CONICET | Buscador de Institutos y Recursos Humanos

Introduction: Natural killer cells (NK) are the cytotoxic cells from the innate immune system. They form specialized junctions withtarget cells referred to as Immune Synapse (IS). IS is characterized by the local reorganization of actin and NK receptors, and thecentrosome and Golgi apparatus (GA) translocation towards this site. In T cells, the GA regulates vesicle trafficking of signalingproteins that are essential for IS maturation. The GA role in NK-IS formation remained unexplored. AKAP350 is a centrosome andGA scaffold-protein that participates in the maintenance of GA integrity and in microtubule nucleation. Our previous resultsshowed that AKAP350 knockdown impaired NK cytotoxic function and inhibited IS maturation. Objective: To evaluate the GAparticipation in IS maturation and AKAP350 relevance in this function. Methodology. YTS were used as NK cell-model, and KT86as susceptible YTS targets. To interfere with the GA function, YTS cells were treated with brefeldin A (BFA). For analysis of YTScytotoxicity, cells were incubated with CFSE-loaded KT86 cells for 4 hours, stained with Iodide Propidium and double positiveevents identified by FACS. Proteins clustering at the IS and localization was analyzed by immunofluorescence confocal microscopy.Results represent mean±s.e.m. in percentage. Paired t-student analysis was used. Results: BFA-treatment impaired YTS cytotoxicactivity (control: 36%±3% BFA: 13%±2%*, n=3) and inhibited LFA-1 clustering at the IS (Control: 58%±5% BFA: 39%±4%*, n=16).AKAP350 localization at the GA increased during NK activation (+44%*). Displacement of AKAP350 from the GA by expression ofits GA binding domain (GABD) impairs GA nucleation of microtubules and inhibited LFA-1 clustering (Control: 48%±4% GABD:34%±3%*, n=16). (*p