Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes

Abstract

T-cell receptor stimulation induces the convergence of multivesicular bodies towards the microtubuleorganizing centre (MTOC) and the polarization of the MTOC to the immune synapse (IS). These events lead to exosome secretion at the IS. We describe here that upon IS formation centrosomal area F-actin decreased concomitantly with MTOC polarization to the IS. PKCδ-interfered T cell clones showed a sustained level of centrosomal area F-actin associated with defective MTOC polarization. We analysed the contribution of two actin cytoskeleton-regulatory proteins, FMNL1 and paxillin, to the regulation of cortical and centrosomal F-actin networks. FMNL1β phosphorylation and F-actin reorganization at the IS were inhibited in PKCδ-interfered clones. F-actin depletion at the central region of the IS, a requirement for MTOC polarization, was associated with FMNL1β phosphorylation at its C-terminal, autoregulatory region. Interfering all FMNL1 isoforms prevented MTOC polarization; nonetheless, FMNL1β re-expression restored MTOC polarization in a centrosomal area F-actin reorganization-independent manner. Moreover, PKCδ-interfered clones exhibited decreased paxillin phosphorylation at the MTOC, which suggests an alternative actin cytoskeleton regulatory pathway. Our results infer that PKCδ regulates MTOC polarization and secretory traffic leading to exosome secretion in a coordinated manner by means of two distinct pathways, one involving FMNL1β regulation and controlling F-actin reorganization at the IS, and the other, comprising paxillin phosphorylation potentially controlling centrosomal area F-actin reorganization.

Role of Actin Cytoskeleton Reorganization in Polarized Secretory Traffic at the Immunological Synapse

“…Instead, it has been proposed that the defect was in part due to impaired F-actin reorganization at the IS produced by centriole deletion (Tamzalit et al, 2020 ), which points out an unexpected role of the intact centrosome in supporting synaptic F-actin architecture and dynamics. Interestingly, CTL lacking centriole formed synapses that lacked an obvious F-actin cleared region at the cSMAC, a similar phenotype to that found in PKCδ-interfered CD4 + Jurkat T lymphocytes (Herranz et al, 2019 ; Bello-Gamboa et al, 2020 ). Polarized secretion is known to occur in IS domains that have been cleared of F-actin and are, thus, accessible to secretion vesicles (Griffiths et al, 2010 ; Huse, 2012 ; Ritter et al, 2015 ; Herranz et al, 2019 ).…”

“…However, centrosome polarization toward the IS was not affected by the absence of PKCδ (Ma et al, 2007 ). In contrast, in the synapses made by CD4 + Jurkat T lymphocytes, multivesicular bodies (MVB, a type of secretory vesicles involved in exosome secretion by CD4 + Jurkat and primary CD8 + and CD4 + T lymphocytes (Alonso et al, 2005 , 2011 )) and centrosome always co-migrated toward the IS, and MVB and centrosome did not polarize in PKCδ-interfered CD4 + Jurkat T lymphocytes and the centrosome/MTOC center of mass (MTOC C ) was coincident or very proximal to the MVB center of mass (MVB C ) regardless of polarization (Herranz et al, 2019 ; Bello-Gamboa et al, 2020 ). In summary, all these results broaden current views of CTL biology by revealing an extremely rapid lytic granule secretion step and by showing that centrosome polarization is dispensable for efficient lytic granule secretion.…”

“…All these processes, most probably acting in a coordinated manner, may facilitate the movement of the centrosome toward the IS and the convergence of MVB toward the F-actin depleted area in the cSMAC, which facilitates MVB fusion at the cSMAC and the subsequent secretion of exosomes (Calvo and Izquierdo, 2020 ) in the synaptic cleft toward the APC. For more details please refer to Herranz et al ( 2019 ) and Bello-Gamboa et al ( 2020 ).…”

The Bardet-Biedl Syndrome complex component BBS1 regulates proteasome-dependent F-actin clearance from the centrosome to enable its translocation to the T cell immune synapse

“…Centrosomal F-actin tethers the centrosome to the nucleus by interacting with nesprins, which cross the outer nuclear membrane and interact with the SUN proteins resident in the nuclear envelope lumen [45]. Similar to B cells [37], F-actin clearance from the centrosome has been recently shown to occur during IS formation in T cells, allowing for centrosome detachment and polarization [46]. Conjugate staining with fluorochrome-labelled phalloidin and anti-g-tubulin antibodies revealed the presence of a centrosomal F-actin pool surrounded by a radial pattern of F-actin + dots (Fig.4a).…”

“…These interactions must be disrupted when the centrosome has to move to a different subcellular localization, such as during IS formation. During IS assembly the centrosomal F-actin pool undergoes depletion [37,46]. In B cells this has been demonstrated to be due to a relocalization of Arp2/3 from the centrosome to the IS, which leads to centrosome detachment from the preprint (which was not certified by peer review) is the author/funder.…”