Tropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

原肌球蛋白通过沿着肌动蛋白丝的两条链形成长链来调节肌动蛋白细胞骨架的动力学和功能，这两条链充当其他肌动蛋白结合蛋白结合的看门人。原肌球蛋白与肌动蛋白结合的基本分子相互作用仍然知之甚少。使用微流体和荧光显微镜，我们实时观察了荧光标记的原肌球蛋白亚型 Tpm1.8 与未标记的肌动蛋白丝的结合。这种方法与数学模型相结合，使我们能够在单丝和单分子水平上量化 Tpm1.8 的成核、组装和分解动力学。我们的分析表明 Tpm1.8 独立装饰肌动蛋白丝的两条链。一旦第一个 Tpm1.8 分子通过添加第二个分子而稳定，生长中的原肌球蛋白结构域就很有可能继续成核，最终导致肌动蛋白丝的完全修饰。此外，Tpm1.8 结构域是不对称的，在朝向肌动蛋白丝带刺末端的边缘处具有增强的动力学。这里介绍的 Tpm1.8 肌动蛋白丝动力学的完整描述提供了对肌动蛋白-原肌球蛋白丝形成以及原肌球蛋白在调节肌动蛋白丝动力学中的作用的分子见解。