Koundinya Neha, Aguilar Rey M, Wetzel Kathryn, Tomasso Meagan R, Nagarajan Priyashree, McGuirk Emma R, Padrick Shae B, Goode Bruce L
Department of Biology, Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts, USA.
Department of Biochemistry and Molecular Biology, Drexel University, Philadelphia, Pennsylvania, USA.
J Biol Chem. 2025 Mar;301(3):108191. doi: 10.1016/j.jbc.2025.108191. Epub 2025 Jan 16.
The rapid turnover of branched actin networks underlies key in vivo processes such as lamellipodial extension, endocytosis, phagocytosis, and intracellular transport. However, our understanding of the mechanisms used to dissociate, or "prune," branched filaments has remained limited. Glia maturation factor (GMF) is a cofilin family protein that binds to the Arp2/3 complex and catalyzes branch dissociation. Here, we show that another ligand of Arp2/3 complex, Saccharomyces cerevisiae coronin (Crn1), enhances Gmf1-mediated debranching by 8- to 10-fold, and that these effects depend on Arp2/3-binding "C" and "A" motifs in Crn1. Further, we show that Crn1 directly binds with high affinity (K = 1.4 nM) to S. cerevisiae GMF (Gmf1), and together they form a stable ternary Crn1-Gmf1-Arp2/3 complex in solution. Using single-molecule analysis, we show that Gmf1 binds transiently and multiple times to F-actin branch junctions prior to debranching. These and other results suggest a mechanism of mutual recruitment, in which Crn1 increases the on-rate of Gmf1 for branch junctions and Gmf1 blocks Crn1 binding to actin filament sides, increasing its availability to bind branch junctions. Taken together, these observations reveal an unanticipated mechanism in which two distinct ligands of the Arp2/3 complex bind to each other and synergize to prune actin branches.
分支肌动蛋白网络的快速周转是诸如片状伪足延伸、胞吞作用、吞噬作用和细胞内运输等关键体内过程的基础。然而，我们对用于解离或“修剪”分支丝的机制的理解仍然有限。神经胶质成熟因子（GMF）是一种与肌动蛋白相关蛋白2/3复合体（Arp2/3复合体）结合并催化分支解离的丝切蛋白家族蛋白。在此，我们表明Arp2/3复合体的另一种配体，酿酒酵母冠蛋白（Crn1），可将Gmf1介导的去分支作用增强8至10倍，且这些效应取决于Crn1中与Arp2/3结合的“C”和“A”基序。此外，我们表明Crn1以高亲和力（K = 1.4 nM）直接与酿酒酵母GMF（Gmf1）结合，并且它们在溶液中共同形成稳定的三元Crn1-Gmf1-Arp2/3复合体。使用单分子分析，我们表明Gmf1在去分支之前会短暂且多次地与F-肌动蛋白分支连接处结合。这些结果及其他结果提示了一种相互招募机制，其中Crn1增加了Gmf1与分支连接处的结合速率，而Gmf1阻止Crn1与肌动蛋白丝侧面结合，从而增加其与分支连接处结合的可用性。综上所述，这些观察结果揭示了一种意想不到的机制，即Arp2/3复合体的两种不同配体相互结合并协同作用以修剪肌动蛋白分支。
