S2.

Figure 2.. Neuronal transmission-independent release of PKD-2::GFP-labeled ciliary EVs.

(A) Representative images showing EV release from the tail at the initial imaging (0 hr) and the second imaging (1 hr) while the animals were mounted on slides, in wild-type, unc-13, and unc-31 males. (B) Schematic diagram depicts the functional roles of UNC-13 and UNC-31 in synaptic vesicle- and dense core vesicle-mediated neuronal transmission in the axon. (C) Quantification of EV release from the tail at the initial imaging (0 hr) and second imaging (1 hr) in wild-type, unc-13, and unc-31 males. The scatter plot with lines indicates the mean ± SEM. Each data point represents the total EV count released by an individual C. elegans male. 12–15 animals were imaged for each genotype. Statistical analysis was performed by two-way ANOVA with Bonferroni correction. ns denotes not significant, * denotes p < 0.05, and ** denotes p < 0.01.

Figure 3.. Continued PKD-2 ciliary EV release relies on ciliary transport by the kinesin 3 protein KLP-6 and the transition zone protein NPHP-4.

(A-C) Representative images of PKD-2::GFP ciliary EV release in male tail of wild-type, klp-6, and nphp-4 males. (D) Quantification of EV counts from wild-type and klp-6 mutant males in the tail regions at 0 and 1 hour. The scatter plot with lines indicates the mean ± SEM. Each data point represents the total EV count released by an individual C. elegans male. (E) Quantification of EV counts from wild-type and nphp-4 mutant males in the tail regions at 0 and 1 hour. The scatter plot with lines indicates the mean ± SEM. Each data point represents the total EV count released by an individual C. elegans male. For D-E, statistical analysis was performed by two-way ANOVA with Bonferroni correction. n = 12–15 animals per genotype. ns denotes not significant, * denotes p < 0.05, and ** denotes p < 0.01., **** p<0.0001. (F) Model of PKD-2 ciliary EV dynamics. The sustained release of PKD-2 ciliary EVs over an hour necessitates the dynamic ciliary replenishment of PKD-2. KLP-6 controls ciliary PKD-2 EV release by concentrating PKD-2 at the ciliary tip. Meanwhile, the transition zone protein NPHP-4 regulates the replenishment of the PKD-2::GFP ciliary tip pool. See also Figure S2–3, Table S1.

Figure 4.. Maintaining the PKD-2 pool at the ciliary tip ensures continuous EV release.

(A-B) Schematics and representative images of RnB cilia in wild-type and nphp-4 mutant animals at 0-hour and 1-hour time points. PKD-2::GFP localizes to the ciliary base, ciliary membrane, and is enriched at the ciliary tip in both wild-type and nphp-4 mutant animals at the initial 0 hr time point. The ciliary tip pool of PKD-2::GFP is maintained at the one-hour time point in the wild type but not in the nphp-4 mutant. Overall, the nphp-4 mutant displays normal ciliary morphology as visualized by TBB-4::tdTomato. However, the localization of PKD-2::GFP at the ciliary base is reduced in the nphp-4 mutant. (C-D) Representative profiling graph of ciliary PKD-2::GFP corresponding to the cilium shown in panels (A-B). In wild type, PKD-2 is replenished at the ciliary tip, but not in the nphp-4 mutant. In wild type, the PKD-2 ciliary tip peak is higher at the 1-hour time point than at the 0-hour time point. In the nphp-4 mutant, the PKD-2 ciliary tip peak at the 0-hour time point is comparable to that of the wild type. However, in the nphp-4 mutant the enrichment at the ciliary tip is lower at the 1-hour time point than at the 0-hour time point in the nphp-4 mutant. Although the PKD-2 ciliary membrane fluorescence intensity is comparable between the wild type and the nphp-4 mutant, the fluorescence intensity of PKD-2::GFP at the ciliary base is lower in the nphp-4 mutant than in the wild type. (E-G) Quantification of PKD-2::GFP dynamic distribution along cilia in wild-type and nphp-4 mutant males. The box plots represent the mean ± SEM. The relative fluorescence intensity of PKD-2 is calculated by normalizing to the distal ciliary PKD-2 fluorescence in the wild type (see methods for details). The sample sizes were 39 cilia from 7 animals for the wild type and 38 cilia from 7 animals for the nphp-4 mutant, respectively. Statistical analyses were conducted using the Kruskal-Wallis test with Dunn's correction. The term 'ns' denotes not significant, while *, **, and *** indicate p-values of < 0.05, < 0.01, and < 0.001, respectively. (E) The ciliary tip pool of PKD-2 is maintained at the 1-hour time point in the wild type but not in the nphp-4 mutant. (F) The ciliary membrane pool of PKD-2 is maintained at a comparable level in both the wild type and the nphp-4 mutant. (G) Overall, PKD-2 is reduced at the ciliary base in the nphp-4 mutant. (H) Schematic representation of the dynamic replenishment of PKD-2 at the ciliary tip, underlying the cilium's capability for sustained EV release. PKD-2 is localized to three pools: the ciliary base membrane, the ciliary membrane, and the ciliary tip, the latter of which supports continuous EV release. Consequently, when the replenishment of the ciliary tip pool is compromised, the cilia release fewer EVs. The diminished PKD-2 pool at the ciliary base may be one mechanism that impairs the replenishment of the ciliary tip pool in the nphp-4 mutant. See also Figure S4.