F-actin binding is essential for coronin 1B function in vivo
F-actin binding is essential for coronin 1B function in vivo
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DOI：
10.1242/jcs.007641
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发表时间：
2007-05-15
影响因子：
4
通讯作者：
Bear, James E.
中科院分区：
文献类型：
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作者：
Cai, Liang;Makhov, Alexander M.;Bear, James E.
冠蛋白是一种保守的F-肌动蛋白结合蛋白，参与成纤维细胞迁移、吞噬和趋化等过程。我们实验室的最新数据表明，冠蛋白1B协调Arp 2/3依赖的肌动蛋白丝成核和cofilin介导的丝周转在迁移成纤维细胞的前沿。冠蛋白功能的分析一直受到阻碍，缺乏一个清晰的了解冠蛋白如何与F-肌动蛋白相互作用。在这里，我们确定了一个表面暴露的保守精氨酸残基的位置30（R30），这是至关重要的冠状蛋白1B结合F-肌动蛋白在体外和体内。使用肌动蛋白共沉降，我们证明冠蛋白1B结合ATP/ADP-P-i-F-肌动蛋白（170 nM）的亲和力高，与ADP-F-肌动蛋白（8 μ M）的亲和力低47倍。与以前的研究相比，我们没有发现证据增强cofilin结合F-肌动蛋白的存在下，无论是冠蛋白1B或冠蛋白1A。相反，我们发现冠蛋白1B保护肌动蛋白丝免受cofilin诱导的解聚。与冠蛋白1B和F-肌动蛋白在体内相互作用的重要作用一致，不结合F-肌动蛋白的R30 D冠蛋白突变体无效地定位于前沿。此外，我们的分析表明，F-肌动蛋白的结合是绝对需要冠蛋白1B发挥其对全细胞运动和板层脂质动力学的影响。
Coronins are conserved F-actin binding proteins that have been implicated in a variety of processes including fibroblast migration, phagocytosis, and chemotaxis. Recent data from our lab indicate that coronin 1B coordinates Arp2/3-dependent actin filament nucleation and cofilin-mediated filament turnover at the leading edge of migrating fibroblasts. Analysis of coronin function has been hampered by the lack of a clear understanding of how coronin interacts with F-actin. Here, we identify a surface-exposed conserved arginine residue at position 30 (R30), which is crucial for coronin 1B binding to F-actin both in vitro and in vivo. Using actin co-sedimentation, we demonstrate that coronin 1B binds with high affinity to ATP/ADP-P-i -F-actin (170 nM) and with 47-fold lower affinity to ADP-F-actin (8 mu M). In contrast to a previous study, we find no evidence for enhanced cofilin binding to F-actin in the presence of either coronin 1B or coronin 1A. Instead, we find that coronin 1B protects actin filaments from cofilin-induced depolymerization. Consistent with an important role for interactions between coronin 1B and F- actin in vivo, an R30D coronin mutant that does not bind F- actin localizes inefficiently to the leading edge. Furthermore, our analysis indicates that F- actin binding is absolutely required for coronin 1B to exert its effects on whole-cell motility and lamellipodial dynamics.