Roles of cytoskektal dynamics in T lymphocyte function

细胞毒性T淋巴细胞(CTL)通过裂解颗粒的极化分泌来杀伤靶细胞，该颗粒含有触发靶细胞程序性细胞死亡的分子。在这里，我们使用快速、多色全内反射显微镜成像了人CD8+CTL与刺激表面接触时的裂解颗粒的胞吐作用。用mGFP标记的Lamp-1跟踪溶解颗粒的限制膜的命运，而用MRFP标记的颗粒酶A、颗粒酶B或丝氨酸跟踪溶解颗粒货物的命运。成像显示，溶解颗粒通过与质膜完全融合而释放，从而在亚秒时间尺度上将所显示的所有三种货物的颗粒的全部内容释放到介质中。然而，GFP-Lamp-1的行为更为复杂。具体地说，尽管它在所有情况下都进入质膜，但随后从胞吐部位扩散出去的程度从几乎完全到高度受限不等。后一种行为见于大多数病例，并可能促进代偿性内吞作用的过程。最后，我们可视化的三种货物释放时的扩散特性对呈现给靶细胞的颗粒酶和丝氨酸氨基大分子复合体的大小施加了上限。

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.