Structure of the Lifeact-F-actin complex.
Structure of the Lifeact-F-actin complex.
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DOI：
10.1371/journal.pbio.3000925
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发表时间：
2020-11
期刊：
影响因子：
9.8
通讯作者：
Raunser S
中科院分区：
文献类型：
--
作者：
Belyy A;Merino F;Sitsel O;Raunser S
Lifeact是一种短小的肌动蛋白结合肽，用于在荧光显微镜下观察活的真核细胞中的丝状肌动蛋白(F-actin)结构。然而，这种流行的探针已经被证明通过影响细胞骨架的结构来改变细胞的形态。人们对这种人工制品的分子基础知之甚少。在这里，我们用电子冷冻显微镜(Cryo-EM)确定了Lifeact-F-肌动蛋白复合体的高分辨结构。结构显示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，稳定闭合构象中肌动蛋白的DNase I结合环(D-环)。有趣的是，疏水结合位点也被肌动蛋白结合蛋白，如cofilin和myosin以及肌动蛋白结合毒素所使用，例如发光杆菌的TccC3的高变区(TccC3HVR)和铜绿假单胞菌的ExoY。体外结合分析和活性测量表明，Lifeact确实与这些蛋白质竞争，为Lifeact对体内细胞形态的改变提供了解释。最后，我们证明了Lifeact对F-肌动蛋白的亲和力可以通过在多肽中引入突变来增加，为设计用于活细胞成像的改进的肌动蛋白探针奠定了基础。Lifeact是一种短的肌动蛋白结合肽，广泛用于在活细胞荧光显微镜下显示丝状肌动蛋白结构。这项冷冻EM研究揭示了Lifeact如何与Cofilin和Myosin等肌动蛋白结合蛋白竞争，从而为Lifeact如何改变细胞形态提供了解释，而结构导引的定点突变表明Lifeact的亲和力是可以调节的。
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 the 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 is a short actin-binding peptide that is widely used to visualize filamentous actin structures in live cell fluorescence microscopy. This cryo-EM study reveals how Lifeact competes with actin-binding proteins such as cofilin and myosin, providing an explanation for how Lifeact alters cell morphology, while structure-guided site directed mutagenesis demonstrates that the affinity of Lifeact can be modulated.
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影响因子：
4.6
作者：
Lopata A;Hughes R;Tiede C;Heissler SM;Sellers JR;Knight PJ;Tomlinson D;Peckham M
通讯作者：
Peckham M
影响因子：
4.8
作者：
Belyy, Alexander;Santecchia, Ignacio;Mechold, Undine
通讯作者：
Mechold, Undine
肌动蛋白去聚合因子（ADF/Cofilin）提高了细丝流动率：基于肌动蛋白的运动的影响。
DOI：
10.1083/jcb.136.6.1307
发表时间：
1997-03-24
期刊：
The Journal of cell biology
影响因子：
--
作者：
Carlier MF;Laurent V;Santolini J;Melki R;Didry D;Xia GX;Hong Y;Chua NH;Pantaloni D
通讯作者：
Pantaloni D
影响因子：
48
作者：
DiMaio, Frank;Song, Yifan;Li, Xueming;Brunner, Matthias J.;Xu, Chunfu;Conticello, Vincent;Egelman, Edward;Marlovits, Thomas C.;Cheng, Yifan;Baker, David
通讯作者：
Baker, David
DOI：
10.1016/0005-2795(75)90196-8
发表时间：
1975-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
DANCKER, P;LOW, I;WIELAND, T
通讯作者：
WIELAND, T