Ikeda Yuka, Kawai Katsuhisa, Ikawa Akira, Kawamoto Kyoko, Egami Youhei, Araki Nobukazu
Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan.
Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan
J Cell Sci. 2017 Aug 1;130(15):2530-2540. doi: 10.1242/jcs.201749. Epub 2017 Jun 9.
Lamellipodia are sheet-like cell protrusions driven by actin polymerization mainly through Rac1, a GTPase molecular switch. In Fcγ receptor-mediated phagocytosis of IgG-opsonized erythrocytes (IgG-Es), Rac1 activation is required for lamellipodial extension along the surface of IgG-Es. However, the significance of Rac1 deactivation in phagosome formation is poorly understood. Our live-cell imaging and electron microscopy revealed that RAW264 macrophages expressing a constitutively active Rac1 mutant showed defects in phagocytic cup formation, while lamellipodia were formed around IgG-Es. Because activated Rac1 reduced the phosphorylation levels of myosin light chains, failure of the cup formation is probably due to inhibition of actin/myosin II contractility. Reversible photo-manipulation of the Rac1 switch in macrophages fed with IgG-Es could phenocopy two lamellipodial motilities: outward-extension and cup-constriction by Rac1 ON and OFF, respectively. In conjunction with fluorescence resonance energy transfer imaging of Rac1 activity, we provide a novel mechanistic model of phagosome formation spatiotemporally controlled by Rac1 switching within a phagocytic cup.
片状伪足是由肌动蛋白聚合驱动的片状细胞突起，主要通过小GTP酶分子开关Rac1发挥作用。在Fcγ受体介导的对IgG调理红细胞（IgG-E）的吞噬作用中，沿IgG-E表面的片状伪足延伸需要Rac1激活。然而，Rac1失活在吞噬体形成中的意义却知之甚少。我们的活细胞成像和电子显微镜研究表明，表达组成型活性Rac1突变体的RAW264巨噬细胞在吞噬杯形成方面存在缺陷，而在IgG-E周围形成了片状伪足。由于激活的Rac1降低了肌球蛋白轻链的磷酸化水平，吞噬杯形成失败可能是由于肌动蛋白/肌球蛋白II收缩性受到抑制。在用IgG-E喂养的巨噬细胞中对Rac1开关进行可逆的光操纵，可以模拟两种片状伪足运动：分别通过Rac1的开启和关闭实现向外延伸和杯状收缩。结合Rac1活性的荧光共振能量转移成像，我们提供了一种由吞噬杯内Rac1开关时空控制的吞噬体形成的新机制模型。
