Label-Free Visualisation of Actin Nucleation and Polymerisation at the Single-Molecule Level using Interferometric Scattering Microscopy
Label-Free Visualisation of Actin Nucleation and Polymerisation at the Single-Molecule Level using Interferometric Scattering Microscopy
复制标题
使用干涉散射显微镜在单分子水平上无标记观察肌动蛋白成核和聚合
DOI：
10.1016/j.bpj.2017.11.2108
复制
发表时间：
2018
影响因子：
3.4
通讯作者：
Kukura P
中科院分区：
文献类型：
--
作者：
Hundt N;Tyler A;Young G;Cole D;Fineberg AJ;Andrecka J;Kukura P
肌动蛋白丝是细胞骨架的主要组成部分，参与细胞迁移、细胞粘附、细胞分裂和肌肉收缩等许多基本过程。肌动蛋白最重要的特性是它由球状亚基（G-肌动蛋白）形成细丝（F-肌动蛋白）。 20 世纪 60 年代，Oosawa 和同事开发了一个 G 肌动蛋白向 F 肌动蛋白转变的模型，其中限速步骤是形成稳定的肌动蛋白核，后来确定该核由 2-4 个肌动蛋白单体组成。通过向细丝末端添加单体来延长核。尽管分子细节永远无法通过实验可视化，但该模型已被广泛接受。在这里，我们使用干涉散射显微镜 (iSCAT) 在单分子水平上监测肌动蛋白丝的成核和伸长。基于最近在无标记单分子灵敏度和质量准确度方面的改进，我们可以揭示 G-肌动蛋白在与成核和丝生长相关的浓度下的多分散性，显示出与上述细胞核大小一致的小蛋白质寡聚体的特征。使用相同的方法，我们可以监测聚合过程中动态附着到细丝尖端和从细丝尖端分离的各个亚基的纳米级到达。与肌动蛋白完全由单体生长的标准模型相反，我们还发现了较大寡聚体被添加到丝中的特征。与我们的多分散性测量一起，这些结果表明肌动蛋白丝的成核和生长机制基于单体和小寡聚物，而不仅仅是单体。这些结果证明了 iSCAT 在无标记单分子成像和研究溶液中介观动力学机制方面的潜力。
Actin filaments are a major component of the cytoskeleton involved in many basic processes such as cell migration, cell adhesion, cell division and muscle contraction. The most important property of actin is that it forms filaments (F-actin) from globular subunits (G-actin). In the 1960s, Oosawa and co-workers developed a model for the G-to F-actin transition where the rate-limiting step is the formation of a stable actin nucleus, later determined to consist of 2-4 actin monomers. Nuclei are elongated by the addition of monomers to the filament ends. This model is widely accepted, although the molecular details could never be visualised experimentally.Here, we used interferometric scattering microscopy (iSCAT) to monitor nucleation and elongation of actin filaments at the single-molecule level. Building on recent improvements in label-free single molecule sensitivity and mass accuracy, we could reveal the polydispersity of G-actin at concentrations relevant to nucleation and filament growth, showing signatures of small protein oligomers in line with the nuclei sizes discussed above. Using the same approach, we could monitor the nanoscopic arrival of individual subunits dynamically attaching to and detaching from the filament tip during polymerisation. Contrary to the standard model, where actin grows exclusively from monomers, we also found signatures of larger oligomers being added to the filament. Together with our polydispersity measurements, these results point towards a nucleation and growth mechanism for actin filaments based on monomers and small oligomers, rather than exclusively monomers. These results demonstrate the potential of iSCAT for label-free single-molecule imaging and for investigating the mechanisms of mesoscopic dynamics in solution.