Shatskiy Dmitry, Sivan Athul, Wedlich-Söldner Roland, Belyy Alexander
Membrane Enzymology Group, Groningen Institute of Biomolecular Sciences and Biotechnology (GBB), Faculty of Science and Engineering, University of Groningen , Groningen, The Netherlands.
Institute of Cell Dynamics and Imaging, and Cells-in-Motion Interfaculty Center (CiMIC), University of Münster , Münster, Germany.
J Cell Biol. 2025 Apr 7;224(4). doi: 10.1083/jcb.202409192. Epub 2025 Feb 10.
F-tractin is a peptide widely used to visualize the actin cytoskeleton in live eukaryotic cells but has been reported to impair cell migration and induce actin bundling at high expression levels. To elucidate these effects, we determined the cryo-EM structure of the F-tractin-F-actin complex, revealing that F-tractin consists of a flexible N-terminal region and an amphipathic C-terminal helix. The N-terminal part is dispensable for F-actin binding but responsible for the bundling effect. Based on these insights, we developed an optimized F-tractin, which eliminates the N-terminal region and minimizes bundling while retaining strong actin labeling. The C-terminal helix interacts with a hydrophobic pocket formed by two neighboring actin subunits, an interaction region shared by many actin-binding polypeptides, including the popular actin-binding probe Lifeact. Thus, rather than contrasting F-tractin and Lifeact, our data indicate that these peptides have analogous modes of interaction with F-actin. Our study dissects the structural elements of F-tractin and provides a foundation for developing future actin probes.
F-肌动蛋白结合肽（F-tractin）是一种广泛用于可视化活真核细胞中肌动蛋白细胞骨架的肽，但据报道，在高表达水平时它会损害细胞迁移并诱导肌动蛋白成束。为了阐明这些影响，我们确定了F-肌动蛋白结合肽-F-肌动蛋白复合物的冷冻电镜结构，揭示F-肌动蛋白结合肽由一个柔性的N端区域和一个两亲性的C端螺旋组成。N端部分对于F-肌动蛋白结合并非必需，但却是成束效应的原因。基于这些见解，我们开发了一种优化的F-肌动蛋白结合肽，它去除了N端区域并在保留强肌动蛋白标记的同时将成束作用最小化。C端螺旋与由两个相邻肌动蛋白亚基形成的疏水口袋相互作用，这是许多肌动蛋白结合多肽（包括常用的肌动蛋白结合探针Lifeact）共有的相互作用区域。因此，我们的数据表明，这些肽与F-肌动蛋白的相互作用模式类似，而不是将F-肌动蛋白结合肽和Lifeact进行对比。我们的研究剖析了F-肌动蛋白结合肽的结构元件，并为开发未来的肌动蛋白探针奠定了基础。
