Architectural regulation of cytotoxic synapse detachment

总结 细胞毒性淋巴细胞，包括细胞毒性T淋巴细胞（CTL）和自然杀伤细胞，通过形成 它们与靶点形成专门的免疫突触，将诱导细胞凋亡的毒性因子导入靶点。 尽管我们对细胞毒性突触的形成方式了解很多，但控制突触形成的细胞和分子机制仍然是未知的。 它们的溶解知之甚少。解决这一知识差距很重要，因为细胞毒性 淋巴细胞必须释放垂死的细胞，以连续的方式杀死多个目标。高效突触拆卸 还通过减弱持续的细胞毒性淋巴细胞活化来预防假性炎症， 促进吞噬细胞对凋亡尸体的清除。以前研究这一过程的尝试集中在 与细胞死亡相关的生化特征的识别。然而我们的初步研究表明 一种替代的和概念上创新的模型，其中淋巴细胞脱离是由生物物理诱导的， 死亡靶细胞的变化。我们特别感兴趣的是凋亡收缩， 就在CTL-靶细胞缀合物溶解之前。此外，遗传学和药理学脱钩， 细胞凋亡引起的收缩延迟了解离反应。基于这些发现，我们假设， CTL使用收缩靶标的特征性生物物理特征来触发释放。我们提出的研究， 分为两个特定的目的，将探讨CTL解离的生物物理基础和CTL的分子生物学基础。 控制这一过程的途径。具体目标1将采用单细胞机械生物学方法来评估 凋亡收缩对皮质硬度和表面配体迁移率的影响。我们还将使用 光遗传学方法来确定细胞骨架收缩是否足以诱导突触 拆卸特异性目标2将检查凋亡收缩的分子基础，并通过 CTL，使用靶向功能丧失和CRISPR/Cas9筛选。我们提出的研究将利用 技术创新方法，包括超分辨率成像，光遗传学和原子力显微镜。 他们还将引入一个新的概念，即机械传感可以确定免疫细胞的寿命-细胞 互动该项目的成功完成将解决淋巴细胞中一个长期存在的谜团， 生物学，并可能揭示新的途径，用于调节细胞毒性在临床上。因此，这 该提案与NIH的使命高度相关，因为它将有助于知识的进步， 改善人类健康。

Summary Cytotoxic lymphocytes, comprising cytotoxic T lymphocytes (CTLs) and natural killer cells, kill by forming specialized immune synapses with their targets, into which they channel toxic factors that induce apoptosis. Although much is known about how cytotoxic synapses form, the cellular and molecular mechanisms that control their dissolution are poorly understood. Addressing this gap in knowledge is important because cytotoxic lymphocytes must let go of dying cells to kill multiple targets in a serial manner. Efficient synapse disassembly also prevents spurious inflammation by attenuating sustained cytotoxic lymphocyte activation and also by facilitating the clearance of apoptotic corpses by phagocytes. Prior attempts to study this process have focused on the recognition of biochemical features associated with cell death. Our preliminary studies, however, suggest an alternative and conceptually innovative model in which lymphocyte detachment is induced by biophysical changes in dying target cells. We are particularly interested apoptotic contraction, which we have found occurs just before the dissolution of CTL-target cell conjugates. In addition, genetic and pharmacological decoupling of contraction from apoptosis delays the dissociation response. Building upon these findings, we hypothesize that CTLs use the characteristic biophysical features of contracting targets to trigger release. Our proposed studies, which are divided into two Specific Aims, will explore the biophysical basis for CTL dissociation and the molecular pathways that govern the process. Specific Aim 1 will employ single cell mechanobiological methods to assess the effects of apoptotic contraction on cortical rigidity and surface ligand mobility. We will also use an optogenetics approach to determine whether cytoskeletal contraction is sufficient to induce synapse disassembly. Specific Aim 2 will examine the molecular bases for apoptotic contraction and its detection by CTLs, using targeted loss-of-function and CRISPR/Cas9 screening. Our proposed studies will leverage technically innovative methods, including super-resolution imaging, optogenetics, and atomic force microscopy. They will also introduce a novel concept, that mechanosensing can determine the lifetime of an immune cell-cell interaction. The successful completion of this project will address a long-standing enigma in lymphocyte cell biology and likely reveal new avenues for the modulation of cellular cytotoxicity in the clinic. As such, this proposal is highly relevant to the NIH mission in that it will contribute to the advancement of knowledge that could improve human health.