A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks.
A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks.
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DOI：
10.1038/s41467-021-25682-5
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发表时间：
2021-09-09
影响因子：
16.6
通讯作者：
Bieling P
中科院分区：
文献类型：
--
作者：
Funk J;Merino F;Schaks M;Rottner K;Raunser S;Bieling P
异源二聚体加帽蛋白（CP/CapZ）是肌动蛋白分支网络组装的重要因素，它推动细胞膜以驱动各种细胞过程。除了终止细丝生长，CP通过不清楚的机制增强了分支肌动蛋白网络中Arp 2/3复合物对肌动蛋白细丝的成核作用。在这里，我们结合联合收割机结构生物学与体外重建证明，CP不仅终止长丝伸长，但间接刺激活性的Arp 2/3激活成核促进因子（NPFs），防止其协会的长丝倒刺结束。这个功能的关键是CP的C-末端“tenomerous”延伸，它在空间上掩盖了末端肌动蛋白原聚体的主要相互作用位点。β延伸酶的缺失仅适度地损害加帽。然而，在一个不断增长的分支肌动蛋白网络的背景下，它的去除有力地抑制成核促进因子通过拴在他们的加帽丝端。NPF的末端拴系防止其负载活化Arp 2/3复合物所需的肌动蛋白单体，从而强烈抑制细胞和重建运动性测定中的分支网络组装。我们的研究结果从机械上解释了CP如何在分支肌动蛋白网络组装中耦合两个相反的过程-加帽和成核。分支肌动蛋白网络的组装依赖于异二聚体加帽蛋白CP/CapZ。结合cryoEM，体外重建和细胞生物学测定，作者表明，CP不仅可以防止肌动蛋白丝的伸长，但也选择性地掩盖肌动蛋白丝末端，以促进成核。
Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP’s C-terminal “tentacle” extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the β tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes—capping and nucleation—in branched actin network assembly. The assembly of branched actin networks depends on the heterodimeric capping protein CP/CapZ. Combining cryoEM, in vitro reconstitution and cell biological assays, the authors show that CP not only prevents actin filament elongation but also selectively masks actin filament ends to promote nucleation.
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