Roles of cytoskektal dynamics in T lymphocyte function

细胞骨架动力学在 T 淋巴细胞功能中的作用

• 批准号: 9157426
• 负责人: JOHN A HAMMER
• 金额: $ 52.5万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9157426
• 关键词: Actins; Actomyosin; Address; Antigen-Presenting Cells; Architecture; Cell Communication; Cell membrane; Centrosome; Cytoplasmic Granules; Cytoskeleton; Cytotoxic T-Lymphocytes; Distal; Employee Strikes; F-Actin; Filament; Fluorescence; Immune; Integrins; Lighting; Lipid Bilayers; Lymphocyte Function; Lytic; Mediating; Microfilaments; Microscopy; Microtubules; Modeling; Mus; Myosin Type II; Peripheral; Play; Role; Structure; Synapses; T-Cell Receptor; T-Lymphocyte; blebbistatin; immunological synapse; immunological synapse formation; inhibitor/antagonist; monomer; polymerization; prevent

The T cells actin cytoskeleton undergoes striking rearrangements upon contact with an antigen presenting cell to form the segregated domains that comprise the immunological synapse (IS) - the distal, peripheral and central supramolecular activation clusters (dSMAC, pSMAC and cSMAC, respectively). Here we addressed the mechanism of formation and function of the concentric actomyosin II arcs that populate the pSMAC. Using total internal reflection fluorescence structured illumination microscopy (TIRF-SIM) and 3D-SIM to visualize the architecture of F-actin networks, we show that actomyosin II arcs are bonafide structures in both Jurkat T cells and primary mouse CD4/8+ T cells. A popular model suggests that the branched actin network comprising the dSMAC, which is created by Arp2/3-dependent branched actin nucleation, is converted into the concentric arcs by debranching and crosslinking1. In contrast to this idea, we observed linear actin filaments that arise at the plasma membrane and project inward such that they are perpendicular to the plasma membrane and embedded in the branched actin network of the dSMAC. Moreover, as these filaments exit the inner aspect of the dSMAC, they splay out in the pSMAC and reorient into concentric arcs with an inherent antiparallel organization that is optimal for supporting contraction by bipolar myosin II filaments. Importantly, the perpendicular actin filaments in the dSMAC are greatly accentuated by inhibiting Arp2/3-dependent actin polymerization, which collapses the branched actin array and provides additional monomer for other actin nucleators like formins2. Consistently, several formins, including Inf2 and mDia1, are concentrated at the tips of these perpendicular structures, and addition of a pan-formin inhibitor disrupts arc architecture and prevents further arc formation in a reversible manner. Importantly, inhibition of myosin II using para-nitro-blebbistatin results in loose, disorganized filaments within the pSMAC that fail to reorient into concentric arcs. Together, these observations argue that arc assembly occurs through a formin-dependent mechanism (i.e. independent of Arp2/3-mediated nucleation), and that myosin II contractility is required for reorienting the perpendicular filaments emanating from the dSMAC into the concentric arcs in the pSMAC. Finally, using supported lipid bilayers, we show that actomyosin II arc structures are required for the proper distribution of T cell receptor (TCR) and LFA-1 integrin microclusters (MCs) in the cSMAC and pSMAC, respectively. Specifically, disrupting actin arc formation/organization by inhibiting either formin or myosin II resulted in less centralized TCR MCs and LFA-1 MCs that are not accumulated at the inner aspect of the pSMAC and are mis-segregated into the cSMAC. We conclude that actomyosin II arcs play important roles in the formation of the mature immune synapse.

T细胞肌动蛋白细胞骨架在与抗原提呈细胞接触后发生显著重排，形成免疫突触(IS)--远端、外周和中央超分子激活簇(分别为dSMAC、pSMAC和cSMAC)。在这里，我们讨论了同心肌动球蛋白II弧的形成机制和功能，它填充在pSMAC中。使用全内反射荧光结构照明显微镜(TIRF-SIM)和3D-SIM显示F-肌动蛋白网络的结构，我们发现肌动球蛋白II弧在Jurkat T细胞和原代小鼠CD4/8+T细胞中都是真实的结构。一个流行的模型认为，由Arp2/3依赖的分枝肌动蛋白成核产生的由dSMAC组成的分枝肌动蛋白网络通过去支化和交联化转化为同心弧1。与这个想法相反，我们观察到在质膜上出现的线性肌动蛋白细丝向内投射，使它们垂直于质膜，嵌入到dSMAC的分支肌动蛋白网络中。此外，当这些细丝离开dSMAC的内部时，它们在pSMAC中展开并重新定向为同心弧形，具有固有的反平行组织，这对于支持双极肌球蛋白II细丝的收缩是最佳的。重要的是，dSMAC中垂直的肌动蛋白细丝通过抑制依赖Arp2/3的肌动蛋白聚合而大大增强，这会破坏分支的肌动蛋白阵列，并为其他肌动蛋白核因子提供额外的单体，如Forins2。始终如一地，包括Inf2和mDia1在内的几种福尔马林集中在这些垂直结构的末端，而泛福尔明抑制剂的加入破坏了电弧的结构，并以可逆的方式防止进一步的电弧形成。重要的是，使用对硝基双抑制素抑制肌球蛋白II会导致pSMAC内疏松、无序的细丝无法重定向为同心圆弧。综上所述，这些观察结果认为，弧形组装是通过一种依赖于形成蛋白的机制发生的(即独立于Arp2/3介导的成核)，并且肌球蛋白II的收缩能力是将dSMAC发出的垂直细丝重新定向为pSMAC中的同心弧所必需的。最后，利用支撑的脂质双层，我们证明了肌动球蛋白II的弧形结构是T细胞受体(TCR)和LFA-1整合素微簇(MC)在cSMAC和pSMAC中正确分布所必需的。具体地说，通过抑制Forin或Myosin II来破坏肌动蛋白弧的形成/组织会导致TCRMCs和LFA-1 MCs不那么集中，它们不会聚集在pSMAC的内侧，而是错误地分离到cSMAC。我们认为肌动球蛋白II ARC在成熟免疫突触的形成中起着重要作用。