Arai Masataka, Shimada Tomoya, Kora Chihiro, Nakashima Kazuhiro, Sera Toshihiro, Kudo Susumu
Department of Mechanical Engineering, Graduate school of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
Division of Mechanical Engineering, Graduate School of Engineering and Science, Shibaura Institute of Technology, 3-7-5 Toyosu, Koto-ku, Tokyo 135-8548, Japan.
Biochem Biophys Rep. 2017 Sep 1;12:91-97. doi: 10.1016/j.bbrep.2017.08.003. eCollection 2017 Dec.
Mechanical wounding of an endothelial monolayer induces an immediate Ca wave. Several hours later, the denuded area is covered by endothelial cells (ECs) that migrate to the wound. This migration process is closely related to protein kinase Cα (PKCα), a Ca-dependent protein that translocates from the cytosol to the cell membrane. Because the cells adjacent to the wounded area are the first to migrate into the wound, we investigated whether a mechanical wound immediately induces PKCα translocation in adjacent cells. We monitored Ca dynamics and PKCα translocation simultaneously using fluorescent microscopy. For this simultaneous observation, we used Fura-2-acetoxymethyl ester to visualize Ca and constructed a green fluorescent protein-tagged fusion protein to visualize PKCα. Mechanical wounding of the endothelial monolayer induced an immediate Ca wave in cells adjacent to the wounded cells before their migration. Almost concurrently, PKCα in the neighboring cells translocated to the cell membrane, then accumulated at the periphery near the wounded cell. This report is the first description of this biphasic and directed translocation of PKCα in cells before cell migration. Our results may provide new insights into the directed migration of ECs.
对内皮细胞单层进行机械损伤会立即引发钙离子波。数小时后，裸露区域会被迁移至伤口处的内皮细胞（ECs）覆盖。这一迁移过程与蛋白激酶Cα（PKCα）密切相关，PKCα是一种依赖钙离子的蛋白，它会从细胞质转移至细胞膜。由于伤口附近的细胞是最先迁移至伤口处的，我们研究了机械损伤是否会立即诱导相邻细胞中的PKCα发生转位。我们使用荧光显微镜同时监测钙离子动态和PKCα转位情况。为了进行这种同步观察，我们使用Fura-2-乙酰甲酯来可视化钙离子，并构建了一种绿色荧光蛋白标记的融合蛋白来可视化PKCα。对内皮细胞单层进行机械损伤会在相邻细胞迁移之前立即在伤口附近的细胞中引发钙离子波。几乎与此同时，相邻细胞中的PKCα转移至细胞膜，然后在伤口附近细胞的周边聚集。本报告首次描述了PKCα在细胞迁移前的这种双相定向转位。我们的结果可能为内皮细胞的定向迁移提供新的见解。
