Identification of intracellular LFA-1 vesicles that polarize to the IS during NK cell activation Pariani AP1,

PARIANI A; MARIN L; HIDALGO F; FUSSI MF; BORINI ETICHETTI C; FAVRE C; LAROCCA MC

Congreso; Reunión Anual Conjunta de la Sociedad Argentina de Fisiología y ALACF; 2021

Identification of intracellular LFA-1 vesicles that polarize to the IS during NK cell activation Pariani AP1, Marin LM1, Hidalgo F1, Fussi F2,3, Borini-Etichetti C1, Favre C1, Larocca MC1 1 Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Rosario, Argentina. 2 Área Farmacología, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario. 3 CONICET Introduction: NK cells are cytotoxic cells from the innate immune system. They form a specialized junction with their target cells called immune synapse (IS). IS formation is highly dependent on NK receptors such as the integrin LFA-1. The mechanisms involved in LFA-1 localization at the IS remains unclear. AKAP350 is a centrosome and Golgi apparatus (GA)-scaffold protein that participates in LFA-1 recruitment to the IS, thus conditioning NK cytolytic activity. Objective: The aim of this work was to elucidate the mechanisms underlying LFA-1 organization during NK cell activation. Methods: YTS (NK) cells, YTS cells with decreased expression of AKAP350 (AKAP350KD) and YTS cells expressing AKAP350 Golgi-binding domain (AKAP350GBD), were exposed to sensible target cells for 30 minutes. LFA-1 distribution in YTS cells was analyzed by immunofluorescence confocal microscopy. Relative distance to the IS (RD) was estimated as the difference between the distance from each LFA-1 vesicle to the IS and the distance from the cell centroid to the IS, related to the latter. Results are expressed as media±standard error. Results: Analysis of activated YTS cells revealed the presence of an intracellular pool of LFA-1 vesicles, which partially colocalized with RAB11 (recycling endosomes) and with GM130 (GA). The assessment of LFA-1 vesicles localization indicated that they polarized to the IS in activated YTS cells, and that this polarization was impaired in AKAP350 KD cells (RD: CONTROL: -0.14±0.01, KD: 0.05±0.02, n=30, p=0.001). Interestingly, the specific displacement of AKAP350 from the GA, which inhibits LFA-1 localization at NK-IS, also impaired LFA-1 polarization towards the IS (RD: Control: -0.22±0.03, AKAP350GBD: 0.03±0.02, n=30, p=0.03). Conclusion: Our results reveal the presence of an intracellular pool of LFA-1 which associates with the GA and might be relevant for NK-lytic IS maturation.