{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T11:38:36Z","timestamp":1779881916411,"version":"3.53.1"},"reference-count":45,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2023,3,30]],"date-time":"2023-03-30T00:00:00Z","timestamp":1680134400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"NIH","doi-asserted-by":"publisher","award":["R01 HD054681"],"award-info":[{"award-number":["R01 HD054681"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>The Caenorhabditis elegans\u00a0spe-38 gene encodes a four-pass transmembrane molecule that is required in sperm for fertilization. In previous work, the localization of the SPE-38 protein was examined using polyclonal antibodies on spermatids and mature amoeboid spermatozoa. SPE-38 is localized to unfused membranous organelles (MOs) in nonmotile spermatids. Different fixation conditions revealed that SPE-38 either localized to fused MOs and the cell body plasma membrane or the pseudopod plasma membrane of mature sperm. To address this localization paradox in mature sperm, CRISPR\/Cas9 genome editing was used to tag endogenous SPE-38 with fluorescent wrmScarlet-I. Homozygous male and hermaphrodite worms encoding SPE-38::wrmScarlet-I were fertile indicating the fluorescent tag does not interfere with SPE-38 function during sperm activation or fertilization. We found that SPE-38::wrmScarlet-I localized to MOs in spermatids consistent with previous antibody localization. In mature and motile spermatozoa we found SPE-38::wrmScarlet-I in fused MOs, the cell body plasma membrane, and the pseudopod plasma membrane. We conclude that the localization pattern observed with SPE-38::wrmScarlet-I represents the complete distribution of SPE-38 in mature spermatozoa and this localization pattern is consistent with a hypothesized role of SPE-38 directly in sperm-egg binding and\/or fusion.<\/jats:p>","DOI":"10.3390\/biom13040623","type":"journal-article","created":{"date-parts":[[2023,3,30]],"date-time":"2023-03-30T05:22:14Z","timestamp":1680153734000},"page":"623","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["CRISPR\/Cas9 Mediated Fluorescent Tagging of Caenorhabditis elegans SPE-38 Reveals a Complete Localization Pattern in Live Spermatozoa"],"prefix":"10.3390","volume":"13","author":[{"given":"Yamei","family":"Zuo","sequence":"first","affiliation":[{"name":"Waksman Institute and Department of Genetics, Rutgers University, Piscataway, NJ 08854, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6498-1261","authenticated-orcid":false,"given":"Xue","family":"Mei","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, St. John\u2019s University, Queens, New York, NY 11439, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5091-0892","authenticated-orcid":false,"given":"Andrew","family":"Singson","sequence":"additional","affiliation":[{"name":"Waksman Institute and Department of Genetics, Rutgers University, Piscataway, NJ 08854, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,3,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1002\/mrd.22634","article-title":"The molecular complexity of fertilization: Introducing the concept of a fertilization synapse","volume":"83","author":"Krauchunas","year":"2016","journal-title":"Mol. Reprod. Dev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e10034","DOI":"10.1002\/ggn2.10034","article-title":"The molecular underpinnings of fertility: Genetic approaches in Caenorhabditis elegans","volume":"2","author":"Mei","year":"2020","journal-title":"Adv. Genet."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"260296","DOI":"10.1242\/jcs.260296","article-title":"Eukaryotic fertilization and gamete fusion at a glance","volume":"135","author":"Lu","year":"2022","journal-title":"J. Cell Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1146\/annurev-cellbio-120219-021751","article-title":"The Fertilization Enigma: How Sperm and Egg Fuse","volume":"37","author":"Deneke","year":"2021","journal-title":"Annu. Rev. Cell Dev. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2795","DOI":"10.1242\/dev.01868","article-title":"The Caenorhabditis elegans spe-38 gene encodes a novel four-pass integral membrane protein required for sperm function at fertilization","volume":"132","author":"Chatterjee","year":"2005","journal-title":"Development"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1006\/dbio.2000.0118","article-title":"Every Sperm Is Sacred: Fertilization in Caenorhabditis elegans","volume":"230","author":"Singson","year":"2001","journal-title":"Dev. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1016\/j.ydbio.2012.02.037","article-title":"The sperm surface localization of the TRP-3\/SPE-41 Ca2+-permeable channel depends on SPE-38 function in Caenorhabditis elegans","volume":"365","author":"Singaravelu","year":"2012","journal-title":"Dev. Biol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-020-15459-7","article-title":"Structural insights into tetraspanin CD9 function","volume":"11","author":"Umeda","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"776","DOI":"10.1093\/biolre\/ioy055","article-title":"Caenorhabditis elegans sperm membrane protein interactome","volume":"98","author":"Marcello","year":"2018","journal-title":"Biol. Reprod."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1186\/1471-213X-1-8","article-title":"Calcium dynamics during fertilization in C. elegans","volume":"1","author":"Samuel","year":"2001","journal-title":"BMC Dev. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1016\/S0092-8674(03)00565-8","article-title":"C. elegans Sperm TRP Protein Required for Sperm-Egg Interactions during Fertilization","volume":"114","author":"Xu","year":"2003","journal-title":"Cell"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1016\/j.celrep.2016.03.040","article-title":"The Sperm TRP-3 Channel Mediates the Onset of a Ca2+ Wave in the Fertilized C. elegans Oocyte","volume":"15","author":"Takayama","year":"2016","journal-title":"Cell Rep."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1016\/0092-8674(80)90520-6","article-title":"Vesicle fusion, pseudopod extension and amoeboid motility are induced in nematode spermatids by the ionophore monensin","volume":"19","author":"Nelson","year":"1980","journal-title":"Cell"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"R988","DOI":"10.1016\/j.cub.2017.07.067","article-title":"Spermatogenesis","volume":"27","author":"Nishimura","year":"2017","journal-title":"Curr. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3959","DOI":"10.1002\/j.1460-2075.1991.tb04966.x","article-title":"Efficient gene transfer in C.elegans: Extrachromosomal maintenance and integration of transforming sequences","volume":"10","author":"Mello","year":"1991","journal-title":"EMBO J."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1007\/978-1-59745-202-1_10","article-title":"Germline Transformation of Caenorhabditis elegans by Injection","volume":"518","author":"Kadandale","year":"2009","journal-title":"Methods Mol. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1217","DOI":"10.1093\/genetics\/157.3.1217","article-title":"Creation of Low-Copy Integrated Transgenic Lines in Caenorhabditis elegans","volume":"157","author":"Praitis","year":"2001","journal-title":"Genetics"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/B978-0-12-544172-8.00006-2","article-title":"Transgenesis in C. elegans","volume":"106","author":"Praitis","year":"2011","journal-title":"Methods Cell Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1016\/B978-0-12-544172-8.00013-X","article-title":"The Genetics and Cell Biology of Fertilization","volume":"106","author":"Geldziler","year":"2011","journal-title":"Methods Cell Biol."},{"key":"ref_20","first-page":"314","article-title":"C. elegans CYLC-2 localizes to sperm","volume":"2020","author":"Krauchunas","year":"2020","journal-title":"Micropubl. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1093\/genetics\/146.1.227","article-title":"Distinct requirements for somatic and germline expression of a generally expressed Caenorhabditis elegans gene","volume":"146","author":"Kelly","year":"1997","journal-title":"Genetics"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1038\/nmeth.1865","article-title":"Improved Mos1-mediated transgenesis in C. elegans","volume":"9","author":"Davis","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"959","DOI":"10.1534\/genetics.119.301506","article-title":"The Caenorhabditis elegans Transgenic Toolbox","volume":"212","author":"Nance","year":"2019","journal-title":"Genetics"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1534\/genetics.115.182162","article-title":"CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering","volume":"202","author":"Dickinson","year":"2016","journal-title":"Genetics"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3385","DOI":"10.1091\/mbc.e16-01-0063","article-title":"Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system","volume":"27","author":"Heppert","year":"2016","journal-title":"Mol. Biol. Cell"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/B978-0-12-394620-1.00003-5","article-title":"Fluorescent Protein Methods: Strategies and Applications","volume":"107","author":"Hutter","year":"2012","journal-title":"Methods Cell Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1038\/nmeth.4074","article-title":"mScarlet: A bright monomeric red fluorescent protein for cellular imaging","volume":"14","author":"Bindels","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/B978-0-12-394620-1.00001-1","article-title":"High-Resolution Imaging of Cellular Processes in Caenorhabditis elegans","volume":"107","author":"Maddox","year":"2012","journal-title":"Methods Cell Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1093\/genetics\/iyab014","article-title":"Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans","volume":"217","author":"Goudeau","year":"2021","journal-title":"Genetics"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1429","DOI":"10.1534\/g3.117.040824","article-title":"Reliable CRISPR\/Cas9 Genome Engineering in Caenorhabditis elegans Using a Single Efficient sgRNA and an Easily Recognizable Phenotype","volume":"7","author":"Lecroisey","year":"2017","journal-title":"G3 Genes Genomes Genet."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1093\/genetics\/77.1.71","article-title":"The genetics of Caenorhabditis elegans","volume":"77","author":"Brenner","year":"1974","journal-title":"Genetics"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1016\/0012-1606(89)90088-2","article-title":"Initiation of spermiogenesis in C. elegans: A pharmacological and genetic analysis","volume":"134","author":"Shakes","year":"1989","journal-title":"Dev. Biol."},{"key":"ref_33","first-page":"e2336","article-title":"Isolation and in vitro activation of Caenorhabditis elegans sperm","volume":"47","author":"Singaravelu","year":"2011","journal-title":"J. Vis. Exp. JoVE"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Mei, X., Druzhinina, M., Dharia, S., Krauchunas, A.R., Ni, J., Singaravelu, G., Gu, S.G., Shakes, D., Grant, B.D., and Singson, A.W. (2021). SPE-51, a sperm secreted protein with an immunoglobulin-like domain, is required for sperm-egg fusion in C. elegans. bioRxiv.","DOI":"10.1101\/2021.07.07.451548"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"85671","DOI":"10.7554\/eLife.85671","article-title":"When fixation creates fiction","volume":"12","year":"2023","journal-title":"Elife"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Krauchunas, A.R., Marcello, M.R., Looper, A.M., Mei, X., Putiri, E., Singaravelu, G., Ahmed, I.I., and Singson, A. (2021). The EGF-motif containing protein SPE-36 is a secreted protein required for sperm function at fertilization in C. elegans. bioRxiv.","DOI":"10.1101\/2021.07.07.451551"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"198","DOI":"10.1007\/s00427-005-0045-4","article-title":"A comparative study of sperm morphology, cytology and activation in Caenorhabditis elegans, Caenorhabditis remanei and Caenorhabditis briggsae","volume":"216","author":"Geldziler","year":"2006","journal-title":"Dev. Genes Evol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1016\/B978-0-12-394620-1.00002-3","article-title":"Immunofluorescence Microscopy","volume":"107","author":"Shakes","year":"2012","journal-title":"Methods Cell Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1046","DOI":"10.1016\/j.cub.2013.04.057","article-title":"Dynamic Localization of G-Actin during Membrane Protrusion in Neuronal Motility","volume":"23","author":"Lee","year":"2013","journal-title":"Curr. Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"702741","DOI":"10.3389\/fcell.2021.702741","article-title":"Wnt Signaling Induces Asymmetric Dynamics in the Actomyosin Cortex of the C. elegans Endomesodermal Precursor Cell","volume":"9","author":"Caroti","year":"2021","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1752","DOI":"10.1016\/j.cub.2009.09.015","article-title":"EGG-4 and EGG-5 Link Events of the Oocyte-to-Embryo Transition with Meiotic Progression in C. elegans","volume":"19","author":"Parry","year":"2009","journal-title":"Curr. Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1083\/jcb.92.1.121","article-title":"Caenorhabditis elegans spermatozoan locomotion: Amoeboid movement with almost no actin","volume":"92","author":"Nelson","year":"1982","journal-title":"J. Cell Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1083\/jcb.39.1.119","article-title":"Zygote Formation in Ascaris Lumbricoides (Nematoda)","volume":"39","author":"Foor","year":"1968","journal-title":"J. Cell Biol."},{"key":"ref_44","unstructured":"Knobil, E., and Neill, J.D. (1994). The Physiology of Reproduction, Raven Press."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1534\/genetics.105.047340","article-title":"spe-10 Encodes a DHHC\u2013CRD Zinc-Finger Membrane Protein Required for Endoplasmic Reticulum\/Golgi Membrane Morphogenesis during Caenorhabditis elegans Spermatogenesis","volume":"172","author":"Gleason","year":"2006","journal-title":"Genetics"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/13\/4\/623\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:07:04Z","timestamp":1760123224000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/13\/4\/623"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,3,30]]},"references-count":45,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2023,4]]}},"alternative-id":["biom13040623"],"URL":"https:\/\/doi.org\/10.3390\/biom13040623","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,3,30]]}}}