Volume dynamics in migrating epithelial cells measured with atomic force microscopy
Volume dynamics in migrating epithelial cells measured with atomic force microscopy
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DOI：
10.1007/s004240050943
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发表时间：
2000-01-01
影响因子：
4.5
通讯作者：
Schwab, A
中科院分区：
文献类型：
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作者：
Schneider, SW;Pagel, P;Schwab, A
转化的肾上皮细胞（转化的 Madin-Darby 犬肾细胞，MDCK-F 细胞）的迁移依赖于 Ca2+ 敏感 K+ 通道（IK 通道）的活性，该通道在这些细胞的后端更为活跃。我们假设间歇性 IK 通道活动会诱导迁移 MDCK-F 细胞后端的局部细胞收缩，从而支持迁移的细胞骨架机制。然而，由于 MDCK-F 细胞的复杂形态，我们还无法直接测量体积变化。本研究的目的是设计一种新技术，利用原子力显微镜 (AFM) 测量 MDCK-F 细胞在其生理环境中的体积，并证明其对 IK 通道活性的依赖性。 MDCK-F 细胞的三维图像的每个像素的空间（x、y 和 z）坐标允许计算给定像素“下方”的柱的体积。因此，总细胞体积是所有像素定义的列的总和。 17个MDCK-F细胞的平均体积为2500+/-300 fl。使用特异性抑制剂 Charybdotoxin (CTX) 阻断 IK 通道可使细胞体积增加 17+/-4%；使用 Ca2+ 离子载体离子霉素升高细胞内 [Ca2+] 来激活 IK，使细胞体积减少 19+/-3%。减影图像（实验负对照）显示肿胀和收缩主要发生在 MDCK-F 细胞的后端。总之，我们的实验表明，AFM 不仅可以测量生理环境中活细胞的总细胞体积，还可以追踪 K+ 通道活性的极化分布引起的局部效应。
Migration of transformed renal epithelial cells (transformed Madin-Darby canine kidney cells, MDCK-F cells) relies on the activity of a Ca2+-sensitive K+ channel (IK channel) that is more active at the rear end of these cells. We have postulated that intermittent IK channel activity induces local cell shrinkage at the rear end of migrating MDCK-F cells and thereby supports the cytoskeletal mechanisms of migration. However, due to the complex morphology of MDCK-F cells we have not yet been able to measure volume changes directly. The aim of the present study was to devise a new technique employing atomic force microscopy (AFM) to measure the volume of MDCK-F cells in their physiological environment and to demonstrate its dependence on IK channel activity. The spatial (x, y and z) co-ordinates of each pixel of the three-dimensional image of MDCK-F cells allow calculation of the volume of the column "underneath" a given pixel. Thus, total cell volume is the sum of all pixel-defined columns. The mean volume of 17 MDCK-F cells was 2500+/-300 fl. Blockade of the IK channel with the specific inhibitor charybdotoxin (CTX) increased cell volume by 17+/-4%; activation of IK by elevating the intracellular [Ca2+] with the Ca2+ ionophore ionomycin decreased cell volume by 19+/-3%. Subtraction images (experimental minus control) reveal that swelling and shrinkage occur predominantly at the rear end of MDCK-F cells. In summary, our experiments show that AFM allows the measurement not only of total cell volume of living cells in their physiological environment but also the tracing of local effects induced by the polarized distribution of K+ channel activity.