Actin fusion proteins alter the dynamics of mechanically induced cytoskeleton rearrangement.
Actin fusion proteins alter the dynamics of mechanically induced cytoskeleton rearrangement.
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DOI：
10.1371/journal.pone.0022941
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Kemkemer R
中科院分区：
文献类型：
--
作者：
Deibler M;Spatz JP;Kemkemer R
机械力可以调节活细胞的各种功能。细胞骨架是细胞内部信号和随后的生物反应中力的转导的关键要素。因此，细胞生物力学的许多研究都集中在收缩性肌动蛋白-肌球蛋白系统在这些过程中的作用。一种广泛使用的观察活细胞中动态肌动蛋白网络的方法是将荧光蛋白融合到肌动蛋白的转基因表达。然而，已经报道了GFP-肌动蛋白融合蛋白对细胞铺展、迁移和细胞粘附强度的不利影响。这些缺点被证明是部分克服的融合肌动蛋白结合肽的荧光蛋白。然而，目前还不清楚是否直接标记的肌动蛋白融合蛋白或间接标记通过这些嵌合体改变细胞和细胞骨架的生物力学响应的力量。我们研究了动态重组肌动蛋白应力纤维在细胞周期性机械负荷下瞬时表达EGFP-Lifeact或EYFP-肌动蛋白在大鼠胚胎成纤维细胞，并观察他们通过活细胞显微镜。我们的研究结果表明，机械诱导的肌动蛋白应力纤维重组表现出非常不同的动力学在EYFP-肌动蛋白细胞和EGFP-Lifeact细胞，后者显示出显着的协议，在相同的实验条件下，非转染细胞的重组动力学。
Mechanical forces can regulate various functions in living cells. The cytoskeleton is a crucial element for the transduction of forces in cell-internal signals and subsequent biological responses. Accordingly, many studies in cellular biomechanics have been focused on the role of the contractile acto-myosin system in such processes. A widely used method to observe the dynamic actin network in living cells is the transgenic expression of fluorescent proteins fused to actin. However, adverse effects of GFP-actin fusion proteins on cell spreading, migration and cell adhesion strength have been reported. These shortcomings were shown to be partly overcome by fusions of actin binding peptides to fluorescent proteins. Nevertheless, it is not understood whether direct labeling by actin fusion proteins or indirect labeling via these chimaeras alters biomechanical responses of cells and the cytoskeleton to forces. We investigated the dynamic reorganization of actin stress fibers in cells under cyclic mechanical loading by transiently expressing either egfp-Lifeact or eyfp-actin in rat embryonic fibroblasts and observing them by means of live cell microscopy. Our results demonstrate that mechanically-induced actin stress fiber reorganization exhibits very different kinetics in EYFP-actin cells and EGFP-Lifeact cells, the latter showing a remarkable agreement with the reorganization kinetics of non-transfected cells under the same experimental conditions.
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