The CIL-1 PI 5-phosphatase localizes TRP Polycystins to cilia and activates sperm in C. elegans

Abstract

Background: C. elegans male sexual behaviors include chemotaxis and response to hermaphrodites, backing, turning, vulva location, spicule insertion, and sperm transfer, culminating in cross-fertilization of hermaphrodite oocytes with male sperm. The LOV-1 and PKD-2 transient receptor potential polycystin (TRPP) complex localizes to ciliated endings of C. elegans male-specific sensory neurons and mediates several aspects of male mating behavior. TRPP complex ciliary localization and sensory function are evolutionarily conserved. A genetic screen for C. elegans mutants with PKD-2 ciliary localization (Cil) defects led to the isolation of a mutation in the cil-1 gene.

Results: Here, we report that a phosphoinositide (PI) 5-phosphatase, CIL-1, regulates TRPP complex ciliary receptor localization and sperm activation. cil-1 does not regulate the localization of other ciliary proteins, including intraflagellar transport (IFT) components, sensory receptors, or other TRP channels in different cell types. Rather, cil-1 specifically controls TRPP complex trafficking in male-specific sensory neurons and does so in a cell-autonomous fashion. In these cells, cil-1 is required for normal PI(3)P distribution, indicating that a balance between PI(3,5)P2 and PI(3)P is important for TRPP localization. cil-1 mutants are infertile because of sperm activation and motility defects. In sperm, the CIL-1 5-phosphatase and a wortmannin-sensitive PI 3-kinase act antagonistically to regulate the conversion of sessile spermatids into motile spermatozoa, implicating PI(3,4,5)P3 signaling in nematode sperm activation.

Conclusion: Our studies identify the CIL-1 5-phosphatase as a key regulator of PI metabolism in cell types that are important in several aspects of male reproductive biology.

Figure 1. cil-1 is required for TRP polycystin complex (PKD-2 and LOV-1) localization. cil-1 acts between lov-1 and stam-1 in ray B neurons

Cell bodies = white arrows, cilia = yellow arrowhead, dendrite = red arrow, axon = green arrowhead. (A-B) Cartoons illustrating locations and structure of pkd-2 expressing neurons in C. elegans male head (A) and tail (B). The head CEMs and tail ray neurons are bilateral, and only one side of the animal is shown. Modified from [37]. (C, D) In a WT male, PKD-2∷GFP localizes to cilia and neuronal cell bodies of CEM, ray B (RnB), and hook B (HOB) neurons. (E, F) In cil-1(my15) males, PKD-2∷GFP is abnormally distributed along neurons including dendrites and axons. PKD-2∷GFP in ciliary regions appears WT. (G) In lov-1 CEMs, PKD-2∷GFP accumulates in cell bodies and weakly labels in cilia. (H) In lov-1 RnBs, PKD-2∷GFP accumulates in cell bodies and is not detectable in cilia. (I) In cil-1; lov-1 CEMs, PKD-2∷GFP aggregates in cell bodies and distributes along dendrites and cilia. (J) In cil-1; lov-1 RnBs, PKD-2∷GFP forms bright aggregates in the cell bodies, similar to the lov-1 single mutant. (K) In stam-1 CEMs, PKD-2∷GFP accumulates in ciliary regions. (L) In stam-1 RnBs, PKD-2∷GFP accumulates in the ciliary regions and distal dendrites [15]. (M) In stam-1; cil-1 CEMs, PKD-2∷GFP localizes to dendrites and axons, and sometimes accumulates ciliary bases. (N) In stam-1; cil-1 RnBs, PKD-2∷GFP is distributed to dendritic and axonal processes, similar to cil-1 single mutants Scale bar, 10um.

Figure 2. cil-1/C50C3.7 encodes a phosphoinositide 5-phosphatase

(A) Genetic and physical maps of the region of LG III encompassing the cil-1 locus. Positions of rescuing genomic fragments are aligned approximately to the physical map. (B) Summary table for rescue effects of injected cil-1(my15) lines. The Spe phenotype was scored by hermaphroditic brood size. (C) C50C3.7 is the gene mutated in cil-1(my15). Six blank boxes indicate exons connected with five introns. my15 is a nonsense mutation in the 5^th exon (301^st amino acid). The black box at the end of the 3^rd exon indicates the position of alternative splicing for the short form C50C3.7b. C50C3.7a encodes a phosphoinositide 5-phosphatase and the catalytic domain is indicated in red.

Figure 3. cil-1 regulates PI(3,5)P2 and PI(3,4,5)P3 subcellular distribution

GFP-tagged PI specific markers in the intestine of adult males are 2×FYVE for PI(3)P, AKT(PH domain) for PI(3,4,5)P3 and PI(3,4)P2, and PLC-delta (PH domain) for PI(4,5)P2. (A-C) In the WT intestine: (A) PI(3)P labels mesh-like, tubulovesicular structures in the cytoplasm without obvious PM labeling. (B) PI(3,4,5)P3 and PI(3,4)P2 label similar tubulovesicular structures as well as the apical (arrowheads) and basolateral (arrow) PM. (C) PI(4,5)P2 predominantly labels the apical (arrowheads) in addition to the basolateral (arrow) PM. (D-F) In the cil-1(my15) intestine: (D) PI(3)P appears soluble in the cytoplasm. (E) PI(3,4,5)P3 and PI(3,4)P2 lose their tubulovesicular pattern, appearing diffuse in the cytoplasm. The PM labeling is less prominent in cil-1 mutants (arrowheads). (F) PI(4,5)P2 remains enriched in the PM. (G-H) tdTomato-tagged 2×FYVE domain (PI(3)P marker) expression in male specific neurons of WT and cil-1(my15). (G) In WT CEMs, the PI(3)P marker is bright in the nuclei (denoted as N), small puncta in the cell bodies, but almost absent from cilia (blank yellow arrowhead). (G′) Similarly, in WT RnBs, the PI(3)P marker is confined to cell bodies (nuclei and small puncta). (H) In cil-1(my15) CEMs, the PI(3)P marker is visible in cilia and dendrites (arrows) in addition to cell bodies. The inset shows PI(3)P marker labeling ciliary and dendritic regions. (H′) In cil-1(my15) RnBs, the PI(3)P marker is occasionally visible in cilia (yellow arrowhead) in addition to cell bodies. Scale bar, 10um.

Figure 4. cil-1 positively regulates sperm activation and motility

(A) C. elegans sperm activation summarized in this cartoon depicting pathways and genes functioning during sperm activation and fertilization. In a spermatid, MOs containing a TRPC receptor TRP-3 are located just below the PM. During WT activation (solid arrow), MOs fuse to the PM and a pseudopod develops, producing a motile spermatozoon. In fer-1 mutant sperm (upper dotted arrow), MOs do not fuse with the PM and a short pseudopod forms, resulting in immotile sperm. A cil-1 mutant sperm (lower dotted arrow) is normal in MO fusion, but develops into immotile spermatozoon with a short pseudopod. TRPC TRP-3 translocation from MO to the PM appear normal in cil-1 mutant sperm. spe-9, spe-38, spe-41/trp-3, and spe-42 encode various membrane proteins required for sperm-egg interactions. Loss of any of these genes results in motile but infertile spermatozoa. (B-C) Nomarski images of isolated male-derived sperm before and after in vitro activation and endogenously activated hermaphrodite-derived sperm. (B) A round WT spermatid. (B′) WT spermatozoa after 15 minute of pronase activation. Spermatozoa extend full-length pseudopods (yellow arrowheads). Yellow bars depict the length of WT spermatozoa measured. (B″) WT hermaphrodite-derived sperm that are endogenously activated. (B‴) WT male-derived sperm are activated to spermatozoa with pseudopods (arrow arrowheads) within 10 minutes upon 100nM wortmannin application. (C) cil-1(my15) mutant spermatids before activation are slightly smaller than WT spermatids. (C′) Upon activation, cil-1 mutant sperm develop stubby pseudopods. The length of sperm is indicated with orange bars shorter than WT (compare to yellow bars). (C″) Hermaphrodite-derived sperm from cil-1(my15) mutant occasionally develop short pseudopods. (C‴) my15 male-derived sperm are not activated by 100nM wortmannin but exhibit subtle morphological changes. (D) Sperm Tracking Assay. The majority of WT male-derived sperm are deposited and retained within the spermatheca in the hermaphroditic reproductive tract at 10 and 16 hours. In contrast, cil-1(my15) male-derived sperm are not found in the spermatheca at 16 hours. (E-E′) Ultrastructure of him-5(e1490) (E) and cil-1(my15) him-5(e1490) (E′) spontaneously activated spermatozoa. Abbreviations: lm, laminar membranes; mo, membranous organelles; n, nucleus; p, pseudopod. While the cytoplasm in this cil-1 pseudopod (compare E to E′) appears denser than that of the WT control, the significance, if any, of this observation is unclear. (F-G) Monitoring MO fusion during sperm activation with a lipophilic FM1-43 dye. (F) The PM of WT spermatids is stained with FM1-43. (F′) In WT spermatozoa, the dye concentrates at the MO fusion sites. MO fusion events are restricted to the PM of cell body (arrowheads) but not pseudopod (bracket). (G) FM1-43 dye marks the PM of cil-1 spermatids. (G′) In short my15 spermatozoa with visible pseudopods, MO fusion sites are concentrated on the cell body (arrowheads) and excluded from the pseudopod PM (bracket). (H-I) Immunohistochemistry of sperm using the MO antibody 1CB4 (red), anti-TRP-3 (green), and DAPI (blue). The triple labeled image were generated by overlaying three confocal images from the same Z-section. (H) In WT spermatids, MOs (red) are located around the cell periphery below the PM. Location of TRP-3 (green) partially overlaps with 1CB4 labeled MOs. (H′) In WT spermatozoa, 1CB4 positive MOs are primarily located around the PM, but absent from pseudopods (compare with bracket area in H″). (H″) In WT spermatozoa, anti-TRP-3 staining is detectable in the cell body and pseudopod PM (bracket). (H‴) Overlay of WT spermatozoa. (I) In my15 spermtids, MOs (red) are localized to the cell periphery just below the PM as in WT. Anti-TRP-3 staining (green) overlaps with MOs. (I′) In my15 spermatozoa, similarly to WT, MOs are found in the cell body but not pseudopod. (I″) In my15 spermatozoa, TRP-3 protein is detected both in the cell body and pseudopod, as in WT. (I‴) Overlay of H′ and H″ with the DAPI image. Scale bar, 5um

Figure 5. Models of CIL-1 functions

(A-B) A model for CIL-1 function in polycystin trafficking in sensory neurons. (A) In WT RnB neurons, polycystins are (1) assembled in the cell body, transported along the dendrite, and (2) exocytosed at the PM, presumably at the ciliary base. Ciliary abundance of polycystins is tightly regulated by dynamic (3) internalization/endocytosis. (4) Pre-early endosomal (pre-EE) vesicles are sorted to PI(3)P enriched (red lining) EE, followed by (5) targeting to MVB. CIL-1 functions to maintain the balance between PI(3)P and PI(3,5)P2 in the EE and MVB membrane. Polycystin sorting from EE to MVB requires the STAM/HRS complex. (6) Lysosomal degradation downregulates the polycystins. In CEM neurons, cil-1 act at least partially in parallel with lov-1 and stam-1, as indicated with green. (B) In cil-1(my15), the Cil defect may occur after endocytosis. Loss of CIL-1 function causes depletion of PI(3)P, which in turn affects EE biogenesis and maturation. As shown in [38], pre-EE vesicles form an excessively tubularized EE along the microtubule network. Alternatively, polycystin-containing pre-EE vesicles may accumulate in neurons as their destination point (PI(3)P enriched EE) is blocked. In my15 CEM neurons, loss of cil-1 affects dendritic targeting and post-endosomal degradation (in green). (C-D) A Model for CIL-1 function in sperm activation. (C) In a WT spermatozoon, lowering PI(3,4,5)P3 (yellow) by CIL-1 action initiates unidentified signaling pathway(s) to coordinate pseudopod extension and sperm movement. An unidentified 3-kinase that generates PI(3,4,5)P3 negatively regulates sperm activation. The balanced action between CIL-1 and 3-kinase maintain low levels of PI(3,4,5)P3, permitting sperm activation. (D) In cil-1 mutant sperm, abnormally high PI(3,4,5)P3 levels inhibit downstream signaling pathways for pseudopod extension and sperm motility.