Extracellular phase separation mediates storage and release of thyroglobulin in the thyroid follicular lumen

Extracellular phase separation mediates storage and release of thyroglobulin in the thyroid follicular lumen | 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 Extracellular phase separation mediates storage and release of thyroglobulin in the thyroid follicular lumen Tuomas Knowles, Yihan Yao, Nadia Erkamp, Tomas Sneideris, Xiqiao Yang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5139555/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Mar, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Thyroid hormones are produced by the thyroid gland and are essential for regulating metabolism, growth and development. Maintenance of circulating thyroid hormone levels within an appropriate range is thus a prerequisite for health. In vivo, this objective is, at least in part, facilitated through an extracellular storage depot of thyroglobulin, the glycoprotein precursor for thyroid hormones, in the thyroid follicular lumen. The molecular basis for how soluble thyroglobulin molecules form such dense depot assemblies remains elusive. Here, we describe in vitro biophysical analysis of thyroglobulin phase behaviour, suggesting that thyroglobulin is prone to undergoing ionic strength-dependent phase separation, leading to the formation of liquid-like condensates. Fluorescence photobleaching measurements further show that these condensates age as a function of time to form reversible gel-like high density storage depots of thyroglobulin. Immunofluorescence experiments on mouse and human thyroid follicles ex vivo reveal that spherical globules of Tg protein dense phase are present in the follicular lumen, consistent with the idea that Tg undergoes phase separation. These findings reveal a molecular mechanism for the last-come-first-served process of thyroglobulin storage and release, suggesting a role for extracellular phase separation in thyroid hormone homeostasis by providing organizational and architectural specificity without requiring membrane-mediated confinement. Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Tgmanuscriptsupplementaryfigures.pdf Cite Share Download PDF Status: Published Journal Publication published 21 Mar, 2025 Read the published version in Communications Biology → 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-5139555","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":366371761,"identity":"21ea49c2-8c6f-4a0f-85bc-2eaf7f9e20ed","order_by":0,"name":"Tuomas Knowles","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYHCCZIYPFXBOAmH1PEAtjDPOkKiFmZm3jRQt9tINjw1459nJ6zYwP/zA2JZGhC0yB5ITJLclG247wGYswdiWQ4QWiYTkA0D1jNsOMJgxMLZVEKklcc4B+20H2L8RryXhYMOBxG0HeEC2EOOwOweSDRuOJSdvO8xTLJFwjgjvs8/uSZb+U2Nnu+14+8YPH8qSCWthkOBJgDCYGYiKFZAW9gNEqRsFo2AUjIIRDABdVTey3WY3AwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7879-0140","institution":"University of Cambridge","correspondingAuthor":true,"prefix":"","firstName":"Tuomas","middleName":"","lastName":"Knowles","suffix":""},{"id":366371762,"identity":"f537df90-e163-4cfa-a1ef-2940eff6e9b7","order_by":1,"name":"Yihan Yao","email":"","orcid":"https://orcid.org/0000-0002-7247-0314","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Yihan","middleName":"","lastName":"Yao","suffix":""},{"id":366371763,"identity":"cfc5e4ba-b73c-43d8-b350-6daf7ef17441","order_by":2,"name":"Nadia Erkamp","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Erkamp","suffix":""},{"id":366371764,"identity":"5a20ac9c-01df-448e-a019-32b1179de332","order_by":3,"name":"Tomas Sneideris","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Tomas","middleName":"","lastName":"Sneideris","suffix":""},{"id":366371765,"identity":"eb7560e2-4407-400e-b1d4-adeb00e4989d","order_by":4,"name":"Xiqiao Yang","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Xiqiao","middleName":"","lastName":"Yang","suffix":""},{"id":366371766,"identity":"9d42f20a-9c5f-4e76-bfa4-0a83ec323d16","order_by":5,"name":"Rob Scrutton","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Rob","middleName":"","lastName":"Scrutton","suffix":""},{"id":366371767,"identity":"9136b4a9-48bb-4d01-99b0-99aef733238a","order_by":6,"name":"Matthias Schneider","email":"","orcid":"https://orcid.org/0000-0002-1894-1859","institution":"Max-Planck-Institute of Biochemistry","correspondingAuthor":false,"prefix":"","firstName":"Matthias","middleName":"","lastName":"Schneider","suffix":""},{"id":366371768,"identity":"8e53c7d8-4edb-4b0b-9040-f19d5b22f133","order_by":7,"name":"Charlotte Fischer","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Charlotte","middleName":"","lastName":"Fischer","suffix":""},{"id":366371769,"identity":"57186386-8902-4287-8019-77bbc428ab94","order_by":8,"name":"Erik Schoenmakers","email":"","orcid":"","institution":"University of Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Erik","middleName":"","lastName":"Schoenmakers","suffix":""},{"id":366371770,"identity":"86b6b7f5-e5a9-449b-8bab-56a41fe7e96c","order_by":9,"name":"Nadia Schoenmakers","email":"","orcid":"","institution":"University of Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Schoenmakers","suffix":""}],"badges":[],"createdAt":"2024-09-23 17:00:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5139555/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5139555/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-07909-z","type":"published","date":"2025-03-21T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79004506,"identity":"7fdc3c34-33a4-46ff-a3f5-e4c9dce55c81","added_by":"auto","created_at":"2025-03-22 07:08:51","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8085196,"visible":true,"origin":"","legend":"","description":"","filename":"Tgmanuscriptmaintext.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5139555/v1_covered_f9c4b5e2-61c4-43f7-b93a-5ac4476d9acb.pdf"},{"id":69863061,"identity":"69715fe1-d131-4855-a528-f3953026f1aa","added_by":"auto","created_at":"2024-11-26 06:20:35","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3189176,"visible":true,"origin":"","legend":"","description":"","filename":"Tgmanuscriptsupplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5139555/v1/611d2a278d029b3710b17579.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Extracellular phase separation mediates storage and release of thyroglobulin in the thyroid follicular lumen","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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-5139555/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5139555/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Thyroid hormones are produced by the thyroid gland and are essential for regulating metabolism, growth and development. Maintenance of circulating thyroid hormone levels within an appropriate range is thus a prerequisite for health. In vivo, this objective is, at least in part, facilitated through an extracellular storage depot of thyroglobulin, the glycoprotein precursor for thyroid hormones, in the thyroid follicular lumen. The molecular basis for how soluble thyroglobulin molecules form such dense depot assemblies remains elusive. Here, we describe in vitro biophysical analysis of thyroglobulin phase behaviour, suggesting that thyroglobulin is prone to undergoing ionic strength-dependent phase separation, leading to the formation of liquid-like condensates. Fluorescence photobleaching measurements further show that these condensates age as a function of time to form reversible gel-like high density storage depots of thyroglobulin. Immunofluorescence experiments on mouse and human thyroid follicles ex vivo reveal that spherical globules of Tg protein dense phase are present in the follicular lumen, consistent with the idea that Tg undergoes phase separation. These findings reveal a molecular mechanism for the last-come-first-served process of thyroglobulin storage and release, suggesting a role for extracellular phase separation in thyroid hormone homeostasis by providing organizational and architectural specificity without requiring membrane-mediated confinement.","manuscriptTitle":"Extracellular phase separation mediates storage and release of thyroglobulin in the thyroid follicular lumen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-26 06:20:31","doi":"10.21203/rs.3.rs-5139555/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bd9626d8-1175-4a8d-a026-56e84077a6dd","owner":[],"postedDate":"November 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-22T07:08:37+00:00","versionOfRecord":{"articleIdentity":"rs-5139555","link":"https://doi.org/10.1038/s42003-025-07909-z","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-03-21 04:00:00","publishedOnDateReadable":"March 21st, 2025"},"versionCreatedAt":"2024-11-26 06:20:31","video":"","vorDoi":"10.1038/s42003-025-07909-z","vorDoiUrl":"https://doi.org/10.1038/s42003-025-07909-z","workflowStages":[]},"version":"v1","identity":"rs-5139555","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5139555","identity":"rs-5139555","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}