Corneal Fibroblasts Respond Rapidly to Changes in Local Mechanical Stress

Abstract

purpose. To investigate the response of corneal fibroblasts to local changes in extracellular matrix (ECM) tension.

methods. Rabbit and human corneal fibroblasts were plated inside fibrillar collagen matrices. After 18 to 72 hours, a glass microneedle was inserted into the ECM and either pushed toward a cell to reduce local tension, or pulled away to increase tension. Time-lapse differential interference contrast (DIC) imaging was performed both before and after needle micromanipulation. ECM displacements were quantified, and strain maps were generated by finite element modeling. In some experiments, cells were treated with the Rho-kinase inhibitor Y-27632 either 30 minutes before, or 1 hour after they were pushed with the microneedle. Changes in focal adhesion organization were also evaluated in a subset of cells expressing green fluorescent protein (GFP)-zyxin, by simultaneous fluorescent and DIC imaging.

results. Pulling on the ECM resulted in initial cell elongation, followed by disengagement and retraction of pseudopodia. In contrast, pushing the ECM toward a cell induced rapid shortening (contraction), presumably since existing cellular forces were no longer counterbalanced by ECM tension. Pseudopodial extension (spreading) was then observed at both ends of the cell. The ECM was pulled inward during this secondary spreading, and rapid turnover of focal adhesions was observed along extending pseudopodia. Preincubation with Y-27632 or cytochalasin D blocked both the initial contractile and secondary spreading responses.

conclusions. Overall, the data suggest that corneal fibroblasts actively respond to increases or decreases in local matrix stress in an attempt to maintain tensional homeostasis (constant tension), and that this response may be mediated by Rho and/or Rac.

It is well established that mechanical stimuli play a key role in regulating growth and function in a variety of cell types. ^{1 2 3 4 5} Mechanical loading has been shown to alter features such as cell morphology ^{6 7} and sensitivity to growth factors. ^{8 9 10} In most cells, the development of focal contacts and stress fibers are tension-dependent processes, ^{11 12 13} and both in vitro and in vivo studies have demonstrated that these structures tend to align along the tensile axis under anisotropic conditions. ^{14 15 16 17} Cell migration and spreading are also influenced by the mechanical stiffness of the substrate. In general, cells on flexible two-dimensional (2-D) substrates are more migratory and have smaller focal adhesions than those on more rigid substrates. ¹⁸ Cells also preferentially spread on more rigid substrates. ¹⁹ Studies of wound healing in vivo and related experimental models in vitro have also shown that cellular force generation is regulated, in part, by the mechanical properties of the extracellular matrix (ECM). ^{6 9 14 20 21 22 23}  

The response of cells to dynamic changes in their mechanical environment has also been investigated. For example, Eastwood et al. ²⁰ used a culture force monitor to measure how dermal fibroblasts within three-dimensional (3-D) collagen matrices respond to changes in tensional loading. ²⁴ Cells were first allowed to develop baseline tension in the 3-D matrix. Subsequent stretching of the entire gel resulted in an initial increase in the measured force. However, this was immediately followed by a gradual cell-dependent reduction in force toward the baseline level. Similarly, compressing the gel caused an initial loss of tension that was followed by a cell-dependent increase in force back to the baseline level. Based on these data the authors conclude that cells within 3-D matrices respond to changes in mechanical loading in a way that maintains “tensional homeostasis” (constant tension) in their surrounding matrix. This model is also supported by studies demonstrating that cellular forces in 3-D matrices reach a constant value that is independent of matrix stiffness. ²⁵ Tensional homeostasis may be fundamental to the regulation of tissue tension under normal conditions, during development and also in response to injury. In the cornea, large shifts in the distribution of ECM tension can be induced by lacerating injury, penetrating keratoplasty, or refractive surgery. The response of corneal fibroblasts to changes in ECM stress may therefore play an important role in both the acute and long-term clinical outcomes after such insults. 

Although the elegant studies by Eastwood et al. ²⁰ have provided important insights into the overall response of an aggregate of dermal fibroblasts to tensional loading, the dynamic response of isolated cells to changes in local stress have yet to be studied in a 3-D system. Thus, the specific changes in mechanical activity (e.g., spreading, contraction, and migration) that underlie the changes in overall ECM stress are not known. Furthermore, the response of corneal fibroblasts to mechanical signals has received little attention. We recently developed a new experimental model to assess directly the cell–matrix mechanical interactions at the cellular level by plating corneal fibroblasts at very low density inside 3-D fibrillar collagen matrices, and performing high-magnification time-lapse differential interference contrast (DIC) and fluorescence imaging. ^{26 27 28} With this approach, pseudopodial extensions and retractions can be directly correlated with local collagen matrix deformation in a 3-D environment. We have used this model to study the behavior of both rabbit and human corneal fibroblasts after serum removal, disruption of the F-actin cytoskeleton, and inhibition of Rho-kinase. ^{26 27 28} However, the response of cells to mechanical stimuli has not yet been evaluated. In this study, we investigated the dynamic response of corneal fibroblasts to tensional loading, by performing time-lapse imaging while locally deforming the collagen matrix adjacent to isolated cells. We demonstrate for the first time that cells inside fibrillar collagen matrices undergo rapid changes in cell mechanical activity that are consistent with the tensional homeostasis model. The data also suggest that these responses may be mediated, in part, by the small GTPases Rho and/or Rac. 

Methods

Cells

Studies were performed using both primary rabbit corneal fibroblasts (NRK) and a previously characterized telomerase-infected, extended lifespan human corneal fibroblast cell line, HTK. ²⁹ NRK cells were harvested from New Zealand White Albino rabbit eyes (Pel-Freez, Rogers, AR) as previously described. ²⁶ Both cell types were cultured in 25-cm² tissue culture flasks (Costar, Cambridge, MA) using complete medium consisting of modified Eagle’s minimum essential media (MEM; Sigma-Aldrich, St. Louis, MO) supplemented with 1% Penicillin, 1% Streptomycin and 1% amphotericin B (Fungizone; BioWhittaker, Inc., Walkersville, MD) and 10% fetal bovine serum (FBS, Sigma-Aldrich). 

For some experiments, NRK cells were transfected to express green fluorescent protein (GFP)-zyxin, using human zyxin in a pEGFP-N1 vector (BD-Clontech Laboratories, Inc., Palo Alto, CA). This probe has been used previously as a marker for focal contacts in goldfish fin fibroblasts, ^{30 31 32} mouse melanoma cells, ³³ and corneal fibroblasts. ^{26 27 28} Transfection was then performed (Lipofectamine PLUS; Invitrogen, Carlsbad, CA) as previously described. ²⁶  

Collagen Matrices

Hydrated collagen matrices were prepared by mixing neutralized bovine dermal collagen (Vitrogen 100; Collagen Corp., Palo Alto, CA) with 10× MEM to achieve a final collagen concentration of 2.48 mg/mL. ²⁶ For plating cells inside the matrix, a 50-μL suspension of NRK or HTK cells was mixed with 500 μL of collagen solution. The cell/collagen mixture was preincubated at 37°C for 5 minutes, and 30-μL aliquots (containing approximately 500 cells) were then poured onto culture dishes (Delta T; Bioptechs, Inc., Butler, PA). Each aliquot was spread over a central 12-mm diameter circular region on the dish and was approximately 100 μm thick. The dish was then placed in a humidified incubator (37°C, 5% CO₂) for 60 minutes for polymerization and overlaid with 2 mL of complete medium. 

Time-Lapse Digital Imaging

Microscopy was performed as previously described. ^{26 27} Briefly, we used an inverted microscope with fluorescence and DIC imaging modules (TE300; Nikon, Tokyo, Japan) two high-speed filter wheels for rapid selection of excitation and emission filters and shuttering of epifluorescent illumination, and a high-resolution cooled CCD camera (CoolSnap HQ; Roper Scientific, Tuscan, AZ). The hardware was controlled by computer running image-analysis software (MetaVue; Universal Imaging Corp., Downingtown, PA). To maintain cell viability during imaging, a microincubation system and objective heater was used (Bioptechs, Inc.). A microperfusion pump (Bioptechs, Inc.) was used to perfuse the cells continuously while on the microscope stage with complete medium containing HEPES buffer at the rate of 6 mL/h. 

Dishes were moved to the microscope stage 18 to 72 hours after seeding on the gel, allowing them to develop a more consistent bipolar spindle-shaped morphology, as is observed during in vivo wound healing. ^{16 34 35 36} In each experiment, cells were allowed to acclimate to the microincubation system for 30 to 60 minutes before time-lapse imaging. The cell density was sparse enough to focus on the mechanical activity of a single cell, minimizing the potential interference caused by neighboring cells. The activity of a single cell was imaged for up to 5 hours using either a 40× or 60× oil-immersion objective. Nomarski DIC images and/or enhanced GFP (EGFP) images were automatically acquired at 1- to 3-minute intervals, using the imaging software (MetaVue; Universal Imaging Corp.). In most experiments, 3-D data sets were obtained at each time point by repeating the acquisition at four to five sequential focal planes in z-steps of 2 to 3 μm. To minimize phototoxicity, neutral-density filters and 2 × 2 on-chip camera binning was used. 

Micromanipulation of ECM

In all experiments, glass microneedles with flame-polished tips attached to a micromanipulator (Narishige Scientific Laboratory, Tokyo, Japan) were used for local deformation of the ECM. Microneedles with ∼35-μm diameter tips were made using a pipette puller. These needles were strong enough to withstand the forces needed to stretch or compress the collagen matrix. The needle was positioned at a 45° angle to the microscope stage. After 30 minutes of time-lapse imaging of a cell of interest, the needle was inserted axially into the collagen lattice 50 to 100 μm from the leading edge of the cell. After the needle was inserted, time-lapse imaging was performed for an additional 30 to 60 minutes. The needle was then either pushed toward the cell (25–50 μm) to compress the collagen ECM, thereby decreasing the effective matrix stiffness, or pulled away from the cell (25–50 μm) to stretch the ECM, thereby increasing the matrix stiffness (Table 1) . All manipulations were visualized using DIC imaging. We generally selected cells that were less than 25 μm from the top of the collagen matrix, to minimize free body motion of the gel. The micropipette was inserted only far enough to bring the tip into the same focal plane as the cell. After the ECM was pushed or pulled, time-lapse imaging was continued for an additional 2 to 4 hours. 

In additional experiments, cells were treated with the Rho-kinase inhibitor Y-27632 either 30 minutes before (10 cells) or 1 hour after the needle push (5 cells). Four microliters of a 5-mM stock solution of Y-27632 was added to the culture dish to achieve a final concentration of 10 μM, and the perfusion medium was simultaneously switched to complete medium containing 10 μM Y-27632. In other experiments, cytochalasin-D (Sigma-Aldrich) was added to the culture dish (final concentration, 25 μM) before ECM micromanipulation, to assess the effect of F-actin on the cellular response. Control experiments were also performed on collagen lattices without cells to determine the effect of needle pushing or pulling alone (i.e., without cellular force generation) on the pattern of ECM deformation. 

Image Processing and Analysis

Image processing was performed using MetaMorph (Universal Imaging Corp.). ECM deformation was quantified by measuring the x, y coordinates of landmarks in DIC images using the “measure pixel” feature in the software. To display the ECM displacements, a custom-written program (Visual Basic; Microsoft, Redmond, WA) program was used. The program generated cross-marks (×) and tracks corresponding with the start points and displacements, respectively, of ECM landmarks from the measured x, y coordinates. Matrix deformation before, during, and after micromanipulation was quantified. A similar approach was used to track adhesion movement in cells expressing GFP-zyxin. ²⁶  

Finite Element Modeling

Finite element modeling (FEM) was used to visualize and quantify the pattern of matrix deformation due to needle pushing, both with and without cells. Finite element models were created using engineering analysis software (Ansys, ver. 7.0; Ansys Inc., Canonsburg, PA), as previously described by us. ^{28 37} Briefly, nodes were defined at coordinates coinciding with ECM landmarks from the DIC images before ECM micromanipulation. Boundary nodes were placed at the periphery of a 600-μm diameter circular field around this central set of nodes. A 2-D plane stress model was created from the nodes with linear elastic triangular elements. For simplicity, the matrix was assumed to be isotropic, with a Young’s modulus of 3.89 × 10⁻¹⁰ N/μm², an effective thickness of 15 μm, and a Poisson’s ratio of 0.3. ^{28 38} To generate maps of ECM deformation, the displacements measured from time-lapse recordings were applied to the corresponding nodes in the model. The resultant strains induced on the matrix were calculated and displayed. 

Results

ECM Stress Induced by Needle Push

To investigate the ECM stress induced by needle micromanipulation, we first tracked the ECM deformation induced by pushing with microneedles using matrices without cells (Fig. 1A , red tracks, cross marks starting position). The FEM strain maps parallel to the direction of movement (x-axis) confirm that there was significant compression in front of needles (Figs. 1B 1C , blue areas). Principal strain vectors reveal that although some tension is produced perpendicular to this axis (Fig. 1D , small white vectors), compression is clearly the dominant effect (large blue vectors) in front of the needle. Note that maximum compression of the ECM was observed just in front of the needle (see Movie 1). We also found that tension was generated by pulling with microneedles (not shown). 

Fibroblast Response to ECM Compression

By 1 day after plating inside 3-D collagen matrices, both NRK and HTK cells generally had a bipolar morphology with thin pseudopodial processes, consistent with previous observations. ^{26 27} Cells were always aligned nearly parallel to the dish on which the collagen matrix was plated. DIC imaging allowed detailed visualization of the cells and the individual collagen fibrils surrounding them. Fibroblasts repeatedly extended and retracted pseudopodia at all time points after plating within 3-D matrices. ²⁶ One to 2 days after the cells were plated, pseudopodial extension generally occurred at the front of the cells, whereas the rear was much less active and underwent intermittent retractions, resulting in cell migration. The initial axial insertion of the microneedle into the ECM did not alter the normal pattern of cell behavior (Movie 2). By 3 days after plating inside collagen matrices, fibroblasts continued to extend and retract pseudopodia, but generally did not undergo significant migration, consistent with previous observations. ²⁸  

Pushing the ECM toward a cell with a microneedle induced rapid cellular shortening (contraction) with corresponding ECM compression along the cell body (Fig. 2B2F , arrows; compare with Figs. 2A2E ). After this initial contraction, rapid cell spreading was observed, and tractional force was generated as indicated by pulling in of the ECM (Figs. 2C2G , black tracks). This secondary spreading response was generally observed at both ends of cells (Fig. 2C) and is best appreciated in time-lapse movies (Movies 3 and 4). Note that during the secondary spreading response, large amounts of ECM displacement were produced for relatively small amounts of pseudopodial extension. “Bundling up” of collagen at the base of the pseudopodia was also often observed as new pseudopodial extensions pulled the collagen fibrils inward. The addition of Y-27632 1 hour after the needle push resulted in dramatic cell elongation and relaxation of tension on the matrix (Fig. 2D ; Movie 5), suggesting an active response to needle pushing that is, in part, Rho-kinase dependent. 

We also followed the dynamic changes in focal adhesion organization in response to ECM micromanipulation, by transfecting cells to express GFP-zyxin. GFP-zyxin was organized into focal adhesions that were most easily visualized along pseudopodial processes (Figs. 3A 3B , arrows), consistent with previous observations. ^{26 27} During cellular contraction after a 35-μm needle push, ECM deformation (Fig. 3B , blue tracks) correlated with the inward movement of existing focal adhesions toward the cell body (Fig. 3B , white tracks). In contrast, the secondary spreading response was associated with the rapid turnover and formation of focal adhesions along extending pseudopodia (Fig. 3C , arrows), which resulted in additional pulling in of the ECM (Fig. 3C , blue tracks; Movie 6). ^{26 27}  

Although there were differences in the magnitude of the initial cell contraction and secondary spreading, the same overall pattern of cell mechanical activity was observed in response to needle pushing in 20 of 21 cells. There was only one exception in which a cell moved away from the needle after the needle push. To quantify the amount of initial cellular contraction in response to ECM micromanipulation, we measured the distance between ECM landmarks at the ends of the cell and calculated the distance between them. Analysis was performed only on experiments in which both ends of the cells and the surrounding matrix were clearly visible (total 14 cells). All cells analyzed showed some amount of contraction immediately after needle push, with an average shortening of 26.9% ± 6.8% (range, 14%–37%; Table 2 ). Note that some amount of passive cellular shortening would be expected due to the effect of the needle push alone. We estimated this shortening to be less than 5%, using data obtained from needle pushing without cells (Fig. 1) . 

Effects of Y-27632 and Cytochalasin D on the Cellular Response to ECM Compression

Addition of the Rho-kinase inhibitor Y-27632 to serum-containing (S⁺) medium prior to pushing with the needle induced dramatic cell elongation and relaxation of tension on the matrix within 5 minutes (Fig. 4B , white arrows and black tracks; compare with 4A ). ²⁸ The active cellular contraction normally observed after needle push was essentially blocked by Y-27632, as indicated by minimal ECM displacements (Fig. 4C , black tracks). For the cell shown in Fig 4C , shortening of only 5.9% was observed; this is much less than the minimum shortening measured with complete medium (14%; Table 2 ). The secondary spreading response was also blocked by pretreatment with Y-27632. As shown is Figure 4D , there was no cell spreading or matrix deformation observed, even 1 hour after pushing with the needle (black tracks; see also Movie 7). 

Similarly, in cells pretreated with cytochalasin D, the normally observed responses to ECM compression were absent. Incubation with cytochalasin D resulted in cell elongation and relaxation of cell-induced ECM stress (Fig. 4F , white arrows and black tracks; compare with 4E ). Cellular shortening of only 8.5% was observed after pushing with the needle (Fig. 4G) . In addition, no secondary cell activity or ECM displacements were observed after the initial needle push (Fig. 4H) . Note that in cells pretreated with either Y-27632 or cytochalasin D, reperfusion with complete medium after the needle push resulted in cellular contraction and tractional force generation (not shown); thus, the cells were still viable. 

FEM Modeling

We also mapped the pattern of ECM deformation after the needle push using FEM (Fig. 5) , for both a large needle push (40 μm; Fig. 5A 5B ), and a smaller needle push (30 μm; Figs. 5C 5D ). The strain maps verify that there was compression along the cell body immediately after pushing the needle in S⁺ medium in both cases (Figs. 5A 5C , blue regions). Cellular shortening of 36% and 24% was measured for the cells in Figures 5A and 5C , respectively. Pulling in of the matrix during the secondary spreading response was also demonstrated in maps showing the strain produced beginning after the initial contraction (Figs. 5B 5D) . This effect was indicated by stretching of the matrix at the ends of cells and additional compression along the cell body. Note that during secondary spreading, maximum compression was usually generated near the base of extending pseudopodia (Figs. 5B 5D , dark blue areas), consistent with the pattern of ECM deformation observed in actively spreading cells both on planar elastic substrates and inside collagen matrices. ^{26 39}  

FEM modeling also confirmed that there was inhibition of both the initial and secondary responses after preincubation with Y-27632 (Figs. 5E 5F ; 30-μm needle push). The compression produced by the needle push is located primarily near the needle tip (Fig. 5E , blue region), not along the cell body as normally observed (compare with Figs. 5A 5C ). Note that a similar pattern of compression was observed in matrices without cells (compare with Fig. 1B ). Furthermore, little change in ECM stress was observed after the initial push (Fig. 5F , yellow indicates strains close to zero). 

Fibroblast Response to ECM Stretch

Inserting a microneedle in front of a cell and pulling it away stretched the ECM (Fig. 6B , red tracks) which resulted in cell elongation (compare small arrows in Figs. 6A6B ). Labeling with GFP-zyxin demonstrated that focal adhesions (Figs. 6E6F , arrows) initially remained attached to the ECM, because ECM displacement and focal adhesion displacements correlated highly (Fig. 6F , compare blue and white tracks). Later, pseudopodia often disengaged and retracted (Figs. 6C6G) . Adhesions were occasionally left behind (Figs. 6F6G , arrowheads), suggesting that they were “torn off” as the cell retracted. After retraction, repeated extension and retraction of pseudopodia were observed as cells partially respread (Movies 8 and 9). New focal adhesions were formed during respreading (Fig. 6H , arrows), but very little ECM displacement was observed (Figs. 6D6H , red and blue tracks). These results were observed in 10 of the 11 cells studied. In the other experiment, when the ECM was stretched by pulling near the trailing end of a migrating cell, the cell migrated away from the needle. 

It should be noted that for all experimental manipulations, similar responses were observed for NRK and HTK cells. Furthermore, there were no apparent differences in the response to micromanipulation between the three time points evaluated. 

Discussion

In this study, we investigated for the first time the response of isolated cells to local mechanical stimulation using a 3-D collagen matrix model. Changing local ECM stress using microneedles induced rapid and reproducible response patterns in both rabbit and human corneal fibroblasts. Pushing the ECM reduced local tension in the matrix and resulted in rapid cellular contraction with corresponding ECM compression. This initial contraction is probably due to the release of preexisting cellular forces, similar to the shortening produced by releasing one end of a rubber band that is under tension. After this initial contraction, respreading was observed at both ends of cells, and tractional force was generated as indicated by pulling in of the ECM. During this secondary spreading response, large amounts of ECM displacement were produced for relatively small amounts of pseudopodial extension, which would be expected for a low-compliance substrate (similar to a tire spinning in the mud). Pulling on the ECM resulted in initial cell elongation, followed by disengagement and retraction of pseudopodia. Raucher and Sheetz ⁴⁰ have demonstrated that the addition of amphiphilic compounds or fluorescent lipids that expand the plasma membrane and decrease membrane tension (as measured using laser tweezers) stimulates spreading of NIH 3T3 cells on rigid substrates. Conversely, increasing membrane tension by osmotically swelling cells reduces the lamellipodial extension rate. Our results are consistent with these findings, in that ECM compression (which reduces membrane tension) induced cell spreading and ECM stretch (which increases membrane tension) caused pseudopodial retraction. 

Many groups have described the existence of a steady level of cell-mediated tension in large aggregates of cells. ^{6 14 20 23 25} Brown et al. ²⁴ investigated the responses of dermal fibroblasts to changes in mechanical loading, using a culture force monitor to measure the cell-mediated changes in mechanical tension across entire 3-D matrices. It was demonstrated that fibroblasts respond to changes in mechanical loading in a way that maintains “tensional homeostasis” in the surrounding matrix. The response of isolated fibroblasts we observed in this study is also consistent with the tensional homeostatic model. After the release of tension due to needle push, cells actively pull in the ECM during respreading to partially decompress the matrix. Similarly, after the initial increase in tension due to pulling with the microneedle, the stretched cells attempt to release tension on the matrix by disengaging their pseudopodia. Thus, in both cases, corneal fibroblasts respond in a way that tends to counteract the initial change in stress. In previous studies, spontaneous rupture of adhesions and retraction of cells have been shown to reduce ECM tension. ^{26 27} However, we were unable to detect such ECM relaxation in this study. This is most likely because the large load being borne by the ECM after needle pulling masked the smaller forces generated by the cells. Future studies using smaller needle displacements during pulling, or more flexible (thinner) needles could be used to overcome this limitation. 

For FEM analysis of fibroblast-induced matrix distortion, linear elastic, isotropic material properties were used. Our previous matrix calibration experiments suggest that this is a reasonable assumption when studying the normal cellular pattern of force generation, ⁴¹ but it should be noted that individual collagen fibrils with which a cell interacts can undergo much larger displacements than neighboring fibrils, particularly at the leading edge. ²⁶ Furthermore, large pushes or pulls with the microneedle may induce stresses that are outside the linear response range for the collagen matrix. For this reason, FEM was used to generate strain maps (to visualize the pattern of ECM deformation), but was not used to estimate forces. 

The cellular response to transient mechanical stimulation with microneedles has been investigated by Lo et al. ¹⁹ and Wang et al., ⁴² who used planar elastic substrates. In their model, pulling near the trailing end of a migrating 3T3 cell caused the cell to reverse direction and move toward the microneedle. In contrast, pushing the substrate toward the leading edge of a cell caused the cell to retract its leading edge and migrate away from the needle. We did not observe this type of migratory behavior in response to mechanical stimulation in this study. However, significant differences exist between these two experimental models. First, most of the cells we studied were not undergoing rapid migration before needle manipulation. Second, the mechanical stiffness of collagen matrices is generally much less than that of polyacrylamide substrates. Finally, cell mechanical interactions on a planar substrate coated with nonfibrillar collagen are likely different from that which occurs within a 3-D fibrillar ECM. 

Previous studies have established that the Rho-family of small guanosine triphosphatases (GTPases) such as Rho, Rac, and Cdc42 play a central role in regulating the cytoskeletal changes associated with cell mechanical activity. These GTP-binding proteins function as molecular switches, alternating between the active GTP-bound state and the inactive GDP-bound state. Activated Rho stimulates the formation of stress fibers, the development of large focal adhesions (focal contacts), and cellular contraction (shortening). ^{43 44 45 46} In contrast, activated Rac induces the creation of smaller focal complexes, actin polymerization, and cell spreading. ^{43 44 45 46 47 48} Recent studies suggest that Rho and Rac may be involved in the cellular response to a variety of mechanical signals. For example, cyclic deformation strain of cultured airway smooth muscle cells induces increased stress fiber and focal adhesion formation and contractility coincident with a fourfold increase in Rho activity. ⁴⁹ In addition, reorientation of vascular endothelial cells in response to shear stress requires Rho-induced depolarization (cell rounding), followed by Rho/Rac mediated migration in the direction of flow. ⁵⁰ In vascular smooth muscle cells, noncyclic uniaxial mechanical stretching was shown to downregulate Rac and suppress cell spreading, whereas decreasing mechanical tension (by inhibiting Rho-kinase or myosin light chain kinase) increased cell spreading through upregulation of Rac. A similar spreading response was observed by inhibiting Rho-kinase in the current study. Increased spreading was also observed after reducing ECM tension by pushing with microneedles. Rac activation can be stimulated by platelet-derived growth factor (PDGF), ^{43 51} and PDGF induces both cell spreading and tractional force generation by corneal fibroblasts (manuscript in preparation), similar to the secondary response observed after needle pushing in the present study. 

In addition to inducing cell elongation and relaxation, inhibition of Rho-kinase also blocked both the initial and secondary responses to ECM compression with a microneedle. This supports our contention that the initial contraction results from preexisting cellular forces. Without these preexisting forces, the change in cellular tension induced by a needle push was apparently too small to induce the secondary spreading response. Taken together, the data suggest that Rho and Rac activation may play a central role in the fibroblast response to local mechanical stimulation. Additional studies more specifically targeting Rho and Rac signaling pathways are needed to clarify the molecular mechanisms underlying these important processes. 

 Supported by Grant EY13322 from the National Institutes of Health and funds from Research to Prevent Blindness, Inc.

 Submitted for publication April 1, 2004; revised May 4, 2004; accepted May 13, 2004.

 Disclosure: W.M. Petroll, None; M. Vishwanath, None; L. Ma, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Summary of Experiments

Needle Maneuver and Reagents	Experiments at Each Time Point (n)					Cell Type (n)
		1 Day	2 Days	3 Days	NRK	HTK
Needle push	9	10	2	12	9
Needle pull	3	4	4	6	5
Y-27632 → needle push	4	5	1	7	3
Cytochalasin-D → needle push	2	2	—	2	2

 NRK, rabbit corneal Keratocytes; HTK, human corneal Fibroblasts.

Figure 1.

 

The effects of needle micromanipulation on the ECM. (A) DIC image of a collagen matrix without cells, being compressed by pushing with a glass microneedle from right to left (ECM displacements are indicated by red tracks; ×, starting position before pushing the needle). (B) Map of strain along the x-axis generated from the ECM displacements using FEM (element solution). Pushing on the ECM with a needle caused matrix compression (blue and green) in front of and tension (red and orange) behind the region of contact of the needle. Scale bars are in dimensionless units ΔL/L (change in length/initial length). Negative values: compression; positive values: tension. Black bar: front of needle. (C) Contour map of strain along the x-axis generated using the nodal solution. (D) Principal strain vectors: blue, compression; white: tension.

Figure 2.

 

Fibroblast response to ECM compression (representative of 20 of 21 experiments). (A–D) Rabbit corneal fibroblast 2 days after plating inside collagen matrix. (A) Before needle push. (B) Pushing the ECM 45 μm toward the cell induced rapid cellular contraction (36% shortening) and ECM compression along the cell body (arrows). (C) This initial contraction was followed by the extension of pseudopodia (arrows) and rapid pulling in of the ECM (traction) at both ends of the cell (black tracks, ×: start position beginning 1 minute after needle push). (D) Subsequent addition of Y-27632 induced cell elongation and dramatic relaxation of cell-induced matrix tension (black tracks, ×: start position after adding Y-27632). This response to Y-27632 was observed in all five cells evaluated. (E–G) Rabbit corneal fibroblast 1 day after plating inside collagen matrix. (E) Before needle push. (F) Cellular contraction (arrows) was observed after a 25-μm needle push. (G) This initial contraction was followed by cell spreading (arrows) and traction (black tracks, ×: start position beginning 1 minute after needle push).

Figure 3.

 

Overlays of GFP-zyxin (green) and DIC (red) images demonstrating the response to ECM compression. (A) Rabbit corneal fibroblast 2 days after plating inside collagen matrix. GFP-zyxin was organized into focal adhesions that were most easily visualized along pseudopodial processes (arrows). (B) During cellular contraction after a 35-μm needle push, ECM deformation (blue tracks) correlated with the inward movement of existing focal adhesions (arrows) toward the cell body (white tracks). (C) The secondary spreading response was associated with the formation of new focal adhesions at pseudopodial tips (arrows), and rearward movement of existing adhesions, which resulted in additional pulling in of the ECM (blue tracks).

Table 2.

 

Cellular Contraction after Needle Push

Cells Analyzed	Shortening (μm)	Range (μm)	Shortening (%)	Range (%)
14	25.3 ± 6.4	13.5–34.8	26.9 ± 6.8	14–37

 Shortening is expressed as the mean ± SD.

Figure 4.

 

Y-27632 and cytochalasin D blocked the cellular response to ECM micromanipulation (representative of 10 Y-27632 experiments and 4 cytochalasin D experiments). (A–D) Rabbit corneal fibroblast 1 day after plating inside collagen matrix. (B) Cell elongation (arrows) and ECM relaxation was observed after adding Y-27632 (black tracks, ×: position just before addition of Y-27632). (C) Subsequent pushing with a needle (note shadow of needle on right) induced little cell contraction (5.9% shortening, black tracks). (D) There was also no secondary spreading or traction after needle push, as indicated by minimal ECM displacement (black tracks, ×: starting position 1 minute after push). (E–H) Human corneal fibroblast 2 days after plating inside collagen matrix. (F) After cytochalasin D was added, elongation of the cell (arrows) and relaxation of cell-induced matrix stress were observed (black tracks). (G) Pushing on the ECM in front of the cell caused ECM compression in front of the needle, but little cell contraction (4.7% shortening, black tracks). (H) There was also no secondary spreading or traction after needle push, as indicated by minimal ECM displacements (black tracks, ×: starting position 1 minute after push).

Figure 5.

 

FEM strain maps of both the initial (A, C, E) and secondary (B, D, F) responses to needle push in S⁺ medium (A–D) and in medium containing Y-27632 (E, F). Scale bars are in dimensionless units ΔL/L (change in length/initial length). Negative values: compression; positive values: tension. Black bars: the front of needle. (A, B) Same cell as in Figures 2A 2B 2C . (A) After a 40-μm needle push, compression was observed along the cell body (blue regions). (B) Additional pulling in of the matrix (traction) was observed over the next 30 minutes. This is indicated by decompression of the matrix at the ends of cells (red, yellow, and green regions) and additional compression along the cell body (blue regions). Only the strain produced after the initial response is shown (starting 1 minute after the needle push). (C, D) Human corneal fibroblast 2 days after plating inside collagen matrix. (C) After a 30-μm needle push, compression was observed along the cell body (blue and green regions). (D) Additional pulling in of the matrix was observed over the next 50 minutes. This is indicated by stretching of the matrix at the ends of cells (red and orange regions) and additional compression along the cell body (blue and green regions). Only the strain produced after the initial response is shown (starting 4 minutes after the needle push). (E, F) Same cell as in Figures 4A 4B 4C 4D . Both the initial and secondary responses to a 30-μm needle push were inhibited by preincubation with Y-27632. The compression produced by the needle push is located primarily near the needle tip (E; blue region); a similar pattern of compression was observed in matrices without cells (compare with Fig. 1B ). Cellular shortening of only 5.9% was detected. Very little ECM stress was generated after the initial push (F; yellow indicates strains close to zero).

Figure 6.

 

Fibroblast response to ECM stretching produced by pulling the ECM away from cells with a glass microneedle (representative of 10/11 experiments). (A–D) Rabbit corneal fibroblast, 2 days after plating inside collagen matrix. Needle pull is 50 μm to the right. (A) Before needle pull. (B) On pulling the ECM in front of the cell with a microneedle, the cells and ECM were stretched, as visualized by matrix displacements (red tracks). (C, D) Later, pseudopodia disengaged and retracted (compare arrows in C and D) and then extended and retracted processes without inducing significant ECM deformation (red tracks, ×: position 1 minute after needle pull). (E–H) GFP-zyxin (green) and DIC (red) overlay of rabbit corneal fibroblast, 1 day after plating inside collagen matrix. Needle pull is 30 μm to the right. (E) Before needle pull. (F) On pulling the ECM in front of the cell with a microneedle, the cells and ECM were stretched, as visualized by matrix displacements (blue tracks). Labeling with GFP-zyxin demonstrated that focal adhesions (E, F, arrows) initially remained attached to the ECM, since ECM displacement and focal adhesion displacements correlated highly (compare blue and white tracks). (G, H) After the initial stretch, the pseudopodia disengaged and retracted. After retraction, repeated extensions and retractions of pseudopodia were observed as cells partially respread. New focal adhesions were formed as the cells respread (H, arrows), but no significant ECM displacement was observed (G, H, blue tracks, ×: position 4 minutes after needle pull). Adhesions were occasionally left behind (F, G, arrowheads), suggesting that they were “torn off” as the cell retracted.

Supplementary Materials

Movie 1 - The effects of needle micromanipulation on the ECM. DIC image of a collagen matrix without cells being compressed by pushing with a glass microneedle from right to left. 

Movie 2 - Time-lapse DIC images of a rabbit corneal fibroblast following axial insertion of needle. Pseudopodial extension occurred at the front of the cell (right), whereas the rear was much less active and underwent intermittent retractions. 

Movie 3 - Rabbit corneal fibroblast 1 day after plating inside collagen matrix. Cellular contraction was observed following a 25 μm needle push. This initial contraction was followed by cell spreading (yellow arrows at end of sequence) and traction (green circled region). Red tracks show measured ECM displacements (crosses mark position 1 minutes after needle push). 

Movie 4 - Rabbit corneal fibroblast 2 days after plating inside collagen matrix. Pushing the ECM 45 μm towards the cell induced rapid cellular contraction (36% shortening) and ECM compression along the cell body. This initial contraction was followed by the extension of pseudopodia (yellow arrows at end of sequence) and rapid pulling in of the ECM (traction) at both ends of the cell (green circled regions). Red tracks show measured ECM displacements (crosses mark position 1 minutes after needle push). 

Movie 5 - Addition of Y-27632 60 minutes after needle push induced cell elongation and dramatic relaxation of cell induced matrix tension (red tracks, cross marks start position after adding Y-27632). 

Movie 6 - Overlays of GFP-zyxin (green) and DIC images (red) demonstrating the response to ECM compression. During cellular contraction following a 35 μm needle push, ECM deformation correlated with the inward movement of existing focal adhesions toward the cell body. The secondary spreading response was associated with the formation of new focal adhesions at pseudopodial tips, and rearward movement of existing adhesions, which resulted in additional pulling in of the ECM (circled regions, blue tracks). 

Movie 7 - Y-27632 blocks the cellular response to ECM micromanipulation. Human corneal fibroblast 1 day after plating inside collagen matrix. Cell elongation and ECM relaxation was observed after adding Y-27632. Subsequent pushing with a needle (note shadow from needle on right) induced little cell contraction. There was also no secondary spreading or traction following needle push as indicated by minimal ECM displacements (red tracks). 

Movie 8 - Fibroblast response to ECM stretch produced by pulling the ECM away from cells with a glass microneedle. Rabbit corneal fibroblast, 2 days after plating inside collagen matrix. Upon pulling on the ECM in front of the cell with a microneedle, the cell and ECM were stretched. Later, pseudopodia disengaged and retracted, then extended and retracted processes without inducing significant ECM deformation. 

Movie 9 - GFP-zyxin (green) and DIC (red) overlay of rabbit corneal fibroblast, 1 day after plating inside collagen matrix. Upon pulling on the ECM in front of the cell with a microneedle, the cells and ECM were stretched. Focal adhesions initially remained attached to the ECM, since ECM displacement and focal adhesion displacements were highly correlated. Following the initial stretch, pseudopodia disengaged and retracted. Following retraction, repeated extension and retraction of pseudopodia were observed as cells partially respread. New focal adhesions were formed as the cells respread, but no significant ECM displacement was observed (blue tracks). 

The authors thank Jürgen Wehland and coworkers (BGF, Braunschweig, Germany) for providing the EGFP-zyxin expression vector, James Jester for providing the HTK cells, and Charles Chuong (University of Texas at Arlington) for advice regarding finite element modeling.