T-cell receptor stimulation (TCR) by antigen bound to the major histocompatibility complex (MHC) on an antigen-presenting cell (APC) induces protein kinase C (PKC) activation and the formation of the immune synapse (IS), followed by depletion of filamentous actin (F-actin) at the central region of the IS (cIS) and the polarization of multivesicular bodies (MVB) and the microtubule-organizing center (MTOC) to the IS. These events lead to polarized exosome secretion at the IS. These exosomes are involved in several crucial immune responses such as autocrine activation-induced cell death (AICD) of T lymphocytes and cytotoxicity. We analysed here how formin-like 1 beta (FMNL1beta), an actin cytoskeleton-regulatory protein, regulates MTOC/MVB polarization and exosome secretion at an IS model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1beta to the IS, which was independent of protein kinase C delta (PKCdelta). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1betaWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCdelta-interfered clones, indicating that S1086 FMNL1beta phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the cIS, which is necessary for MTOC/MVB polarization. FMNL1betaWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1beta variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in TCR and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1beta activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.
