Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis
Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis
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DOI：
10.1091/mbc.e03-11-0847
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发表时间：
2004-08-01
影响因子：
3.3
通讯作者：
Swanson, JA
中科院分区：
文献类型：
--
作者：
Hoppe, AD;Swanson, JA
小 G 蛋白 Cdc42、Rac1 和 Rac2 调节肌动蛋白和膜的重排，这是巨噬细胞 Fcgamma 受体介导的吞噬作用所必需的。激活的、GTP 结合的 Cdc42、Rac1 和 Rac2 与 PAK1 的 p21 结合域 (PBD) 结合，这种相互作用为微观方法在细胞内定位这些 G 蛋白的激活提供了基础。基于荧光共振能量转移的肌动蛋白、PBD、Cdc42、Rac1 和 Rac2 荧光嵌合体的化学计量学用于量化 IgG 调理红细胞吞噬过程中相对于肌动蛋白运动的 G 蛋白激活。使用黄色荧光蛋白标记的 PBD 定位的内源性 G 蛋白的激活动力学仅限于吞噬杯，在吞噬杯底部的肌动蛋白贫乏区域上有明显的激活峰值。荧光共振能量转移化学计量的改进允许计算形成吞噬体的活化 GTP 酶的分数。 Cdc42 的激活仅限于细胞的前缘，而 Rac1 在整个吞噬杯中都活跃。在吞噬体关闭期间，Rac1 和 Rac2 的激活在吞噬体膜的肌动蛋白缺乏区域均匀且短暂地增加。 Cdc42、Rac1 和 Rac2 在吞噬作用的组成活动中的这些不同作用表明了它们的差异调节协调肌动蛋白和膜重排的机制。
The small G proteins Cdc42, Rac1, and Rac2 regulate the rearrangements of actin and membrane necessary for Fcgamma receptor-mediated phagocytosis by macrophages. Activated, GTP-bound Cdc42, Rac1, and Rac2 bind to the p21-binding domain (PBD) of PAK1, and this interaction provided a basis for microscopic methods to localize activation of these G proteins inside cells. Fluorescence resonance energy transfer-based stoichiometry of fluorescent chimeras of actin, PBD, Cdc42, Rac1, and Rac2 was used to quantify G protein activation relative to actin movements during phagocytosis of IgG-opsonized erythrocytes. The activation dynamics of endogenous G proteins, localized using yellow fluorescent protein-labeled PBD, was restricted to phagocytic cups, with a prominent spike of activation over an actin-poor region at the base of the cup. Refinements of fluorescence resonance energy transfer stoichiometry allowed calculation of the fractions of activated GTPases in forming phagosomes. Cdc42 activation was restricted to the leading margin of the cell, whereas Rac1 was active throughout the phagocytic cup. During phagosome closure, activation of Rac1 and Rac2 increased uniformly and transiently in the actin-poor region of phagosomal membrane. These distinct roles for Cdc42, Rac1, and Rac2 in the component activities of phagocytosis indicate mechanisms by which their differential regulation coordinates rearrangements of actin and membranes.