Roles of cytoskektal dynamics in T lymphocyte function

细胞毒性T淋巴细胞（ctl）通过极化分泌含有触发靶细胞程序性死亡分子的溶解颗粒来杀死靶细胞。在这里，我们使用快速，多色全内反射显微镜对人CD8+ ctl与刺激表面接触的溶解颗粒的胞吐进行了成像。使用mRFP标记的Lamp-1跟踪裂解颗粒的限制膜的命运，而使用mRFP标记的颗粒酶A，颗粒酶B或serglycin跟踪裂解颗粒货物的命运。成像显示，溶解颗粒通过与质膜的完全融合而释放出来，因此可见的所有三种货物的颗粒的全部内容在亚秒的时间尺度上释放到介质中。然而，GFP-Lamp-1的行为更为复杂。具体地说，在所有病例中，当它进入质膜时，它从胞吐部位向外扩散的程度从几乎完全到高度限制不等。后一种行为在大多数情况下可见，可能促进代偿性内吞过程。最后，我们可视化的三种物质释放时的扩散特性对颗粒酶和serglycin大分子复合物的大小提出了上限，这些大分子复合物呈现在目标细胞中。

Cytotoxic T lymphocytes (CTLs) kill target cells by the polarized secretion of lytic granules containing molecules that trigger programmed cell death in the target cell. Here we imaged the exocytosis of lytic granules from human CD8+ CTLs in contact with stimulatory surfaces using rapid, multicolor Total Internal Refection microscopy. The fate of the limiting membrane of the lytic granule was followed using mGFP-tagged Lamp-1, while the fate of lytic granule cargo was followed using granzyme A, granzyme B or serglycin tagged with mRFP. Imaging revealed that lytic granules are released by full fusion with the plasma membrane such that the entire content of the granule for all three cargos visualized was released into the media on a subsecond time scale. The behavior of GFP-Lamp-1 was, however, more complex. Specifically, while it entered the plasma membrane in all cases, the extent to which it then diffused away from the site of exocytosis varied from nearly complete to highly restricted. This latter behavior was seen in the majority of cases and may facilitate a process of compensatory endocytosis. Finally, the diffusion properties upon release of the three cargos we visualized put an upper limit on the size of the macromolecular complex of granzyme and serglycin that is presented to the target cell.