Concurrent diffusion of nicotinic acetylcholine receptors and fluorescent cholesterol disclosed by two-colour sub-millisecond MINFLUX-based single-molecule tracking

Concurrent diffusion of nicotinic acetylcholine receptors and fluorescent cholesterol disclosed by two-colour sub-millisecond MINFLUX-based single-molecule tracking | 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 Concurrent diffusion of nicotinic acetylcholine receptors and fluorescent cholesterol disclosed by two-colour sub-millisecond MINFLUX-based single-molecule tracking Christian Eggeling, Francisco Barrantes, Francesco Reina, Lucas Saavedra This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5619606/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Nicotinic acetylcholine receptors (nAChRs) are ubiquitous neurotransmitter receptors predominantly located at the cell-surface of neurons and muscle cells. Their dynamics affect synaptogenesis at neurodevelopmental stages and the efficacy of synaptic transmission in the adult synapse. Here we exploit the enhanced capabilities of superresolution fluorescence MINFLUX microscopy to track for minute-long periods with nanometric precision and sub-millisecond time resolution the 2D translational dynamics of the bungarotoxin-labelled adult muscle-type nAChR in tandem with a fluorescent cholesterol analogue. To this end, we implemented a multiplexing procedure in continuous MINFLUX microscopy that enabled the simultaneous excitation of the two molecules using a single wavelength, followed by discrimination of their emissions via differential ratiometric recording. Single-molecule trajectories displayed a heterogeneous spectrum of diffusive behaviours (subdiffusive, Brownian and superdiffusive), with a predominance of the subdiffusive component, which became less pronounced upon cholesterol depletion. nAChRs spent most of their lifetime in confined areas of characteristic size (~ 0.005 µm 2 ) lasting for ~ 100 ms. Further, MINFLUX captured regions where nAChR and fluorescent cholesterol moved jointly, both in confinement sojourns and along the free Brownian walks, which strongly indicated mutual interactions between the receptor macromolecule and the neutral lipid. To the best of our knowledge, this study constitutes the first series of experiments showing the diffusion dynamics of a transmembrane protein -a functionally important neurotransmitter receptor- together with a key membrane lipid in the native plasma membrane of a live cell at such high detail, thanks to the MINFLUX-based recordings. Biological sciences/Biophysics/Nanoscale biophysics Biological sciences/Biological techniques/Microscopy/Super-resolution microscopy acetylcholine receptor cholesterol dynamics diffusion translational motion superresolution microscopy multiplexed MINFLUX co-tracking Full Text Additional Declarations There is NO Competing Interest. Supplementary Files ReinaMinfluxSupplementaryMaterial.docx Supplementary Material Cite Share Download PDF Status: Published Journal Publication published 09 Jul, 2025 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. 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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-5619606","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":398489840,"identity":"dffd8ca0-0f0d-4081-9da9-fdf630c6c82b","order_by":0,"name":"Christian Eggeling","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-3698-5599","institution":"Friedrich Schiller University Jena","correspondingAuthor":true,"prefix":"","firstName":"Christian","middleName":"","lastName":"Eggeling","suffix":""},{"id":398489841,"identity":"8534553f-e883-40c9-9fc4-d2cf82ea35de","order_by":1,"name":"Francisco Barrantes","email":"","orcid":"https://orcid.org/0000-0002-4745-681X","institution":"Laboratory of Molec. 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Their dynamics affect synaptogenesis at neurodevelopmental stages and the efficacy of synaptic transmission in the adult synapse. Here we exploit the enhanced capabilities of superresolution fluorescence MINFLUX microscopy to track for minute-long periods with nanometric precision and sub-millisecond time resolution the 2D translational dynamics of the bungarotoxin-labelled adult muscle-type nAChR in tandem with a fluorescent cholesterol analogue. To this end, we implemented a multiplexing procedure in continuous MINFLUX microscopy that enabled the simultaneous excitation of the two molecules using a single wavelength, followed by discrimination of their emissions via differential ratiometric recording. 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