Costa Kevin D, Hucker William J, Yin Frank C-P
Department of Biomedical Engineering, Columbia University, New York, New York 10027, USA.
Cell Motil Cytoskeleton. 2002 Aug;52(4):266-74. doi: 10.1002/cm.10056.
Intracellular tension is considered an important determinant of cytoskeletal architecture and cell function. However, many details about cytoskeletal tension remain poorly understood because these forces cannot be directly measured in living cells. Therefore, we have developed a method to characterize the magnitude and distribution of pre-extension of actin stress fibers (SFs) due to resting tension in the cytoskeleton. Using a custom apparatus, human aortic endothelial cells (HAECs) were cultured on a pre-stretched silicone substrate coated with a fibronectin-like polymer. Release of the substrate caused SFs aligned in the shortening direction in adhered cells to buckle when compressed rapidly (5% shortening per second or greater) beyond their unloaded slack length. Subsequently, the actin cytoskeleton completely disassembled in 5 sec and reassembled within 60 sec. Quantification of buckling in digital fluorescent micrographs of cells fixed and stained with rhodamine phalloidin indicated a nonuniform distribution of 0-26% pre-extension of SFs in non-locomoting HAECs. Local variability suggests heterogeneity of cytoskeletal tension and/or stiffness within individual cells. These findings provide new information about the magnitude and distribution of cytoskeletal tension and the dynamics of actin stress fibers, and the approach offers a novel method to elucidate the role of specific cytoskeletal elements and crosslinking proteins in the force generating apparatus of non-muscle cells.
细胞内张力被认为是细胞骨架结构和细胞功能的重要决定因素。然而，由于无法在活细胞中直接测量这些力，关于细胞骨架张力的许多细节仍知之甚少。因此，我们开发了一种方法来表征由于细胞骨架中的静息张力而导致的肌动蛋白应力纤维（SFs）预拉伸的大小和分布。使用定制装置，将人主动脉内皮细胞（HAECs）培养在涂有纤连蛋白样聚合物的预拉伸硅树脂底物上。当底物快速压缩（每秒缩短5%或更多）超过其无负载松弛长度时，底物的释放会导致粘附细胞中沿缩短方向排列的SFs发生弯曲。随后，肌动蛋白细胞骨架在5秒内完全解体，并在60秒内重新组装。用罗丹明鬼笔环肽固定和染色的细胞的数字荧光显微照片中弯曲的量化表明，在非运动性HAECs中，SFs的预拉伸为0-26%，分布不均匀。局部变异性表明单个细胞内细胞骨架张力和/或刚度的异质性。这些发现提供了关于细胞骨架张力的大小和分布以及肌动蛋白应力纤维动力学的新信息，并且该方法提供了一种新的方法来阐明特定细胞骨架元件和交联蛋白在非肌肉细胞力产生装置中的作用。
