Roles of cytoskektal dynamics in T lymphocyte function

细胞骨架动力学在 T 淋巴细胞功能中的作用

• 批准号: 8344916
• 负责人: JOHN A HAMMER
• 金额: $ 37.51万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8344916
• 关键词: Actins; Apoptosis; Behavior; CD8B1 gene; Cell Communication; Cell membrane; Cells; Centrosome; Complex; Cytoplasmic Granules; Cytoskeleton; Cytotoxic T-Lymphocytes; Diffuse; Diffusion; Endocytosis; Exocytosis; Granzyme; Human; Image; Lymphocyte Function; Lytic; Macromolecular Complexes; Membrane; Microscopy; Microtubules; Process; Property; Role; Site; Surface; T-Lymphocyte; Time; granzyme A; granzyme B; immunological synapse formation; killings; polarized cell; serglycin

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.

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细胞死亡的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与刺激表面接触的人 CD8+ CTL 的裂解颗粒的胞吐作用进行了成像。 使用 mGFP 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 或血清甘氨酸跟踪裂解颗粒货物的命运。 成像显示，溶解颗粒通过与质膜完全融合而释放，使得可视化的所有三种货物的颗粒的全部内容物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。 具体来说，虽然在所有情况下它都进入质膜，但它随后扩散离开胞吐作用位点的程度从几乎完全到高度受限不等。 后一种行为在大多数情况下都可见，并且可能促进补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甘氨酸的大分子复合物的大小设定了上限。