Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.
PLoS One. 2012;7(1):e30033. doi: 10.1371/journal.pone.0030033. Epub 2012 Jan 18.
Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor that is required for macrophages to directionally migrate towards various chemoattractants. The chemotaxis defect of WASp-deficient cells and its activation by Cdc42 in vivo suggest that WASp plays a role in directional sensing, however, its precise role in macrophage chemotaxis is still unclear. Using shRNA-mediated downregulation of WASp in the murine monocyte/macrophage cell line RAW/LR5 (shWASp), we found that WASp was responsible for the initial wave of actin polymerization in response to global stimulation with CSF-1, which in Dictyostelium discoideum amoebae and carcinoma cells has been correlated with the ability to migrate towards chemoattractants. Real-time monitoring of shWASp cells, as well as WASp⁻/⁻ bone marrow-derived macrophages (BMMs), in response to a CSF-1 gradient revealed that the protrusions from WASp-deficient cells were directional, showing intact directional sensing. However, the protrusions from WASp-deficient cells demonstrated reduced persistence compared to their respective control shRNA and wild-type cells. Further examination showed that tyrosine phosphorylation of WASp was required for both the first wave of actin polymerization following global CSF-1 stimulation and proper directional responses towards CSF-1. Importantly, the PI3K, Rac1 and WAVE2 proteins were incorporated normally in CSF-1 - elicited protrusions in the absence of WASp, suggesting that membrane protrusion driven by the WAVE2 complex signaling is intact. Collectively, these results suggest that WASp and its phosphorylation play critical roles in coordinating the actin cytoskeleton rearrangements necessary for the persistence of protrusions required for directional migration of macrophages towards CSF-1.
Wiskott-Aldrich 综合征蛋白（WASp）是一种肌动蛋白成核促进因子，对于巨噬细胞向各种趋化因子定向迁移是必需的。WASp 缺陷细胞的趋化缺陷及其在体内被 Cdc42 激活表明，WASp 在定向感应中发挥作用，然而，其在巨噬细胞趋化中的精确作用仍不清楚。我们使用小鼠单核细胞/巨噬细胞系 RAW/LR5 中的 shRNA 介导的 WASp 下调（shWASp），发现 WASp 负责对 CSF-1 的全刺激做出反应的初始肌动蛋白聚合波，在变形虫 Dictyostelium discoideum 和癌细胞中，这与向趋化因子迁移的能力相关。实时监测 shWASp 细胞以及 WASp⁻/⁻骨髓来源的巨噬细胞（BMM）对 CSF-1 梯度的反应表明，WASp 缺陷细胞的突起是定向的，表现出完整的定向感应。然而，与各自的对照 shRNA 和野生型细胞相比，WASp 缺陷细胞的突起显示出减少的持久性。进一步的检查表明，WASp 的酪氨酸磷酸化对于 CSF-1 全刺激后第一波肌动蛋白聚合以及朝向 CSF-1 的适当定向反应都是必需的。重要的是，在没有 WASp 的情况下，PI3K、Rac1 和 WAVE2 蛋白在 CSF-1 引发的突起中正常整合，这表明由 WAVE2 复合物信号驱动的膜突起是完整的。总之，这些结果表明，WASp 及其磷酸化在协调肌动蛋白细胞骨架重排方面起着关键作用，这对于巨噬细胞朝向 CSF-1 的定向迁移所必需的突起的持久性是必需的。
