RhoC regulates the actin remodeling required for phagosome formation during FcγR-mediated phagocytosis
RhoC regulates the actin remodeling required for phagosome formation during FcγR-mediated phagocytosis
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DOI：
10.1242/jcs.202739
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发表时间：
2017-12-01
影响因子：
4
通讯作者：
Araki, Nobukazu
中科院分区：
文献类型：
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作者：
Egami, Youhei;Kawai, Katsuhisa;Araki, Nobukazu
吞噬体的形成是一个复杂的过程，需要时空调控的肌动蛋白重组。我们发现RhoC GTPase是巨噬细胞中Fc γ r介导的吞噬的关键调节因子。我们的活细胞成像显示RhoC，而不是RhoA，被招募到吞噬igg活化红细胞（IgG-Es）的吞噬杯中。通过RNAi、CRISPR/ cas介导的RhoC敲除以及表达显性阴性或组成型活性RhoC突变体抑制igg - e的吞噬作用来沉默RhoC。此外，RhoC- gtp下拉实验表明，内源性RhoC在吞噬体形成过程中被短暂激活。值得注意的是，在表达组成型活性突变体RhoC-G14V的细胞中，肌动蛋白驱动的假足延伸（形成吞噬杯所必需的）严重受损，这导致了质膜下异常的f -肌动蛋白积累。rho依赖性肌动蛋白成核因子mDia1（由DIAPH1编码）和RhoC共定位于吞噬杯。与RhoC类似，通过RNAi沉默mDia1抑制吞噬体的形成。此外，mDia1与组成型活性突变体RhoC-G14V或活性突变体mDia1- delta N3的共表达可显著抑制IgG-Es的摄取。这些数据表明RhoC通过mDia1调节肌动蛋白细胞骨架重塑来调节吞噬体的形成。
Phagosome formation is a complicated process that requires spatiotemporally regulated actin reorganization. We found that RhoC GTPase is a critical regulator of Fc gamma R-mediated phagocytosis in macrophages. Our live-cell imaging revealed that RhoC, but not RhoA, is recruited to phagocytic cups engulfing IgG-opsonized erythrocytes (IgG-Es). RhoC silencing through RNAi, CRISPR/Cas-mediated RhoC knockout, and the expression of dominant-negative or constitutively active RhoC mutants suppressed the phagocytosis of IgG-Es. Moreover, RhoC-GTP pulldown experiments showed that endogenous RhoC is transiently activated during phagosome formation. Notably, actin-driven pseudopod extension, which is required for the formation of phagocytic cups, was severely impaired in cells expressing the constitutively active mutant RhoC-G14V, which induced abnormal F-actin accumulation underneath the plasma membrane. mDia1 (encoded by DIAPH1), a Rho-dependent actin nucleation factor, and RhoC were colocalized at the phagocytic cups. Similar to what was seen for RhoC, mDia1 silencing through RNAi inhibited phagosome formation. Additionally, the coexpression of mDia1 with constitutively active mutant RhoC-G14V or expression of active mutant mDia1-Delta N3 drastically inhibited the uptake of IgG-Es. These data suggest that RhoC modulates phagosome formation be modifying actin cytoskeletal remodeling via mDia1.