Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), CSIC-UAM, Madrid, Spain
Alfonso Navarro-Zapata
Translational Research in Pediatric Oncology, Hematopoietic Transplantation and Cell Therapy, IdiPAZ, La Paz University Hospital, Madrid, Spain; Pediatric Onco-Hematology Clinical Research Unit, Spanish National Cancer Center (CNIO), Madrid, Spain
Antonio Pérez-Martínez
Translational Research in Pediatric Oncology, Hematopoietic Transplantation and Cell Therapy, IdiPAZ, La Paz University Hospital, Madrid, Spain; Pediatric Onco-Hematology Clinical Research Unit, Spanish National Cancer Center (CNIO), Madrid, Spain; Department of Pediatric Hemato-Oncology, La Paz University Hospital, Madrid, Spain; Pediatric Department, Autonomous University of Madrid, Madrid, Spain
Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), CSIC-UAM, Madrid, Spain; CIBER de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain; Instituto de Investigación Sanitaria del Hospital Universitario La Paz (IdiPAZ), Madrid, Spain
Víctor Calvo
Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), CSIC-UAM, Madrid, Spain
We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT 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 PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT 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 FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.
