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呈递细胞接触时经历显著重排以形成包含免疫突触（IS）的分离结构域-远端、外周和中央超分子活化簇（分别为dSMAC、pSMAC和cSMAC）。 在这里，我们解决了同心肌动球蛋白II弧，填充pSMAC的形成和功能的机制。 使用全内反射荧光结构照明显微镜（TIRF-SIM）和3D-SIM可视化的F-肌动蛋白网络的架构，我们表明，肌动球蛋白II弧在Jurkat T细胞和原代小鼠CD 4/8+ T细胞是真正的结构。 一个流行的模型表明，由Arp 2/3依赖性分支肌动蛋白成核产生的包含dSMAC的分支肌动蛋白网络通过脱支和交联转化为同心弧1。 与此相反，我们观察到线性肌动蛋白丝出现在质膜和项目向内，使他们垂直于质膜和嵌入在分支肌动蛋白网络的dSMAC。 此外，当这些细丝离开dSMAC的内部时，它们在pSMAC中展开并重新定向成具有固有反平行组织的同心弧，该组织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中的垂直肌动蛋白丝通过抑制Arp 2/3依赖性肌动蛋白聚合而大大增强，这使分支肌动蛋白阵列崩溃，并为其他肌动蛋白成核剂如formins 2提供额外的单体。 一致的是，几个formins，包括Inf 2和mDia 1，集中在这些垂直结构的尖端，并添加泛抑制剂破坏弧架构，并防止进一步的电弧形成在一个可逆的方式。 重要的是，使用对硝基-blebbistatin抑制肌球蛋白II导致pSMAC内的松散、无序的细丝不能重新定向成同心弧。 总而言之，这些观察结果表明，弧组装是通过福明依赖性机制（即独立于Arp 2/3介导的成核）发生的，并且需要肌球蛋白II收缩性来将从dSMAC发出的垂直细丝重新定向到pSMAC中的同心弧。 最后，使用支持的脂质双层，我们表明，肌动球蛋白II弧结构所需的T细胞受体（TCR）和LFA-1整合素微簇（MC）的cSMAC和pSMAC中的适当分布，分别。 具体地，通过抑制cSMAC或肌球蛋白II来破坏肌动蛋白弧形成/组织导致不太集中的TCR MC和LFA-1 MC，其不积聚在pSMAC的内部并且被错误地分离到cSMAC中。 我们的结论是肌动球蛋白II弧在成熟的免疫突触的形成中发挥重要作用。