Control of SNARE-driven vesicle fusion by synapsin condensates on freestanding membranes

Control of SNARE-driven vesicle fusion by synapsin condensates on freestanding membranes | 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 Control of SNARE-driven vesicle fusion by synapsin condensates on freestanding membranes Min Ju Shon, Minkwon Cha, Jaehyeon Shin, Hyun-Ro Lee, Taehyun Yang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6900968/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Single-molecule observations of membrane protein dynamics have long been challenging due to the limitations of traditional model membrane systems. Here, we developed a simple method to prepare freestanding lipid bilayers (FLBs) for studying protein diffusion and clustering. An array of large-area, protein-embedded membrane patches were stably formed on an electron microscopy grid for high-resolution fluorescence imaging. Applying this technique to SNARE-driven vesicle fusion, the core mechanism for synaptic vesicle release, we dissected vesicle docking and membrane fusion activities through diffusion-based analysis at the single-particle level, revealing distinct intermediate species on membranes. Upon adding complexin and synapsin, well-established regulators of the synaptic vesicle cycle, we observed their unique effects on the membrane-bound populations. Notably, synapsin condensates not only clustered VAMP2-containing vesicles but also engaged closely with the target membrane, releasing the vesicles for immediate fusion upon dispersal. This suggests a more direct role for synapsin in vesicle release than previously assumed. The FLB platform enables the study of diverse protein–membrane activities, with future applications in membrane deformation and tension. Biological sciences/Biophysics/Membrane biophysics Biological sciences/Biophysics/Single-molecule biophysics Biological sciences/Neuroscience/Synaptic transmission/Synaptic vesicle exocytosis Biological sciences/Biochemistry/Biophysical chemistry Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryMaterialsChaetal.pdf Supplementary Information VideoS1.Formationoffreestandinglipidbilayersoveranelectronmicroscopygrid.avi Video S1. Formation of freestanding lipid bilayers over an electron microscopy grid VideoS2.AsymmetricquenchingofNBDfluorescenceinafreestandinglipidbilayer.avi Video S2. Asymmetric quenching of NBD fluorescence in a freestanding lipid bilayer VideoS3.ComparisonofXXNcomplexdiffusiononfreestandingandsupportedlipidbilayers.avi Video S3. Comparison of ΔN complex diffusion on freestanding and supported lipid bilayers VideoS4.ProbingtheorientationofXXNcomplexeswithinafreestandinglipidbilayer.avi Video S4. Probing the orientation of ΔN complexes within a freestanding lipid bilayer VideoS5.ComparisonofVAMP2diffusionintheabsenceandpresenceofcomplexin.avi Video S5. Comparison of VAMP2 diffusion in the absence and presence of complexin VideoS6.EffectofsynapsinonVAMP2vesiclebindingtoXXNSNAREcomplexes.avi Video S6. Effect of synapsin on VAMP2 vesicle binding to ΔN-SNARE complexes VideoS7.VAMP2vesiclesinthepresenceofsynapsinbeforeandaftercondensatedispersal.avi Video S7. VAMP2 vesicles in the presence of synapsin before and after condensate dispersal Cite Share Download PDF Status: Posted 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. 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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-6900968","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":471997886,"identity":"a45df5b7-9bbd-465c-a23c-940e24d0d759","order_by":0,"name":"Min Ju Shon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYFCCA2xAwgbCZmwAkWxEaUkDYmaitYBVHCZBi27j8WcPPu44n8fPf/7gg587GOT5G9jSPuDTYnbgjLnhzDO3iyVnJDMb9p5hMJxxgO3wDAJa2KR5224nbrjBzCbN2MbAuIGBvRmvw8wOHH8G1HIuccP5w+y/gVrsidBywAyo5UDihgPJbMxALYkbGNgOE9ByxkxyZlsyyC/Gkr1tEskzDrMl49dy4/gziY9tdsAQO/jww882G9v+9jZjvFoYJA6AqQQYFxw/+AF/A4qWUTAKRsEoGAWYAACC3UqofIjabQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-0333-1150","institution":"POSTECH","correspondingAuthor":true,"prefix":"","firstName":"Min","middleName":"Ju","lastName":"Shon","suffix":""},{"id":471997887,"identity":"5779c019-d5cd-41e9-bffe-6461fd3fba17","order_by":1,"name":"Minkwon Cha","email":"","orcid":"","institution":"POSTECH","correspondingAuthor":false,"prefix":"","firstName":"Minkwon","middleName":"","lastName":"Cha","suffix":""},{"id":471997888,"identity":"5054fcfd-ab28-4ae3-b752-5827711d1e51","order_by":2,"name":"Jaehyeon Shin","email":"","orcid":"https://orcid.org/0009-0003-7141-2360","institution":"POSTECH","correspondingAuthor":false,"prefix":"","firstName":"Jaehyeon","middleName":"","lastName":"Shin","suffix":""},{"id":471997889,"identity":"c37883c0-80dd-44d5-be47-9b06634ff525","order_by":3,"name":"Hyun-Ro Lee","email":"","orcid":"","institution":"POSTECH","correspondingAuthor":false,"prefix":"","firstName":"Hyun-Ro","middleName":"","lastName":"Lee","suffix":""},{"id":471997890,"identity":"e0794229-e5d5-4c2d-a039-036fc6432d33","order_by":4,"name":"Taehyun Yang","email":"","orcid":"","institution":"POSTECH","correspondingAuthor":false,"prefix":"","firstName":"Taehyun","middleName":"","lastName":"Yang","suffix":""},{"id":471997891,"identity":"3a3a041f-0715-4b12-919b-c8ffd726fbca","order_by":5,"name":"Eunji Cho","email":"","orcid":"https://orcid.org/0000-0002-8486-3641","institution":"Seoul National University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Eunji","middleName":"","lastName":"Cho","suffix":""},{"id":471997892,"identity":"5e76c8b1-fb48-497e-be78-2d0feb1c2b02","order_by":6,"name":"Sunghoe Chang","email":"","orcid":"https://orcid.org/0000-0002-3446-7288","institution":"Seoul National University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sunghoe","middleName":"","lastName":"Chang","suffix":""},{"id":471997893,"identity":"d0693243-e12a-4a2c-b9f5-cc4b35341273","order_by":7,"name":"Seung Soo Oh","email":"","orcid":"https://orcid.org/0000-0001-8045-080X","institution":"Pohang University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Seung","middleName":"Soo","lastName":"Oh","suffix":""}],"badges":[],"createdAt":"2025-06-16 02:50:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6900968/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6900968/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85675594,"identity":"73ce0078-50b4-4513-ba75-a9d395ec6670","added_by":"auto","created_at":"2025-06-30 14:23:31","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1056732,"visible":true,"origin":"","legend":"Article File","description":"","filename":"ManuscriptChaetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6900968/v1_covered_98ae348f-ffc3-48fe-ab45-016c836590ec.pdf"},{"id":85326600,"identity":"6f0115a4-b5c2-4e66-b766-3ac57666fac1","added_by":"auto","created_at":"2025-06-24 16:40:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":365432,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryMaterialsChaetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6900968/v1/6e93017e7f9898cc9023d9b0.pdf"},{"id":85326608,"identity":"7923632a-9437-4ccb-b82c-44b5d53f6c3b","added_by":"auto","created_at":"2025-06-24 16:40:35","extension":"avi","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7783942,"visible":true,"origin":"","legend":"Video S1. 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