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: 7321445
• 负责人: Jordan Scott Orange
• 金额: $ 41.25万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7321445
• 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）是免疫细胞与所识别细胞之间的动态接口。 IS的主要功能是定向分泌，该分泌允许特定靶向免疫效应子功能。我们的长期目标是使用自然杀手（NK）细胞，这些细胞是宿主防御至关重要的先天免疫系统的淋巴细胞，以机械化定义与分泌的形成和功能有关的基本阶段。我们的假设是，关键的细胞骨架依赖性事件控制IS串联的顺序形成，以允许分泌功能并促进免疫防御。我们基于我们的发现，即：1）细胞溶解的NK细胞取决于Wiskott-Aldrich综合征蛋白指导的肌动蛋白聚合； 2）在NK细胞处的肌动蛋白重组是在微管功能之前所必需的； 3）需要微管才能将裂解颗粒转移到NK细胞的中心为，但不形成肌动蛋白依赖性结构。根据我们的观察，该提案的具体目的是双重的。 1）确定在IS的肌动蛋白依赖性复合物（称为肌动蛋白体）与裂解颗粒极化和定向分泌所需的微管之间的联系。我们最初的候选cdc42相互作用蛋白4（CIP4）至少部分负责这种活动，如我们的初步结果所示。通过质谱法鉴定出的CIP4和其他人将进一步评估其功能性地将阳式体与微管连接到微管的能力。 2）确定在裂解颗粒通过微管传递到IS之后，阳离子体如何通过IS进行分泌。初步数据表明，在细胞溶解过程中的较晚时间点，还需要在阳光蛋白体内的非肌肉肌球蛋白蛋白，以使裂解颗粒的胞吐通过IS。因此，肌球蛋白在使用显微镜，生化，遗传和无细胞的方法中确定肌球蛋白的裂解裂解颗粒以及炎症外泌体的能力。相关性：我们旨在定义通过IS分泌功能获得免疫所需的2个关键检查点的机制。这将实现治疗方法来操纵IS中的新型策略，以增加分泌并改善宿主防御，减少分泌并减少炎症。