differential rhoa dynamics in migratory and stationary cells measured by fret and automated image analysis微分rhoa动力学在迁徙和固定细胞由烦恼和自动图像分析测量.pdfVIP

Differential RhoA Dynamics in Migratory and Stationary Cells Measured by FRET and Automated Image Analysis 1. 2. 2 1,2,3 John Paul Eichorst , Shaoying Lu , Jing Xu , Yingxiao Wang * 1 Center of Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America, 2 Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America, 3 Beckman Institute for Advanced Science and Technology, Department of Molecular and Integrative Physiology and Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America Abstract 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 t