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. 2022 Oct 4;25(11):105262.
doi: 10.1016/j.isci.2022.105262. eCollection 2022 Nov 18.

Kinesin-2 motors differentially impact biogenesis of extracellular vesicle subpopulations shed from sensory cilia

Michael Clupper  1 , Rachael Gill  1 , Malek Elsayyid  1 , Denis Touroutine  1 , Jeffrey L Caplan  1   2 , Jessica E Tanis  1
Affiliations

Kinesin-2 motors differentially impact biogenesis of extracellular vesicle subpopulations shed from sensory cilia

Michael Clupper et al. iScience. .

Abstract

Extracellular vesicles (EVs) are bioactive lipid-bilayer enclosed particles released from nearly all cells. One specialized site for EV shedding is the primary cilium. Here, we discover the conserved ion channel CLHM-1 as a ciliary EV cargo. Imaging of EVs released from sensory neuron cilia of Caenorhabditis elegans expressing fluorescently tagged CLHM-1 and TRP polycystin-2 channel PKD-2 shows enrichment of these cargoes in distinct EV subpopulations that are differentially shed in response to mating partner availability. PKD-2 alone is present in EVs shed from the cilium distal tip, whereas CLHM-1 EVs bud from a secondary site(s), including the ciliary base. Heterotrimeric and homodimeric kinesin-2 motors have discrete impacts on PKD-2 and CLHM-1 colocalization in both cilia and EVs. Total loss of kinesin-2 activity decreases shedding of PKD-2 but not CLHM-1 EVs. Our data demonstrate that anterograde intraflagellar transport is required for selective enrichment of protein cargoes into heterogeneous EVs with different signaling potentials.

Keywords: Molecular neuroscience; Natural sciences; Neuroscience.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
CLHM-1 is cargo in EVs released from male and hermaphrodite ciliated neurons (A) A Pclhm-1:gfp transgene (top) drives GFP expression in male head (left) and tail (right) neurons. Colocalization with a Pklp-6:mCherry reporter (middle) shows that clhm-1 is expressed in IL2, CEM, RnB, and HOB EVNs (merge, bottom) plus additional sensory neurons. Scale bars, 10 μm. (B) Schematic of EVs released from a C. elegans cilium into the environment. (C) CLHM-1:tdT (top) localizes to neuronal cilia in the adult male head (left) and tail (right). EVNs are filled out with KLP-6:GFP (middle). Bottom, merge; scale bars, 10 μm. (D) CLHM-1 localizes to the ciliary base (triangle) and cilium proper (bracket), but is excluded from the distal tip (arrow); R4B and R5B (box, panel C); scale bars, 3 μm. (E) CLHM-1:tdT (top) is excluded from the transition zone, labeled with MKS-2:mNG (middle) in R4B and R5B cilia. Labels and scale as in (D). (F) Average number of PKD-2:GFP EVs released from the male tail does not change between wild type and clhm-1(tm4071) animals. Data are represented as mean ± SEM, Mann-Whitney test; n ≥ 15. (G) Structured illumination microscopy shows CLHM-1:GFP localized to RnB cilia and released in EVs (arrows). Scale bar, 5 μm. (H–J) TIRF microscopy shows CLHM-1:GFP EVs (arrows) released from the male tail (H), male head (I), and hermaphrodite head (J). Scale bars, 3 μm. (K) Percent of males and hermaphrodites releasing CLHM-1 EVs. n ≥ 23 animals. See also Figure S1.
Figure 2
Figure 2
CLHM-1 and PKD-2 are enriched in distinct EV subpopulations released from male tail EVNs (A) CLHM-1:tdT (henSi2) and CLHM-1:GFP (drSi33) colocalize in EVs released into the environment. Boxed region (left) is enlarged in subsequent images to clearly show EVs. (B) PKD-2:tdT (henSi26) and PKD-2:GFP (henSi20) colocalize in EVs. (C) PKD-2:GFP (henSi20) and CLHM-1:tdT (henSi17) display less frequent colocalization in EVs (compare to A,B). (D) Average number of CLHM-1:tdT EVs released is not different between animals expressing different CLHM-1:tdT transgenes (henSi2, henSi17) or when CLHM-1:tdT is paired with different PKD-2:GFP SCI transgenes (henSi20, henSi21); n ≥ 35 animals. (E) Probability of GFP cargo colocalization with CLHM-1:tdT in EVs. CLHM-1:GFP is more likely than PKD-2:GFP to coincide with CLHM-1:tdT; n ≥ 35 animals. (F) Probability of tdTomato cargo colocalization with PKD-2:GFP in EVs. PKD-2:tdT is more likely than CLHM-1:tdT to coincide with PKD-2:GFP; n ≥ 29 animals. Scale bars, 10 μm. Data are represented as mean ± SEM. Kruskal-Wallis test used for (D,E). Mann-Whitney test used for (F). ∗∗ = p<0.01, ∗∗∗ = p<0.001. See also Figure S2.
Figure 3
Figure 3
Abundance of CLHM-1 and PKD-2 containing EVs is dependent on mating partner availability (A) Average number of CLHM-1:tdT EVs released per virgin male tail is lower compared to males raised with mating partners, consistent between transgenes (henSi2, henSi17); n ≥ 22. (B) Average number of PKD-2:GFP EVs released per virgin male tail is higher compared to males raised with mating partners, consistent between transgenes (henSi20, henSi21); n ≥ 22. (C) Probability of PKD-2:GFP being present in a CLHM-1:tdT-containing EV is greater in EVs released from virgin male tails compared to males raised with mating partners; n ≥ 20. (D) Availability of mating partners does not affect release of CLHM-1:tdT (henSi17) EVs from the male head; n ≥ 29. (E) Average number of PKD-2:GFP (henSi21) EVs released per virgin adult male head is not statistically different compared to males raised with mating partners; n ≥ 29. Data are represented as mean ± SEM; Mann-Whitney test ∗ = p<0.05, ∗∗ = p<0.01. See also Figure S3.
Figure 4
Figure 4
EVs released into the environment are shed from multiple ciliary subcompartments (A) CLHM-1:tdT (left) and CLHM-1:GFP (middle) localize to the ciliary base (triangle) and the middle segment (bracket). Representative R4B and R5B cilia shown; scale bars, 2 μm. (B) CLHM-1:tdT (left) and PKD-2:GFP (middle) colocalize in the ciliary base (triangle) and middle segment (bracket). PKD-2:GFP localizes to the distal tip (arrow) of R4B and R5B cilia; scale bars, 2 μm. (C) In the cilium proper, the M1 colocalization coefficient is higher when CLHM-1:tdT is expressed with CLHM-1:GFP rather than PKD-2:GFP; n ≥ 25 cilia. (D) Normalized fluorescence intensity of PKD-2:GFP and CLHM-1:tdT along RnB cilia, measured from base to tip (left to right) shows PKD-2, but not CLHM-1, enters the distal tip; n = 16 cilia. (E) PKD-2:GFP (middle), but not CLHM-1:tdT (top) is present in EVs (arrowheads) shed from the distal tip (arrow). Scale bars, 2 μm. (F) The M2 colocalization coefficient in the cilium proper is higher when CLHM-1:tdT is expressed with CLHM-1:GFP rather than PKD-2:GFP; n ≥ 25 cilia. (G-H) M1 (G) and M2 (H) are higher in the ciliary base when CLHM-1:tdT is expressed with CLHM-1:GFP compared to PKD-2:GFP; n ≥ 25 cilia. (I) An EV (arrowhead) containing CLHM-1:tdT (top), but not PKD-2:GFP (middle) is shed from the PCMC of the ciliary base (triangle) in relation to the cilium proper (bracket) and distal tip (arrow); scale bars, 2 μm. (J) EVs containing CLHM-1:tdT (top) are shed from the PCMC of the ciliary base and observed adjacent to the middle segment of the cilium proper. EVNs are filled out with KLP-6:GFP (middle). Labels and scale as in I. (K) An EV containing CLHM-1:tdT is observed in the extracellular space adjacent to the transition zone labeled with MKS-2:mNG. Labels and scale as in I. (L) EVs containing CLHM-1:tdT (top) are located in the luminal space surrounded by an Rnst support cell marked by the Pmir-228:GFP transgene (middle). Labels and scale as in I. (M) Schematic of discrete ciliary membrane regions shedding EVs containing only CLHM-1 (magenta), only PKD-2 (green), or both (yellow) cargoes. Data are represented as mean ± SEM; Mann-Whitney test, ∗∗∗ = p<0.001.
Figure 5
Figure 5
Loss of OSM-3, but not KLP-11, affects enrichment of ciliary EV cargo (A) Average number of PKD-2:GFP EVs released from male tail EVNs does not change in klp-11 or osm-3 mutants compared to wild type; n ≥ 24 animals. (B and C) Normalized fluorescence intensity of PKD-2:GFP and CLHM-1:tdT along RnB cilia in klp-11 (B) or osm-3 (C) mutants. PKD-2, but not CLHM-1, enters the distal tip as in wild type; n = 15 cilia. (D) Average number of released CLHM-1:tdT EVs increases in klp-11 and osm-3 mutants compared to wild type; n ≥ 24 animals. (E) CLHM-1:tdT fluorescence intensity in the cilium proper of klp-11 and osm-3 mutants is the same as wild type; n ≥ 16 cilia. (F) CLHM-1:tdT fluorescence intensity in the ciliary base is lower in klp-11 and osm-3 mutants compared to wild type; n ≥ 16 cilia. (G) Probability of PKD-2:GFP being present in a CLHM-1:tdT-containing EV does not change in klp-11 mutants but increases in osm-3 mutants; n ≥ 24 animals. (H and I) The M2 coefficient significantly decreases in the cilium proper (H) and ciliary base (I) of klp-11 mutant males, indicating reduced colocalization of CLHM-1:tdT with PKD-2:GFP. M2 was unchanged in osm-3 mutants compared to wild type; n ≥ 21 cilia. Data are represented as mean ± SEM; Kruskal-Wallis test ∗ = p<0.05, ∗∗ = p<0.01, ∗∗∗ = p<0.005. See also Figure S4.
Figure 6
Figure 6
Loss of kinesin-2 activity reduces release of PKD-2, but not CLHM-1 EVs (A and B) Representative images of CLHM-1:tdT (henSi17) and PKD-2:GFP (henSi21) EVs released from wild type (A) and klp-11 osm-3 (B) male tails. Boxed region (left) is enlarged in subsequent images; scale bars, 10 μm. (C) CLHM-1:tdT EV release is not altered in klp-11 osm-3 mutants; n ≥ 28. (D) Release PKD-2:GFP EVs decreases in klp-11 osm-3 mutants; n ≥ 28. (E) Probability of PKD-2:GFP presence in CLHM-1:tdT EVs decreases in klp-11 osm-3 mutants; n ≥ 28. (F and G) PKD-2:GFP localizes to the distal tip (arrow) in wild type, but not klp-11 osm-3 mutants. CLHM-1:tdT localizes to the PCMC (triangle) and cilium proper (bracket) in both wild type and klp-11 osm-3 animals. Representative R2B and R3B cilia shown; scale bars, 3 μm. (H) Normalized fluorescence intensity of PKD-2:GFP and CLHM-1:tdT along RnB cilia in klp-11 osm-3 mutants; compare to wild type (Figure 4D); n = 14 cilia. (I) CLHM-1:tdT fluorescence intensity in the ciliary base is significantly reduced in the klp-11 osm-3 mutant compared to wild type; n ≥ 16 cilia. Data are represented as mean ± SEM; Mann-Whitney test, ∗∗∗ = p<0.001. See also Figure S5.
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