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Galectins, a family of \u03b2-galactoside\u2013binding proteins, control cell membrane organization, exerting crucial roles in multiple physiological processes. Here, we report a novel mechanism underlying cell polarity and uropod retraction by demonstrating that galectin-9 regulates basal and chemokine-driven DC migration in humans and mice. Galectin-9 depletion caused a defect in RhoA signaling that resulted in impaired cell rear contractility. Mechanistically, galectin-9 interacts with and organizes CD44 at the cell surface, in turn modulating RhoA binding to GEF-H1 and the initiation of downstream signaling. Analysis of DC motility in the 3D tumor microenvironment revealed galectin-9 is also required for DC recruitment and infiltration. Exogenous galectin-9 rescued the motility of tumor-immunocompromised human blood DCs, validating the physiological relevance of galectin-9 in DC migration. Our results identify galectin-9 as a necessary mechanistic component for DC motility by regulating cell polarity and contractility, and underscore its implications for DC-based immunotherapies.<\/jats:p>","DOI":"10.1083\/jcb.202404079","type":"journal-article","created":{"date-parts":[[2025,8,15]],"date-time":"2025-08-15T06:21:09Z","timestamp":1755238869000},"update-policy":"https:\/\/doi.org\/10.1083\/jcb.crossmarkpolicy","source":"Crossref","is-referenced-by-count":4,"title":["Galectin-9 regulates dendritic cell polarity and uropod contraction by modulating RhoA activity"],"prefix":"10.1083","volume":"224","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-0905-6373","authenticated-orcid":true,"given":"Guus A.","family":"Franken","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05wg1m734","id-type":"ROR","asserted-by":"publisher"}],"name":"Radboud University Medical Center 1 Department of Medical BioSciences, , Nijmegen,","place":["The 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