当前位置: X-MOL 学术J. Extracell. Vesicles › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes.
Journal of Extracellular Vesicles ( IF 16.0 ) Pub Date : 2020-06-19 , DOI: 10.1080/20013078.2020.1759926
Ana Bello-Gamboa 1, 2 , Marta Velasco 1, 2 , Solange Moreno 1, 2 , Gonzalo Herranz 1, 2, 3 , Roxana Ilie 1, 2 , Silvia Huetos 1, 2 , Sergio Dávila 1, 2, 4 , Alicia Sánchez 1, 2, 5 , Jorge Bernardino De La Serna 6, 7 , Víctor Calvo 1, 2 , Manuel Izquierdo 1, 2
Affiliation  

ABSTRACT

T-cell receptor stimulation induces the convergence of multivesicular bodies towards the microtubule-organizing 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. FMNL1 β 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 FMNL1 β phosphorylation at its C-terminal, autoregulatory region. Interfering all FMNL1 isoforms prevented MTOC polarization; nonetheless, FMNL1 β 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 FMNL1 β regulation and controlling F-actin reorganization at the IS, and the other, comprising paxillin phosphorylation potentially controlling centrosomal area F-actin reorganization.

Abbreviations

Ab, antibody; AICD, activation-induced cell death; AIP, average intensity projection; APC, antigen-presenting cell; BCR, B-cell receptor for antigen; C, centre 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, microtubule-organizing centre; 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 supramolecular activation cluster; PSF, point spread function; ROI, region of interest; SD, standard deviation; shRNA, short hairpin RNA; SEE, Staphylococcus enterotoxin E; SMAC, supramolecular activation cluster; TCR, T-cell receptor for antigen; T-helper (Th); TRANS, transmittance; WB, Western blot.



中文翻译:

中心体区域的肌动蛋白重组和免疫突触调节 T 淋巴细胞中多泡体的极化分泌交通。

摘要

T 细胞受体刺激诱导多泡体向微管组织中心 (MTOC) 的会聚和 MTOC 向免疫突触 (IS) 的极化。这些事件导致 IS 处的外泌体分泌。我们在此描述了在 IS 形成后,中心体区域 F-肌动蛋白随着 MTOC 向 IS 极化而降低。PKCδ 干扰的 T 细胞克隆显示出与 MTOC 极化缺陷相关的持续水平的中心体区域 F-肌动蛋白。我们分析了两种肌动蛋白细胞骨架调节蛋白 FMNL1 和桩蛋白对皮质和中心体 F-肌动蛋白网络的调节作用。FMNL1 β 在 PKCδ 干扰的克隆中,IS 处的磷酸化和 F-肌动蛋白重组受到抑制。IS 中心区域的 F-肌动蛋白消耗是 MTOC 极化的要求,与 FMNL1 相关 β 磷酸化在其 C 末端,自动调节区域。干扰所有 FMNL1 亚型可防止 MTOC 极化;尽管如此,FMNL1 β 重新表达以与中心体区域 F-肌动蛋白重组无关的方式恢复了 MTOC 极化。此外,PKCδ 干扰的克隆在 MTOC 处表现出降低的桩蛋白磷酸化,这表明另一种肌动蛋白细胞骨架调节途径。我们的研究结果推断,PKCδ 通过两种不同的途径以协调的方式调节 MTOC 极化和分泌交通,导致外泌体分泌,一个涉及 FMNL1 β 调节和控制 IS 处的 F-肌动蛋白重组,另一个包括桩蛋白磷酸化,可能控制中心体区域 F-肌动蛋白重组。

缩写

Ab,抗体;AICD,激活诱导的细胞死亡;AIP,平均强度投影;APC,抗原呈递细胞;BCR,抗原的 B 细胞受体;C, 质心; 中心2,中心2;cIS,免疫突触的中心区域;CMAC,CellTracker™ Blue(7-氨基-4-氯甲基香豆素);cSMAC,中央超分子激活簇;CTL,细胞毒性 T 淋巴细胞;DAG,甘油二酯;DGKα,二酰基甘油激酶α;直径1,透明-1;dSMAC,远端超分子激活簇;ECL,增强化学发光;ESCRT,交通所需的内体分选复合体;F-肌动蛋白,丝状肌动蛋白;位于免疫突触中心的 Fact-low cIS、F-actin-low 区域;FasL,Fas配体;FMNL1,仿甲醛 1;fps,每秒帧数;GFP,绿色荧光蛋白;HBSS,汉克平衡盐溶液;HRP,辣根过氧化物酶;ILV,腔内囊泡;IS,免疫突触;MFI,平均荧光强度;MHC,主要组织相容性复合物;MIP,最大强度投影;MV, 多泡体;MTOC,微管组织中心;NS,不显着;PBL,外周血淋巴细胞;PKC,蛋白激酶 C;PKCδ,蛋白激酶 C δ 异构体;PLC,磷脂酶 C;PMA,醋酸佛波醇肉豆蔻酸酯;波尔。指数,极化指数;pSMAC,外周超分子激活簇;PSF,点扩散函数;ROI,感兴趣区域;SD,标准差;shRNA,短发夹 RNA;见,葡萄球菌肠毒素E;SMAC,超分子活化簇;TCR,抗原的 T 细胞受体;T-助手(Th);TRANS,透光率;WB,蛋白质印迹。外周超分子活化簇;PSF,点扩散函数;ROI,感兴趣区域;SD,标准差;shRNA,短发夹 RNA;见,葡萄球菌肠毒素E;SMAC,超分子活化簇;TCR,抗原的 T 细胞受体;T-助手(Th);TRANS,透光率;WB,蛋白质印迹。外周超分子活化簇;PSF,点扩散函数;ROI,感兴趣区域;SD,标准差;shRNA,短发夹 RNA;见,葡萄球菌肠毒素E;SMAC,超分子活化簇;TCR,抗原的 T 细胞受体;T-助手(Th);TRANS,透光率;WB,蛋白质印迹。

更新日期:2020-06-19
down
wechat
bug