CONICET | Buscador de Institutos y Recursos Humanos

Natural killer cells (NK) are the cytotoxic cells from the innate immune system, which eliminates viral-infected and neoplastic cells.They form a specialized junction with their target cells referred toas Lytic (L) Immune Synapse (IS). LIS maturation is characterizedby the local reorganization of actin filaments and NK receptors, andcentrosome (Ct) and Golgi apparatus (GA) translocation towardsthis site. AKAP350 is an A-kinase anchoring protein that participates in MT nucleation at the Ct and at the GA. Our previous resultsshowed that AKAP350 participates in NK cytotoxic activity. The aimof this work was to characterize MT nucleation during NK activationand to evaluate MT´ role in the initial events of LIS maturation. In order to characterize MT nucleation, YTS cells were activated throughLFA-1 and CD28 receptors for 30 minutes at 37°C and then subjected to an ice-recovery assay for detection of newly nucleated microtubules. Confocal microscopy analysis of a-tubulin and GM130 (aGA marker) showed that not only the Ct (commonly regarded as theexclusive microtubule organizing center, MTOC, in lymphocytes) butalso the GA were responsible for MT nucleation. Our results furthershowed that MT nucleation at the GA was diminished in AKAP350knockdown cells (-50%*). Previous works indicated that NK cytotoxicity is reduced by Nocodazole (Noc) or Taxol (Tx) treatment.YTS cells were subjected to Noc or Tx treatment and incubated withKT86 target cells at 37° for 30 minutes. Confocal microscopy analysis of LFA-1 staining of those cells showed that LFA-1 organizationwas inhibited both by disruption of MT filaments (Noc: -42%*) and byMT stabilization (Tx: -40%*). On the other hand, actin organizationat the LIS was exclusively impaired by Noc treatment (-30%*). Overall, our studies characterize for the first time the GA participation asa MTOC in NK cells and demonstrate the relevance of MT dynamicsfor LFA-1 clustering at NK LIS. (*p