Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium.
Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium.
复制标题
DOI：
10.1016/j.bpj.2015.06.066
复制
发表时间：
2015-08-18
影响因子：
3.4
通讯作者：
Fels J
中科院分区：
文献类型：
--
作者：
Kronlage C;Schäfer-Herte M;Böning D;Oberleithner H;Fels J
接触模式原子力显微镜 (AFM) 已被证明可以揭示皮质肌动蛋白结构。使用活内皮细胞，我们通过 AFM 和共焦荧光显微镜同时观察皮质肌动蛋白动态。我们提出了一种量化皮质肌动蛋白网机械超微结构动态变化的方法。我们认为，AFM 中常用的所谓误差信号成像可以对皮质肌动蛋白动力学进行定性而非定量分析。我们使用的方法包括基于快速力曲线的形貌成像和随后增强局部高度差的图像处理。可以通过表面粗糙度计算和自动形态测量来观察和量化细胞骨架网络组织的动态变化。用低浓度的肌动蛋白不稳定剂细胞松弛素 D 处理后，皮质细胞骨架网络变薄，平均网格尺寸增加。相比之下，jasplakinolide是一种增强肌动蛋白聚合的药物，可以巩固细胞骨架网络并减少平均网格面积。总之，可以量化活细胞中的皮质肌动蛋白动力学。据我们所知，这为对顶端质膜下方的内皮肌动蛋白网进行定量结构功能分析开辟了一条新途径。
Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane.