Department of Chemistry, Center for NanoScience, Nanosystems Initiative Munich (NIM) and Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians University Munich, Munich, Germany.
Department of Chemistry, Center for NanoScience, Nanosystems Initiative Munich (NIM) and Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians University Munich, Munich, Germany; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
Biophys J. 2022 Jan 18;121(2):327-335. doi: 10.1016/j.bpj.2021.12.011. Epub 2021 Dec 9.
Actin filament dynamics underlie key cellular processes. Although the elongation of actin filaments has been extensively studied, the mechanism of nucleation remains unclear. The micromolar concentrations needed for filament formation have prevented direct observation of nucleation dynamics on the single molecule level. To overcome this limitation, we have used the attoliter excitation volume of zero-mode waveguides to directly monitor the early steps of filament assembly. Immobilizing single gelsolin molecules as a nucleator at the bottom of the zero-mode waveguide, we could visualize the actin filament nucleation process. The process is surprisingly dynamic, and two distinct populations during gelsolin-mediated nucleation are observed. The two populations are defined by the stability of the actin dimers and determine whether elongation occurs. Furthermore, by using an inhibitor to block flattening, a conformational change in actin associated with filament formation, elongation was prevented. These observations indicate that a conformational transition and pathway competition determine the nucleation of gelsolin-mediated actin filament formation.
肌动蛋白丝动力学是细胞关键过程的基础。尽管肌动蛋白丝的延伸已被广泛研究，但成核机制仍不清楚。形成丝状所需的微摩尔浓度阻止了在单分子水平上直接观察成核动力学。为了克服这一限制，我们使用零模波导的 attoliter 激发体积直接监测丝状组装的早期步骤。将单个凝胶蛋白分子作为成核物固定在零模波导的底部，我们可以可视化肌动蛋白丝成核过程。该过程出人意料地具有动态性，并且在凝胶蛋白介导的成核过程中观察到两种不同的群体。这两个群体由肌动蛋白二聚体的稳定性定义，并决定是否发生延伸。此外，通过使用抑制剂来阻止扁平，这是与丝状形成相关的肌动蛋白的构象变化，从而阻止了延伸。这些观察结果表明，构象转变和途径竞争决定了凝胶蛋白介导的肌动蛋白丝形成的成核。
