Junctional and Actomyosin Dynamics Drive Endothelial Cell Rearrangements during Vascular Tube Formation

Junctional and Actomyosin Dynamics Drive Endothelial Cell Rearrangements during Vascular Tube Formation | 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 Junctional and Actomyosin Dynamics Drive Endothelial Cell Rearrangements during Vascular Tube Formation Ludovico Maggi, Jianmin Yin, Ilkka Paatero, Cora Wiesner, Julian Malchow, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7065344/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Lumen formation is a key process during the morphogenesis of tubular organs such as the vertebrate vascular network. At the cellular level, lumen formation can be achieved by cell shape changes or cell rearrangements. We have previously shown that such cell rearrangements are driven by oscillating membrane protrusions, called junction-based lamellipodia (JBL), which provide a ratchet mechanism driving convergent cell movements to join local lumens and generate vascular patency. By performing in vivo time-lapse imaging at high spatiotemporal resolution, we have analyzed the cytoskeletal and junctional dynamics, which underlie JBL formation and function. We show that JBL formation requires the activity of the F-actin nucleation complex Arp2/3. We further show that a novel junction is formed at the distal end of the JBL from a pool of VE-cadherin, originating from outside the initial JBL area. Subsequently, proximal and distal junctions merge and fuse, a process driven by actomyosin contractility. Prior to this fusion we observe a specific recruitment Myl9 within the interjunctional space. Furthermore, inhibition of actomyosin contractility abrogates junctional merging. Taken together, our analyses demonstrate that JBL constitute a module, which by alternate generation of pushing forces (JBL formation) and pulling forces (junctional merging) provide the physical means of endothelial cells to elongate and to rearrange thereby generating a continuous vascular lumen. Biological sciences/Cell biology/Cell adhesion/Adherens junctions Biological sciences/Developmental biology/Angiogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Full Text Additional Declarations The authors declare no competing interests. Supplementary Files videos1.mp4 JBL drive endothelial cell movements. videos1.mp4 JBL drive endothelial cell movements. videos3.mp4 JBL form adjacent to the apical cell compartment. videos4.mp4 Formation of a new junction at the distal end of the JBL. videos4.mp4 Formation of a new junction at the distal end of the JBL. videos6.mp4 Formation of a new junction at the distal end of the JBL. videos7.mp4 The distal junction forms de novo . videos7.mp4 The distal junction forms de novo . videos9.mp4 Arpc1b localizes at the distal end of JBL. videos10.mp4 Arp2/3 activity is required for JBL formation (control). videos11.mp4 Arp2/3 activity is required for JBL formation (CK666). videos12.mp4 CK666 mediated JBL inhibition is reversible upon washout. videos12.mp4 CK666 mediated JBL inhibition is reversible upon washout. videos14.mp4 Arp2/3 activity is required for junction elongation (control). videos15.mp4 Arp2/3 activity is required for junction elongation (CK666). videos16.mp4 Proximal and distal junctions merge at the end of JBL cycle. videos17.mp4 Myl9 is enriched within the JBL. videos18.mp4 Dynamic redistribution of Myl9 during JBL formation and junctional merging. videos18.mp4 Dynamic redistribution of Myl9 during JBL formation and junctional merging. videos20.mp4 Myosin light-chain recruitment at the inter-junctional space during merging. videos21.mp4 Myosin light-chain recruitment at the inter-junctional space during merging. videos21.mp4 Myosin light-chain recruitment at the inter-junctional space during merging. videos23.mp4 Junction elongation during DLAV formation requires actomyosin contractility (control). videos24.mp4 Junction elongation during DLAV formation requires actomyosin contractility (Y-27632). videos25.mp4 Merging of proximal and distal junctions requires actomyosin contractility (control). videos26.mp4 Merging of proximal and distal junctions requires actomyosin contractility (Y-27632). videos27.mp4 Animation depicting the molecular mechanisms of junction elongation by junction-based lamellipodia (JBL). sFigure1.png Supplementary Figure 1: The distal junction forms de novo . sFigure1.png Supplementary Figure 1: The distal junction forms de novo . sFigure3.png Supplementary Figure 3: CK666 mediated JBL inhibition is reversible upon washout. sFigure4.png Supplementary Figure 4: Myosin light-chain recruitment at the inter-junctional space during merging. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions 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-7065344","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":597676319,"identity":"44e00c82-d2d9-4097-a7fb-93666abde3bf","order_by":0,"name":"Ludovico Maggi","email":"","orcid":"https://orcid.org/0000-0002-9543-7840","institution":"Biozentrum University of Basel","correspondingAuthor":false,"prefix":"","firstName":"Ludovico","middleName":"","lastName":"Maggi","suffix":""},{"id":597676320,"identity":"8a8222c9-e929-4e4e-ad2e-86de6207fa13","order_by":1,"name":"Jianmin 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Formation","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research 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At the cellular level, lumen formation can be achieved by cell shape changes or cell rearrangements. We have previously shown that such cell rearrangements are driven by oscillating membrane protrusions, called junction-based lamellipodia (JBL), which provide a ratchet mechanism driving convergent cell movements to join local lumens and generate vascular patency. By performing \u003cem\u003ein vivo\u003c/em\u003e time-lapse imaging at high spatiotemporal resolution, we have analyzed the cytoskeletal and junctional dynamics, which underlie JBL formation and function. We show that JBL formation requires the activity of the F-actin nucleation complex Arp2/3. We further show that a novel junction is formed at the distal end of the JBL from a pool of VE-cadherin, originating from outside the initial JBL area.\u003c/p\u003e\u003cp\u003eSubsequently, proximal and distal junctions merge and fuse, a process driven by actomyosin contractility. Prior to this fusion we observe a specific recruitment Myl9 within the interjunctional space. Furthermore, inhibition of actomyosin contractility abrogates junctional merging. Taken together, our analyses demonstrate that JBL constitute a module, which by alternate generation of pushing forces (JBL formation) and pulling forces (junctional merging) provide the physical means of endothelial cells to elongate and to rearrange thereby generating a continuous vascular lumen.\u003c/p\u003e","manuscriptTitle":"Junctional and Actomyosin Dynamics Drive Endothelial Cell Rearrangements during Vascular Tube Formation","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2026-03-27 19:24:51","doi":"10.21203/rs.3.rs-7065344/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2025-07-18 14:25:18","doi":"10.21203/rs.3.rs-7065344/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e8cebc99-872c-4f80-8292-fd685934bd9d","owner":[],"postedDate":"March 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63604479,"name":"Biological sciences/Cell biology/Cell adhesion/Adherens junctions"},{"id":63604480,"name":"Biological sciences/Developmental biology/Angiogenesis"}],"tags":[],"updatedAt":"2025-09-03T17:10:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-27 19:24:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-7065344","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7065344","identity":"rs-7065344","version":["v2"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}