Directing Function at the Natural Killer Cell Secretory Immunological Synapse

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，其允许特异性靶向免疫效应子功能。我们的长期目标是使用自然杀伤（NK）细胞，这是先天免疫系统的淋巴细胞，对宿主防御至关重要，机械地定义参与分泌性IS的形成和功能的基本阶段。我们的假设是，关键的细胞因子依赖性事件控制顺序形成的IS系列，使分泌功能，从而促进免疫防御。我们的研究结果是基于以下发现：1）溶细胞NK细胞IS依赖于Wiskott-Aldrich综合征蛋白质指导的肌动蛋白聚合; 2）NK细胞IS处的肌动蛋白重组先于微管功能并且是微管功能所需的;以及3）微管需要将溶解颗粒移位到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，这项建议的具体目标有两个方面。1)确定IS处的肌动蛋白依赖性复合物（称为肌动蛋白体）与微管之间的联系，微管是溶解颗粒极化和定向分泌所必需的。我们最初的候选人，Cdc 42相互作用蛋白4（CIP 4）是至少部分负责这种活动，在我们的初步结果表明。CIP 4和其他通过质谱鉴定的将进一步评估其使用显微镜、过表达、显性负表达和小干扰RNA将放线菌体与微管功能性连接的能力。2)确定在裂解颗粒通过微管被递送到IS后，放线菌体如何引导分泌通过IS。初步数据表明，在细胞溶解过程的后期时间点，也需要放线菌体内的非肌肉肌球蛋白，以使溶解颗粒通过IS胞吐。因此，肌球蛋白穿梭溶解颗粒以及炎性外泌体通过IS的能力将使用显微镜、生物化学、遗传学和无细胞方法来确定。相关性：我们的目的是确定通过IS的分泌功能获得免疫所需的2个关键检查点的机制。这将使治疗操纵疾病中的IS的新策略能够增加分泌并改善宿主防御，或减少分泌并减少炎症。

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.