The intracellular events underlying phagocytosis, a crucial event for innate immunity, are still unresolved. In order to test whether the reservoir of membrane for the formation of the phagocytic pseudopodia is maintained by cortical ezrin, and that its cleavage is a key step in releasing this membrane, the cleavage of cortical ezrin was monitored within living phagocytes (phagocytically-competent cell line RAW264.7), by expressing two ezrin constructs with fluorescent protein tags located either inside the FERM or at the actin binding domains. When ezrin is cleaved in the linker region by the Ca2+ activated protease, calpain, separation of the two fluo-Tags would result. Experimentally-induced Ca2+influx triggered cleavage of peripherally located ezrin; which was temporally associated with cell expansion. Ezrin cleavage was also observed during phagocytosis, localised to the phagocytic pseudopodia. Thus, our data demonstrates that peripheral ezrin is cleaved during Ca2+-influx induced "membrane expansion" and locally within the extending pseudopodia during phagocytosis. This is consistent with a role for intact ezrin in maintaining folded membrane on the cell surface which becomes available for cell spreading and phagocytosis.

中文翻译：

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在细胞表面积扩张过程中，活髓细胞中 Ca2+ 激活的埃兹蛋白裂解动态可视化。

吞噬作用是先天免疫的关键事件，其背后的细胞内事件仍未得到解决。为了测试形成吞噬伪足的膜库是否由皮质埃兹蛋白维持，并且其裂解是释放该膜的关键步骤，在活体吞噬细胞（吞噬细胞）内监测皮质埃兹蛋白的裂解。线RAW264.7），通过表达两个带有荧光蛋白标签的ezrin构建体，该荧光蛋白标签位于FERM内部或肌动蛋白结合域处。当埃兹蛋白在连接区被 Ca2+ 激活的蛋白酶、钙蛋白酶切割时，就会导致两个氟标签的分离。实验诱导的 Ca2+ 内流触发了外周埃兹蛋白的裂解；这在时间上与细胞扩张相关。在吞噬过程中也观察到埃兹蛋白裂解，位于吞噬细胞伪足。因此，我们的数据表明，外周埃兹蛋白在Ca2+流入诱导的“膜扩张”期间被裂解，并且在吞噬作用期间在延伸的伪足内局部裂解。这与完整的埃兹蛋白在细胞表面维持折叠膜的作用一致，该折叠膜可用于细胞扩散和吞噬作用。