High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells.
High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells.
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DOI：
10.3791/54265
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发表时间：
2016-08-13
期刊：
影响因子：
--
通讯作者：
Myers KA
中科院分区：
文献类型：
--
作者：
Braun A;Caesar NM;Dang K;Myers KA
新的脉管系统从现有脉管系统形成的生理过程需要触发个体内皮细胞（EC）内的形态学变化的特定信号传导事件。这些过程对于伤口愈合等稳态维持至关重要，对于促进肿瘤生长和转移也至关重要。EC形态由细胞骨架的组织来定义，细胞骨架是一种严格调节的肌动蛋白和微管（MT）动力学系统，已知其控制EC分支、极性和定向迁移，血管生成的基本组分。为了研究MT动力学，我们使用高分辨率荧光显微镜结合荧光标记的MT加端的计算图像分析来研究MT生长动力学和EC分支形态和定向迁移的调节。在用荧光标记的MT末端结合蛋白3（EB3）和有丝分裂着丝粒相关驱动蛋白（MCAK）特异性cDNA构建体转染后，对活的人脐静脉内皮细胞（HUVEC）进行延时成像，以评估对MT动力学的影响。使用PlusTipTracker软件追踪EB 3标记的MT+末端，以测量延时图像中的MT生长速度和MT生长寿命。这种方法允许MT动力学的研究和鉴定如何局部调节MT动力学的亚细胞区域内的EC分支和迁移的血管生成过程作出贡献。
The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.
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