Purchase this article with an account.
W. Petroll, L. Ma, J.V. Jester; Assessment of the Mechanical Interactions between Corneal Fibroblasts and Collagen Fibrils inside 3-D Extracellular Matrix . Invest. Ophthalmol. Vis. Sci. 2003;44(13):875.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: We and other investigators have used green fluorescent protein (GFP) to correlate focal adhesion dynamics with force generation by cells plated on top of deformable substrates. However, cells reside within 3-D extracellular matrices (ECM) in vivo. In this study we investigate cell-ECM interactions within more physiologic 3-D collagen matrices. Methods: Primary cultures of rabbit corneal fibroblasts (NRK) and a corneal fibroblast cell line (TRK-36) were transfected using a vector encoding EGFP-zyxin (gift from J. Wehland and coworkers) to allow visualization of focal adhesions. Cells were plated at low density inside 100 µm thick fibrillar collagen matrices. Time-lapse imaging was performed at high magnification for 3-5 hours. At each 1-3 minute interval, EGFP and Nomarski DIC images were acquired in rapid succession; 3-D datasets (10-15 microns thick) were obtained in some cases. Results: Cells inside the ECM had a bipolar morphology with long thin pseudopodial processes, and did not form lamellipodia as is typically observed in cells plated on top of the ECM. EGFP-zyxin was localized to focal adhesions as confirmed using vinculin counterstaining. Focal adhesions were predominantly localized along the pseudopodia, and were oriented parallel to the long axis of the processes. Adhesions did not fan out at the tip of the processes as is typically observed on planar substrates. The fibrillar ECM structure was clearly visualized using DIC. Tension was generated during extension and partial retraction of pseudopodia as indicated by pulling in of collagen fibrils. During extension, new focal adhesions formed at the front edge of pseudpodia while existing adhesions moved backward. Displacement and/or realignment of collagen fibrils by focal adhesions could be directly visualized in many cases. Adhesions near the base of pseudopodia often moved toward those at the tip, resulting in contractile-like shortening and local ECM compression. Cytochalasin D induced rapid disassembly of focal adhesions, cell elongation and ECM relaxation. Conclusions: This is the first experimental model to allow direct, dynamic assessment of cell-matrix interactions in a 3-D fibrillar ECM. Fibroblasts inside the matrix (3-D) demonstrate important differences in cell morphology, adhesion organization and mechanical behavior as compared to cells in a 2-D environment.
This PDF is available to Subscribers Only