Mechanical Distortion of Single Actin Filaments Induced by External Force: Detection by Fluorescence Imaging
Mechanical Distortion of Single Actin Filaments Induced by External Force: Detection by Fluorescence Imaging
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DOI：
10.1016/j.bpj.2008.09.056
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发表时间：
2009-02-04
影响因子：
3.4
通讯作者：
Ishiwata, Shin'ichi
中科院分区：
文献类型：
--
作者：
Shimozawa, Togo;Ishiwata, Shin'ichi
肌动蛋白是细胞骨架的主要成分，它在肌肉细胞和非肌肉细胞中传递机械应力。作为开发一种能够报告施加在肌动蛋白丝上的机械应力的“生物 - 纳米应变仪”的第一步，我们定量检测了在各种张力（5 - 20皮牛）下连接到单个肌动蛋白丝的染料的荧光强度。张力是通过两个光学捕获的塑料珠子施加的，这些珠子共价涂覆有化学修饰的重酶解肌球蛋白分子，这些分子连接到肌动蛋白丝的两端区域。结果，我们发现，在肌动蛋白丝中，20%的单体在半胱氨酸（374）处用四甲基罗丹明（TMR） - 5 - 马来酰亚胺标记，并且丝状结构用非荧光鬼笔环肽稳定，其荧光强度每施加10皮牛的力降低约6%，而在半胱氨酸（374）处用BODIPY TMR尸胺 - 碘乙酰胺或罗丹明 - 鬼笔环肽标记的肌动蛋白丝的荧光强度每施加10皮牛的力仅降低约2%。另一方面，对肌动蛋白溶液的光谱测量表明，TMR - 肌动蛋白的荧光强度在聚合（G - F转变）时增加了1.65倍，而BODIPY - 肌动蛋白的荧光强度仅增加了1.06倍。这些结果表明外力使丝结构变形，使得半胱氨酸（374）周围的微环境接近G - 肌动蛋白中的情况。因此我们得出结论，掺入肌动蛋白适当位点的荧光染料能够报告结合位点的机械变形，这是生物 - 纳米应变仪的必要条件。
Actin is a major component of the cytoskeleton that transmits mechanical stress in both muscle and nonmuscle cells. As the first step toward developing a "bio-nano strain gauge" that would be able to report the mechanical stress imposed on an actin filament, we quantitatively examined the fluorescence intensity of dyes attached to single actin filaments under various tensile forces (5-20 pN). Tensile force was applied via two optically trapped plastic beads covalently coated with chemically modified heavy meromyosin molecules that were attached to both end regions of an actin filament. As a result, we found that the fluorescence intensity of an actin filament, where 20% of monomers were labeled with tetramethylrhodamine (TMR)5-maleimide at Cys(374) and the filamentous structure was stabilized with nonfluorescent phalloidin, decreased by similar to 6% per 10 pN of the applied force, whereas the fluorescence intensity of an actin filament labeled with either BODIPY TMR cadaverin-iodoacetamide at Cys(374) or rhodamine-phalloidin showed only an similar to 2% decrease per 10 pN of the applied force. On the other hand, spectroscopic measurements of actin solutions showed that the fluorescence intensity of TMR-actin increased 1.65-fold upon polymerization (G-F transformation), whereas that of BODIPY-actin increased only 1.06-fold. These results indicate that the external force distorts the filament structure, such that the microenvironment around Cys(374) approaches that in G-actin. We thus conclude that the fluorescent dye incorporated into an appropriate site of actin can report the mechanical distortion of the binding site, which is a necessary condition for the bio-nano strain gauge.