LipoTag: A minimal motif for live and functional imaging of plant cell membranes.

LipoTag: A minimal motif for live and functional imaging of plant cell 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 LipoTag: A minimal motif for live and functional imaging of plant cell membranes. Joris Sprakel, Maarten Besten, Tanguy Heesemans, Rik Froeling, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9470172/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 plant plasma membrane is a highly dynamic structure that is crucial for cell compartmentalization, the maintenance of (bio)chemical gradients, signaling and cell growth and responses to stress. In plants, plasma membranes are tightly connected to the cell walls that encase them. These cell walls can act as diffusion barriers and prevent the use of a wide range of synthetic fluorescent probes that have been developed to study animal cell membranes, which lack a cell wall, with live functional imaging. Here, we introduce LipoTag, a minimal chemical motif that, upon chemical conjugation, transforms hydrophobic fluorophores into water-soluble, membrane-targeted probes that can permeate plant cell walls to reach their intended location. LipoTag uses a localized positive charge in combination with a short aliphatic spacer to direct cargo to the plasma membrane. We used LipoTag to develop a suite of membrane-specific fluorescent probes that work in walled organisms beyond the plant kingdom. In addition, we used LipoTag to develop functional reporters for the quantitative imaging of membrane density, lipid order and membrane oxidation in living plant tissues. LipoTag forms a modular platform for exploring the plant plasma membrane with a suite of contemporary imaging modalities. Biological sciences/Biological techniques/Imaging/Fluorescence imaging Biological sciences/Plant sciences/Plant cell biology Full Text Additional Declarations There is NO Competing Interest. Supplementary Files BestenetalLipoTag2026SI.pdf Supplementary Information Bestenetalreportingsummary.pdf Reporting Summary 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9470172","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":630646588,"identity":"6a4bd46c-b8b0-48d1-9872-a67b6ebb7ee6","order_by":0,"name":"Joris Sprakel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIie2OsQrCMBCGTwK6VF0zKL5CioOvk0OIW5cuHUQ6tUvdMxQfQ9dIoC59A5e66Fq3btpSQafYUTDfcPdz3Ac/gMXyg/RCANXGPkARtEfVXeF5G43KB7WCUQeFxNvLsYKNt4gzLHCn8RAOlblYcmLaAe1PcqEZ7jWmasTNihSg6yYoYRVR3J/r4LCvSlMM5fgWV5h2VJQDBCUVGWDYRUky0A7TPqXXJeXZYy7JF8WNI3Kvgo1Hx8Ity7WYykHiFkYlbCYD/j4R03/N7LW56clisVj+nCf+lk7fInhPkAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-6532-4514","institution":"Wageningen University \u0026 Research","correspondingAuthor":true,"prefix":"","firstName":"Joris","middleName":"","lastName":"Sprakel","suffix":""},{"id":630646589,"identity":"a0f27df7-7b66-4862-b2d9-6c44d0a5c773","order_by":1,"name":"Maarten Besten","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Maarten","middleName":"","lastName":"Besten","suffix":""},{"id":630646590,"identity":"2cdd6543-3716-4a8b-8a86-0e5f8d91215e","order_by":2,"name":"Tanguy Heesemans","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Tanguy","middleName":"","lastName":"Heesemans","suffix":""},{"id":630646591,"identity":"de0e817e-4225-47a8-bded-0a79a1610081","order_by":3,"name":"Rik Froeling","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Rik","middleName":"","lastName":"Froeling","suffix":""},{"id":630646592,"identity":"bf33585f-fabc-4ff6-ac13-d8f025d44d8e","order_by":4,"name":"Marie Zilliox","email":"","orcid":"","institution":"ENS Lyon","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"","lastName":"Zilliox","suffix":""},{"id":630646593,"identity":"60ff34e0-82ae-4bc9-be1b-582e560855d6","order_by":5,"name":"Youri Peeters","email":"","orcid":"","institution":"Wageningen University and Research, Wageningen, Netherlands","correspondingAuthor":false,"prefix":"","firstName":"Youri","middleName":"","lastName":"Peeters","suffix":""},{"id":630646594,"identity":"7965e81b-7815-4fe0-89d4-8c0c0f1b2b7f","order_by":6,"name":"Robin Romein","email":"","orcid":"","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Robin","middleName":"","lastName":"Romein","suffix":""},{"id":630646595,"identity":"33052609-7aff-4941-b4fa-39bbf47ce8cd","order_by":7,"name":"Hui Tian","email":"","orcid":"","institution":"University of Cologne","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Tian","suffix":""},{"id":630646596,"identity":"c7df117c-ba4c-4358-83af-1bba34032c4b","order_by":8,"name":"Jasper Lamers","email":"","orcid":"https://orcid.org/0000-0001-9807-5489","institution":"Wageningen University","correspondingAuthor":false,"prefix":"","firstName":"Jasper","middleName":"","lastName":"Lamers","suffix":""},{"id":630646597,"identity":"bd3e2039-37c3-4b85-81c9-c4a3588122d2","order_by":9,"name":"Daan Vorselen","email":"","orcid":"https://orcid.org/0000-0002-7800-4023","institution":"Wageningen University \u0026 Research","correspondingAuthor":false,"prefix":"","firstName":"Daan","middleName":"","lastName":"Vorselen","suffix":""},{"id":630646598,"identity":"72f29819-6808-4e91-b9af-f4dc053a7151","order_by":10,"name":"Bénédicte Charrier","email":"","orcid":"","institution":"ENS Lyon","correspondingAuthor":false,"prefix":"","firstName":"Bénédicte","middleName":"","lastName":"Charrier","suffix":""},{"id":630646599,"identity":"f9d02043-5b9b-45df-ab11-e5ca1e6a2a95","order_by":11,"name":"Jan Willem Borst","email":"","orcid":"","institution":"Wageningen University","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"Willem","lastName":"Borst","suffix":""}],"badges":[],"createdAt":"2026-04-20 09:46:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9470172/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9470172/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108181587,"identity":"9ea3877e-0ca1-4703-a245-dc3d6d9feec9","added_by":"auto","created_at":"2026-04-30 08:58:46","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1972649,"visible":true,"origin":"","legend":"Article File","description":"","filename":"BestenetalLipoTag2026main.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9470172/v1_covered_6e26acde-eeeb-491b-9a10-51fd92ec431d.pdf"},{"id":108076846,"identity":"b84c901b-3a8a-42f8-823b-5b21e6b46f5f","added_by":"auto","created_at":"2026-04-29 07:04:20","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6529864,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"BestenetalLipoTag2026SI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9470172/v1/8e8e006d46bb0021a54ffdc8.pdf"},{"id":108076847,"identity":"2698a61d-bacf-4237-80a0-ceb453c1b35e","added_by":"auto","created_at":"2026-04-29 07:04:20","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1667889,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"Bestenetalreportingsummary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9470172/v1/027055699657a6f024a04820.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"LipoTag: A minimal motif for live and functional imaging of plant cell membranes.","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":"","lastPublishedDoi":"10.21203/rs.3.rs-9470172/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9470172/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The plant plasma membrane is a highly dynamic structure that is crucial for cell compartmentalization, the maintenance of (bio)chemical gradients, signaling and cell growth and responses to stress. In plants, plasma membranes are tightly connected to the cell walls that encase them. These cell walls can act as diffusion barriers and prevent the use of a wide range of synthetic fluorescent probes that have been developed to study animal cell membranes, which lack a cell wall, with live functional imaging. Here, we introduce LipoTag, a minimal chemical motif that, upon chemical conjugation, transforms hydrophobic fluorophores into water-soluble, membrane-targeted probes that can permeate plant cell walls to reach their intended location. LipoTag uses a localized positive charge in combination with a short aliphatic spacer to direct cargo to the plasma membrane. We used LipoTag to develop a suite of membrane-specific fluorescent probes that work in walled organisms beyond the plant kingdom. In addition, we used LipoTag to develop functional reporters for the quantitative imaging of membrane density, lipid order and membrane oxidation in living plant tissues. LipoTag forms a modular platform for exploring the plant plasma membrane with a suite of contemporary imaging modalities.","manuscriptTitle":"LipoTag: A minimal motif for live and functional imaging of plant cell membranes.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-29 07:04:16","doi":"10.21203/rs.3.rs-9470172/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":"edc09511-eee3-48ee-9bea-bbfde6e0c339","owner":[],"postedDate":"April 29th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-10T13:04:56+00:00","index":2,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67121539,"name":"Biological sciences/Biological techniques/Imaging/Fluorescence imaging"},{"id":67121540,"name":"Biological sciences/Plant sciences/Plant cell biology"}],"tags":[],"updatedAt":"2026-04-29T07:04:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-29 07:04:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9470172","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9470172","identity":"rs-9470172","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}