Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture

Abstract

The purpose of this study was to investigate the role of microtubules in regulating corneal fibroblast structure and mechanical behavior using static (3-D) and dynamic (4-D) imaging of both cells and their surrounding matrix. Human corneal fibroblasts transfected to express GFP-zyxin (to label focal adhesions) or GFP-tubulin (to label microtubules) were plated at low density inside 100 μm thick type I collagen matrices. After 24 hours, the effects of nocodazole (to depolymerize microtubules), cytochalasin D (to disrupt f-actin), and/or Y-27632 (to block Rho-kinase) were evaluated using 3-D and 4-D imaging of both cells and ECM. After 24 hours of incubation, cells had well organized microtubules and prominent focal adhesions, and significant cell-induced matrix compaction was observed. Addition of nocodazole induced rapid microtubule disruption which resulted in Rho activation and additional cellular contraction. The matrix was pulled inward by retracting pseudopodial processes, and focal adhesions appeared to mediate this process, when present. Following 24 hour exposure to nocodazole, there was an even greater increase in both the number of stress fibers and the amount of matrix compaction and alignment at the ends of cells. When Rhokinase was inhibited, disruption of microtubules resulted in retraction of dendritic cell processes, and rapid formation and extension of lamellipodial processes at random locations along the cell body, eventually leading to a convoluted, disorganized cell shape. These data suggest that microtubules modulate both cellular contractility and local collagen matrix reorganization via regulation of Rho/ Rho kinase activity. In addition, microtubules appear to play a central role in dynamic regulation of cell spreading mechanics, morphology and polarity in 3-D culture.

Morphological Study of Fibroblasts Treated with Cytochalasin D and Colchicine Using a Confocal Laser Scanning Microscopy

“…7). The decrease in the adhesion area is in agreement with results reported in previous studies [10,12]. The most likely reason for the decrease in the adhesion area is a disturbance of the mechanical balance in the cell after microtubule disruption.…”

“…Many studies have focused on the role of the cytoskeleton in cellular mechanics [5][6][7][10][11][12], and Ingber [13] proposed a cellular mechanical model, the tensegrity model, which focused on the cytoskeletal system. This model can account for some of the mechanical behavior of living cells [7,14,15].…”

“…The infl uence of the extracellular matrix [5][6][7][8] and the effects of the degree of polymerization of the cytoskeleton [6,[9][10][11][12] on cell shape have been examined using a variety of methods. Many studies have focused on the role of the cytoskeleton in cellular mechanics [5][6][7][10][11][12], and Ingber [13] proposed a cellular mechanical model, the tensegrity model, which focused on the cytoskeletal system.…”

Techniques for assessing 3-D cell–matrix mechanical interactions in vitro and in vivo

“…In contrast to lamellipodial adhesions on 2-D substrates, adhesions in 3-D matrices tended to form and treadmill in a linear pattern along individual collagen fibrils, consistent with studies showing that alignment of collagen fibrils can provide contact guidance for cell spreading and migration [55, 88–90]. Subsequent studies have used DIC imaging to evaluate the role of Rho and Rac in regulating sub-cellular mechanical activity during fibroblast spreading and migration [40, 84, 91, 92]. Such studies have demonstrated that while Rho kinase plays a central role in mediating overall force generation by fibroblasts within 3-D matrices (Fig.…”

Effect of Photobiomodulation on Wound Healing of the Corneal Epithelium through Rho-GTPase

“…15,16 Rho kinases (ROCKs) were initially characterized for their roles in mediating the formation of RhoA-induced stress fibers and focal adhesions through their effects on the phosphorylation of myosin light chain. 17 In the cornea, ROCKs have been suggested to be involved in epithelial differentiation, 18 cell cycle progression, 11 cell-cell adhesion, 15 endothelial barrier integrity, 19 stromal cell phenotype conversion, 20 cytoskeleton reorganization, contractility, 21 and cell-matrix interaction. 22 Thus, we investigated the Rho-GTPase activity by PBM and found that the expression of RhoA and RhoC was significantly increased after PBM treatment.…”