Spectral demixing-enhanced dual-color pair correlation function: Application to the study of host-pathogen protein interaction

Spectral demixing-enhanced dual-color pair correlation function: Application to the study of host-pathogen protein interaction | 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 Spectral demixing-enhanced dual-color pair correlation function: Application to the study of host-pathogen protein interaction Laura Estrada, Natalia Philipp, Melina Magalnik, Berta Pozzi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7615108/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 Pair correlation functon(pCF) is a powerful approach that has gained increasing popularity in recent years for studying protein dynamics in living cells; however, its application remains limited by methodological constraints and the absence of standardized protocols. In this work, we optimize key aspects of pCF and dual color-pCF analysis. We demonstrate that spectral bleed-through as low as 1% produces spurious cross-correlations, underscoring the need for rigorous correction. To address this, we introduce a robust signal demixing strategy that removes false positive correlations while preserving genuine molecular interactions. This advance expands the reliability of correlation analyses in complex live-cell environments. Applying this approach, we investigated the nucleocytoplasmic shuttling of the splicing factor RBM10 in living cells expressing dengue virus (DENV) NS5 polymerase under poly (I:C)-triggered innate immune response. Our findings reveal that RBM10 and NS5 not only interact but also co-shuttle between the nuclear and cytoplasmic compartments. Furthermore, RBM10 transport is differentially modulated by both NS5 and innate immune induction, reinforcing the role of DENV in altering host nuclear transport. Overall, this work establishes signal demixing as a key advance for live-cell correlation techniques and demonstrates its potential for uncovering complex host-virus interactions. Biological sciences/Biophysics/Biological fluorescence Physical sciences/Physics/Biological physics Full Text Additional Declarations There is NO Competing Interest. Supplementary Files waiverNatComm2025.pdf waiver request SupplementaryMaterialNatComm2025.pdf Supplementary Material 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. 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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-7615108","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":525594043,"identity":"823617d9-4ae5-4513-99c9-1c80c66dc5db","order_by":0,"name":"Laura Estrada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACPgjFzMDPkPiAgbGBCC1sDGB1zAySDckGJGoxOEC0Fvb2548raqzljY8nMzDz7rBj4J99gIAWnjOGjWeOpRtuO/MYqOVMMoPEuQQCWiRyGBsb2A4zbruRf4CZt42ZgeEMIYfJP3/Y2PDvsP3mGSCHtdUzyBPUIsFg2NjYdjhxgwRYy2EGA4JaeHIMZzb2pSfPAPrl4Nwzx3kMCWnhZz/+4GPDN2vb/vZkxgdvd1TLyRHSggIOADEPKRpGwSgYBaNgFOAAADHvQKDrVjlNAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9881-0201","institution":"Universidad de Buenos Aires","correspondingAuthor":true,"prefix":"","firstName":"Laura","middleName":"","lastName":"Estrada","suffix":""},{"id":525594044,"identity":"603f5887-2fe1-4ae2-9852-fa044007a511","order_by":1,"name":"Natalia Philipp","email":"","orcid":"https://orcid.org/0000-0003-2001-4656","institution":"Physics department University of Buenos Aires","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Philipp","suffix":""},{"id":525594045,"identity":"ffb72696-3b6e-45d2-89a7-c55df6042314","order_by":2,"name":"Melina Magalnik","email":"","orcid":"","institution":"School of Science, Univesity of Buenos Aires and IFIBYNE-UBA-CONICET","correspondingAuthor":false,"prefix":"","firstName":"Melina","middleName":"","lastName":"Magalnik","suffix":""},{"id":525594046,"identity":"6a90cfb2-847e-4c4c-a73c-954cdbe6e7db","order_by":3,"name":"Berta Pozzi","email":"","orcid":"","institution":"School of Science, Univesity of Buenos Aires and IFIBYNE-UBA-CONICET","correspondingAuthor":false,"prefix":"","firstName":"Berta","middleName":"","lastName":"Pozzi","suffix":""},{"id":525594047,"identity":"159c8e33-1eff-45dc-867a-299ddfdf1b1b","order_by":4,"name":"Annabella Srebrow","email":"","orcid":"https://orcid.org/0000-0002-1468-4736","institution":"a Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE, UBA- CONICET); 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