Architectural regulation of cytotoxic synapse detachment

摘要 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 专门化的免疫突触与它们的靶标相连，它们将诱导细胞凋亡的有毒因子导入其中。 虽然关于细胞毒性突触是如何形成的已知很多，但控制细胞和分子机制的 人们对它们的解体知之甚少。解决这一知识差距很重要，因为细胞毒性 淋巴细胞必须放弃死亡细胞，才能以连续的方式杀死多个目标。高效拆解突触 还通过减弱持续的细胞毒性淋巴细胞激活来防止假性炎症，还通过 促进吞噬细胞对凋亡身体的清除。以前对这一过程的研究主要集中在 关于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， 一种替代的概念创新的生物物理诱导淋巴细胞分离模型 垂死的靶细胞的变化。我们特别感兴趣的是我们已经发现的细胞凋亡性收缩。 就在CTL-靶细胞结合物溶解之前。此外，遗传和药物脱钩 由细胞凋亡引起的收缩会延迟解离反应。根据这些发现，我们假设 CTL使用收缩目标的特有生物物理特征来触发释放。我们提议的研究， 分为两个特定的目的，将探索CTL解离的生物物理基础和分子 管理这一过程的途径。特殊目标1将使用单细胞机械生物学方法来评估 细胞凋亡性收缩对皮质刚性和表面配体流动性的影响。我们还将使用 确定细胞骨架收缩是否足以诱导突触的光遗传学方法 拆卸。《特殊目标2》将研究细胞凋亡性收缩的分子基础，并通过 CTL，使用靶向功能丧失和CRISPR/Cas9筛查。我们提议的研究将利用 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，即机械感应可以决定免疫细胞的寿命 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，并可能在临床上揭示调节细胞细胞毒性的新途径。因此，这是 该提案与美国国立卫生研究院的任务高度相关，因为它将有助于促进知识的进步， 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.