Discipline of Medical Imaging Science, School of Health Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia.
Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
Biomolecules. 2024 Sep 30;14(10):1240. doi: 10.3390/biom14101240.
Tropomyosins (Tpms) are rod-shaped proteins that interact head-to-tail to form a continuous polymer along both sides of most cellular actin filaments. Head-to-tail interaction between adjacent Tpm molecules and the formation of an overlap complex between them leads to the assembly of actin filaments with one type of Tpm isoform in time and space. Variations in the affinity of tropomyosin isoforms for different actin structures are proposed as a potential sorting mechanism. However, the detailed mechanisms of the spatio-temporal sorting of Tpms remain elusive. In this study, we investigated the early intermediates during actin-tropomyosin filament assembly, using a skeletal/cardiac Tpm isoform (Tpm1.1) and a cytoskeletal isoform (Tpm1.6) that differ only in the last 27 amino acids. We investigated how the muscle isoform Tpm1.1 and the cytoskeletal isoform Tpm1.6 nucleate domains on the actin filament, and tested whether (1) recruitment is affected by the actin isoform (muscle vs. cytoskeletal) and (2) whether there is specificity in recruiting the same isoform to a domain at these early stages. To address these questions, actin filaments were exposed to low concentrations of fluorescent tropomyosins in solution. The filaments were immobilized onto glass coverslips and the pattern of decoration was visualized by TIRF microscopy. We show that at the early assembly stage, tropomyosins formed multiple distinct fluorescent domains (here termed "cluster") on the actin filaments. An automated image analysis algorithm was developed and validated to identify clusters and estimate the number of tropomyosins in each cluster. The analysis showed that tropomyosin isoform sorting onto an actin filament is unlikely to be driven by a preference for nucleating on the corresponding muscle or cytoskeletal actin isoforms, but rather is facilitated by a higher probability of incorporating the same tropomyosin isoforms into an early assembly intermediate. We showed that the 27 amino acids at the end of each tropomyosin seem to provide enough molecular information for the attachment of the same tropomyosin isoforms adjacent to each other on an actin filament. This results in the formation of homogeneous clusters composed of the same isoform rather than clusters with mixed isoforms.
原肌球蛋白（Tpms）是一种杆状蛋白，它沿着大多数细胞肌动蛋白丝的两侧头对头地相互作用形成连续聚合物。相邻 Tpm 分子之间的头对头相互作用以及它们之间重叠复合物的形成，导致具有一种类型的 Tpm 同工型的肌动蛋白丝在时间和空间上的组装。肌球蛋白同工型与不同肌动蛋白结构的亲和力的变化被认为是一种潜在的分拣机制。然而，Tpms 的时空分拣的详细机制仍不清楚。在这项研究中，我们使用一种骨骼肌/心肌 Tpm 同工型（Tpm1.1）和一种细胞骨架同工型（Tpm1.6）研究了肌动球蛋白丝组装过程中的早期中间产物，这两种同工型仅在最后 27 个氨基酸上有所不同。我们研究了肌球蛋白同工型 Tpm1.1 和细胞骨架同工型 Tpm1.6 如何在肌动蛋白丝上成核域，以及测试 1）招募是否受肌动蛋白同工型（肌肉与细胞骨架）的影响，2）是否存在将相同同工型招募到这些早期阶段的同一域的特异性。为了解决这些问题，将肌动蛋白丝暴露于溶液中的低浓度荧光肌球蛋白中。将丝固定在玻璃载玻片上，并通过 TIRF 显微镜观察装饰图案。我们表明，在早期组装阶段，肌球蛋白在肌动蛋白丝上形成多个不同的荧光域（此处称为“簇”）。开发并验证了一种自动图像分析算法来识别簇并估计每个簇中的肌球蛋白数量。分析表明，肌球蛋白同工型在肌动蛋白丝上的分拣不太可能是由于优先在相应的肌肉或细胞骨架肌动蛋白同工型上成核驱动的，而是由于将相同的肌球蛋白同工型纳入早期组装中间体的可能性更高而得到促进。我们表明，每个肌球蛋白末端的 27 个氨基酸似乎为相邻肌球蛋白同工型在肌动蛋白丝上的附着提供了足够的分子信息。这导致形成由相同同工型组成的同质簇，而不是具有混合同工型的簇。
