Differential RhoA dynamics in migratory and stationary cells measured by FRET and automated image analysis.
Differential RhoA dynamics in migratory and stationary cells measured by FRET and automated image analysis.
复制标题
DOI：
10.1371/journal.pone.0004082
复制
发表时间：
2008
期刊：
影响因子：
3.7
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Eichorst JP;Lu S;Xu J;Wang Y
基于荧光共振能量转移（FRET）的基因编码生物传感器已被广泛应用于高分辨率地研究活细胞中分子活性的时空调控。从这些单细胞FRET测量的大量成像数据的有效和准确的定量需要强大的和自动化的数据分析。然而，活细胞的非线性运动对这项任务提出了巨大的挑战。基于单细胞运动的图像配准，我们开发了自动图像分析方法来跟踪和量化用户定义的亚细胞区域内的FRET信号。此外，亚细胞像素根据其相关的FRET信号和分析的簇的动力学进行分类。结果表明，EGF诱导的RhoA活性的降低在迁移的HeLa细胞中是显着小于在静止的细胞。此外，RhoA活性在迁移细胞中被极化，极性梯度朝向细胞迁移的相反方向。相反，在静止细胞中RhoA极性缺乏一致的偏好。因此，我们的图像分析方法可以提供强有力的工具，高通量和系统的调查的时空分子活动，在调节功能的活细胞，其形状和位置不断变化的时间。
Genetically-encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to study the spatiotemporal regulation of molecular activity in live cells with high resolution. The efficient and accurate quantification of the large amount of imaging data from these single-cell FRET measurements demands robust and automated data analysis. However, the nonlinear movement of live cells presents tremendous challenge for this task. Based on image registration of the single-cell movement, we have developed automated image analysis methods to track and quantify the FRET signals within user-defined subcellular regions. In addition, the subcellular pixels were classified according to their associated FRET signals and the dynamics of the clusters analyzed. The results revealed that the EGF-induced reduction of RhoA activity in migratory HeLa cells is significantly less than that in stationary cells. Furthermore, the RhoA activity is polarized in the migratory cells, with the gradient of polarity oriented toward the opposite direction of cell migration. In contrast, there is a lack of consistent preference in RhoA polarity among stationary cells. Therefore, our image analysis methods can provide powerful tools for high-throughput and systematic investigation of the spatiotemporal molecular activities in regulating functions of live cells with their shapes and positions continuously changing in time.
登录
查看更多内容
影响因子：
3.3
作者：
Kurokawa, K;Matsuda, M
通讯作者：
Matsuda, M
影响因子：
64.8
作者：
Pertz, O;Hodgson, L;Hahn, KM
通讯作者：
Hahn, KM
影响因子：
3.7
作者：
El-Sibai, Mirvat;Pertz, Olivier;Backer, Jonathan M.
通讯作者：
Backer, Jonathan M.
影响因子：
5.5
作者：
Mott, Rosalind E.;Helmke, Brian P.
通讯作者：
Helmke, Brian P.
影响因子：
64.8
作者：
LEE, J;ISHIHARA, A;JACOBSON, K
通讯作者：
JACOBSON, K