PDF(Pubmed)
Abstract：
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 (IC50 = 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 ×107 s-1 M-1) and off-rate (0.03 s-1) 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 (Kd = 0.9 nM) and filament sides (Kd = 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.
摘要：
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,和其他细胞过程仍然没有被很好地理解。这里,我们研究了Arp2/3复合物的两个结合伴侣之间的机制关系，胶质细胞成熟因子（GMF）和皮质肌动蛋白。个别地,GMF和cortactin对肌动蛋白丝分支的稳定性有相反的影响，但它是未知的，他们如何协同工作，以管理分支机构的营业额。使用TIRF显微镜，我们观察到GMF的分支去稳定活性被cortactin（IC50=1.3nM）有效阻断，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的模型是结合Arp2/3复合物的竞争。然而,我们发现cortactin和GMF在溶液中不竞争游离的Arp2/3复合物。Further,我们使用单分子分析表明，分支连接处的cortactin的结合率（3×107s-1M-1）和解离率（0.03s-1）受过量GMF的影响最小。一起,这些结果表明，皮质肌动蛋白以高亲和力与分支连接结合，它阻止了GMF的不稳定影响，可能是通过一种本质上是变构的机制。此外,我们在肌动蛋白丝分支连接处(Kd=0.9nM)和丝侧(Kd=206nM)测量的cortactin亲和力比以前报道的强约20倍.这些观察结果有助于对Arp2/3复合物如何通过多个输入的整合来调节的分子复杂性的新观点。
