Dynamic three‐dimensional visualization of collagen matrix remodeling and cytoskeletal organization in living corneal fibroblasts

Abstract

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.

Quantitative assessment of local collagen matrix remodeling in 3-D Culture: The role of Rho kinase

“…This technique allows detailed visualization of the cells and the fibrillar collagen surrounding them [41][42][43]. By overlaying the corresponding reflected light and fluorescent optical section images, cell-matrix interactions could be directly visualized (Fig.…”

Mechanotransduction of mesenchymal melanoma cell invasion into 3D collagen lattices: Filopod-mediated extension–relaxation cycles and force anisotropy

“…For fibroblasts embedded in 3D fibrillar collagen, confocal reflectance microscopy has revealed cytoskeletal focalization and actomyosin mediated contractility as the basis of migration and structural remodeling of tissue [6,24,25]. In mesenchymal tumor cells, the mechanotransduction to collagen depends upon integrinmediated adhesion to collagen fibrils [26,27], myosin-II mediated contraction of actin filaments [20,21,24], and intracellular mechanocoupling between adhesion receptors and the actin cytoskeleton by focal adhesion kinase, talin and p130Cas [16]. The main force vector may either result from a single protrusion persisting over extended time periods [26], or multiple protrusions that form and retract to generate a combinatorial net vector of translocation over time [16].…”

Collagen Fibril Flow and Tissue Translocation Coupled to Fibroblast Migration in 3D Collagen Matrices

“…Three-dimensional (3D) matrices prepared with type I collagen exhibit mechanical properties that resemble connective tissue (Barocas et al, 1995;Wakatsuki et al, 2000;Roeder et al, 2002;Silver et al, 2002;Ahlfors and Billiar, 2007). Unlike ECM-coated material surfaces, fibroblasts can mechanically remodel collagen matrices both locally and globally (Brown et al, 1998;Tomasek et al, 2002;Grinnell, 2003;Petroll, 2004; Tranquillo, 1999Such mechanical remodeling of connective tissue ECM is believed to be important for tissue homeostasis (Silver et al, 2002;Wiig et al, 2003;Goldsmith et al, 2004;Langevin et al, 2004), aging (Varani et al, 2004), repair (Tonnesen et al, 2000Tomasek et al, 2002;Grinnell, 2003), fibrosis (Eckes et al, 2000;Desmouliere et al, 2005), and tumorigenesis (Beacham and Cukierman, 2005;Gaggioli et al, 2007;Yamada and Cukierman, 2007).…”