Rho Plays a Central Role in Regulating Local Cell-Matrix Mechanical Interactions in 3D Culture N. Lakshman, 1 A. Kim, 1 K. J. Bayless, 2 G. E. Davis, 2 and W. M. Petroll 1 * 1 Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas 2 Department of Pathology and Laboratory Medicine, Texas A&M Health Science Center College of Medicine, College Station, Texas The purpose of this study was to assess quantitatively the role of the small GTPase Rho on cell morphology, f-actin organization, and cell-induced matrix remodeling in 3D culture. Human corneal fibroblasts (HTK) were infected with adenoviruses that express green fluorescent protein (GFP) or GFP-N19Rho (dominant negative Rho). One day later cells were plated inside collagen matrices and allowed to spread for 24 h. Cells were fixed and stained for f-actin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) images were acquired using confocal microscopy. Fourier transform analysis was used to assess local collagen fibril alignment, and changes in cell morphology and collagen density were measured using MetaMorph. The decrease in matrix height was used as an indicator of global matrix contraction. HTK and HTK-GFP cells induced significant global matrix contraction; this was inhibited by N19Rho. HTK and HTK-GFP fibroblasts generally had a bipolar mor- phology and occasional intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. In contrast, HTK-GFPN19 cells were elongated, and had a more cortical f-actin distribution. Numerous small exten- sions were also observed along the cell body. In addition, both local collagen fibril density and alignment were significantly reduced. Rho plays a key role in regulating both the morphology and mechanical behavior of corneal fibroblasts in 3D culture. Overall, the data suggest that Rho-kinase dependent cell contractility contributes to global and local matrix remodeling, whereas Rho dependent activation of mDia and/ or other downstream effectors regulates the structure and number of cell processes. Cell Motil. Cytoskeleton 64:434–445, 2007. ' 2007 Wiley-Liss, Inc. Key words: Rho; Rho kinase; collagen matrices; confocal microscopy; cytoskeleton INTRODUCTION The mechanical interactions that occur between cells and the surrounding matrix play an important role in a number of biological processes including develop- mental morphogenesis and wound healing. A key media- tor of cell mechanical behavior is the cytoskeleton. Cell adhesion, spreading and migration all require changes in cytoskeletal organization. The Rho family of small GTPases (Rho, Rac and Cdc42) are well known modula- tors of the signal transduction pathways that mediate cytoskeletal dynamics [Hall, 1994]. In fibroblasts on rigid 2D substrates, activated Rho regulates several proc- esses including actomyosin contractility, stress fiber for- This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0886-1544/suppmat *Correspondence to: W. Matthew Petroll, Ph.D., Department of Ophthalmology, Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390-9057, USA. E-mail: matthew.petroll@utsouthwestern.edu Contract grant sponsor: NIH; Contract grant number: R01 EY013322; Contract grant sponsor: NIH; Contract grant number: R24 EY016664; Contract grant sponsor: Research to Prevent Blindness, NY. Received 8 September 2006; Accepted 16 January 2007 Published online 6 March 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/cm.20194 ' 2007 Wiley-Liss, Inc. Cell Motility and the Cytoskeleton 64:434–445 (2007)