Single molecule visualization of tropomyosin isoform organization in the mammalian actin cytoskeleton.

Maria L Cagigas, Nicholas Ariotti, Jeff Hook, James Rae, Robert G Parton, Nicole S Bryce, Peter W Gunning, Edna C Hardeman
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Abstract

The actin cytoskeleton is composed of both branched and unbranched actin filaments. In mammals, the unbranched actin filaments are primarily copolymers of actin and tropomyosin. Biochemical and imaging studies indicate that different tropomyosin isoforms are segregated to different actin filament populations in cells and tissues, providing isoform-specific functionality to the actin filament. Intrinsic to this model is the prediction that single-molecule imaging of tropomyosin isoforms would confirm homopolymer formation along the length of single actin filaments, a knowledge gap that remains unaddressed in the cellular environment. We combined chemical labeling of genetically engineered tropomyosin isoforms with electron tomography to locate individual tropomyosin molecules in fibroblasts. We find that the organization of two non-muscle tropomyosins, Tpm3.1 with Tpm4.2, can be distinguished from each other using light and electron microscopy. Visualization of single tropomyosin molecules associated with actin filaments supports the hypothesis that tropomyosins form continuous homopolymers, instead of heteropolymers, in the presence of all physiologically native actin-binding proteins. This is true for both isoforms tested. Furthermore, the data suggest that the tropomyosin molecules on one side of an actin filament may not be in register with those on the opposite side, indicating that each tropomyosin polymer may assembly independently.

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哺乳动物肌动蛋白细胞骨架中肌球蛋白同工酶组织的单分子可视化。
肌动蛋白细胞骨架由分枝和不分枝肌动蛋白丝组成。在哺乳动物中,不分枝的肌动蛋白丝主要是肌动蛋白和肌球蛋白的共聚物。生化和成像研究表明,在细胞和组织中,不同的肌球蛋白同工酶被分离到不同的肌动蛋白丝群中,从而为肌动蛋白丝提供了同工酶特异性功能。这一模型的本质是预测肌球蛋白同工酶的单分子成像将确认沿单肌动蛋白丝长度的同聚物形成,而这一知识空白在细胞环境中仍未得到解决。我们将基因工程肌球蛋白同工酶的化学标记与电子断层扫描相结合,以确定成纤维细胞中单个肌球蛋白分子的位置。我们发现,两种非肌肉肌球蛋白(Tpm3.1 和 Tpm4.2)的组织可通过光镜和电子显微镜相互区分。与肌动蛋白丝相关的单个肌球蛋白分子的可视化支持了这样一种假设,即肌球蛋白在所有生理原生肌动蛋白结合蛋白的存在下形成连续的均聚物,而不是杂聚物。测试的两种同工酶都是如此。此外,数据还表明,肌动蛋白丝一侧的肌球蛋白分子可能与另一侧的肌球蛋白分子不一致,这表明每种肌球蛋白聚合物都可能独立组装。
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