Simon de Beco, Charles Gueudry, Francois Amblard, Sylvie Coscoy (2011) CIL:24069, Homo sapiens, epithelial cell, invasive breast ductal carcinoma, mammary gland. CIL. Dataset

E-cadherin local dynamics were studied in mature junctions, that is, junctions engaged in adhesion for many hours, in which cadherin expression level is stable. After stable transfection with E-cadherin-GFP, E-cadherin dynamics were studied by 2-photon single-particle tracking and fluorescence recovery after photobleaching (FRAP) combined with 3D wide-field fluorescence microscopy, which allowed the recovery process to be analyzed from series of image stacks in the entire 3D region surrounding the photobleached volume. Image analysis of fluorescence recovery indicates that most E-cadherin did not diffuse in the membrane along mature junctions, but followed a first order turn-over process that was rate-limited by endocytosis. This z-stack (0.3 µm) of images is of a FRAP experiment on MCF7 cell junctions expressing E-cadherin-GFP before photobleaching. Also available is the z-stack immediately following photobleaching (CIL# 24070) and the time-lapse video of FRAP experiment (CIL# 24068). Photobleached regions are indicated by arrows. Scale bar: 10 µm.

本研究针对成熟连接处的E-钙粘蛋白局部动态进行了探究，即参与数小时粘附作用的连接，其中钙粘蛋白的表达水平保持稳定。通过将E-钙粘蛋白-GFP进行稳定转染后，采用二光子单粒子追踪技术以及光漂白后的荧光恢复（FRAP）实验，结合三维宽场荧光显微镜，对光漂白体积周围整个三维区域的恢复过程进行了分析。荧光恢复的图像分析表明，大多数E-钙粘蛋白在成熟连接的膜上并未发生扩散，而是遵循一级周转过程，该过程受内吞作用速率限制。此Z栈（0.3 µm）图像为对表达E-钙粘蛋白-GFP的MCF7细胞连接进行FRAP实验前的图像。此外，还提供了光漂白后的Z栈（CIL# 24070）以及FRAP实验的时间间隔视频（CIL# 24068）。光漂白区域由箭头标出。比例尺：10 µm。