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 microm thick type I collagen matrices. After 24h, 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 24h 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. Following 24h 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 Rho-kinase 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.

Figure 1

Dynamic assessment of the effects of nocodazole and/or cytochalasin D on cell mechanical activity. Following 24 hours of incubation in serum-free media (A–F) or serum-containing media (G–L), matrices were transferred to the microscope stage, and time-lapse DIC imaging was performed. A–C: Large retractions of the pseudopodial processes were observed following treatment with nocodazole, but much smaller displacements of the ECM were induced (cyan tracks). D–F: Zoomed DIC images from square region in A–C. G–L: Following culture in serum-containing media, nocodazole treatment induced cellular contraction, and collagen was simultaneously pulled inward (H, cyan tracks). Cytochalasin D reversed the process and induced cell elongation, and corresponding matrix relaxation (I, cyan tracks). J–L: FEM strain maps along the cell axis generated from matrix displacements (cyan tracks) in G–I. Color legend is in dimensionless units ΔL/L (change in length/initial length). Nocadozole treatment induced compression along the cell body, and tension at the end of the cells. In contrast, subsequent treatment with CytoD resulted in decompression of the ECM under the cell, and reduction of tension at the ends.

Figure 2

Dynamic assessment of the effects of nocodazole and/or cytochalasin D on corneal fibroblast mechanical activity. Following 24 hours of incubation in serum-containing media, matrices were transferred to the microscope stage, and time-lapse confocal imaging was performed. Color overlays of GFP-tubulin (green, B–C) or GFP-zyxin (green, A, D–H) and collagen fibils (red) allow interaction between cells and the extracellular matrix to be directly visualized. A: Corneal fibroblast cultured for 24 hours in serum-containing media. GFP-zyxin is organized into focal adhesions along the pseudopodia, which appear to be attached to the collagen fibrils. B: Microtubules emanated from a microtubule organizing center, and were generally aligned with the long axis of the cells. C: Same cell as in B. Nocodazole induced microtubule disruption and retraction of cell processes. D–F: GFP-zyxin labeling revealed centripetal movement of focal adhesions and matrix reorganization (compaction and alignment) surrounding the cell following treatment with nocodazole. G–I: A subset of focal adhesions (G, white arrows) was tracked following addition of nocodazole. The relationship between movement of focal adhesions (I, green arrows) and individual collagen fibrils (I, red arrows) is demonstrated.

Figure 3

Dynamic assessment of nocodazole reversibility using time-lapse confocal imaging. Same cell as shown in Fig. 2D–F. After 50 minutes of nocodazole treatment (A), reperfusion with basal media (i.e. without nocodazole) induced re-extension of cellular processes and matrix relaxation (B and C).

Figure 4

Rho activation data. Graph shows the mean and standard deviation of three independent experiments. Data shown is absorbance over background signal (wells incubated with lysis buffer alone instead of cell lysates). Rho activation was significantly increased following 30 minutes of nocodazole treatment (P < 0.05, ANOVA with Holm-Sidak method for multiple comparisons) as compared to untreated control cells.

Figure 5

Time-lapse DIC images before and after nocodazole treatment in the presence of Y-27632. A: Cells treated with Y-27632 for 24 hours were elongated and had dendritic processes (arrowheads). B,C: Nocodazole induced retraction of these dendritic processes without force generation (i.e. no associated pulling in of the ECM), followed by formation and extension of lamellipodial processes from random locations along the cell body (B and C, arrows).

Figure 6

Assessment of the role of microtubules on initial cell spreading and polarization in 3-D culture. Fibroblasts were incubated for 24 hours in basal (serum-containing media), Y-27632, nocodazole, or both Y-27632 and nocodazole, stained for f-actin, and imaged using 3-D LSCM. A: Following culture in basal media, corneal fibroblasts had a bipolar morphology, and stress fibers were sometimes observed along the cell body. Collagen fibrils were compacted and aligned nearly parallel to the pseudopodial tips at the end of cells. B. Cells treated with Y-27632 were more elongated, had a dendritic morphology, and did not induce matrix reorganization. C. 24 hours of incubation with nocodazole reduced the amount of cell spreading and induced larger and more numerous stress fibers. Collagen fibrils were also more compacted and aligned along the cell body as compared to basal media. D. When treated with both nocodazole and Y-27632, cells lost their polarity and developed numerous convoluted branching processes. A more random collagen organization was also observed, with no preferred matrix alignment at the ends of the cell and isolated areas of matrix compaction around the cell body.

Figure 7

Quantitative analysis of cell morphology following 24 hours of culture in serum (control), Y-27632, nocodazole (Noc) or Noc + Y-27632. Each bar shows mean and standard deviation from four independent experiments. A. Cell length was significantly increased when Rho-kinase was inhibited, and decreased when microtubules were disrupted. B. The breadth/length ratio was also higher when microtubules were disrupted, confirming a less bipolar morphology.