Research

Cell Mechanics

Overview

The organization of extracellular matrices by cells through the exertion of mechanical forces drives fundamental processes such as developmental morphogenesis, wound healing, and the organization of bioengineered tissues. Historically, our ability to investigate cell mechanical behavior has been limited by the technical challenges associated with measuring the sub-cellular origins of cellular force generation and local matrix patterning in a 3-D environment. Thus, our understanding of these fundamental processes is limited, especially in ocular tissues. Over the last several years, our research team has addressed these challenges through the development of new experimental models, use of emerging imaging technologies, and the application of quantitative analysis techniques. An overview of some of these approaches is provided below and additional examples are provided separately in the form of time-lapse movies and 3-D reconstructions.

Experimental Models

We first developed an experimental model for dynamic investigation of cell-matrix mechanical interactions by plating corneal fibroblasts transfected to express GFP-tagged cytoskeletal or adhesive proteins inside fibrillar collagen matrices, and performing high-magnification time-lapse differential interference contrast (DIC) and fluorescent imaging. As shown in Figure 1, DIC imaging allowed detailed visualization of the cells and the fibrillar collagen surrounding them.

Figure 1: Three DIC images from a five-image z-series of an untransfected corneal fibroblast plated inside 3-D collagen matrix. The z position shown is relative to the bottom of the collagen matrix. Individual collagen fibrils are easily discerned adjacent to the cell (arrows), and also crossing above (C, arrowheads) and below (A, arrowheads) the cell body. A different population of collagen fibrils is observed in each image.

A corneal fibroblast transfected to express GFP-zyxin (which labels focal adhesions) is shown in Figure 2.

Figure 2: Image processing and display of 3-D datasets. DIC (A), GFP (B-E), and combined (F) images of a corneal fibroblast expressing GFP-zyxin are shown. A: A single DIC image showing the fibrillar collagen organization surrounding the cell. B, C: The top and bottom GFP images from a 5-image z-series. GFP-zyxin was organized into adhesions that were most concentrated along pseudopodia at the ends of the cell. D: Color-coded reconstruction produced by performing a flatten background operation on each image in the z-series, then overlaying the bottom three images in red and the top two images in green. Adhesions are visualized on both the ventral (red) and dorsal (green) surface of the cell body in some regions (circle). E: 3-D reconstruction produced using a maximum intensity projection. F: Color overlay of E (green) and A (red) allows focal adhesion and collagen organization to be directly compared.

Color-coded reconstructions demonstrate that adhesions were present on both the ventral (red) and dorsal (green) surfaces of cells (2D); whereas overlaying of GFP-zyxin maximum intensity projections and individual DIC images allowed the organization of focal adhesions and collagen fibrils to be directly compared (2F). This unique approach allows dynamic changes in the sub-cellular organization of cytoskeletal and adhesive proteins to be quantitatively correlated with the pattern of cell-induced matrix deformation. By tracking matrix deformation and applying Finite Element Modeling (FEM), maps of ECM strain can be also generated. (Figure 3)

Figure 3: FEM strain maps generated using ANSYS, showing regions of matrix tension and compression. Stress on the matrix in serum containing media (A) is reduced when the cell is switched to Y-27632, which inhibits Rho Kinase (B). Stress is reestablished after washing out the Y-27632 (C). Note the reduction in stress fibers within the cell (as indicated by GFP-α-actinin labeling) following Y-27632 treatment. Strain is shown relative to the “relaxed” matrix configuration determined by treating cells with Cytochalasin D and TritonX-100.

In subsequent studies, we further extended this paradigm to quantify the detailed 3-D pattern of local collagen matrix remodeling by using confocal reflection imaging. Using this approach, the 3-D structural organization of the fibrillar collagen can be directly correlated with temporal changes in the organization of key cytoskeletal, adhesive or regulatory proteins (Figure 4).

Figure 4: Color overlay of f-actin (green) and collagen fibils (red) allows interaction between cells and the extracellular matrix to be directly visualized. Increased compaction and alignment of collagen fibrils parallel to the pseudopodial tips is generally observed at the ends of corneal fibroblasts. Collagen was visualized using reflected light confocal imaging and f-actin was visualized using fluorescent labeling (Alexaflour phallodin 488).

By using Fourier Transform analysis, quantitative assessment of cell-induced collagen remodeling can be performed (Figure 5).

Figure 5: Example of Fourier Transform approach to quantifying local collagen fibril alignement. Sub-regions were taken from the ends of pseudopodia following culture in serum (left) and the Rho Kinase inhibitor Y-27632 (right). The pseudopodia tips were located at the bottom left corner of each image. A: Collagen fibrils are aligned nearly parallel to the pseudopodial tip. C: After rotating 90 degrees, the power spectrum reveals a bright band-like signal aligned nearly parallel to the group of collagen fibrils. E: The line average plot has one distinct peak near θpseud. The magnitude of the orientation index (OI) at θpseud is 54 percent, indicating substantial compaction and alignment of collagen parallel to the pseudopodial axis. B: Collagen is more randomly oriented in Y-27632, and the rotated FT power spectrum (D) is more dispersed. F: The corresponding line average plot has a more uniform distribution without a single dominant peak. The magnitude of the OI at θpseud is only 1.2 percent, indicating little compaction and alignment of collagen parallel to the pseudopodial axis.

Ongoing Research

Overall, the combination of DIC and reflected light confocal imaging allows us to assess both rapid changes in cellular contractility which can occur within minutes, and more gradual changes in cell-induced compaction and alignment of ECM which can occur over hours or even days. These novel approaches are being used to investigate the roles of the small GTPases Rho and Rac in regulating the sub-cellular pattern of force generation and extracellular matrix reorganization by corneal fibroblasts (Figure 6), as well as the cellular responses to local changes in mechanical stress (see Publications).

Recently, we have expanded our techniques to include: 1) experimental models for assessing how specific growth factors expressed following injury or surgery alter the mechanical differentiation of quiescent corneal keratocytes within 3-D collagen matrices, 2) novel approaches for assessing the response of fibroblasts to large scale changes in ECM mechanical properties (as might be observed during development or wound healing), and 3) 3-D constructs for assessing the mechanical interplay between fibroblast migration, sub-cellular force generation, and ECM patterning. Together, these experimental models are providing unique insights into the underlying biochemical and biomechanical signaling mechanisms controlling corneal fibroblast migration, contraction, and matrix reorganization in response to specific growth factors. This is fundamental information which can not be obtained using standard 2-D culture models, and may eventually lead to improved strategies for modulating cell mechanical activity during wound healing, and for designing artificial matrices and directing cell behavior during corneal tissue engineering.

Figure 6: Cell-matrix mechanical interactions in response to PDGF, which activates Rac. Cell-induced displacement and realignment of collagen fibrils was observed during PDGF-induced spreading. Tracking of the ECM displacements showed minimal collagen displacement prior to the addition of PDGF (B, red tracks, crosses mark position at time 0:00). However, following addition of PDGF, the matrix in front of the cell was pulled inward by the extending pseudopodial processes (C), resulting in compression of the ECM (yellow arrows).