Dynamics of integrin clustering at focal contacts of endothelial cells studied by multimode imaging microscopy.
Dynamics of integrin clustering at focal contacts of endothelial cells studied by multimode imaging microscopy.
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发表时间：
2001-09
影响因子：
4
通讯作者：
K. Kawakami;H. Tatsumi;M. Sokabe
中科院分区：
文献类型：
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作者：
K. Kawakami;H. Tatsumi;M. Sokabe
用fitc标记的抗β(1)整合素抗体对人脐静脉内皮细胞进行染色，并将其镀在玻璃片上，以阐明整合素在局灶接触形成过程中的聚集机制。利用延时全内反射荧光显微镜观察整合素聚集的过程，该显微镜可以选择性地观察活细胞基表面标记的整合素。整合素的聚集始于镀后1小时，单个聚集持续生长约6小时。大多数整合素簇（约80%）以0.29+/-0.24微米分钟（-1）的速率向细胞中心或沿细胞边缘伸长。光漂白和瞬时光照恢复实验表明，簇延伸尖端的整合素是由细胞内空间提供的。整合素囊泡胞吐和局灶接触延长的同时延时成像显示，大多数胞吐发生在局灶接触处或其附近，随后是局灶接触延长。f -肌动蛋白和整合素双染色表明，应力纤维位于整合素簇附近，细胞内点状整合素与这些应力纤维相关。这些结果表明，整合素的聚集是由肌动蛋白纤维依赖的整合素转运到焦点接触的延伸尖端介导的。
Human umbilical vein endothelial cells were stained with FITC-labeled anti-beta(1) integrin antibody and plated on a glass cover slip to elucidate the mechanism of integrin clustering during focal contact formation. The process of integrin clustering was observed by time-lapse total-internal-reflection fluorescence microscopy, which can selectively visualize the labeled integrins at the basal surface of living cells. The clustering of integrins at focal contacts started at 1 hour after plating and individual clusters kept growing for approximately 6 hours. Most integrin clusters (approximately 80%) elongated towards the cell center or along the cell margin at a rate of 0.29+/-0.24 microm minute(-1). Photobleaching and recovery experiments with evanescent illumination revealed that the integrins at the extending tip of the clusters were supplied from the intracellular space. Simultaneous time-lapse imaging of exocytosis of integrin-containing vesicles and elongating focal contacts showed that most exocytosis occurred at or near the focal contacts followed by their elongation. Double staining of F-actins and integrins demonstrated that stress fibers were located near the integrin clusters and that intracellular punctate integrins were associated with these stress fibers. These results suggest that the clustering of integrins is mediated by actin-fiber-dependent translocation of integrins to the extending tip of focal contacts.