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)
Showing the most recent 10 out of 26 publications
