Department of Biochemistry, Brandeis University, Waltham, MA 02454-9110, USA.
Proc Natl Acad Sci U S A. 2013 Jan 22;110(4):1285-90. doi: 10.1073/pnas.1211164110. Epub 2013 Jan 4.
Actin filament nucleation by actin-related protein (Arp) 2/3 complex is a critical process in cell motility and endocytosis, yet key aspects of its mechanism are unknown due to a lack of real-time observations of Arp2/3 complex through the nucleation process. Triggered by the verprolin homology, central, and acidic (VCA) region of proteins in the Wiskott-Aldrich syndrome protein (WASp) family, Arp2/3 complex produces new (daughter) filaments as branches from the sides of preexisting (mother) filaments. We visualized individual fluorescently labeled Arp2/3 complexes dynamically interacting with and producing branches on growing actin filaments in vitro. Branch formation was strikingly inefficient, even in the presence of VCA: only ~1% of filament-bound Arp2/3 complexes yielded a daughter filament. VCA acted at multiple steps, increasing both the association rate of Arp2/3 complexes with mother filament and the fraction of filament-bound complexes that nucleated a daughter. The results lead to a quantitative kinetic mechanism for branched actin assembly, revealing the steps that can be stimulated by additional cellular factors.
肌动蛋白相关蛋白（Arp）2/3 复合物引发的肌动蛋白丝成核是细胞运动和胞吞作用的关键过程，但由于缺乏实时观察到 Arp2/3 复合物在成核过程中的情况，其机制的关键方面仍不清楚。Wiskott-Aldrich 综合征蛋白（WASp）家族蛋白的肌动蛋白丝结合重复（VCA）区域触发 Arp2/3 复合物从预先存在的（母）肌动蛋白丝的侧面产生新的（子）丝。我们在体外可视化了单个荧光标记的 Arp2/3 复合物与生长中的肌动蛋白丝动态相互作用并在其上产生分支。分支形成的效率非常低，即使存在 VCA 也是如此：只有约 1%的与丝结合的 Arp2/3 复合物产生了一个子丝。VCA 在多个步骤中起作用，增加了 Arp2/3 复合物与母丝的结合速率，以及与丝结合的复合物中引发子丝的部分。结果得到了一个分支肌动蛋白组装的定量动力学机制，揭示了可以被其他细胞因子刺激的步骤。
