Directing Function at the Natural Killer Cell Secretory Immunological Synapse

自然杀伤细胞分泌免疫突触的指导功能

• 批准号: 7650556
• 负责人: Jordan Scott Orange
• 金额: $ 6万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7650556
• 关键词: Actins; Activated Natural Killer Cell; Activities of Daily Living; Biochemical; Biochemical Genetics; Cell membrane; Cells; Complex; Cytoplasmic Granules; Cytoskeleton; Data; Disease; Dominant-Negative Mutation; Event; Exocytosis; Goals; Health; Host Defense; Human; Immune; Immune Targeting; Immune system; Immunity; Inflammation; Inflammatory; Kinetics; Life; Link; Lymphocyte; Lytic; Maintenance; Mass Spectrum Analysis; Membrane; Microfilaments; Microscopy; Microtubules; Molecular; Muscle; Myosin ATPase; Myosin Type II; Natural Killer Cells; Pathway interactions; Play; Process; Protein Overexpression; Proteins; Role; Series; Small Interfering RNA; Sorting - Cell Movement; Staging; Structure; Time; Tubulin; Wiskott-Aldrich Syndrome; Work; actin 2; base; cell cortex; cellular imaging; cytotoxicity; immunological synapse; improved; novel; novel strategies; polymerization; receptor; synaptic function

DESCRIPTION (provided by applicant): Project Summary: The immunological synapse (IS) is the dynamic interface between an immune cell and the cell that it is recognizing. A major function of the IS is directed secretion, which allows specific targeting of immune effector function. Our long-term goal is to use natural killer (NK) cells, which are lymphocytes of the innate immune system critical for host defense, to mechanistically define essential stages involved in formation and function of the secretory IS. Our hypothesis is that critical cytoskeleton-dependent events control sequential formation of the IS in series to allow secretory function and thus promote immune defense. We base this upon our findings that: 1) The cytolytic NK cell IS is dependent upon Wiskott-Aldrich syndrome protein-directed actin polymerization; 2) Actin reorganization at the NK cell IS precedes and is required for microtubule function; and 3) Microtubules are needed to translocate lytic granules to the center of the NK cell IS, but not form the actin dependent structures. Based upon our observations, the specific aims of this proposal are twofold. 1) Determine the link between the actin-dependent complex (termed the actinosome) at the IS and the microtubules that is required for lytic granule polarization and directed secretion. Our initial candidate, Cdc42 interacting protein 4 (CIP4) is at least in part responsible for this activity as demonstrated in our preliminary results. CIP4 and others identified through mass spectrometry will be further evaluated for their ability to functionally link the actinosome to microtubules using microscopy, overexpression, dominant negative expression and small-interfering RNA. 2) Determine how the actinosome directs secretion through the IS after lytic granules have been delivered to the IS via microtubules. Preliminary data demonstrate non-muscle myosin proteins within the actinosome are also needed at late time points in the cytolytic process to allow the exocytosis of lytic granules through the IS. Thus, the ability of myosin to shuttle lytic granules as well as inflammatory exosomes though the IS will be determined using microscopy, biochemical, genetic and cell-free approaches. Relevance: We aim to define the mechanism underlying 2 critical checkpoints required to access immunity through secretory function of the IS. This will enable novel strategies for therapeutically manipulating the IS in disease to increase secretion and improve host defense, or decrease secretion and reduce inflammation.

描述（由申请人提供）： 项目摘要：免疫突触 (IS) 是免疫细胞与其识别的细胞之间的动态界面。 IS 的主要功能是定向分泌，从而可以特异性靶向免疫效应子功能。我们的长期目标是利用自然杀伤 (NK) 细胞（对宿主防御至关重要的先天免疫系统的淋巴细胞）来机械地定义参与分泌型 IS 形成和功能的重要阶段。我们的假设是，关键的细胞骨架依赖性事件控制 IS 的连续形成，以实现分泌功能，从而促进免疫防御。我们基于以下发现：1) 溶细胞性 NK 细胞 IS 依赖于 Wiskott-Aldrich 综合征蛋白定向肌动蛋白聚合； 2) NK 细胞 IS 的肌动蛋白重组先于微管功能，并且是微管功能所必需的； 3) 需要微管将溶解颗粒转移到 NK 细胞 IS 的中心，但不形成肌动蛋白依赖性结构。根据我们的观察，该提案的具体目标是双重的。 1) 确定 IS 处的肌动蛋白依赖性复合物（称为肌动体）与溶解颗粒极化和定向分泌所需的微管之间的联系。我们的初步结果表明，我们最初的候选者 Cdc42 相互作用蛋白 4 (CIP4) 至少部分负责这种活性。通过显微镜、过表达、显性失活表达和小干扰 RNA，将进一步评估 CIP4 和通过质谱法鉴定的其他物质将放线体与微管功能连接的能力。 2) 确定在裂解颗粒通过微管递送至 IS 后，放线体如何通过 IS 引导分泌。初步数据表明，在细胞溶解过程的后期，还需要放线体内的非肌肉肌球蛋白，以允许溶解颗粒通过 IS 进行胞吐。因此，肌球蛋白通过 IS 穿梭溶解颗粒和炎性外泌体的能力将使用显微镜、生化、遗传和无细胞方法来确定。相关性：我们的目标是定义通过 IS 的分泌功能获得免疫力所需的 2 个关键检查点的潜在机制。这将为治疗性操纵疾病中的 IS 来增加分泌和改善宿主防御，或减少分泌和减少炎症提供新策略。