MICROTUBULES STABILIZATION AT THE trans-GOLGI IS NECESSARY FOR IMMUNE SYNAPSE MATURATION

PARIANI A; HUHN V; RIVABELLA MAKNIS T; VENA R; FAVRE C; ALONSO V; LAROCCA MC

Congreso; Reunión Anual de la Sociedad Argentina de Fisiología; 2023

Introduction: Natural Killer Cells (NK) are cytotoxic cells from the innate immune system. They form a specialized junction with their target cells, the immune synapse (IS). Along the IS formation, one of the key events is the polarization of the centrosome (Ct) and the Golgi apparatus (GA). We have previously demonstrated that the GA participates in the leukocyte functional antigen (LFA)-1 organization at NK-IS. Besides those studies, GA role in IS maturation in NK cells has hardly been investigated. CLASP1/2 are plusend binding proteins that are associated with the trans-GA, where they stabilize microtubules (Mt). Objective: To analyze the participation of GA associated proteins,CLASP1/2, in the IS maturation in NK cells. Methods: NK-YTS cells with decreased expression of CLASP1/2 (CLASPKD) were prepared by transduction with lentiviral particles for expression of CLASP1 and CLASP2 specific shRNAs. YTS were exposed to erythroleukemia KT86 cells (2:1 ratio) for 30 minutes. LFA-1 clustering at IS and Ct and GA polarization were analyzed by confocal microscopy. Relative distance to the IS (RD) was estimated as the difference between the distance from Ct to the IS and the distance from the cell centroid to the IS, related to the latter. The Area Weighted Distance (AWD) was estimated as the average distance of GA particles to the IS, weighted by each particle area. At least 10 IS were analyzed for each experiment. Results are expressed as media±standard error. Results: Our results revealed the inhibition of LFA-1 accumulation at the IS in CLASPKD cells (Control: 54±8%, CLASPKD: 37±9%, p < 0.05). Furthermore, the AWD of the GA was increased in CLASPKD cells (Control: 3,6±0,9, CLASPKD:6,9±1,3, p < 0.05). Concomitantly, CLASPKD cells showed an impaired Ct polarization (Control: 0,35±0,2, CLASPKD:0,2±0,1, p < 0.05). Conclusion: These results point out the importance of GA mt stabilization by CLASP1/2 in the organization of LFA-1 during the IS formation and brings out the first evidence that, similarly to what has been demonstrated in migratory cells, the GA acts as a regulator of Ct polarization throughout the course of SI formation in NK cells.