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oai:uvadoc.uva.es:10324/843182026-04-28T19:01:53Zcom_10324_32522com_10324_952com_10324_894col_10324_32523

2026-04-28T07:29:48Z urn:hdl:10324/84318 Spatial regulation of Lck activation at the CD8 immune synapse revealed by a FRET-Based biosensor Meana González, Clara San José Rodríguez, Gonzalo Balboa García, María Ángeles Casas Requena, Javier Biología molecular Biofísica Bioquímica Producción Científica T cell receptor (TCR) signaling is critically dependent on the Src-family kinase Lck, whose activation is tightly regulated both spatially and conformationally during antigen recognition. Here, we employ an improved second-generation FRET-based biosensor (TqLckV2.3) to visualize Lck conformational dynamics in live T cells with high spatial resolution. Upon TCR engagement, we observe a paradoxical increase in whole-cell FRET signal, which immunolabeling reveals to be due to selective internalization of inactive Lck (pY505), while active Lck (pY394) remains membrane-associated and enriched at the immune synapse (IS). Using CD8α mutants that disrupt Lck binding, we demonstrate that free Lck undergoes more pronounced conformational activation than CD8-bound Lck. Furthermore, we show that C-terminal Src kinase (Csk) preferentially phosphorylates free Lck at Y505, while CD45 suppresses its activation via dephosphorylation of Y394, suggesting a dual regulatory mechanism that maintains free Lck in an inactive state under resting conditions. High-resolution imaging confirms sustained activation of Lck at the IS and transient inactivation at the periphery, revealing a spatially confined signaling architecture. These findings uncover a novel regulatory mechanism involving selective internalization and spatial segregation of Lck conformations and establish TqLckV2.3 as a powerful tool for dissecting TCR signaling dynamics. 2026-04-28T07:29:48Z 2026-04-28T07:29:48Z 2026 info:eu-repo/semantics/article Cellular and Molecular Life Sciences, 2026 (in press) 1420-682X https://uvadoc.uva.es/handle/10324/84318 10.1007/s00018-026-06209-x Cellular and Molecular Life Sciences 1420-9071 eng https://link.springer.com/article/10.1007/s00018-026-06209-x info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/4.0/ © 2026 The Author(s) Atribución 4.0 Internacional Springer Nature