Garcia-Ortiz, AlmudenaMartin-Cofreces, Noa B.Ibiza, SalesOrtega, AngelIzquierdo-Alvarez, AliciaTrullo, AntonioVictor, Victor ManuelCalvo, EnriqueSot, BegonaMartinez-Ruiz, AntonioVazquez, JesusSanchez-Madrid, FranciscoSerrador, Juan M.2017-10-202017-10-202017PLoS Biol. 2017; 15(4):e20006531545-7885http://hdl.handle.net/20.500.12105/5124The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-theta (PKC-theta) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of beta-actin and PKC-theta from the lamellipodium-like distal (d)-SMAC, promoting PKC-theta. activation. Furthermore, eNOS-derived NO S-nitrosylated beta-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-theta. was corroborated by overexpression of PFN1- and actin-binding defective mutants of beta-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-theta. at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.engVoRhttp://creativecommons.org/licenses/by/4.0/NITRIC-OXIDE SYNTHASET-CELL-ACTIVATIONIMMUNOLOGICAL SYNAPSERETROGRADE FLOWARP2/3 COMPLEXPROTEINRECEPTORPOLYMERIZATIONPOLARIZATIONCYTOSKELETONAtribución 4.0 Internacional283949351510.1371/journal.pbio.2000653Plos Biologyopen access