TARGET CELL LYSIS BY CYTOLYTIC EFFECTOR CELLS

这项建议的目标是分析导致目标的事件 小鼠克隆的杀伤细胞裂解细胞。以前的实验： 克隆的小鼠自然杀伤(NK)细胞表明，在 NK细胞与其靶细胞的随机结合，重新定位 微管组织中心(MTOC)和高尔基体(GA) 出现靶标结合位点。接下来，颗粒迁移到 靶结合部位并产生携带细胞溶解的小泡 分子到达靶膜。本提案的目的是分析 NK细胞和特异性T细胞杀伤细胞的杀伤作用。这个 该项目关注三个单独的问题：(1)是否有GA-MTOC T-杀伤细胞的重定向及其诱导信号 目标结合部位的重定向和粒状流量？；(2)是 有可能证明细胞溶解分子是在 无细胞系统中诱导的杀伤细胞颗粒和颗粒 裂解靶标，组装细胞溶解囊泡和细胞溶解分子？ (3)能否对可能的细胞溶解分子进行纯化和鉴定？ 以便在无细胞系统中测试它们的功能？一次尝试将是 按顺序提纯和鉴定假定的细胞溶解分子 在无细胞系统中测试它们的功能。克隆的细胞系将被 用于大多数实验。细胞内细胞器将通过 荧光特异性抗体。颗粒将被Percoll分离 梯度离心法及其功能和蛋白质组成 分析过了。细胞溶解的管状复合体将被洗涤剂分离 增溶，然后凝胶过滤。它们的子单元结构将是 分析和使用针对复合体的特异性抗体 用于功能和生化分析。(Sr)

The objective of this proposal is to analyze the events that lead to target cell lysis by cloned killer cells from mice. Previous experiments with cloned murine natural killer (NK) cells had shown that subsequent to an apparently random binding of NK cells to their targets, a repositioning of the microtubule organizing center (MTOC) and Golgi apparatus (GA) toward the target-binding site occurs. Next, granules migrate to the target-binding site and give rise to vesicles that carry cytolytic molecules to the target membrane. The present proposal's aim is to analyze the cytolytic killing by NK cells and by specific T-killer cells. The project focuses on three individual questions: (1) Is there GA-MTOC reorientation in T-killer cells and what are the signals that induce reorientation and granular traffic to the target-binding site?; (2) Is it possible to demonstrate that cytolytic molecules are generated in the granules of killer cells and can granules be induced in a cell-free system to lyse targets, assemble cytolytic vesicles and cytolytic molecules?; and (3) Can the putative cytolytic molecules be purified and characterized in order to test their function in a cell-free system? An attempt will be made to purify and characterize the putative cytolytic molecules in order to test their function in a cell-free system. Cloned cell lines will be used for most experiments. Intracellular organelles will be visualized by fluoresceinated-specific antibody. Granules will be isolated by percoll gradient centrifugation and their function and protein composition analyzed. Cytolytic tubular complexes will be isolated by detergent solubilization followed by gel filtration. Their subunit structure will be analyzed and antibodies specific for the complexes will be raised and used for functional and biochemical analysis. (SR)