Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University, Homburg, Germany.
Front Immunol. 2020 May 29;11:1080. doi: 10.3389/fimmu.2020.01080. eCollection 2020.
Granules of cytotoxic T lymphocytes (CTL) are derived from the lysosomal compartment. Synaptotagmin7 (Syt7) appears to be the calcium sensor triggering fusion of lysosomes in fibroblasts. Syt7 has been proposed to control cytotoxic granule (CG) fusion in lymphocytes and mice lacking Syt7 have reduced ability to clear infections. However, fusion of CG persists in the absence of Syt7. To clarify the role of Syt7 in CTL function, we have examined the fusion of cytotoxic granules of CD8 T-lymphocytes from Syt7 knock-out mice. We have recorded granule fusion in living CTL, using total internal reflection microscopy. Since Syt7 is considered a high affinity calcium-sensor specialized for fusion under low calcium conditions, we have compared cytotoxic granule fusion under low and high calcium conditions in the same CTL. There was no difference in latencies or numbers of fusion events per CTL under low-calcium conditions, indicating that Syt7 is not required for cytotoxic granule fusion. A deficit of fusion in Syt7 KO CTL was seen when a high-calcium solution was introduced. Expressing wild type Syt7 in Syt7 KO lymphocytes reversed this deficit, confirming its Syt7-dependence. Mutations of Syt7 which disrupt calcium binding to its C2A domain reduced the efficacy of this rescue. We counted the cytotoxic granules present at the plasma membrane to determine if the lack of fusion events in the Syt7 KO CTL was due to a lack of granules. In low calcium there were no differences in fusion events per CTL, and granule numbers were similar. In high calcium, granule number was similar though wild type CTL exhibited significantly more fusion than Syt7 KO CTL. The modest differences in granule counts do not account for the lack of fusion in high calcium in Syt7 KO CTL. In Syt7 KO CTL expressing wild type Syt7, delivery of cytotoxic granules to the plasma membrane was comparable to that of wild type CTL. Syt7 KO CTL expressing Syt7 with deficient calcium binding in the C2A domain had significantly less fusion and fewer CG at the plasma membrane. These results indicate that Syt7 is involved in trafficking of CG to the plasma membrane.
细胞毒性 T 淋巴细胞 (CTL) 的颗粒来源于溶酶体区室。突触结合蛋白 7 (Syt7) 似乎是触发成纤维细胞中溶酶体融合的钙传感器。Syt7 被提出控制淋巴细胞中的细胞毒性颗粒 (CG) 融合，并且缺乏 Syt7 的小鼠清除感染的能力降低。然而，在缺乏 Syt7 的情况下 CG 融合仍然持续。为了阐明 Syt7 在 CTL 功能中的作用，我们检查了 Syt7 敲除小鼠的 CD8 T 淋巴细胞的细胞毒性颗粒的融合。我们使用全内反射显微镜记录活 CTL 中的颗粒融合。由于 Syt7 被认为是一种高亲和力钙传感器，专门用于低钙条件下的融合，因此我们比较了同一 CTL 下低钙和高钙条件下细胞毒性颗粒的融合。在低钙条件下，CTL 中融合的潜伏期或融合事件数量没有差异，表明 Syt7 不是细胞毒性颗粒融合所必需的。当引入高钙溶液时，Syt7 KO CTL 中的融合缺陷可见。在 Syt7 KO 淋巴细胞中表达野生型 Syt7 逆转了这种缺陷，证实了其对 Syt7 的依赖性。破坏 Syt7 钙结合其 C2A 结构域的突变降低了这种挽救的效果。我们计算了存在于质膜上的细胞毒性颗粒的数量，以确定 Syt7 KO CTL 中融合事件的缺乏是否是由于缺乏颗粒。在低钙条件下，每个 CTL 的融合事件没有差异，颗粒数量相似。在高钙条件下，颗粒数量相似，尽管野生型 CTL 表现出比 Syt7 KO CTL 更多的融合。在 Syt7 KO CTL 中，高钙条件下缺乏融合并不是由于颗粒计数的适度差异造成的。在表达野生型 Syt7 的 Syt7 KO CTL 中，细胞毒性颗粒递送到质膜与野生型 CTL 相当。在表达 C2A 结构域钙结合缺陷的野生型 Syt7 的 Syt7 KO CTL 中，融合和质膜上的 CG 明显较少。这些结果表明 Syt7 参与 CG 向质膜的运输。
