Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan.
Sci Rep. 2013;3:2385. doi: 10.1038/srep02385.
Macropinocytosis, a fluid-phase endocytosis, is a crucial pathway for antigen uptake and presentation in macrophages. We attempted to characterise the activation and deactivation of a small GTPase molecular switch, Rac1, in macropinocytosis using microscopic photo-manipulation. Expression of genetically encoded photoactivatable-Rac1 (PA-Rac1) in RAW264 macrophages enabled the local, reversible control of macropinocytosis using blue laser irradiation. Marked membrane ruffling and unclosed pre-macropinosomes were observed in the irradiated region of macrophages under the persistent activation of PA-Rac1. Although phosphatidylinositol 4,5-bisphosphate and actin were also localised to this region, the recruitment of maturating endosome markers, such as phosphatidylinositol 3-phosphate and Rab21, was restricted until PA-Rac1 deactivation. After deactivating PA-Rac1 by ceasing irradiation, membrane ruffling immediately receded, and the macropinosomes acquired maturation markers. These data suggest that activation of Rac1 is sufficient to induce membrane ruffling and macropinocytic cup formation, but subsequent deactivation of Rac1 is required for macropinosome closure and further maturation.
巨胞饮作用是一种液相内吞作用，是巨噬细胞中抗原摄取和呈递的关键途径。我们试图通过微观光操纵来描述小 GTPase 分子开关 Rac1 在巨胞饮作用中的激活和失活。在 RAW264 巨噬细胞中表达遗传编码的光激活 Rac1（PA-Rac1），可以使用蓝色激光照射实现局部、可逆的巨胞饮作用控制。在持续激活 PA-Rac1 的情况下，观察到巨噬细胞受照射区域出现明显的细胞膜皱襞和未闭合的前巨胞饮体。虽然磷脂酰肌醇 4,5-二磷酸和肌动蛋白也定位于该区域，但成熟内体标志物（如磷酸肌醇 3-磷酸和 Rab21）的募集受到限制，直到 PA-Rac1 失活。停止照射使 PA-Rac1 失活后，细胞膜皱襞立即回缩，巨胞饮体获得成熟标志物。这些数据表明，Rac1 的激活足以诱导细胞膜皱襞和巨胞饮杯形成，但 Rac1 的后续失活对于巨胞饮体的闭合和进一步成熟是必需的。
