Measuring the regulation of keratin filament network dynamics
Measuring the regulation of keratin filament network dynamics
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DOI：
10.1073/pnas.1306020110
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发表时间：
2013-06-25
影响因子：
11.1
通讯作者：
Windoffer, Reinhard
中科院分区：
文献类型：
--
作者：
Moch, Marcin;Herberich, Gerlind;Windoffer, Reinhard
角蛋白中间丝细胞骨架的组织与上皮功能密切相关。为了研究角蛋白网络可塑性及其在不同水平上的调节，需要工具来定位和测量局部网络动力学。在本文中，我们提出了图像分析方法，旨在确定角蛋白丝运动的速度和方向，并确定在亚细胞分辨率角蛋白丝聚合和解聚的位置。使用这些方法，我们分析了荧光角蛋白13在人外阴癌衍生的A431细胞的延时荧光记录。荧光角蛋白整合到内源性角蛋白细胞骨架中，从而作为角蛋白动力学的可靠标记。我们发现，播种后的时间增加与向内定向角蛋白丝运动的下调。整体流动分析进一步揭示，角蛋白丝聚合在细胞周边和角蛋白解聚在更中央的细胞质都减少。用EGF处理这些细胞和其他人角质形成细胞衍生的细胞在几分钟内逆转了所有这些过程，同时增加了角蛋白磷酸化。这些结果突出了新开发的工具的价值，用于识别角蛋白丝网络动力学的调节剂和表征其作用模式，这反过来又有助于理解角蛋白丝网络可塑性和上皮生理学之间的密切联系。
The organization of the keratin intermediate filament cytoskeleton is closely linked to epithelial function. To study keratin network plasticity and its regulation at different levels, tools are needed to localize and measure local network dynamics. In this paper, we present image analysis methods designed to determine the speed and direction of keratin filament motion and to identify locations of keratin filament polymerization and depolymerization at subcellular resolution. Using these methods, we have analyzed time-lapse fluorescence recordings of fluorescent keratin 13 in human vulva carcinoma-derived A431 cells. The fluorescent keratins integrated into the endogenous keratin cytoskeleton, and thereby served as reliable markers of keratin dynamics. We found that increased times after seeding correlated with down-regulation of inward-directed keratin filament movement. Bulk flow analyses further revealed that keratin filament polymerization in the cell periphery and keratin depolymerization in the more central cytoplasm were both reduced. Treating these cells and other human keratinocyte-derived cells with EGF reversed all these processes within a few minutes, coinciding with increased keratin phosphorylation. These results highlight the value of the newly developed tools for identifying modulators of keratin filament network dynamics and characterizing their mode of action, which, in turn, contributes to understanding the close link between keratin filament network plasticity and epithelial physiology.