SMNK-1 and VHA-18 are essential for FB-MO complex assembly and transport during spermatogenesis in C. elegans

SMNK-1 and VHA-18 are essential for FB-MO complex assembly and transport during spermatogenesis in C. elegans | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SMNK-1 and VHA-18 are essential for FB-MO complex assembly and transport during spermatogenesis in C. elegans Peng Wang, Xiaojing Ma, Xinyan Chen, Lianwan Chen, Xiaolong Zhang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6000630/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The spatiotemporal control of asymmetric positioning of cellular cargoes such as macromolecules and organelles during spermatogenesis is essential to produce functional sperm in most animal species. Yet, the regulatory mechanisms that govern cargo sorting and transport remain largely unknown. Here we show that the casein-like kinase SMNK-1 localized on the surface of membranous organelle (MO) is required to recruit IDR-containing SPE-18 and facilitate the assembly of Major Sperm Protein (MSP) condensate, named Fibrous Body (FB) in spermatocyte during spermatogenesis in C. elegans. Meanwhile, the MO-localized VHA-18, the H subunit of the v-ATPase, is required for pumping the cytoplasmic protons into MOs, generating an alkaline cytoplasmic microenvironment adjacent to MOs, favorable for packaging MSP filaments into fibrous body (FB) and thus guaranteeing the correct cargo sorting during spermatogenesis. Thus, the interplay between membraneless condensate FBs mediated by SMNK-1 and membrane-bound organelle MOs regulated by VHA-18 provides a novel yet uncovered mechanism for cargo sorting and vectorial transport during spermatogenesis in C. elegans. Biological sciences/Cell biology/Organelles Biological sciences/Developmental biology Casein kinase MSP assembly v-ATPase spermatogenesis C. elegans Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Additional Declarations Table 1 is available in the Supplementary Files section. There is NO Competing Interest. Supplementary Files table.pdf table VideoS1.mp4 Video S1 VideoS2.mp4 Video S2 Supplementaryfigure.pdf Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6000630","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":421718822,"identity":"44bfd921-61ce-40a9-9a7e-7611b1f31e43","order_by":0,"name":"Peng Wang","email":"data:image/png;base64,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","orcid":"","institution":"Institute of Biophysics, Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Peng","middleName":"","lastName":"Wang","suffix":""},{"id":421718823,"identity":"5c1e654e-05e9-419f-9709-3cfa8227e673","order_by":1,"name":"Xiaojing Ma","email":"","orcid":"","institution":"College of Life Sciences, Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiaojing","middleName":"","lastName":"Ma","suffix":""},{"id":421718824,"identity":"623cee43-75ae-4741-9d33-75806093e93b","order_by":2,"name":"Xinyan Chen","email":"","orcid":"","institution":"Institute of Biophysics, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xinyan","middleName":"","lastName":"Chen","suffix":""},{"id":421718825,"identity":"d7373add-8fad-4978-b322-f9827215e74f","order_by":3,"name":"Lianwan Chen","email":"","orcid":"","institution":"Institute of Biophysics, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lianwan","middleName":"","lastName":"Chen","suffix":""},{"id":421718826,"identity":"1da1148f-18fc-42d1-aad5-1741d5f08d24","order_by":4,"name":"Xiaolong Zhang","email":"","orcid":"","institution":"College of Life Sciences, Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiaolong","middleName":"","lastName":"Zhang","suffix":""},{"id":421718827,"identity":"d04ea5e2-380d-4a23-a1a9-e52a0b5d86ca","order_by":5,"name":"Ying Chen","email":"","orcid":"","institution":"College of Life Sciences, Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Chen","suffix":""},{"id":421718828,"identity":"b7117d03-7df9-40fa-a890-ed51bdd1b663","order_by":6,"name":"Ning Wang","email":"","orcid":"","institution":"Institute of Biophysics, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Wang","suffix":""},{"id":421718829,"identity":"e2a7fdab-7a63-43bc-91a5-c8e61681cc63","order_by":7,"name":"Qiushi Wang","email":"","orcid":"","institution":"Institute of Biophysics, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qiushi","middleName":"","lastName":"Wang","suffix":""},{"id":421718830,"identity":"8a7fed56-22e4-4a0a-a606-c1901c1d0692","order_by":8,"name":"Zheng Cao","email":"","orcid":"","institution":"Institute of Biophysics, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Cao","suffix":""},{"id":421718831,"identity":"7fc11db7-2a40-48c8-8797-01664df5b247","order_by":9,"name":"Yong Liu","email":"","orcid":"","institution":"College of Life Sciences, Fuyang Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Liu","suffix":""},{"id":421718832,"identity":"0baca906-57ee-4a99-b177-1c509e3dfe06","order_by":10,"name":"Yanmei Zhao","email":"","orcid":"","institution":"Institute of Biophysics, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yanmei","middleName":"","lastName":"Zhao","suffix":""},{"id":421718833,"identity":"6d570610-4e4a-4537-8286-5e222d0bd9ee","order_by":11,"name":"Long Miao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Long","middleName":"","lastName":"Miao","suffix":""}],"badges":[],"createdAt":"2025-02-10 15:52:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6000630/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6000630/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77384196,"identity":"d3b06224-e29b-4812-8a1d-8d0236f09470","added_by":"auto","created_at":"2025-02-28 04:47:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":203980,"visible":true,"origin":"","legend":"\u003cp\u003eCasein-like kinase SMNK-1 is required for C. elegans fertility. (A) RT-PCR showed the relative expression of smnk-1 in N2 and fem-1 (hc17), \u0026nbsp;fem-3(q96 gf) and glp-4(bn2) mutants, with act-1 serving as the control. \u0026nbsp;fem-1 (hc17) and fem-3 (q96 gf) worms only produced oocytes and sperm, \u0026nbsp;respectively, while glp-4(bn2) worms displayed defective germline \u0026nbsp;development and do not produce either oocytes or sperm. \u0026nbsp;(B) Quantification of relative smnk-1 expression shown in (A). values are \u0026nbsp;means ± SEM. ****, P\u0026lt;0.0001. NS, not significant. (C) Genomic structure of smnk-1 gene. The boxes represent exons, and the \u0026nbsp;solid lines represent introns. The mutations are labeled at their \u0026nbsp;corresponding positions. smnk-1 (ibp107) is a frameshift mutation caused \u0026nbsp;by a 7 bp insertion in the first exon. smnk-1(ibp129) is a point mutation at \u0026nbsp;the putative active site residue T195; smnk-1(ibp116) is terminated at the \u0026nbsp;294th amino acid, thus a C-terminus deletion mutant. smnk-1(ibp129) and \u0026nbsp;smnk-1(ibp116) are two alleles that might affect kinase activity of SMNK-1. (D) The smnk-1 mutant hermaphrodites sired fewer progeny compared with \u0026nbsp;control group, and the loss of fertility in smnk-1 hermaphrodite could be \u0026nbsp;rescued by mating with him-5 males. \u0026nbsp;(E) smnk-1 mutant males had impaired fertility. The number of cross progeny \u0026nbsp;(ND, non-Dumpy) and self (D, Dumpy) progeny produced by spe-8;dpy-4 \u0026nbsp;hermaphrodites was counted after mating with control him-5 or smnk-1; him-5 males. Values in (D) and (E) are presented as the mean ± SEM. ****p \u0026nbsp;\u0026lt; 0.0001, in comparison to the control. (F) Sperm ejaculated from smnk-1(ibp107) males failed to migrate toward and \u0026nbsp;accumulate in the spermatheca, unlike the behavior of sperm from control \u0026nbsp;males. Males were stained with MitoTracker Red before mating with L4 \u0026nbsp;virgin hermaphrodites. The arrow showed the MitoTracker Red-labeled \u0026nbsp;male sperm from smnk-1;him-5 males located outside of the hermaphrodite \u0026nbsp;uterus, which might be pushed out of the uterus during egg-laying. Scale: \u0026nbsp;50 μm.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6000630/v1/822458ebe1755b7474da306d.png"},{"id":77384198,"identity":"99c2caa8-e01d-49ae-9bd5-b425bc9513e3","added_by":"auto","created_at":"2025-02-28 04:47:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1098558,"visible":true,"origin":"","legend":"\u003cp\u003esmnk-1 affects sperm activation and motility. (A) Loss of smnk-1 slightly affected hermaphrodite sperm activation, whichwas reflected by the number of GFP puncta on spermatozoa located in the \u0026nbsp;spermatheca. Scale bars in left and right panels represent 20 and 10 μm, \u0026nbsp;respectively. (B) The dissected sperm from male gonad of smnk-1;swm-1;gfp::nkb-2;him-5 \u0026nbsp;and control group. In vivo activated smnk-1 sperm nearly had no \u0026nbsp;pseudopod, though GFP::NKB-2 displaying a polarized pattern. Scale: 5 \u0026nbsp;μm. (C) smnk-1 affected sperm activation in vitro with extracellular sperm activators. \u0026nbsp;SM represented the sperm medium, serving as the inactivated control; the \u0026nbsp;known sperm activators, such as weak bases (NH4Cl and TEA), the proton \u0026nbsp;carrier monensin, trace element Zn2+ , and Pronase, were used to trigger \u0026nbsp;sperm activation. The MO fusions were indicated by the fluorescent puncta \u0026nbsp;of membrane impermeable probe FM 1-43. Scale: 5 μm. \u0026nbsp;(D) smnk-1 spermatids contained normal MOs as seen in control. The acidic \u0026nbsp;MOs were labeled with LysoTracker Red. Scale: 5 μm. \u0026nbsp;(E) Quantification of the number of the MOs indicated by LysoTracker Red \u0026nbsp;shown in (D). \u0026nbsp;(F) smnk-1 affected MO fusion and pseudopod extension when sperm were \u0026nbsp;activated by Pronase. The fluorescent puncta of GFP::NKB-2 was used to \u0026nbsp;indicate the fused MOs. Scale: 5 μm. \u0026nbsp;(G) Quantification of the number of fused MOs indicated by GFP::NKB-2 puncta \u0026nbsp;fluorescence in (F). Values in (E) and (G) are presented as the mean ± \u0026nbsp;SEM. ****p \u0026lt; 0.0001. NS, not significant\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6000630/v1/46801d268e6ebf67b41c60d0.png"},{"id":77385028,"identity":"cd7c3dd6-b8bb-445b-b309-74931114228c","added_by":"auto","created_at":"2025-02-28 04:55:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2034602,"visible":true,"origin":"","legend":"\u003cp\u003esmnk-1 affects FB assembly in spermatocytes. \u0026nbsp;(A) The anti-MSP immunofluorescence microscopy assay of spermatids from \u0026nbsp;smnk-1(ibp107) and control males. Scale: 5 μm. \u0026nbsp;(B) Quantification of MSP fluorescence shown in (A). Values are presented as \u0026nbsp;the mean ± SEM. ****p \u0026lt; 0.0001. (C) The anti-MSP immunofluorescence microscopy assay of spermatocytes \u0026nbsp;from smnk-1(ibp107) and control males in different stages. Green puncta \u0026nbsp;observed in control cells indicate FBs. MSP fluorescence appeared \u0026nbsp;diffusive in the cortical area of smnk-1(ibp107) spermatocytes in anaphase \u0026nbsp;I stage and in the cortex of RB during budding stage Scale: 5 μm. (D) The anti-MSP immunofluorescence microscopy assay of male gonads of \u0026nbsp;smnk-1(ibp116), smnk-1(ibp129), and control wroms. MSP \u0026nbsp;immunofluorescence appeared diffusive in smnk-1(ibp116) and smnk-1(ibp129) gonad, unlike the fluorescent puncta (FBs) observed in the \u0026nbsp;control. Scale: 10 μm. \u0026nbsp;(E) Quantification of MSP fluorescence shown in (D). Values are presented as \u0026nbsp;the mean ± SEM. ****p \u0026lt; 0.0001. \u0026nbsp;(F) EM images of spermatocytes from smnk-1(ibp107) and control males. \u0026nbsp;White arrows: tubule-like MOs; red arrows in the left panels: FBs semi-enclosed by MOs; m: mitochondria. Scale: 500 nm. (G) EM images of sperm and RB in the budding stage from smnk-1(ibp107) and \u0026nbsp;control males. Red arrows: FB-MO complex in control spermatids; The \u0026nbsp;white dashed ellipse on the right showed the MO components, typically \u0026nbsp;partitioned to spermatids, were lagged in RB in smnk-1;him-5. m: \u0026nbsp;mitochondrion; N: nucleus. Scale: 1 μm.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6000630/v1/e0fe25c8c48f7502895d22ed.png"},{"id":77384208,"identity":"cbda6848-2f17-4396-9105-33b5f8f9267a","added_by":"auto","created_at":"2025-02-28 04:47:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1315752,"visible":true,"origin":"","legend":"\u003cp\u003ePunctate SMNK-1 is associated with FBs in spermatocytes. \u0026nbsp;(A) The shape and size of the nuclei labeled by Hoechst illustrated the various \u0026nbsp;developmental stages in the male gonad. Scale bars in the left and right \u0026nbsp;panels represent 50 μm and 20 μm, respectively. (B) The expression and localization pattern of SMNK-1::GFP in the male gonad. \u0026nbsp;The arrow indicated the boat-like structure of GFP puncta. The nuclei were labeled by Hoechst. Scale bars in the upper and lower panels represent 10 \u0026nbsp;μm. (C) Immunofluorescence microscopy assay showed that SMNK-1(green) was localized to the periphery of FBs (red) in meiotically dividing spermatocytes. \u0026nbsp;The nuclei were labeled by DAPI. Scale: 10 μm. (D) Quantification of MSP and SMNK-1::GFP fluorescence in the dashed line \u0026nbsp;shown in (C).\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6000630/v1/1fdc7feca67a9e52915acd29.png"},{"id":77384203,"identity":"e279f52b-773b-4472-a620-43ba60a60131","added_by":"auto","created_at":"2025-02-28 04:47:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2149228,"visible":true,"origin":"","legend":"\u003cp\u003eSMNK-1 functions upstream of SPE-18 and is required for \u0026nbsp;recruiting SPE-18 to MOs. (A) Live-cell imaging of SMNK-1::GFP and SPE-18::mCherry in different \u0026nbsp;stages of spermatogenesis indicated by the nuclear size and structure. The \u0026nbsp;appearance of SMNK-1::GFP occurred earlier than SPE-18::mCherry in the \u0026nbsp;male gonad. The boxed areas in the upper panelwere magnified on the \u0026nbsp;lower panels. Scale bars in the upper and lower panels represent 10 and 5 \u0026nbsp;μm, respectively. (B) Quantification of SPE-18::mCherry and SMNK-1::GFP fluorescence \u0026nbsp;intensity along the dashed line shown in (A).The upper and lower images \u0026nbsp;correspond to the dashed lines in magnified b and c, respectively. (C) Inserting mCherry into the C-terminal of SPE-18 affected the fertility of \u0026nbsp;hermaphrodites. values are presented as the mean ± SEM. ****p \u0026lt; 0.0001. (D) Loss of SMNK-1 affected the localization of SPE-18::mCherry during \u0026nbsp;spermatogenesis. In the control, SPE-18::mCherry appeared as puncta, \u0026nbsp;while in any of the three smnk-1 mutants, SPE-18::mCherry appeared \u0026nbsp;diffusive in the cytoplasm. The boxed areas in the left are magnified on the \u0026nbsp;right. Scale bars in left and right panels represent 20 and 10 μm, \u0026nbsp;respectively. (E) Quantification of SPE-18::mCherry fluorescence intensity in the dashed \u0026nbsp;lines shown in (D).\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6000630/v1/1058bca83564621bd456b6d4.png"},{"id":77384205,"identity":"60ab9d01-abfd-49a4-8070-43cd6c15f1bb","added_by":"auto","created_at":"2025-02-28 04:47:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1576888,"visible":true,"origin":"","legend":"\u003cp\u003eThe acidification of MO lumen mediated by v-ATPase is required \u0026nbsp;for FB-MO morphogenesis. (A) SMNK-1::GFP puncta were localized on the surface of acidic MOs probed \u0026nbsp;by the LysoTracker Red. Scale: 5 μm. (B) Quantification of the SMNK-1::GFP fluorescence intensity in the dashed \u0026nbsp;line shown in (A). (C) BFA treatment neutralized the lumen of MOs and caused the puncta of \u0026nbsp;SMNK-1::GFP to diffuse in spermatocytes. Scale: 20 μm. (D) Loss of VHA-18 caused abnormal vacuoles scattered in the cytoplasm and \u0026nbsp;the failure of SMNK-1::GFP condensation in spermatocytes. Many big \u0026nbsp;round and scattered vacuoles were not stained by LysoTracker Red in vha-18 spermatocyte. Meanwhile, the cytoplasmic space among vacuoles was \u0026nbsp;stained heavily by LysoTracker Red, instead of the MOs lumen observed in \u0026nbsp;the control. The fluorescence of SMNK-1::GFP appeared as puncta in the \u0026nbsp;distal end, but decreased dramatically and failed to colocalize with \u0026nbsp;LysoTracker Red in the area of spermatocyte in the proximal germline of \u0026nbsp;vha-18 male. The white boxes indicate the spermatocytes undergoing \u0026nbsp;meiotic divisions. Scale: 5 μm.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6000630/v1/e17d17958f1a64d54d4565ca.png"},{"id":77384201,"identity":"acfedad0-f3ed-4201-8edd-c96a0440c587","added_by":"auto","created_at":"2025-02-28 04:47:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1806272,"visible":true,"origin":"","legend":"\u003cp\u003eVHA-18 is required for MO-FB complex morphogenesis and male \u0026nbsp;fertility. (A)EM images of spermatocytes in dissected male gonad from vha-18;him-8 or \u0026nbsp;him-8 control. Many vesicles were observed in vha-18;him-8 spermatocytes. \u0026nbsp;At the early stage of spermatocytes in vha-18;him-8 (Panel c), some \u0026nbsp;assembled FBs were associated with the abnormal vesicles, unlike that \u0026nbsp;seen in control where tubule-like MOs semi-enclose the FBs (Panels a and \u0026nbsp;b); at the late stage of spermatocytes in vha-18;him-8 (Panel d), FBs \u0026nbsp;expanded in both length and width were naked and not associated with the \u0026nbsp;vacuoles. FB and MO membranes are labeled with white and yellow arrow, \u0026nbsp;respectively. Scale: 500 nm. \u0026nbsp;(B) Cryo-immuno EM analysis of vha-18;him-8 and him-8 control \u0026nbsp;spermatocytes. Both MO membrane and vacuole membrane were recognized by 1CB4 (Panels a and b) and serpin antibody (Panel d). A \u0026nbsp;smaller MO head (Panel b, the white dashed circle), and a swollen MO body \u0026nbsp;(Panels b, c and d) could be observed in vha-18;him-8 spermatocytes, \u0026nbsp;unlike that seen in control spermatocytes where normal sized MO head was \u0026nbsp;connected via neck region to well-organized interrelated MO body (Panel a). \u0026nbsp;The boxed areas in the upper panel were magnified on the lower panels. \u0026nbsp;The antibody-conjugated colloidal gold particle can be distinctly observed in \u0026nbsp;the bottom magnified panels a, b and d, but not panel c, which is the control \u0026nbsp;without the primary antibody included. m: mitochondria; N: nucleus. The \u0026nbsp;scale bar upper, 400 nm; the lower, 200 nm (C)VHA-18 was necessary for MSP sorting after anaphase II. \u0026nbsp;Immunofluorescence microscopy assay with MSP antibody showed that \u0026nbsp;though some big MSP puncta could be observed in the budding spermatids, \u0026nbsp;dramatic amount of MSP was retained in the RB in vha-18;him-8. MSP \u0026nbsp;puncta appeared relatively similar in size and number in the budding \u0026nbsp;spermatids and barely visible in RBs in the control. The white arrow and \u0026nbsp;arrowhead indicated the dividing spermatids and RB, respectively. Scale: 5 \u0026nbsp;μm. (D)Loss of VHA-18 causeed sperm activation defects. TEA was used as \u0026nbsp;extracellular agent to activate sperm. In the vha-18 mutant sperm, a \u0026nbsp;diminished presence of fused MOs (as indicated by reduced GFP::NKB-2 puncta) and shorter pseudopods or spiky projections were observed in \u0026nbsp;comparison to the control group. Scale: 5 μm. (E) Loss of VHA-18 affected hermaphrodite fertility. The reduced fertility of vha-18 mutant hermaphrodites was rescued by mating with him-5 males. (F) Loss of VHA-18 affected male fertility. The number of cross progeny (ND, \u0026nbsp;non-dumpy) or self-progeny (D, dumpy) produced by spe-8;dpy-4 \u0026nbsp;hermaphrodites are counted after mating with him-8 or vha-18;him-8 mutant \u0026nbsp;males.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6000630/v1/df11bdb6b6b28dd98e374987.png"},{"id":77385139,"identity":"25e42ccb-2ff7-4038-ba3b-27e5ca6c143d","added_by":"auto","created_at":"2025-02-28 05:03:56","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2003855,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6000630/v1_covered_3409ef49-a171-4653-a6a4-43b2049bfa5c.pdf"},{"id":77384197,"identity":"a1178dba-3647-4d24-aea7-a58256d8f964","added_by":"auto","created_at":"2025-02-28 04:47:46","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":40948,"visible":true,"origin":"","legend":"table","description":"","filename":"table.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6000630/v1/737fc6235bfc0f1cd91de93b.pdf"},{"id":77384199,"identity":"69e8734f-4707-4122-afbb-e2ef68cd6a25","added_by":"auto","created_at":"2025-02-28 04:47:46","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":809873,"visible":true,"origin":"","legend":"Video S1","description":"","filename":"VideoS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6000630/v1/c14e88827326e7e580ac5668.mp4"},{"id":77384202,"identity":"2230a2bf-486b-4ac1-8848-99d16dd50942","added_by":"auto","created_at":"2025-02-28 04:47:46","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":424255,"visible":true,"origin":"","legend":"Video S2","description":"","filename":"VideoS2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6000630/v1/ef30bfb66e6a0a3d78cd97f9.mp4"},{"id":77384210,"identity":"f8f4db56-76f0-49b4-b0f1-3c0811cd1299","added_by":"auto","created_at":"2025-02-28 04:47:46","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":9112342,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6000630/v1/a3481edbc3c180eb5b5b6d6d.pdf"}],"financialInterests":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e\n\u003cp\u003eThere is \u003cstrong\u003eNO\u003c/strong\u003e Competing Interest.\u003c/p\u003e","formattedTitle":"SMNK-1 and VHA-18 are essential for FB-MO complex assembly and transport during spermatogenesis in C. elegans","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Casein kinase, MSP assembly, v-ATPase, spermatogenesis, C. elegans","lastPublishedDoi":"10.21203/rs.3.rs-6000630/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6000630/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The spatiotemporal control of asymmetric positioning of cellular cargoes such as macromolecules and organelles during spermatogenesis is essential to produce functional sperm in most animal species. Yet, the regulatory mechanisms that govern cargo sorting and transport remain largely unknown. Here we show that the casein-like kinase SMNK-1 localized on the surface of membranous organelle (MO) is required to recruit IDR-containing SPE-18 and facilitate the assembly of Major Sperm Protein (MSP) condensate, named Fibrous Body (FB) in spermatocyte during spermatogenesis in C. elegans. Meanwhile, the MO-localized VHA-18, the H subunit of the v-ATPase, is required for pumping the cytoplasmic protons into MOs, generating an alkaline cytoplasmic microenvironment adjacent to MOs, favorable for packaging MSP filaments into fibrous body (FB) and thus guaranteeing the correct cargo sorting during spermatogenesis. Thus, the interplay between membraneless condensate FBs mediated by SMNK-1 and membrane-bound organelle MOs regulated by VHA-18 provides a novel yet uncovered mechanism for cargo sorting and vectorial transport during spermatogenesis in C. elegans.","manuscriptTitle":"SMNK-1 and VHA-18 are essential for FB-MO complex assembly and transport during spermatogenesis in C. elegans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-28 04:47:41","doi":"10.21203/rs.3.rs-6000630/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"77a860de-c19f-4ed5-a19f-bd943ab28dae","owner":[],"postedDate":"February 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":44950271,"name":"Biological sciences/Cell biology/Organelles"},{"id":44950272,"name":"Biological sciences/Developmental biology"}],"tags":[],"updatedAt":"2026-05-07T09:18:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-28 04:47:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6000630","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6000630","identity":"rs-6000630","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}