Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as a hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

Lifeact是一种短的肌动蛋白结合肽，可用于使用荧光显微镜观察真核生物活细胞中的丝状肌动蛋白（F-actin）结构。然而，已经显示出这种流行的探针通过影响细胞骨架的结构来改变细胞形态。此类人工制品的分子基础知之甚少。在这里，我们使用电子冷冻显微镜（cryo-EM）确定了Lifeact-F-肌动蛋白复合物的高分辨率结构。该结构表明Lifeact与F-肌动蛋白上的疏水结合口袋相互作用，并延伸到2个相邻的肌动蛋白亚基上，从而使肌动蛋白的DNase I结合环（D环）稳定在闭合构象中。有趣的是，疏水结合位点也被肌动蛋白结合蛋白（例如cofilin和肌球蛋白）以及肌动蛋白结合毒素所利用，铜绿假单胞菌（Pseudomonas aeruginosa）的Photorhabdus luminescens和ExoY 。体外结合测定和活性测量表明，Lifeact确实与这些蛋白质竞争，为Lifeact对体内细胞形态变化的影响提供了解释。最后，我们证明可以通过将突变引入肽中来提高Lifeact对F-肌动蛋白的亲和力，为设计用于活细胞成像的改良肌动蛋白探针奠定基础。