Developing a Bioanalytical Toolkit to Study the Mechanobiology of Juxtacrine Signaling

开发生物分析工具包来研究近分泌信号传导的力学生物学

• 批准号: 9894683
• 负责人: Khalid S Salaita
• 金额: $ 7.35万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9894683
• 关键词: Adaptive Immune System; Amino Acids; Antigens; Area; Atomic Force Microscopy; Autoimmune Diseases; Biochemical Reaction; Biological Assay; CD3 Antigens; Cancerous; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Cellular biology; Chemistry; Complex; Coupling; Cytoplasmic Tail; Cytoskeleton; DNA; Data; Enzymes; Fluorescence Polarization; Fluorescence Spectrometry; Goals; Image; Immobilization; Immune response; Immunology; Immunotherapy; Intercellular Junctions; Intuition; Label; Lateral; Ligands; Link; Liquid substance; Literature; Location; Major Histocompatibility Complex; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mechanics; Membrane; Methods; Modeling; Molecular; Molecular Immunology; Molecular Probes; Mutate; Pathway interactions; Peptide Fragments; Peptides; Pharmaceutical Preparations; Receptor Activation; Receptor Signaling; Research Personnel; Resolution; Scanning; Signal Transduction; Signaling Molecule; Signaling Protein; Specificity; Spectrum Analysis; Surface; T cell regulation; T cell therapy; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Techniques; Technology; Testing; Time; Tumor stage; Virus; Work; adaptive immune response; base; biophysical chemistry; cancer cell; chimeric antigen receptor; design; experience; fighting; fluorescence lifetime imaging; high resolution imaging; imaging approach; immune function; immunoengineering; immunological synapse; improved; interdisciplinary approach; interest; mechanical force; mechanotransduction; molecular mechanics; mutant; pathogen; protein complex; public health relevance; ratiometric; receptor; receptor binding; recruit; single molecule; tool; transmission process; vector; virtual; Adaptive Immune System; Amino Acids; Antigens; Area; Atomic Force Microscopy; Autoimmune Diseases; Biochemical Reaction; Biological Assay; CD3 Antigens; Cancerous; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Cellular biology; Chemistry; Complex; Coupling; Cytoplasmic Tail; Cytoskeleton; DNA; Data; Enzymes; Fluorescence Polarization; Fluorescence Spectrometry; Goals; Image; Immobilization; Immune response; Immunology; Immunotherapy; Intercellular Junctions; Intuition; Label; Lateral; Ligands; Link; Liquid substance; Literature; Location; Major Histocompatibility Complex; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mechanics; Membrane; Methods; Modeling; Molecular; Molecular Immunology; Molecular Probes; Mutate; Pathway interactions; Peptide Fragments; Peptides; Pharmaceutical Preparations; Receptor Activation; Receptor Signaling; Research Personnel; Resolution; Scanning; Signal Transduction; Signaling Molecule; Signaling Protein; Specificity; Spectrum Analysis; Surface; T cell regulation; T cell therapy; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Techniques; Technology; Testing; Time; Tumor stage; Virus; Work; adaptive immune response; base; biophysical chemistry; cancer cell; chimeric antigen receptor; design; experience; fighting; fluorescence lifetime imaging; high resolution imaging; imaging approach; immune function; immunoengineering; immunological synapse; improved; interdisciplinary approach; interest; mechanical force; mechanotransduction; molecular mechanics; mutant; pathogen; protein complex; public health relevance; ratiometric; receptor; receptor binding; recruit; single molecule; tool; transmission process; vector; virtual

Project Summary/Abstract The long-term goal of this proposal is to better understand how T cells defend against pathogens and eradicate cancerous cells within our bodies. To achieve this goal, T cells continuously crawl seeking evidence of foreign peptide fragments on the surface of other cells. Once the T cell encounters a target cell with foreign or mutant peptides, then it initiates activation mechanisms that unleash a potent immune response. Malfunctions in T cell activation are linked with autoimmune disease, while drugs that enhance T cell activation are used to treat cancer. Therefore, there is much interest in understanding the molecular mechanisms of T cell activation. The very first step in T cell activation involves recognition between the T cell receptor (TCR) and the short peptides (8-11 amino acids) presented by the major histocompatibility complex (pMHC) protein. Because T cells are highly migratory and antigen recognition occurs when the T cell physically contacts a target cell, there are long standing questions of whether T cells transmit defined forces to their TCR complex and if chemo-mechanical coupling influences immune function. These questions cannot be answered using conventional imaging approaches. The central hypothesis of the proposed work is that advanced mechano- imaging and mechano-analytical approaches will reveal the TCR forces involved in regulation of T cell signaling. Building on our recent breakthroughs in developing high-resolution imaging approaches to map the forces transmitted by cell surface receptors, we will aim to close this gap in our understanding and unravel the mechanical basis of T cell activation. Our preliminary data clearly shows that we have successfully developed the first molecular probes to image the piconewton forces transmitted by the TCR to its ligand during TCR activation. We will test the central hypothesis by first developing molecular force microscopy for the TCR. These probes will test whether the TCR is an anisotropic mechanosensor as proposed in the literature. Next we will map TCR forces within membrane-membrane junctions where the receptor is free to assemble into signaling microclusters. Fluorescence lifetime imaging microscopy (FLIM) and ratiometric probes will be used to map these forces in space and time. Finally, we will use mechanically-triggered enzymes to quantify TCR forces with ultrahigh sensitivity and to tag proximal molecules that are recruited following transmission of TCR forces. The work requires multidisciplinary approaches combining expertise from three investigators that cover the areas of biophysical chemistry, cell biology, and molecular immunology. Importantly, not only will the imaging and quantification techniques developed for this proposal be critical for better understanding the specificity of the adaptive immune system, we expect important implications for the optimal design and implementation of adoptive T cell transfer and chimeric antigen receptors (CARs) in immunotherapy as well as understanding the causes of autoimmune disease.

项目摘要/摘要 该提案的长期目标是更好地了解T细胞如何抗病病原体和 根除我们体内的癌细胞。为了实现这一目标，T细胞不断爬网寻求证据 在其他细胞表面上的异物碎片。一旦T细胞遇到异物的目标细胞 或突变肽，然后启动释放有效免疫反应的激活机制。 T细胞激活中的故障与自身免疫性疾病有关，而增强T细胞激活的药物 用于治疗癌症。因此，有很大的兴趣了解T的分子机制 细胞激活。 T细胞激活的第一步涉及T细胞受体（TCR）和 主要的组织相容性复合物（PMHC）蛋白提出的短肽（8-11氨基酸）。 因为T细胞是高度迁移的，并且当T细胞物理接触A时会发生抗原识别 靶细胞，长期存在的问题是T细胞是否将定义力传输到其TCR复合物 如果化学机械耦合影响免疫功能。这些问题无法使用 传统的成像方法。拟议工作的中心假设是先进的机械 成像和机械分析方法将揭示与T细胞调节有关的TCR力 信号。以我们最近在开发高分辨率成像方法来映射的基础上建立了突破 通过细胞表面受体传输的力，我们将旨在缩小我们的理解差距，并揭开 T细胞激活的机械基础。我们的初步数据清楚地表明我们已经成功开发了 第一个分子探针成像TCR在TCR期间传播到其配体的Piconewton力量成像 激活。我们将通过首先为TCR开发分子力显微镜来检验中心假设。 这些探针将测试TCR是否是文献中提出的各向异性机械传感器。下一个 我们将在膜膜连接处绘制TCR力，其中受体可以自由组装成 信号微群体。荧光寿命成像显微镜（FLIM）和比率探针将使用 在时空中绘制这些力。最后，我们将使用机械触发的酶来量化TCR 具有超高灵敏度和标记TCR传播后募集的近端分子的力 力量。这项工作需要三名调查人员的专业知识的多学科方法，这些方法涵盖了 生物物理化学，细胞生物学和分子免疫学领域。重要的是，不仅 为此提案开发的成像和量化技术对于更好地理解 自适应免疫系统的特异性，我们期望对最佳设计和 在免疫疗法和 了解自身免疫性疾病的原因。