Dynamics of cytoskeletal proteins during Fcγ receptor-mediated phagocytosis in macrophages
Dynamics of cytoskeletal proteins during Fcγ receptor-mediated phagocytosis in macrophages
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DOI：
10.1091/mbc.01-05-0273
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发表时间：
2002-02-01
影响因子：
3.3
通讯作者：
Swanson, JA
中科院分区：
文献类型：
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作者：
Diakonova, M;Bokoch, G;Swanson, JA
颗粒吞噬的结果从顺序重排的肌动蛋白细胞骨架和覆盖膜。为了组装吞噬体形成过程中的分子事件年表，并检查磷酸肌醇3-激酶（PI 3-激酶）对这些动力学的贡献，开发了一种用于同步小鼠巨噬细胞Fc γ受体介导的吞噬作用的方法。用补体成分C3 bi调理的红细胞在37 ℃下与巨噬细胞结合，该条件不利于颗粒吞噬作用。添加可溶性抗红细胞IgG导致结合红细胞的快速调理作用，随后通过吞噬作用立即内化。细胞内的F-肌动蛋白的含量，通过结合罗丹明鬼笔环肽，增加瞬时吞噬过程中，这种增加并没有减少PI 3-激酶的抑制剂。在吞噬过程中的不同时间固定的巨噬细胞中的肌球蛋白的免疫荧光定位表明，肌球蛋白II和IXb集中在早期吞噬体，肌球蛋白IC增加后，和肌球蛋白V出现后，吞噬体关闭。其他细胞骨架蛋白在吞噬体中出现的时间也有类似的变化。PI 3-激酶抑制剂渥曼青霉素没有改变PI 3-激酶或ezrin定位的动力学，但防止了PAK 1从吞噬体的丢失。这些结果表明PI 3-激酶使PAK 1失活，并且这可能是吞噬体关闭所需要的。
Particle ingestion by phagocytosis results from sequential rearrangements of the actin cytoskeleton and overlying membrane. To assemble a chronology of molecular events during phagosome formation ana to examine the contributions of phosphoinositide 3-kinase (PI 3-kinase) to these dynamics, a method was developed for synchronizing Fcgamma receptor-mediated phagocytosis by murine macrophages. Erythrocytes opsonized with complement component C3bi were bound to macrophages at 37degreesC, a condition that does not favor particle phagocytosis. Addition of soluble anti-erythrocyte IgG resulted in rapid opsonization of the bound erythrocytes, followed by their immediate internalization via phagocytosis. Cellular content of F-actin, as measured by binding of rhodamine-phalloidin, increased transiently during phagocytosis, and this increase was not diminished by inhibitors of PI 3-kinase. Immunofluorescence localization of myosins in macrophages fixed at various times during phagocytosis indicated that myosins II and IXb were concentrated in early phagosomes, myosin IC increased later, and myosin V appeared after phagosome closure. Other cytoskeletal proteins showed similar variations in the timing of their appearance in phagosomes. The PI 3-kinase inhibitor wortmannin did not change the dynamics of PI 3-kinase or ezrin localization but prevented the loss of PAK1 from phagosomes. These results suggest that PI 3-kinase deactivates PAK1, and that this may be needed for phagosome closure.