How cells tightly control the formation and turnover of branched actin filament arrays to drive cell motility, endocytosis, and other cellular processes is still not well understood. Here, we investigated the mechanistic relationship between two binding partners of the Arp2/3 complex, glia maturation factor (GMF) and cortactin. Individually, GMF and cortactin have opposite effects on the stability of actin filament branches, but it is unknown how they work in concert with each other to govern branch turnover. Using TIRF microscopy, we observe that GMF’s branch destabilizing activities are potently blocked by cortactin (IC = 1.3 nM) and that this inhibition requires direct interactions of cortactin with Arp2/3 complex. The simplest model that would explain these results is competition for binding Arp2/3 complex. However, we find that cortactin and GMF do not compete for free Arp2/3 complex in solution. Further, we use single molecule analysis to show that cortactin’s on-rate (3 ×10 s M) and off-rate (0.03 s) at branch junctions are minimally affected by excess GMF. Together, these results show that cortactin binds with high affinity to branch junctions, where it blocks the destabilizing effects of GMF, possibly by a mechanism that is allosteric in nature. In addition, the affinities we measure for cortactin at actin filament branch junctions (K = 0.9 nM) and filament sides (K = 206 nM) are approximately 20-fold stronger than previously reported. These observations contribute to an emerging view of molecular complexity in how Arp2/3 complex is regulated through the integration of multiple inputs.

中文翻译：

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直接观察 cortactin 保护 Arp2/3-肌动蛋白丝分支连接免受 GMF 介导的不稳定

细胞如何严格控制分支肌动蛋白丝阵列的形成和周转以驱动细胞运动、内吞作用和其他细胞过程仍不清楚。在这里，我们研究了 Arp2/3 复合物的两个结合伙伴——神经胶质成熟因子（GMF）和皮质素之间的机制关系。单独而言，GMF 和 cortactin 对肌动蛋白丝分支的稳定性具有相反的影响，但尚不清楚它们如何协同作用来控制分支周转。使用 TIRF 显微镜，我们观察到 GMF 的分支不稳定活性被 cortactin 有效阻断 (IC = 1.3 nM)，并且这种抑制需要 cortactin 与 Arp2/3 复合物的直接相互作用。解释这些结果的最简单模型是结合 Arp2/3 复合体的竞争。然而，我们发现 cortactin 和 GMF 在溶液中不会竞争游离的 Arp2/3 复合物。此外，我们使用单分子分析表明 Cortactin 在分支连接处的结合速率 (3 ×10 s M) 和解离速率 (0.03 s) 受过量 GMF 的影响最小。总之，这些结果表明 cortactin 以高亲和力与分支连接点结合，在分支连接处它可能通过本质上变构的机制阻止 GMF 的不稳定作用。此外，我们测量的肌动蛋白丝分支连接处 (K = 0.9 nM) 和丝侧面 (K = 206 nM) 的 cortactin 亲和力比之前报道的大约强 20 倍。这些观察结果有助于对 Arp2/3 复合物如何通过多种输入的整合进行调节的分子复杂性形成新的观点。