Petroll W Matthew, Ma Lisha
Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9057, USA.
Cell Motil Cytoskeleton. 2003 Aug;55(4):254-64. doi: 10.1002/cm.10126.
Cell mechanical behavior has traditionally been studied using 2-D planar elastic substrates. The goal of this study was to directly assess cell-matrix mechanical interactions inside more physiologic 3-D collagen matrices. Rabbit corneal fibroblasts transfected to express GFP-zyxin were plated at low density inside 100 micro m-thick type I collagen matrices. 3-D datasets of isolated cells were acquired at 1-3-min intervals for up to 5 h using fluorescent and Nomarski DIC imaging. Unlike cells on 2-D substrates, cells inside the collagen matrices had a bipolar morphology with thin pseudopodial processes, and without lamellipodia. The organization of the collagen fibrils surrounding each cell was clearly visualized using DIC. Using time-lapse color overlays of GFP and DIC images, displacement and/or realignment of collagen fibrils by focal adhesions could be directly visualized. During pseudopodial extension, new focal adhesions often formed in a line along collagen fibrils in front of the cell, while existing adhesions moved backward. This process generated tractional forces as indicated by the pulling in of collagen fibrils in front of the cell. Meanwhile, adhesions on both the dorsal and ventral surface of the cell body generally moved forward, resulting in contractile shortening along the pseudopodia and localized extracellular matrix (ECM) compression. Cytochalasin D induced rapid disassembly of focal adhesions, cell elongation, and ECM relaxation. This experimental model allows direct, dynamic assessment of cell-matrix interactions inside a 3-D fibrillar ECM. The data suggest that adhesions organize along actin-based contractile elements that are much less complex than the network of actin filaments that mechanically links lamellar adhesions on 2-D substrates.
传统上，细胞力学行为是使用二维平面弹性基质进行研究的。本研究的目的是直接评估在更接近生理状态的三维胶原基质中细胞与基质的力学相互作用。将转染以表达绿色荧光蛋白-桩蛋白（GFP-zyxin）的兔角膜成纤维细胞以低密度接种在100微米厚的I型胶原基质内。使用荧光和诺马斯基微分干涉对比（Nomarski DIC）成像，以1至3分钟的间隔获取分离细胞的三维数据集，持续长达5小时。与二维基质上的细胞不同，胶原基质内的细胞具有双极形态，有细的伪足样突起，且无片状伪足。使用DIC可以清晰地观察到围绕每个细胞的胶原纤维的组织情况。通过对GFP和DIC图像进行延时彩色叠加，可以直接观察到粘着斑对胶原纤维的位移和/或重新排列。在伪足伸展过程中，新的粘着斑常常沿着细胞前方的胶原纤维排成一行形成，而现有的粘着斑则向后移动。这一过程产生了牵引力，表现为细胞前方的胶原纤维被拉近。同时，细胞体背侧和腹侧表面的粘着斑通常向前移动，导致沿伪足的收缩性缩短和局部细胞外基质（ECM）压缩。细胞松弛素D诱导粘着斑迅速解体、细胞伸长和ECM松弛。该实验模型允许直接、动态地评估三维纤维状ECM内的细胞-基质相互作用。数据表明，粘着斑沿着基于肌动蛋白的收缩元件排列，这些元件比在二维基质上机械连接片状粘着斑的肌动蛋白丝网络要简单得多。
