Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages.
Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages.
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RAB35和ARF6的激活灭活循环是RAW264巨噬细胞中Zymosan的吞噬作用所必需的。
DOI：
10.1155/2015/429439
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发表时间：
2015
影响因子：
4.1
通讯作者：
Araki N
中科院分区：
文献类型：
--
作者：
Egami Y;Fujii M;Kawai K;Ishikawa Y;Fukuda M;Araki N
吞噬细胞对酵母聚糖的吞噬作用是先天免疫系统广泛使用的微生物识别模型。活细胞成像显示，荧光蛋白融合的 Rab35 在吞噬杯的膜中积累，然后从新形成的吞噬体的膜上解离。通过我们新颖的 Rab35 活性下拉测定，我们发现 Rab35 在酵母聚糖内化到细胞中后立即失活。在表达 GDP 或 GTP 锁定的 Rab35 突变体的细胞中，吞噬体形成受到抑制。此外，ACAP2（一种 Rab35 效应蛋白）与 GTP 锁定的 Rab35 的同时表达或质膜靶向的 ACAP2 的表达通过 ACAP2 的 GAP 活性使 ARF6 失活，显示出对吞噬作用的显着抑制作用。 ARF6 是 ACAP2 的底物，也定位于吞噬杯并与内化吞噬体的膜分离。为了支持显微镜观察，ARF6-GTP 下拉实验表明 ARF6 在吞噬体形成过程中被短暂激活。此外，GDP 或 GTP 锁定的 ARF6 突变体的表达也会抑制酵母聚糖的摄取。这些数据表明，Rab35 和 ARF6 的激活-失活循环是酵母聚糖摄取所必需的，并且 ACAP2 是在酵母聚糖吞噬过程中连接 Rab35/ARF6 信号传导的重要组成部分。
Phagocytosis of zymosan by phagocytes is a widely used model of microbial recognition by the innate immune system. Live-cell imaging showed that fluorescent protein-fused Rab35 accumulated in the membranes of phagocytic cups and then dissociated from the membranes of newly formed phagosomes. By our novel pull-down assay for Rab35 activity, we found that Rab35 is deactivated immediately after zymosan internalization into the cells. Phagosome formation was inhibited in cells expressing the GDP- or GTP-locked Rab35 mutant. Moreover, the simultaneous expression of ACAP2—a Rab35 effector protein—with GTP-locked Rab35 or the expression of plasma membrane-targeted ACAP2 showed a marked inhibitory effect on phagocytosis through ARF6 inactivation by the GAP activity of ACAP2. ARF6, a substrate for ACAP2, was also localized on the phagocytic cups and dissociated from the membranes of internalized phagosomes. In support of the microscopic observations, ARF6-GTP pull-down experiments showed that ARF6 is transiently activated during phagosome formation. Furthermore, the expression of GDP- or GTP-locked ARF6 mutants also suppresses the uptake of zymosan. These data suggest that the activation-inactivation cycles of Rab35 and ARF6 are required for the uptake of zymosan and that ACAP2 is an important component that links Rab35/ARF6 signaling during phagocytosis of zymosan.