Architectural regulation of cytotoxic synapse detachment

细胞毒性突触脱离的结构调节

• 批准号: 10467438
• 负责人: Morgan A Huse
• 金额: $ 26.55万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10467438
• 关键词: Address; Adhesives; Apoptosis; Apoptotic; Architecture; Atomic Force Microscopy; Attenuated; Biochemical; Biophysics; CRISPR screen; Cancerous; Cell Communication; Cell Death; Cell-Mediated Cytolysis; Cells; Cellular biology; Cessation of life; Clinic; Complex; Contracts; Cytoskeleton; Cytotoxic T-Lymphocytes; Data; Detection; Dissociation; Down-Regulation; Exhibits; Experimental Models; F-Actin; Fluorescence; Fluorescent Probes; Foundations; Funding Mechanisms; Future; Genetic; Granzyme; Health; Human; Image; Immune; Immune response; Immunity; Inflammation; Investigation; Knowledge; Ligands; Lymphocyte; Lymphocyte Activation; Lymphocyte Function; Mechanics; Methods; Mission; Modality; Modeling; Molecular; Morphology; Myosin Type II; Pathway interactions; Peptide Hydrolases; Phagocytes; Pharmacology; Photobleaching; Positioning Attribute; Process; Proteins; Regulation; Resolution; Rho-associated kinase; Stereotyping; Surface; Synapses; T-Lymphocyte and Natural Killer Cell; Testing; Therapeutic; Time; United States National Institutes of Health; Up-Regulation; VDAC1 gene; Work; apoptosis inducing factor; base; biophysical properties; biophysical techniques; cell killing; cell type; cytotoxic; falls; high resolution imaging; immune function; immunological synapse; improved; innovation; insight; interdisciplinary approach; interest; interfacial; loss of function; mechanical properties; mechanotransduction; neoplasm immunotherapy; novel; optogenetics; pathogen; perforin; prevent; recruit; response; tumor

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.

概括 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，通过形成杀伤细胞 特殊的免疫突触及其目标，将诱导细胞凋亡的有毒因子引入其中。 尽管人们对细胞毒性突触如何形成了解很多，但控制细胞毒性突触的细胞和分子机制 人们对它们的解散知之甚少。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才能连续杀死多个目标。高效突触拆卸 还可以通过减弱持续的细胞毒性淋巴细胞激活来预防虚假炎症 促进吞噬细胞清除凋亡尸体。先前研究该过程的尝试集中于 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 一种替代性且概念上创新的模型，其中通过生物物理诱导淋巴细胞脱离 死亡靶细胞的变化。我们对细胞凋亡收缩特别感兴趣，我们发现它发生了 就在 CTL-靶细胞缀合物溶解之前。此外，遗传和药理学脱钩 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 利用收缩目标的生物物理特征来触发释放。我们提出的研究， 分为两个具体目标，将探索 CTL 解离的生物物理基础和分子 控制过程的途径。具体目标 1 将采用单细胞力学生物学方法来评估 细胞凋亡收缩对皮质刚性和表面配体迁移率的影响。我们还将使用一个 光遗传学方法确定细胞骨架收缩是否足以诱导突触 拆卸。具体目标 2 将检查细胞凋亡收缩的分子基础及其检测 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概念，即机械传感可以确定免疫细胞的寿命 相互作用。该项目的成功完成将解开淋巴细胞长期存在的谜团 生物学并可能揭示临床调节细胞毒性的新途径。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。