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 microtubule-organizing center (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. 3 Keywords: T lymphocytes / immune synapse / actin cytoskeleton/ protein kinase C δ / centrosome / multivesicular bodies / FMNL1 / paxillin / Abbreviations: Ab, antibody; AICD, activation-induced cell death; AIP, average intensity projection; APC, antigen-presenting cell; BCR, B-cell receptor for antigen; C , center of mass; cent2, centrin 2; cIS, central region of the immune synapse; CMAC, CellTracker™ Blue (7-amino-4-chloromethylcoumarin); cSMAC, central supramolecular activation cluster; CTL, cytotoxic T lymphocytes; DAG, diacylglycerol; DGKα, diacylglycerol kinase α; Dia1, Diaphanous-1; dSMAC, distal supramolecular activation cluster; ECL, enhanced chemiluminescence; ESCRT, endosomal sorting complex required for traffic; F-actin, filamentous actin; Fact-low cIS, F-actin-low region at the centre of the immune synapse; FasL, Fas ligand; FMNL1, formin-like 1; fps, frames per second; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; HRP, horseradish peroxidase; ILV, intraluminal vesicles; IS, immune synapse; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; MIP, maximal intensity projection; MVB, multivesicular bodies; MTOC, microtubuleorganizing center; NS, not significant; PBL, peripheral blood lymphocytes; PKC, protein kinase C; PKCδ, protein kinase C δ isoform; PLC, phospholipase C; PMA, phorbol myristate acetate; Pol. Index, polarization index; pSMAC, peripheral sup...

Analysis of centrosomal area actin reorganization and centrosome polarization upon lymphocyte activation at the immunological synapse

“…Included among non-cortical actin networks, centrosomal area F-actin depletion appears to be crucial to allow centrosome polarization towards the IS in B lymphocytes stimulated with BCRligand-coated beads, used as a B lymphocyte synapse model (Obino et al, 2016). These data do not directly allow inferring either the sufficiency or the relative contribution of each F-actin network (cortical and centrosomal area) to centrosome polarization, unless specific experimental approaches are designed to address this point (Bello-Gamboa et al, 2020). The above-mentioned results on B lymphocytes, together with our results showing that PKCδ interference affects cortical F-actin at the IS (Herranz et al, 2019), prompted us to study the involvement of centrosomal area F-actin in centrosome polarization in cell-to-cell synapses made by T lymphocytes and its potential regulation by PKCδ (Bello-Gamboa et al, 2020).…”

“…These data do not directly allow inferring either the sufficiency or the relative contribution of each F-actin network (cortical and centrosomal area) to centrosome polarization, unless specific experimental approaches are designed to address this point (Bello-Gamboa et al, 2020). The above-mentioned results on B lymphocytes, together with our results showing that PKCδ interference affects cortical F-actin at the IS (Herranz et al, 2019), prompted us to study the involvement of centrosomal area F-actin in centrosome polarization in cell-to-cell synapses made by T lymphocytes and its potential regulation by PKCδ (Bello-Gamboa et al, 2020). The experimental approach described here allows the simultaneous evaluation of centrosome polarization and centrosomal area F-actin, at the single cell level.…”

“…Draw the regions of interest (ROI) as seen in Fig. 1 as previously described (Obino et al, 2017) (Bello-Gamboa et al, 2020. Saving all the ROIs by using the "ROI Manager" Image J submenu will allow to recover the ROIs when required (see Note 4).…”

“…To develop these analyses, modifications of the methods previously described in B lymphocytes to measure normalized centrosome polarization (Obino et al, 2017) (Saez et al, 2019) and centrosomal area F-actin (Obino et al, 2016) (Ibanez-Vega et al, 2019 were used. Following this approach, we have shown that, upon Th lymphocyte IS formation, centrosomal area F-actin decreased concomitantly with centrosome polarization to the IS, and a linear correlation between these two parameters exists (Bello-Gamboa et al, 2020).…”

“…Determine the "Z" substack in which the centrosome is visually displayed. The appropriate volume to quantify the amount of F-actin around the centrosome is a 2 µm height cylinder with a 2 µm diameter circular base, as previously described (Ibanez-Vega et al, 2019) (Bello-Gamboa et al, 2020). Therefore, adjust the "Z" substack selection to this 2 µm height.…”