Functional domains and temperature-sensitive mutations in SPE-9, an EGF repeat-containing protein required for fertility in Caenorhabditis elegans.

The spe-9 gene is required for fertility in Caenorhabditis elegans and encodes a sperm transmembrane protein with an extracellular domain (ECD) that contains 10 epidermal growth factor (EGF) repeats. Deletion analysis reveals that the EGF repeats and the transmembrane domain are required for fertilization. In contrast, the cytoplasmic region of SPE-9 is not essential for fertilization. Individual point mutations in all 10 EGF motifs uncover a differential sensitivity of these sequences to alteration. Some EGF repeats cannot tolerate mutation leading to a complete lack of fertility. Other EGF repeats can be mutated to create animals with temperature-sensitive (ts) fertility phenotypes. All ts mutations were generated by changing either conserved cysteine or glycine residues in the EGF motifs. For two endogenous ts alleles of spe-9, loss of function at nonpermissive temperatures is not due to protein mislocalization or degradation. Additionally, the proper localization of SPE-9 in sperm is not altered in a genetically interacting fertility mutant (spe-13) or a mutant that affects sperm vesicle-plasma membrane fusion (fer-1). Like the EGF repeats in the Notch/LIN-12/GLP-1 receptors and their ligands, the EGF repeats in SPE-9 may carry out different functions. Because EGF motifs are found in many proteins in different species, similar experimental strategies could be used to generate useful temperature-sensitive mutations in other EGF motif-containing molecules.

Dynamic localization of SPE-9 in sperm: a protein required for sperm-oocyte interactions in Caenorhabditis elegans.

BACKGROUND: Fertilization in Caenorhabditis elegans requires functional SPE-9 protein in sperm. SPE-9 is a transmembrane protein with a predicted extracellular domain that contains ten epidermal growth factor (EGF)-like motifs. The presence of these EGF-like motifs suggests that SPE-9 is likely to function in gamete adhesive and/or ligand-receptor interactions. RESULTS: We obtained specific antisera directed against different regions of SPE-9 in order to determine its subcellular localization. SPE-9 is segregated to spermatids with a pattern that is consistent with localization to the plasma membrane. During spermiogenesis, SPE-9 becomes localized to spiky projections that coalesce to form a pseudopod. This leads to an accumulation of SPE-9 on the pseudopod of mature sperm. CONCLUSIONS: The wild type localization patterns of SPE-9 provide further evidence that like the sperm of other species, C. elegans sperm have molecularly mosaic and dynamic regions. SPE-9 is redistributed by what is likely to be a novel mechanism that is very fast (approximately 5 minutes) and is coincident with dramatic rearrangements in the major sperm protein cytoskeleton. We conclude that SPE-9 ends up in a location on mature sperm where it can function during fertilization and this localization defines the sperm region required for these interactions.

Calcineurin, a calcium/calmodulin-dependent protein phosphatase, is involved in movement, fertility, egg laying, and growth in Caenorhabditis elegans.

Calcineurin is a Ca(2+)-calmodulin-dependent serine/threonine protein phosphatase that has been implicated in various signaling pathways. Here we report the identification and characterization of calcineurin genes in Caenorhabditis elegans (cna-1 and cnb-1), which share high homology with Drosophila and mammalian calcineurin genes. C. elegans calcineurin binds calcium and functions as a heterodimeric protein phosphatase establishing its biochemical conservation in the nematode. Calcineurin is expressed in hypodermal seam cells, body-wall muscle, vulva muscle, neuronal cells, and in sperm and the spermatheca. cnb-1 mutants showed pleiotropic defects including lethargic movement and delayed egg-laying. Interestingly, these characteristic defects resembled phenotypes observed in gain-of-function mutants of unc-43/Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and goa-1/G(o)-protein alpha-subunit. Double mutants of cnb-1 and unc-43(gf) displayed an apparent synergistic severity of movement and egg-laying defects, suggesting that calcineurin may have an antagonistic role in CaMKII-regulated phosphorylation signaling pathways in C. elegans.