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Here, we used stimulated emission depletion (STED) microscopy to perform the first super-resolution analysis of actin remodeling at the IS in single- and dual (CD19\/CD22) CAR-T cells, activated on supported lipid bilayers across different time points. Quantitative imaging reveals that CAR-T cells form structurally distinct synapses to untransduced cells, characterized by reduced actin-depleted regions, fewer actin foci, and persistent microvilli-like protrusions. These features indicate incomplete cytoskeletal contraction and impaired actin network reorganization, leading to partial synapse maturation. Our findings highlight fundamental differences in actin dynamics between CAR- and TCR-mediated signaling and suggest that defective actin remodeling may contribute to unstable synapse formation, altered signaling integration, and dysregulated responses or off-target effects. These insights could inform future CAR-T engineering strategies to enhance safety and efficacy.<\/jats:p>","DOI":"10.1083\/jcb.202506119","type":"journal-article","created":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T06:21:07Z","timestamp":1783750867000},"update-policy":"https:\/\/doi.org\/10.1083\/jcb.crossmarkpolicy","source":"Crossref","is-referenced-by-count":0,"title":["Super-resolution imaging reveals altered actin architecture at CAR-T cell immune synapses"],"prefix":"10.1083","volume":"225","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0545-2092","authenticated-orcid":true,"given":"Olivia P.L.","family":"Dalby","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02jx3x895","id-type":"ROR","asserted-by":"publisher"}],"name":"London Centre for Nanotechnology, University College London 1 , London,","place":["UK"]},{"id":[{"id":"https:\/\/ror.org\/02jx3x895","id-type":"ROR","asserted-by":"publisher"}],"name":"University College London 2 Department of Chemistry, , London,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3368-7331","authenticated-orcid":true,"given":"Efstratios","family":"Kirtsios","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02jx3x895","id-type":"ROR","asserted-by":"publisher"}],"name":"Great Ormond Street Institute of Child Health, UCL 3 Molecular and Cellular Immunology Section, , London,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-1040-1150","authenticated-orcid":true,"given":"Hale-Seda","family":"Radoykova","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02jx3x895","id-type":"ROR","asserted-by":"publisher"}],"name":"London Centre for Nanotechnology, University College London 1 , London,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8206-0178","authenticated-orcid":true,"given":"Cecilia","family":"Zaza","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02jx3x895","id-type":"ROR","asserted-by":"publisher"}],"name":"London Centre for Nanotechnology, University College London 1 , London,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8313-8467","authenticated-orcid":true,"given":"Megan D.","family":"Joseph","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02jx3x895","id-type":"ROR","asserted-by":"publisher"}],"name":"London Centre for Nanotechnology, University College London 1 , London,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0480-3911","authenticated-orcid":true,"given":"Persis J.","family":"Amrolia","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02jx3x895","id-type":"ROR","asserted-by":"publisher"}],"name":"Great Ormond Street Institute of Child Health, UCL 3 Molecular and Cellular Immunology Section, , London,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8733-8594","authenticated-orcid":true,"given":"Alice","family":"Giustacchini","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02jx3x895","id-type":"ROR","asserted-by":"publisher"}],"name":"Great Ormond Street Institute of Child Health, UCL 3 Molecular and Cellular Immunology Section, , London,","place":["UK"]},{"name":"Human Technopole 4 , Milan,","place":["Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7089-7667","authenticated-orcid":true,"given":"Sabrina","family":"Simoncelli","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02jx3x895","id-type":"ROR","asserted-by":"publisher"}],"name":"London Centre for Nanotechnology, University College London 1 , London,","place":["UK"]},{"id":[{"id":"https:\/\/ror.org\/02jx3x895","id-type":"ROR","asserted-by":"publisher"}],"name":"University College London 2 Department of Chemistry, , London,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[2026,8,5]]},"reference":[{"key":"2026080509500292400_bib1","doi-asserted-by":"publisher","first-page":"1703","DOI":"10.1016\/j.bpj.2017.01.038","article-title":"Live-cell super-resolution reveals F-Actin and plasma membrane dynamics at the T cell synapse","volume":"112","author":"Ashdown","year":"2017","journal-title":"Biophys. 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