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）通过含有裂解颗粒的极化分泌杀死靶细胞，这些分子含有触发靶细胞中编程细胞死亡的分子。 在这里，我们使用快速的多色总内反射显微镜对人CD8+ CTL的裂解颗粒的胞吐作用与刺激表面接触。 使用MGFP标记的LAMP-1跟随裂解颗粒的极限膜的命运，同时使用用MRFP标记的Granzyme A，Granzyme B或Serglycin随后遵循裂解颗粒货物的命运。 成像表明，裂解颗粒是通过与质膜完全融合释放的，因此所有三个cargos的颗粒的全部含量都以次代时尺度释放到媒体中。 但是，GFP-LAMP-1的行为更为复杂。 具体而言，尽管在所有情况下都进入质膜，但从几乎完全限制到高度限制的情况下，它从胞吞作用部位散开的程度不等。 在大多数病例中都可以看到后一种行为，并可能促进代偿性内吞作用的过程。 最后，我们可视化的三颗嘉戈斯释放时的扩散特性对呈谷物酶和塞格兰霉素的大分子络合物的大小呈上限，并呈现给靶细胞。