Characterization of AKAP350 participation in actin reorganization at the immune synapse in natural killer cells.

PARIANI A; ALMADA E; HIDALGO F; MARIN L; FAVRE C; LAROCCA MC

Rosario

Congreso; Reunión Anual de la Sociedad Argentina de Fisiología (SAFIS); 2019

SAFIS

The immune synapse (IS) refers to a specialized cell-celljunction established between an immune cell and itstarget cell (TC). When a natural killer cell (NK) is activatedby a susceptible TC, the actin cytoskeleton reorganizes atthe IS, facilitating integrin and receptor clustering.Subsequently, the centrosome polarizes to the IS, enablingthe directional secretion of cytotoxic components, which triggers the lysis of the TC. AKAP350 is an A-kinaseanchoring protein essential in the regulation ofmicrotubule dynamics. AKAP350 interacts with themicrotubule plus end binding protein 1 (EB1), whichparticipates in the crosstalk between microtubule andactin cytoskeleton. In non-lytic T cell IS, AKAP350participates in LFA-1 clustering, whereas EB1 is involved inT cell receptor activation. Our previous results indicatethat AKAP350 participates in NK cytotoxic response, byregulating actin accumulation and LFA-1 clustering at theIS. The aim of our work was to demonstrate if AKAP350participation occurs exclusively downstream LFA-1activation and to evaluate EB1 as a possible mediator. YTSNK cells with decreased expression of AKAP350(AKAP350KD) were activated for 30 min using specificligands for LFA-1 or CD28. Actin accumulation and LFA-1clustering were analyzed by confocal microscopy. Actinaccumulation at the IS was impaired in AKAP350KD cellsactivated via LFA-1 (-37%*) or CD28 (-73%*).Concomitantly, AKAP350KD cells showed decreased LFA-1localization at the IS in cells activated via either LFA-1 (-40%*) or CD28 (-60 %*). In order to analyze AKAP350 rolein EB1 recruitment to microtubules at the IS, YTSAKAP350KD cells were incubated with KT86 cells (TC) for30 min. EB1 localization was analyzed by confocalmicroscopy. YTS AKAP350KD showed decreased EB1localization at the IS edge (-45%*) and at IS microtubules(-55%*). The latter results were confirmed by western blotanalysis of microtubule fractions of YTS cells activated viaLFA-1. Overall, our results suggest that AKAP350participates in actin accumulation and receptor clusteringdownstream both LFA-1 and CD28activations and positionEB1 as a putative mediator of this effect. *p