Dynamic three-dimensional visualization of collagen matrix remodeling and cytoskeletal organization in living corneal fibroblasts.
The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing, as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the dynamic process of 3-D matrix remodeling at the cellular and subcellular level. We recently developed an experimental model for investigating cell-matrix mechanical interactions by plating green fluorescen protein (GFP)-zyxin transfected cells inside fibrillar collagen matrices and performing high-magnification time-lapse differential interference microscopy (DIC) and wide-field fluorescent imaging. In this study, we extend this experimental model by performing four-dimensional (4-D) reflected light and fluorescent confocal imaging (using either visible light or multiphoton excitation) of living corneal fibroblasts transfected to express GFP-zyxin or GFP-alpha-actinin, 18 h after plating inside 3-D collagen matrices. Reflected light confocal imaging allowed detailed visualization of the cells and the fibrillar collagen surrounding them. By overlaying maximum intensity projections of reflected light and GFP-zyxin or GFP-alpha-actinin images and generating stereo pair reconstructions, 3-D interactions between focal adhesions and collagen fibrils in living cells could be visualized directly. Focal adhesions were generally oriented parallel to the direction of collagen fibril alignment in front of the cell. Killing the cells induced relaxation of transient cell-induced tension on the matrix; however, significant permanent remodeling always remained. Time-lapse 3-D imaging demonstrated an active response to the Rho-kinase inhibitor Y-27632, as indicated by cell elongation, extracellular matrix relaxation, and extension of pseudopodial processes. It is interesting that, at higher cell densities, groups of collagen fibrils were compacted and aligned into straps between neighboring cells. Overall, the continued development and application of this new approach should provide important insights into the basic underlying biochemical and biomechanical regulatory mechanisms controlling matrix remodeling by corneal fibroblasts.
Petroll
W Matthew
WM
Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9057, USA. matthew.petroll@utsouthwestern.edu
Cavanagh
H Dwight
HD
Jester
James V
JV
D000818:Animals; D002460:Cell Line; D003094:Collagen / Q000378:metabolism / Q000648:ultrastructure; D003315:Cornea / Q000166:cytology; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D005109:Extracellular Matrix / Q000378:metabolism* / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D022001:Focal Adhesions / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D011817:Rabbits
D003094:Collagen