During exocytosis vesicle fusion with the membrane relies on SNARE and SEC1/MUNC18 (SM) proteins. Although mutation of the human SM protein STXBP1 is associated with neuronal disease, mutations in STXBP2, and -3 primarily affect the function of non-neuronal cell types. Here we characterize non-neuronal functions of the C. elegans SM protein, UNCP-18. Endogenously tagged UNCP-18 localizes to the membrane consistent with a role in exocytosis. Loss of uncp-18 causes reduced brood size which can be partially rescued by mating. Our results demonstrate that UNCP-18 function is required for full fertility and suggest that C. elegans might provide a useful model for understanding the non-neuronal functions of SM proteins.
","acknowledgements":"Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440)
","authors":[{"affiliations":["College of New Jersey, Ewing, NJ, US"],"departments":["Department of Biology"],"credit":["investigation","writing_reviewEditing","dataCuration","formalAnalysis"],"email":"jacobsi2@tcnj.edu","firstName":"Isabella K","lastName":"Jacobs","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["College of New Jersey, Ewing, NJ, US"],"departments":["Department of Biology"],"credit":["conceptualization","dataCuration","formalAnalysis","fundingAcquisition","investigation","writing_originalDraft"],"email":"peeln@tcnj.edu","firstName":"Nina","lastName":"Peel","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4277-4106"}],"awards":[{"awardId":"","funderName":"National Institutes of Health (United States)","awardRecipient":"Peel"}],"conflictsOfInterest":null,"dataTable":null,"extendedData":[],"funding":"This work was supported by a Sherman Fairchild Foundation Award (IKJ) and by the National Institute of General Medical Sciences under the National Institutes of Health (Award Numbers R15 GM114727-01 and R15 GM135886-01 to NP). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/ff4211de172e015bd6b902834642e612.jpg"},"imageCaption":"A) Brood size and B) viability of WT and uncp-18(tm530) hermaphrodites at indicated culture temperatures. C) Brood size of hermaphrodites of the stated genotype when mated with him-5 males. D) Brood size of fog-1 females mated with males of the indicated genotypes. E) Summary of relative sperm count in 1day old adult hermaphrodite spermathecae, and correlation with germline phenotypes. uncp-18 heterozygous worms were used as a control. F&G) UNCP-18::mNG-expressing worms. F) Plasma membrane localization in the germline, G) A two-cell embryo at telophase. Arrows indicate likely vesicular association. Arrowheads indicate perinuclear localization. ** p<0.01; * p<0.05, Student's t test.
","imageTitle":"uncp-18(tm530) mutants have a reduced brood size due to reduced sperm number and germline defects.
","methods":"C. elegans strains were cultured on agar plates seeded with OP50 bacteria at 20°C unless stated otherwise. For brood size and viability assays single L4 hermaphrodites were transferred onto a 35mm plates. After 24 hours, each worm was transferred to a new plate and this was repeated until embryo production had ceased. Each plate was scored 24h after adults were removed, and the number of viable worms and embryos was recorded. Brood size was calculated by summing viable worms and unhatched embryos. Viability was calculated as viable worms/broodsize. The ability of mating to rescue uncp-18 brood size was tested by putting four him-5 males and one L4 hermaphrodite onto 35mm plates. After 24 hours, worms were transferred to a new plate and this was repeated until embryo production ceased. Each plate was scored 24h after adults were removed and the number of viable worms and embryos was recorded. To test fertility of uncp-18 males, fog-1 females were generated by moving L4 hermaphrodites to 25°C whereby all offspring are feminized. One male and one fog-1 female were transferred onto 35mm plates and after 24 hours worms were transferred to a new plate. This was repeated for 3days. Each plate was scored 24h after embryos were laid and the number of viable worms and embryos was recorded. The uncp-18::mNG strain was made by CRISPR-mediated integration of the neonGreen ORF at the endogenous uncp-1 locus (SUNY Biotech). For imaging, the worms were anesthetized in 20mM sodium azide, mounted on a 2% agarose pad and viewed on a Diskovery spinning disk confocal system (Andor) mounted on a Nikon Eclipse Ti microscope with a 60×/1.4 NA objective. Images were captured using an ImagE Mx2 EM-CCD camera (Hamamatsu Photonics, Shizuoka, Japan). Metamorph software was used for Image capture.
","reagents":"Strain | Genotype | Available from |
Wild type | CGC | |
CGC | ||
oxIs318 II; unc-119(ed3 or e2498) ruIs32 III; ddIs6 V; dnIs17 | CGC (Johnston et al., 2010) | |
CGC (Dejima et al., 2018) | ||
oxIs318 [spe-11p::mCherry::histone + unc-119(+)] II ; uncp-18(tm530)/dpy-9(tm9713) kvs-5(tmIs1245) IV; him-5(e1490) V | authors | |
authors | ||
uncp-18(tm530)/dpy-9(tm9713) kvs-5(tmIs1245) IV; him-5(e1490) V | authors | |
authors; SUNY Biotech | ||
CGC |
Exocytosis is important for delivery of proteins and lipids to the cell surface, and for the release of cargo to the cell exterior. Multiple steps in exocytosis are mediated by conserved SNARE proteins and by SEC1/MUNC18 (SM) proteins. Once vesicles arrive at the membrane, vSNAREs on the vesicle membrane, and tSNARES on the plasma membrane assemble into a trans-SNARE complex which facilitates vesicle docking. The SM-family proteins act as chaperones to ensure the fast and accurate assembly of the trans-SNARE complex aiding efficient membrane fusion.
Humans have three SM family proteins that regulate exocytosis, and their dysfunction is associated with disease. STXBP1 mutations cause early infantile epileptic encephalopathy (EIEE) (Saitsu et al., 2010); STXBP2 mutations cause familial hemophagocytic lymphohistiocytosis 5 (F-HLH5) (Côte et al., 2009; Spessott et al., 2017); and STXBP3 mutations cause very early onset inflammatory bowel disease (VEOIBD) (Ouahed et al., 2021). In each case disease phenotypes arise due to impaired exocytosis. Neurons require STXBP1 for synaptic vesicle release. Immune cells use STXBP2 for cytotoxic granule extrusion. And gut epithelial cells require STXPB2/3 for apical cargo delivery (Ouahed et al., 2021; Vogel et al., 2017).
The C. elegans genome encodes two SM family proteins, UNC-18 and UNCP-18 (Boeglin et al., 2023). Of these, UNC-18, is important for synaptic transmission, and its role in neurotransmitter release suggests a functional similarity to STXBP1 (Hosono et al., 1992). In comparison, the role of the paralog, UNCP-18, is less well studied. At the outset of this project uncp-18 was uncharacterized, but a recent paper described a role for UNCP-18 in neurons where it functions redundantly with UNC-18 (Boeglin et al., 2023). Given that STXBP2 and -3 play important non-neuronal roles we sought to characterize UNCP-18 functions in non-neuronal contexts focusing on the germline.
We obtained the uncp-18(tm530) mutant allele and although viable, it was difficult to maintain due to low numbers of offspring. Because of this, we crossed the mutation to the marked dpy-9(tm9713) kvs-5(tmIs1245) chromosome and subsequently maintained it as a balanced strain, selecting uncp-18(tm530) homozygotes for each analysis. A brood size analysis showed that homozygous uncp-18 mutants had a striking decrease in fertility (Figure 1A), with brood size reaching a maximum of ~30% of wild-type. This phenotype was evident across all assayed temperatures indicating that the phenotype was not temperature-sensitive. In contrast we found that embryonic viability was similar to wild type (Figure 1B).
Since C. elegans are self-fertilizing hermaphrodites, we reasoned that the reduced brood size could result from either sperm or oocyte defects. To differentiate between these possibilities, we mated control males with uncp-18 hermaphrodites. Mating significantly increased the brood size of the uncp-18 hermaphrodites (Figure 1C), suggesting that the reduction in fertility was at least partially attributable to a sperm defect. In parallel, we tested sperm function in uncp-18 males by mating them with feminized worms. While control him-5 males produced upward of 100 offspring, most uncp-18 males produced none. Only three males sired offspring, and in each case fewer than 13 embryos were produced (Figure 1D). This reduced fertility of uncp-18 males, points to sperm deficiency as a major contributor to the reduced brood size phenotype. Our results largely concur with a recent analysis of an independently derived uncp-18 null allele (Boeglin et al., 2023).
To investigate the sperm defect further, we crossed a spe-11-promoter-driven fluorescent transgene (which is expressed specifically in sperm) with uncp-18 worms. This sperm-marked strain allowed us to score relative sperm abundance in one day old hermaphrodites. We found that more than 50% of uncp-18 worms completely lacked sperm (Figure 1E). Of these, approximately one third possessed structurally intact germlines, one third had germlines that appeared abnormal and one third lacked any discernable germline, indicating the sperm deficiency has pleiotropic causes. The remaining ~40% of uncp-18 worms which did possess sperm, tended to show a lower sperm abundance than control worms (Fig 1E). These data suggest that UNCP-18 contributes to fertility at multiple levels, including in germline development, and in germ cell production.
Finally, to assess whether, similar to homologs, UNCP-18 plays a role in endocytosis, we integrated a neon green tag into the endogenous locus. The fusion protein was widely expressed including in the germline and embryos. We found that UNCP-18::mNG localized at the plasma membrane throughout the germline (Figure 1F). It also shows a strong signal between adjacent cells in the two-cell embryo (Figure 1G), and can be seen in small cytoplasmic particles that move towards the membrane at cytokinesis, (arrows). In addition, a particulate localization in close proximity to the nucleus can be observed (arrowheads), suggestive of localization to internal membranes. This localization is consistent with UNCP-18 contributing to vesicle trafficking and exocytosis.
Given its homology and subcellular localization, it is likely that UNCP-18 functions in exocytosis in C. elegans, however our data suggest it is not essential in the embryo. At fertilization the regulated exocytosis of cortical granules is required for eggshell formation and failure to establish this permeability barrier leads to embryonic lethality (Sato et al., 2008). In addition, exocytosis is essential later in embryogenesis with impairment of secretory pathways leading to defects in cuticle and pharynx formation (Roberts et al., 2003). Although our expression/localization data show that UNCP-18 is present in the embryo, we found that uncp-18 mutant embryos were viable and survived at levels comparable to wild type. These embryos were produced by mutant hermaphrodites so lacked any maternal UNCP-18 contribution, therefore the observed viability suggests that UNCP-18 is not essential for exocytosis in the embryo. It has been reported that unc-18(e81); uncp-18(syb6377) double mutants are embryonic lethal, indicating that UNCP-18 functions redundantly with UNC-18 in the embryo (Boeglin et al., 2023).
In contrast, loss of UNCP-18 leads to a range of germline phenotypes from complete germline loss, to a reduction in gamete number. This defect in germline development and maintenance may result from altered signaling between the distal tip cell (DTC) and germline stem cells. Normally, the germline stem cell pool is maintained by notch signaling from the DTC niche which activates the GLP-1 receptor on the surface of adjacent germline cells (Albert Hubbard and Schedl, 2019). However, disrupting secretory pathways leads to impaired trafficking of GLP-1 to the membrane (Roberts et al., 2003). Thus, in the absence of UNCP-18 impaired exocytosis may disrupt the membrane localization of GLP-1, reduce notch signaling, and lead to premature meiotic entry, ultimately depleting the proliferative pool of cells from the germline.
Interestingly, our data show that a majority of hermaphrodites have some rescue of brood size upon mating, indicating that a sperm deficit is a key contributor to the brood size phenotype (Figure 1C). Since sperm production precedes oocyte production during development, depletion of the stem cell pool cannot explain why sperm numbers are low and limiting for brood size. Instead, impaired repatriation of sperm expelled from the spermathecae as the fertilized oocyte moves into the uterus may be to blame. These misplaced sperm should migrate back to the spermathecae guided by prostaglandins synthesized in the oocytes (Edmonds et al., 2010). However, when exocytosis is blocked oocytes fail to import yolk, which is a precursor for prostaglandin synthesis (Roberts et al., 2003). We speculate that in the uncp-18 mutants exocytosis defects lead to impaired yolk accumulation in the oocytes, resulting in reduced prostaglandin availability, which leads to defects in sperm retention in the spermatheca.
In summary, we have characterized germline phenotypes associated with loss of UNCP-18. Our data show that UNCP-18 is membrane associated, consistent with a role in exocytosis, and that UNCP-18 is required for full fertility. Determining the exact roles UNCP-18 plays in the germline will require further investigation. Importantly, our initial analyses have identified C. elegans as a model for understanding the non-neuronal functions of STXBP proteins with the potential to illuminate the etiology of associated diseases.
","references":[{"reference":"Hubbard EJA, Schedl T. 2019. Biology of the Caenorhabditis elegans Germline Stem Cell System. Genetics 213(4): 1145-1188.
","pubmedId":"31796552","doi":""},{"reference":"Boeglin M, Leyva-Díaz E, Hobert O. 2023. Expression and function of Caenorhabditis elegans UNCP-18, a paralog of the SM protein UNC-18. Genetics 225(4): 10.1093/genetics/iyad180.
","pubmedId":"37793339","doi":""},{"reference":"Côte M, Ménager MM, Burgess A, Mahlaoui N, Picard C, Schaffner C, et al., de Saint Basile G. 2009. Munc18-2 deficiency causes familial hemophagocytic lymphohistiocytosis type 5 and impairs cytotoxic granule exocytosis in patient NK cells. J Clin Invest 119(12): 3765-73.
","pubmedId":"19884660","doi":""},{"reference":"Dejima K, Hori S, Iwata S, Suehiro Y, Yoshina S, Motohashi T, Mitani S. 2018. An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans. Cell Rep 22(1): 232-241.
","pubmedId":"29298424","doi":""},{"reference":"Edmonds JW, Prasain JK, Dorand D, Yang Y, Hoang HD, Vibbert J, Kubagawa HM, Miller MA. 2010. Insulin/FOXO signaling regulates ovarian prostaglandins critical for reproduction. Dev Cell 19(6): 858-71.
","pubmedId":"21145501","doi":""},{"reference":"Hosono R, Hekimi S, Kamiya Y, Sassa T, Murakami S, Nishiwaki K, et al., Kodaira KI. 1992. The unc-18 gene encodes a novel protein affecting the kinetics of acetylcholine metabolism in the nematode Caenorhabditis elegans. J Neurochem 58(4): 1517-25.
","pubmedId":"1347782","doi":""},{"reference":"Johnston WL, Krizus A, Dennis JW. 2010. Eggshell chitin and chitin-interacting proteins prevent polyspermy in C. elegans. Curr Biol 20(21): 1932-7.
","pubmedId":"20971008","doi":""},{"reference":"Ouahed J, Kelsen JR, Spessott WA, Kooshesh K, Sanmillan ML, Dawany N, et al., Snapper SB. 2021. Variants in STXBP3 are Associated with Very Early Onset Inflammatory Bowel Disease, Bilateral Sensorineural Hearing Loss and Immune Dysregulation. J Crohns Colitis 15(11): 1908-1919.
","pubmedId":"33891011","doi":""},{"reference":"Roberts B, Clucas C, Johnstone IL. 2003. Loss of SEC-23 in Caenorhabditis elegans causes defects in oogenesis, morphogenesis, and extracellular matrix secretion. Mol Biol Cell 14(11): 4414-26.
","pubmedId":"14551256","doi":""},{"reference":"Saitsu H, Kato M, Okada I, Orii KE, Higuchi T, Hoshino H, et al., Matsumoto N. 2010. STXBP1 mutations in early infantile epileptic encephalopathy with suppression-burst pattern. Epilepsia 51(12): 2397-405.
","pubmedId":"20887364","doi":""},{"reference":"Sato M, Grant BD, Harada A, Sato K. 2008. Rab11 is required for synchronous secretion of chondroitin proteoglycans after fertilization in Caenorhabditis elegans. J Cell Sci 121(Pt 19): 3177-86.
","pubmedId":"18765566","doi":""},{"reference":"Spessott WA, Sanmillan ML, McCormick ME, Kulkarni VV, Giraudo CG. 2017. SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity. Proc Natl Acad Sci U S A 114(11): E2176-E2185.
","pubmedId":"28265073","doi":""},{"reference":"Vogel GF, van Rijn JM, Krainer IM, Janecke AR, Posovszky C, Cohen M, et al., Huber LA. 2017. Disrupted apical exocytosis of cargo vesicles causes enteropathy in FHL5 patients with Munc18-2 mutations. JCI Insight 2(14): pii: 94564. 10.1172/jci.insight.94564.
","pubmedId":"28724787","doi":""}],"title":"UNCP-18 is a C. elegans STXBP homolog that is required for fertility","reviews":[{"reviewer":{"displayName":"Peter Kropp"},"openAcknowledgement":null,"status":{"submitted":false}},{"reviewer":{"displayName":"Laura Thomas"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"bf46e08e-6910-45f2-a740-e30d95c09a0b","decision":"accept","abstract":"Exocytosis supplies the plasma membrane with lipids and proteins, and is essential for release of molecules to the cell exterior. The capture and efficient fusion of vesicles with the plasma membrane relies on SNARE and SEC1/MUNC18 (SM) proteins. The human SM protein STXBP1 is required for exocytosis of neurotransmitters, and mutation is associated with human neuronal disease. In contrast, mutations in STXBP2 and -3 primarily affect the function of non-neuronal cell types. Here we characterize the role of the C. elegans SM protein UNCP-18 in the germline. Endogenously tagged UNCP-18 is broadly expressed and primarily localized to the plasma membrane, consistent with a role in exocytosis. Loss of UNCP-18 led to reduced brood size which can be partially rescued by mating. Additional germline defects are evident, however, suggesting a role beyond the sperm. Our results demonstrate that UNCP-18 plays non-neuronal roles and suggest that C. elegans might provide a useful model for understanding the non-neuronal functions of SM proteins.
","acknowledgements":"Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440)
","authors":[{"affiliations":["College of New Jersey, Ewing, NJ, US"],"departments":["Department of Biology"],"credit":["investigation","writing_reviewEditing","dataCuration","formalAnalysis"],"email":"jacobsi2@tcnj.edu","firstName":"Isabella K","lastName":"Jacobs","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["College of New Jersey, Ewing, NJ, US"],"departments":["Department of Biology"],"credit":["conceptualization","dataCuration","formalAnalysis","fundingAcquisition","investigation","writing_originalDraft"],"email":"peeln@tcnj.edu","firstName":"Nina","lastName":"Peel","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4277-4106"}],"awards":[{"awardId":"","funderName":"National Institutes of Health (United States)","awardRecipient":"Peel"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by a Sherman Fairchild Foundation Award (IKJ). Research in the Peel lab is funded by the National Institute of General Medical Sciences under the National Institutes of Health (Award Number R15 GM135886-01 to NP). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/ff4211de172e015bd6b902834642e612.jpg"},"imageCaption":"A) Brood size and B) viability of WT and uncp-18(tm530) hermaphrodites at indicated culture temperatures. C) Brood size of hermaphrodites of the stated genotype when mated with him-5 males. D) Brood size of fog-1 females mated with males of the indicated genotypes. E) Summary of relative sperm count in 1day old adult hermaphrodite spermathecae, and correlation with germline phenotypes. uncp-18 heterozygous worms were used as a control. F&G) UNCP-18::mNG-expressing worms. F) Plasma membrane localization in the germline, G) A two-cell embryo at telophase. Arrows indicate likely vesicular association. Arrowheads indicate perinuclear localization. ** p<0.01; * p<0.05, Student's t test.
","imageTitle":"uncp-18(tm530) mutants have a reduced brood size due to reduced sperm number and germline defects.
","methods":"C. elegans strains were cultured on agar plates seeded with OP50 bacteria at 20°C unless stated otherwise. For brood size and viability assays, single L4 hermaphrodites were transferred onto 35mm plates. After 24 hours, each worm was transferred to a new plate, and this was repeated until embryo production had ceased. Each plate was scored 24h after adults were removed, and the number of viable worms and embryos was recorded. Brood size was calculated by summing viable worms and unhatched embryos. Viability was calculated as viable worms/broodsize. The ability of mating to rescue uncp-18 brood size was tested by putting four him-5 males and one L4 hermaphrodite onto 35mm plates. After 24 hours, worms were transferred to a new plate, and this was repeated until embryo production ceased. Each plate was scored 24h after adults were removed and the number of viable worms and embryos was recorded. To test fertility of uncp-18 males, fog-1 females were generated by moving L4 hermaphrodites to 25°C whereby all offspring are feminized. One male and one fog-1 female were transferred onto 35mm plates and after 24 hours worms were transferred to a new plate. This was repeated for 3days. Each plate was scored 24h after embryos were laid, and the number of viable worms and embryos was recorded. The uncp-18::mNG strain was made by CRISPR-mediated integration of the neonGreen ORF at the endogenous uncp-1 locus (SUNY Biotech). For imaging, the worms were anesthetized in 20mM sodium azide, mounted on a 2% agarose pad and viewed on a Diskovery spinning disk confocal system (Andor) mounted on a Nikon Eclipse Ti microscope with a 60×/1.4 NA objective. Images were captured using an ImagE Mx2 EM-CCD camera (Hamamatsu Photonics, Shizuoka, Japan). Metamorph software was used for Image capture.
","reagents":"Strain | Genotype | Available from |
Wild type | CGC | |
CGC | ||
oxIs318 II; unc-119(ed3 or e2498) ruIs32 III; ddIs6 V; dnIs17 | CGC (Johnston et al., 2010) | |
CGC (Dejima et al., 2018) | ||
oxIs318 [spe-11p::mCherry::histone + unc-119(+)] II ; uncp-18(tm530)/dpy-9(tm9713) kvs-5(tmIs1245) IV; him-5(e1490) V | authors | |
authors | ||
uncp-18(tm530)/dpy-9(tm9713) kvs-5(tmIs1245) IV; him-5(e1490) V | authors | |
authors; SUNY Biotech | ||
CGC |
Exocytosis is important for delivery of proteins and lipids to the cell surface, and for the release of cargo to the cell exterior. Multiple steps in exocytosis are mediated by conserved SNARE proteins and by SEC1/MUNC18 (SM) proteins. Once vesicles arrive at the membrane, vSNAREs on the vesicle membrane and tSNARES on the plasma membrane assemble into a trans-SNARE complex which facilitates vesicle docking. The SM-family proteins act as chaperones to ensure the fast and accurate assembly of the trans-SNARE complex, aiding efficient membrane fusion.
Humans have three SM family proteins that regulate exocytosis, and their dysfunction is associated with disease. STXBP1 mutations cause early infantile epileptic encephalopathy (EIEE) (Saitsu et al., 2010); STXBP2 mutations cause familial hemophagocytic lymphohistiocytosis 5 (F-HLH5) (Côte et al., 2009; Spessott et al., 2017); and STXBP3 mutations cause very early onset inflammatory bowel disease (VEOIBD) (Ouahed et al., 2021). In each case, disease phenotypes arise due to impaired exocytosis. Neurons require STXBP1 for synaptic vesicle release. Immune cells use STXBP2 for cytotoxic granule extrusion. And gut epithelial cells require STXPB2/3 for apical cargo delivery (Ouahed et al., 2021; Vogel et al., 2017).
The C. elegans genome encodes two SM family proteins, UNC-18 and UNCP-18 (Boeglin et al., 2023). Of these, UNC-18 is important for synaptic transmission, and its role in neurotransmitter release suggests a functional similarity to STXBP1 (Hosono et al., 1992). In comparison, the role of the paralog, UNCP-18, is less well studied. At the outset of this project uncp-18 was uncharacterized, but a recent paper described a role for UNCP-18 in neurons where it functions redundantly with UNC-18 (Boeglin et al., 2023). Given that STXBP2 and -3 play important non-neuronal roles, we sought to characterize UNCP-18 functions in non-neuronal contexts focusing on the germline.
We obtained the uncp-18(tm530) mutant allele, a 336bp deletion that is predicted to cause an in-frame 48 amino acid deletion (p.310-358), in a region of the protein that is highly conserved with human STXBP proteins (Boeglin et al., 2023). Although viable, the strain was difficult to maintain due to low numbers of offspring. Because of this, we crossed the mutation to the marked dpy-9(tm9713) kvs-5(tmIs1245) chromosome, and subsequently maintained it as a balanced strain, selecting uncp-18(tm530) homozygotes for each analysis. A brood size analysis showed that homozygous uncp-18 mutants had a striking decrease in fertility (Figure 1A), with brood size reaching a maximum of ~30% of wild-type. This phenotype was evident across all assayed temperatures indicating that the phenotype was not temperature-sensitive. In contrast, we found that embryonic viability was similar to wild type (Figure 1B).
Since C. elegans are self-fertilizing hermaphrodites, we reasoned that the reduced brood size could result from either sperm or oocyte defects. To differentiate between these possibilities, we mated control males with uncp-18 hermaphrodites. Mating significantly increased the brood size of the uncp-18 hermaphrodites (Figure 1C), suggesting that the reduction in fertility was at least partially attributable to a sperm defect. In parallel, we tested sperm function in uncp-18 males by mating them with feminized worms. While control him-5 males produced upward of 100 offspring, most uncp-18 males produced none. Only three males sired offspring, and in each case fewer than 13 embryos were produced (Figure 1D). This reduced fertility of uncp-18 males points to sperm deficiency as a major contributor to the reduced brood size phenotype. Our results largely concur with a recent analysis of an independently derived uncp-18 null allele (Boeglin et al., 2023).
To investigate the sperm defect further, we crossed a spe-11-promoter-driven fluorescent transgene (which is expressed specifically in sperm) with uncp-18 worms. This sperm-marked strain allowed us to score relative sperm abundance in one day old hermaphrodites. We found that more than 50% of uncp-18 worms completely lacked sperm (Figure 1E). Of these, approximately one third possessed structurally intact germlines, one third had germlines that appeared abnormal, and one third lacked any discernible germline, indicating the sperm deficiency has pleiotropic causes. The remaining ~40% of uncp-18 worms which did possess sperm, tended to show a lower sperm abundance than control worms (Fig 1E). These data suggest that UNCP-18 contributes to fertility at multiple levels, including in germline development, and in germ cell production.
Finally, to assess whether, similar to homologs, UNCP-18 plays a role in exocytosis, we integrated a neon green tag into the endogenous locus. The fusion protein was widely expressed including in the germline and embryos. We found that UNCP-18::mNG localized at the plasma membrane throughout the germline (Figure 1F). It also shows a strong signal between adjacent cells in the two-cell embryo (Figure 1G), and can be seen in small cytoplasmic particles that move towards the membrane at cytokinesis (arrows). In addition, a particulate localization in close proximity to the nucleus can be observed (arrowheads), suggestive of localization to internal membranes. This localization is consistent with UNCP-18 contributing to vesicle trafficking and exocytosis.
Given its homology and subcellular localization, it is likely that UNCP-18 functions in exocytosis in C. elegans. Our data suggest, however, that it is not essential in the embryo. At fertilization the regulated exocytosis of cortical granules is required for eggshell formation, and failure to establish this permeability barrier leads to embryonic lethality (Sato et al., 2008). In addition, exocytosis is essential later in embryogenesis with impairment of secretory pathways leading to defects in cuticle and pharynx formation (Roberts et al., 2003). Although our expression/localization data show that UNCP-18 is present in the embryo, we found that uncp-18 mutant embryos were viable and survived at levels comparable to wild type. These embryos were produced by mutant hermaphrodites so they lacked any maternal UNCP-18 contribution. The observed viability therefore suggests that UNCP-18 is not essential for exocytosis in the embryo. It has been reported that unc-18(e81); uncp-18(syb6377) double mutants are embryonic lethal, indicating that UNCP-18 functions redundantly with UNC-18 in the embryo (Boeglin et al., 2023).
In contrast, loss of UNCP-18 leads to a range of germline phenotypes from a reduction in gamete number to complete germline loss. A majority of hermaphrodites have partial rescue of brood size upon mating (Figure 1C), indicating that a sperm deficit contributes to the brood size phenotype. In addition to reduced sperm number, however, structural defects in the germline were also observed. Our data suggest, therefore, that UNCP-18 is required for both germline development/maintenance, and germ cell production. The underlying basis for this germline defect remains unknown.
In summary, we have characterized germline phenotypes associated with loss of UNCP-18. Our data show that UNCP-18 is membrane-associated, consistent with a role in exocytosis, and that UNCP-18 is required for full fertility. Determining the exact roles UNCP-18 plays in the germline will require further investigation. Importantly, our initial analyses have identified C. elegans as a model for understanding the non-neuronal functions of STXBP proteins with the potential to illuminate the etiology of associated diseases.
","references":[{"reference":"
Boeglin M, Leyva-Díaz E, Hobert O. 2023. Expression and function of Caenorhabditis elegans UNCP-18, a paralog of the SM protein UNC-18. Genetics 225(4): 10.1093/genetics/iyad180.
","pubmedId":"37793339","doi":""},{"reference":"Côte M, Ménager MM, Burgess A, Mahlaoui N, Picard C, Schaffner C, et al., de Saint Basile G. 2009. Munc18-2 deficiency causes familial hemophagocytic lymphohistiocytosis type 5 and impairs cytotoxic granule exocytosis in patient NK cells. J Clin Invest 119(12): 3765-73.
","pubmedId":"19884660","doi":""},{"reference":"Dejima K, Hori S, Iwata S, Suehiro Y, Yoshina S, Motohashi T, Mitani S. 2018. An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans. Cell Rep 22(1): 232-241.
","pubmedId":"29298424","doi":""},{"reference":"Hosono R, Hekimi S, Kamiya Y, Sassa T, Murakami S, Nishiwaki K, et al., Kodaira KI. 1992. The unc-18 gene encodes a novel protein affecting the kinetics of acetylcholine metabolism in the nematode Caenorhabditis elegans. J Neurochem 58(4): 1517-25.
","pubmedId":"1347782","doi":""},{"reference":"Johnston WL, Krizus A, Dennis JW. 2010. Eggshell chitin and chitin-interacting proteins prevent polyspermy in C. elegans. Curr Biol 20(21): 1932-7.
","pubmedId":"20971008","doi":""},{"reference":"Ouahed J, Kelsen JR, Spessott WA, Kooshesh K, Sanmillan ML, Dawany N, et al., Snapper SB. 2021. Variants in STXBP3 are Associated with Very Early Onset Inflammatory Bowel Disease, Bilateral Sensorineural Hearing Loss and Immune Dysregulation. J Crohns Colitis 15(11): 1908-1919.
","pubmedId":"33891011","doi":""},{"reference":"Roberts B, Clucas C, Johnstone IL. 2003. Loss of SEC-23 in Caenorhabditis elegans causes defects in oogenesis, morphogenesis, and extracellular matrix secretion. Mol Biol Cell 14(11): 4414-26.
","pubmedId":"14551256","doi":""},{"reference":"Saitsu H, Kato M, Okada I, Orii KE, Higuchi T, Hoshino H, et al., Matsumoto N. 2010. STXBP1 mutations in early infantile epileptic encephalopathy with suppression-burst pattern. Epilepsia 51(12): 2397-405.
","pubmedId":"20887364","doi":""},{"reference":"Sato M, Grant BD, Harada A, Sato K. 2008. Rab11 is required for synchronous secretion of chondroitin proteoglycans after fertilization in Caenorhabditis elegans. J Cell Sci 121(Pt 19): 3177-86.
","pubmedId":"18765566","doi":""},{"reference":"Spessott WA, Sanmillan ML, McCormick ME, Kulkarni VV, Giraudo CG. 2017. SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity. Proc Natl Acad Sci U S A 114(11): E2176-E2185.
","pubmedId":"28265073","doi":""},{"reference":"Vogel GF, van Rijn JM, Krainer IM, Janecke AR, Posovszky C, Cohen M, et al., Huber LA. 2017. Disrupted apical exocytosis of cargo vesicles causes enteropathy in FHL5 patients with Munc18-2 mutations. JCI Insight 2(14): pii: 94564. 10.1172/jci.insight.94564.
","pubmedId":"28724787","doi":""}],"title":"UNCP-18 is a C. elegans STXBP homolog that is required for full fertility","reviews":[{"reviewer":{"displayName":"Laura Thomas"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":"1777854824530"}]},{"id":"21a4aef4-23f0-43ca-8848-6253aba28a74","decision":"publish","abstract":"Exocytosis supplies the plasma membrane with lipids and proteins, and is essential for release of molecules to the cell exterior. The capture and efficient fusion of vesicles with the plasma membrane relies on SNARE and SEC1/MUNC18 (SM) proteins. The human SM protein STXBP1 is required for exocytosis of neurotransmitters, and mutation is associated with human neuronal disease. In contrast, mutations in STXBP2 and -3 primarily affect the function of non-neuronal cell types. Here we characterize the role of the C. elegans SM protein UNCP-18 in the germline. Endogenously tagged UNCP-18 is broadly expressed and primarily localized to the plasma membrane, consistent with a role in exocytosis. Loss of UNCP-18 led to reduced brood size which can be partially rescued by mating. Additional germline defects are evident, however, suggesting a role beyond the sperm. Our results demonstrate that UNCP-18 plays non-neuronal roles and suggest that C. elegans might provide a useful model for understanding the non-neuronal functions of SM proteins.
","acknowledgements":"Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
","authors":[{"affiliations":["College of New Jersey, Ewing, NJ, US"],"departments":["Department of Biology"],"credit":["investigation","writing_reviewEditing","dataCuration","formalAnalysis"],"email":"jacobsi2@tcnj.edu","firstName":"Isabella K","lastName":"Jacobs","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["College of New Jersey, Ewing, NJ, US"],"departments":["Department of Biology"],"credit":["conceptualization","dataCuration","formalAnalysis","fundingAcquisition","investigation","writing_originalDraft"],"email":"peeln@tcnj.edu","firstName":"Nina","lastName":"Peel","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4277-4106"}],"awards":[{"awardId":"","funderName":"National Institutes of Health (United States)","awardRecipient":"Peel"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by a Sherman Fairchild Foundation Award (IKJ). Research in the Peel lab is funded by the National Institute of General Medical Sciences under the National Institutes of Health (Award Number R15 GM135886-01 to NP). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/ff4211de172e015bd6b902834642e612.jpg"},"imageCaption":"A) Brood size and B) viability of WT and uncp-18(tm530) hermaphrodites at indicated culture temperatures. C) Brood size of hermaphrodites of the stated genotype when mated with him-5 males. D) Brood size of fog-1 females mated with males of the indicated genotypes. E) Summary of relative sperm count in 1day old adult hermaphrodite spermathecae, and correlation with germline phenotypes. uncp-18 heterozygous worms were used as a control. F&G) UNCP-18::mNG-expressing worms. F) Plasma membrane localization in the germline, G) A two-cell embryo at telophase. Arrows indicate likely vesicular association. Arrowheads indicate perinuclear localization. ** p<0.01; * p<0.05, Student's t test.
","imageTitle":"uncp-18(tm530) mutants have a reduced brood size due to reduced sperm number and germline defects
","methods":"C. elegans strains were cultured on MYOB plates seeded with OP50 bacteria at 20°C unless stated otherwise. For brood size and viability assays, single L4 hermaphrodites were transferred onto 35mm plates. After 24 hours, each worm was transferred to a new plate, and this was repeated until embryo production had ceased. Each plate was scored 24h after adults were removed, and the number of viable worms and embryos was recorded. Brood size was calculated by summing viable worms and unhatched embryos. Viability was calculated as viable worms/broodsize. The ability of mating to rescue uncp-18 brood size was tested by putting four him-5 males and one L4 hermaphrodite onto 35mm plates. After 24 hours, worms were transferred to a new plate, and this was repeated until embryo production ceased. Each plate was scored 24h after adults were removed and the number of viable worms and embryos was recorded. To test fertility of uncp-18 males, fog-1 females were generated by moving L4 hermaphrodites to 25°C whereby all offspring are feminized. One male and one fog-1 female were transferred onto 35mm plates and after 24 hours worms were transferred to a new plate. This was repeated for 3days. Each plate was scored 24h after embryos were laid, and the number of viable worms and embryos was recorded. The uncp-18::mNG strain was made by CRISPR-mediated integration of the neonGreen ORF at the endogenous uncp-1 locus (SUNY Biotech). For imaging, the worms were anesthetized in 20mM sodium azide, mounted on a 2% agarose pad and viewed on a Diskovery spinning disk confocal system (Andor) mounted on a Nikon Eclipse Ti microscope with a 60×/1.4 NA objective. Images were captured using an ImagE Mx2 EM-CCD camera (Hamamatsu Photonics, Shizuoka, Japan). Metamorph software was used for Image capture.
","reagents":"Strain | Genotype | Available from |
Wild type | CGC | |
CGC | ||
oxIs318 II; unc-119(ed3 or e2498) ruIs32 III; ddIs6 V; dnIs17 | CGC (Johnston et al., 2010) | |
CGC (Dejima et al., 2018) | ||
oxIs318 [spe-11p::mCherry::histone + unc-119(+)] II ; uncp-18(tm530)/dpy-9(tm9713) kvs-5(tmIs1245) IV; him-5(e1490) V | authors | |
authors | ||
uncp-18(tm530)/dpy-9(tm9713) kvs-5(tmIs1245) IV; him-5(e1490) V | authors | |
authors; SUNY Biotech | ||
CGC |
Exocytosis is important for delivery of proteins and lipids to the cell surface, and for the release of cargo to the cell exterior. Multiple steps in exocytosis are mediated by conserved SNARE proteins and by SEC1/MUNC18 (SM) proteins. Once vesicles arrive at the membrane, vSNAREs on the vesicle membrane and tSNARES on the plasma membrane assemble into a trans-SNARE complex which facilitates vesicle docking. The SM-family proteins act as chaperones to ensure the fast and accurate assembly of the trans-SNARE complex, aiding efficient membrane fusion.
Humans have three SM family proteins that regulate exocytosis, and their dysfunction is associated with disease. STXBP1 mutations cause early infantile epileptic encephalopathy (EIEE) (Saitsu et al., 2010); STXBP2 mutations cause familial hemophagocytic lymphohistiocytosis 5 (F-HLH5) (Côte et al., 2009; Spessott et al., 2017); and STXBP3 mutations cause very early onset inflammatory bowel disease (VEOIBD) (Ouahed et al., 2021). In each case, disease phenotypes arise due to impaired exocytosis. Neurons require STXBP1 for synaptic vesicle release. Immune cells use STXBP2 for cytotoxic granule extrusion. And gut epithelial cells require STXPB2/3 for apical cargo delivery (Ouahed et al., 2021; Vogel et al., 2017).
The C. elegans genome encodes two SM family proteins, UNC-18 and UNCP-18 (Boeglin et al., 2023). Of these, UNC-18 is important for synaptic transmission, and its role in neurotransmitter release suggests a functional similarity to STXBP1 (Hosono et al., 1992). In comparison, the role of the paralog UNCP-18 is less well studied. At the outset of this project uncp-18 was uncharacterized, but a recent paper described a role for UNCP-18 in neurons where it functions redundantly with UNC-18 (Boeglin et al., 2023). Given that STXBP2 and -3 play important non-neuronal roles, we sought to characterize UNCP-18 functions in non-neuronal contexts focusing on the germline.
We obtained the uncp-18(tm530) mutant allele, a 336bp deletion that is predicted to cause an in-frame 48 amino acid deletion (p.310-358) in a region of the protein that is highly conserved with human STXBP proteins (Boeglin et al., 2023; Harris et al. 2020). Although viable, the strain was difficult to maintain due to low numbers of offspring. Because of this, we crossed the mutation to the marked dpy-9(tm9713) kvs-5(tmIs1245) chromosome, and subsequently maintained it as a balanced strain, selecting uncp-18(tm530) homozygotes for each analysis. A brood size analysis showed that homozygous uncp-18 mutants had a striking decrease in fertility (Figure 1A), with brood size reaching a maximum of ~30% of wild-type. This phenotype was evident across all assayed temperatures indicating that the phenotype was not temperature-sensitive. In contrast, we found that embryonic viability was similar to wild type (Figure 1B).
Since C. elegans are self-fertilizing hermaphrodites, we reasoned that the reduced brood size could result from either sperm or oocyte defects. To differentiate between these possibilities, we mated control males with uncp-18 hermaphrodites. Mating significantly increased the brood size of the uncp-18 hermaphrodites (Figure 1C), suggesting that the reduction in fertility was at least partially attributable to a sperm defect. In parallel, we tested sperm function in uncp-18 males by mating them with feminized worms. While control him-5 males produced upward of 100 offspring, most uncp-18 males produced none. Only three males sired offspring, and in each case fewer than 13 embryos were produced (Figure 1D). This reduced fertility of uncp-18 males points to sperm deficiency as a major contributor to the reduced brood size phenotype. Our results largely concur with a recent analysis of an independently derived uncp-18 null allele (Boeglin et al., 2023).
To investigate the sperm defect further, we crossed a spe-11-promoter-driven fluorescent transgene (which is expressed specifically in sperm) with uncp-18 worms. This sperm-marked strain allowed us to score relative sperm abundance in one day old hermaphrodites. We found that more than 50% of uncp-18 worms completely lacked sperm (Figure 1E). Of these, approximately one third possessed structurally intact germlines, one third had germlines that appeared abnormal, and one third lacked any discernible germline, indicating the sperm deficiency has pleiotropic causes. The remaining ~40% of uncp-18 worms which did possess sperm tended to show a lower sperm abundance than control worms (Fig 1E). These data suggest that UNCP-18 contributes to fertility at multiple levels, including in germline development and in germ cell production.
Finally, to assess whether, similar to homologs, UNCP-18 plays a role in exocytosis, we integrated a neon green tag into the endogenous locus. The fusion protein was widely expressed including in the germline and embryos. We found that UNCP-18::mNG localized at the plasma membrane throughout the germline (Figure 1F). It also shows a strong signal between adjacent cells in the two-cell embryo (Figure 1G), and can be seen in small cytoplasmic particles that move towards the membrane at cytokinesis (arrows). In addition, a particulate localization in close proximity to the nucleus can be observed (arrowheads), suggestive of localization to internal membranes. This localization is consistent with UNCP-18 contributing to vesicle trafficking and exocytosis.
Given its homology and subcellular localization, it is likely that UNCP-18 functions in exocytosis in C. elegans. Our data suggest, however, that it is not essential in the embryo. At fertilization the regulated exocytosis of cortical granules is required for eggshell formation, and failure to establish this permeability barrier leads to embryonic lethality (Sato et al., 2008). In addition, exocytosis is essential later in embryogenesis with impairment of secretory pathways leading to defects in cuticle and pharynx formation (Roberts et al., 2003). Although our expression/localization data show that UNCP-18 is present in the embryo, we found that uncp-18 mutant embryos were viable and survived at levels comparable to wild type. These embryos were produced by mutant hermaphrodites so they lacked any maternal UNCP-18 contribution. The observed viability therefore suggests that UNCP-18 is not essential for exocytosis in the embryo. It has been reported that unc-18(e81); uncp-18(syb6377) double mutants are embryonic lethal, indicating that UNCP-18 functions redundantly with UNC-18 in the embryo (Boeglin et al., 2023).
In contrast, loss of UNCP-18 leads to a range of germline phenotypes, from a reduction in gamete number to complete germline loss. A majority of hermaphrodites have partial rescue of brood size upon mating (Figure 1C), indicating that a sperm deficit contributes to the brood size phenotype. In addition to reduced sperm number, however, structural defects in the germline were also observed. Our data suggest, therefore, that UNCP-18 is required for both germline development/maintenance, and germ cell production. The underlying basis for this germline defect remains unknown.
In summary, we have characterized germline phenotypes associated with loss of UNCP-18. Our data show that UNCP-18 is membrane-associated, consistent with a role in exocytosis, and that UNCP-18 is required for full fertility. Determining the exact roles UNCP-18 plays in the germline will require further investigation. Importantly, our initial analyses have identified C. elegans as a model for understanding the non-neuronal functions of STXBP proteins with the potential to illuminate the etiology of associated diseases.
","references":[{"reference":"
Boeglin M, Leyva-Díaz E, Hobert O. 2023. Expression and function of Caenorhabditis elegans UNCP-18, a paralog of the SM protein UNC-18. Genetics 225(4): 10.1093/genetics/iyad180.
","pubmedId":"37793339","doi":""},{"reference":"Côte M, Ménager MM, Burgess A, Mahlaoui N, Picard C, Schaffner C, et al., de Saint Basile G. 2009. Munc18-2 deficiency causes familial hemophagocytic lymphohistiocytosis type 5 and impairs cytotoxic granule exocytosis in patient NK cells. J Clin Invest 119(12): 3765-73.
","pubmedId":"19884660","doi":""},{"reference":"Dejima K, Hori S, Iwata S, Suehiro Y, Yoshina S, Motohashi T, Mitani S. 2018. An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans. Cell Rep 22(1): 232-241.
","pubmedId":"29298424","doi":""},{"reference":"Hosono R, Hekimi S, Kamiya Y, Sassa T, Murakami S, Nishiwaki K, et al., Kodaira KI. 1992. The unc-18 gene encodes a novel protein affecting the kinetics of acetylcholine metabolism in the nematode Caenorhabditis elegans. J Neurochem 58(4): 1517-25.
","pubmedId":"1347782","doi":""},{"reference":"Johnston WL, Krizus A, Dennis JW. 2010. Eggshell chitin and chitin-interacting proteins prevent polyspermy in C. elegans. Curr Biol 20(21): 1932-7.
","pubmedId":"20971008","doi":""},{"reference":"Ouahed J, Kelsen JR, Spessott WA, Kooshesh K, Sanmillan ML, Dawany N, et al., Snapper SB. 2021. Variants in STXBP3 are Associated with Very Early Onset Inflammatory Bowel Disease, Bilateral Sensorineural Hearing Loss and Immune Dysregulation. J Crohns Colitis 15(11): 1908-1919.
","pubmedId":"33891011","doi":""},{"reference":"Roberts B, Clucas C, Johnstone IL. 2003. Loss of SEC-23 in Caenorhabditis elegans causes defects in oogenesis, morphogenesis, and extracellular matrix secretion. Mol Biol Cell 14(11): 4414-26.
","pubmedId":"14551256","doi":""},{"reference":"Saitsu H, Kato M, Okada I, Orii KE, Higuchi T, Hoshino H, et al., Matsumoto N. 2010. STXBP1 mutations in early infantile epileptic encephalopathy with suppression-burst pattern. Epilepsia 51(12): 2397-405.
","pubmedId":"20887364","doi":""},{"reference":"Sato M, Grant BD, Harada A, Sato K. 2008. Rab11 is required for synchronous secretion of chondroitin proteoglycans after fertilization in Caenorhabditis elegans. J Cell Sci 121(Pt 19): 3177-86.
","pubmedId":"18765566","doi":""},{"reference":"Spessott WA, Sanmillan ML, McCormick ME, Kulkarni VV, Giraudo CG. 2017. SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity. Proc Natl Acad Sci U S A 114(11): E2176-E2185.
","pubmedId":"28265073","doi":""},{"reference":"Vogel GF, van Rijn JM, Krainer IM, Janecke AR, Posovszky C, Cohen M, et al., Huber LA. 2017. Disrupted apical exocytosis of cargo vesicles causes enteropathy in FHL5 patients with Munc18-2 mutations. JCI Insight 2(14): pii: 94564. 10.1172/jci.insight.94564.
","pubmedId":"28724787","doi":""},{"reference":"Harris TW, Arnaboldi V, Cain S, Chan J, Chen WJ, Cho J, et al., Sternberg. 2019. WormBase: a modern Model Organism Information Resource. Nucleic Acids Research : 10.1093/nar/gkz920.
","pubmedId":"","doi":"10.1093/nar/gkz920 "}],"title":"UNCP-18 is a C. elegans STXBP homolog that is required for full fertility","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon 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