Department of Bioengineering & Biophysics Program, University of California, Berkeley, Berkeley, United States.
Division of Biological Systems & Engineering, Lawrence Berkeley National Laboratory, Berkeley, United States.
Elife. 2022 Jun 24;11:e73145. doi: 10.7554/eLife.73145.
Branched actin networks are self-assembling molecular motors that move biological membranes and drive many important cellular processes, including phagocytosis, endocytosis, and pseudopod protrusion. When confronted with opposing forces, the growth rate of these networks slows and their density increases, but the stoichiometry of key components does not change. The molecular mechanisms governing this force response are not well understood, so we used single-molecule imaging and AFM cantilever deflection to measure how applied forces affect each step in branched actin network assembly. Although load forces are observed to increase the density of growing filaments, we find that they actually decrease the rate of filament nucleation due to inhibitory interactions between actin filament ends and nucleation promoting factors. The force-induced increase in network density turns out to result from an exponential drop in the rate constant that governs filament capping. The force dependence of filament capping matches that of filament elongation and can be explained by expanding Brownian Ratchet theory to cover both processes. We tested a key prediction of this expanded theory by measuring the force-dependent activity of engineered capping protein variants and found that increasing the size of the capping protein increases its sensitivity to applied forces. In summary, we find that Brownian Ratchets underlie not only the ability of growing actin filaments to generate force but also the ability of branched actin networks to adapt their architecture to changing loads.
分支肌动蛋白网络是自我组装的分子马达，能够移动生物膜并驱动许多重要的细胞过程，包括吞噬作用、内吞作用和伪足突起。当面临相反的力时，这些网络的生长速度会减慢，密度会增加，但关键成分的化学计量不变。控制这种力响应的分子机制尚未得到很好的理解，因此我们使用单分子成像和原子力显微镜悬臂挠度来测量施加的力如何影响分支肌动蛋白网络组装的每个步骤。尽管负载力被观察到增加了生长丝的密度，但我们发现由于肌动蛋白丝末端和核促进因子之间的抑制相互作用，它们实际上降低了丝核的速率。网络密度的力诱导增加是由于控制丝帽化的速率常数呈指数下降所致。丝帽化的力依赖性与丝延伸的力依赖性相匹配，并且可以通过扩展布朗棘轮理论来涵盖这两个过程来解释。我们通过测量工程化帽蛋白变体的力依赖性活性来测试该扩展理论的一个关键预测，并发现帽蛋白的尺寸增加会增加其对施加力的敏感性。总之，我们发现布朗棘轮不仅是生长肌动蛋白丝产生力的能力的基础，也是分支肌动蛋白网络适应不断变化的负载的结构的基础。
