School of Biosciences, University of Kent, Canterbury, United Kingdom.
Department of Mathematical Sciences, University of Essex, Colchester, United Kingdom.
Elife. 2021 Sep 27;10:e69184. doi: 10.7554/eLife.69184.
Regulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which enables tropomyosin to expose myosin-binding sites on actin. Myosin binding holds tropomyosin in an open position, exposing more myosin-binding sites on actin, leading to cooperative activation. At lower calcium levels, troponin and tropomyosin turn off the thin filament; however, this is antagonised by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation, we used single-molecule imaging of green fluorescent protein (GFP)-tagged myosin-S1 (S1-GFP) to follow the activation of RTF tightropes. In sub-maximal activation conditions, RTFs are not fully active, enabling direct observation of deactivation in real time. We observed that myosin binding occurs in a stochastic step-wise fashion; however, an unexpectedly large probability of multiple contemporaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated active process.
调节性细肌丝 (RTFs) 通过钙结合肌钙蛋白来紧密控制横纹肌收缩，这使得肌球蛋白结合蛋白能够暴露肌动蛋白上的肌球蛋白结合位点。肌球蛋白结合将肌球蛋白结合蛋白保持在开放状态，暴露出更多的肌球蛋白结合位点在肌动蛋白上，导致协同激活。在较低的钙水平下，肌钙蛋白和肌球蛋白结合蛋白会关闭细肌丝；然而，这被肌球蛋白的局部高浓度所拮抗，这就提出了一个问题，即细肌丝如何松弛。为了提供失活的分子细节，我们使用绿色荧光蛋白 (GFP) 标记的肌球蛋白 S1 (S1-GFP) 的单分子成像来跟踪 RTF 紧绳的激活。在亚最大激活条件下，RTFs 不是完全活跃的，这使得可以实时直接观察失活过程。我们观察到肌球蛋白的结合是一种随机的逐步方式发生的；然而，观察到多个同时脱离的概率异常大。这表明细肌丝的失活是一个协调的主动过程。
