Single-molecule localization microscopy reveals the ultrastructural root constitution of distal appendages in expanded mammalian centrioles

Single-molecule localization microscopy reveals the ultrastructural root constitution of distal appendages in expanded mammalian centrioles | 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 Single-molecule localization microscopy reveals the ultrastructural root constitution of distal appendages in expanded mammalian centrioles Ting-Jui Chang, Jimmy Hsu, T. Tony Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1838177/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Mar, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Distal appendages (DAPs) are vital in cilia formation, mediating vesicular and ciliary docking to the plasma membrane during early ciliogenesis. Although numerous DAP proteins arranging a nine-fold symmetry have been studied using superresolution microscopy analyses, the extensive ultrastructural understanding of the DAP root structure developing from the centriole wall remains elusive owing to insufficient resolution. Here, we proposed a pragmatic imaging strategy for two-color single-molecule localization microscopy of swellable mammalian DAP proteins. Importantly, our imaging workflow enables us to push the resolution limit of a light microscope well close to an electron microscopy level, thus achieving an unprecedented λ/200 mapping precision inside intact cells. Upon this workflow, we unravel the ultra-resolved higher-order protein complexes of the core DAP. Intriguingly, C2CD3, microtubule triplet, and ODF2 jointly constitute the spatial basis of DAP, suggesting a unique configuration of the DAP assembly. Moreover, our results show that the distal-layered ODF2 labeled at the N- and C-terminus construct a fastening unit encircling the microtubule triplets. Together, we develop an organelle-based drift correction protocol and a two-color imaging solution with minimum crosstalk, allowing a robust localization microscopy imaging of expanded cellular structures deep into the gel-specimen composites. Full Text Additional Declarations There is NO Competing Interest. Supplementary Files ChangDAPExdSTORMSI.pdf Supporting Information Cite Share Download PDF Status: Published Journal Publication published 27 Mar, 2023 Read the published version in Nature Communications → 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-1838177","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":120135760,"identity":"da9e416e-a502-411c-abe9-043bf5088c87","order_by":0,"name":"Ting-Jui Chang","email":"","orcid":"","institution":"Institute of Atomic and Molecular Sciences, Academia Sinica","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ting-Jui","middleName":"","lastName":"Chang","suffix":""},{"id":120135761,"identity":"a6977336-3a5f-4289-8b45-3128514e160f","order_by":1,"name":"Jimmy Hsu","email":"","orcid":"","institution":"National Taiwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jimmy","middleName":"","lastName":"Hsu","suffix":""},{"id":120135762,"identity":"e773e317-48f9-425e-8aa7-f84b06d31337","order_by":2,"name":"T. 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