January 2008
Volume 49, Issue 1
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Glaucoma  |   January 2008
Substrate Rigidity Modulates Cell–Matrix Interactions and Protein Expression in Human Trabecular Meshwork Cells
Author Affiliations
  • Günther Schlunck
    From the Division of Experimental Ophthalmology and the
  • Hong Han
    From the Division of Experimental Ophthalmology and the
  • Thomas Wecker
    From the Division of Experimental Ophthalmology and the
  • Daniel Kampik
    From the Division of Experimental Ophthalmology and the
    Glaucoma Center, Würzburg University Eye Hospital, Würzburg, Germany.
  • Tobias Meyer-ter-Vehn
    From the Division of Experimental Ophthalmology and the
    Glaucoma Center, Würzburg University Eye Hospital, Würzburg, Germany.
  • Franz Grehn
    From the Division of Experimental Ophthalmology and the
    Glaucoma Center, Würzburg University Eye Hospital, Würzburg, Germany.
Investigative Ophthalmology & Visual Science January 2008, Vol.49, 262-269. doi:10.1167/iovs.07-0956
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      Günther Schlunck, Hong Han, Thomas Wecker, Daniel Kampik, Tobias Meyer-ter-Vehn, Franz Grehn; Substrate Rigidity Modulates Cell–Matrix Interactions and Protein Expression in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(1):262-269. doi: 10.1167/iovs.07-0956.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. Extracellular matrix (ECM) composition, tension, and rigidity modulate cell–ECM interactions and have substantial impact on cell functions. The authors studied the effects of ECM rigidity on human trabecular meshwork (HTM) cells to assess ECM rigidity as a possible pathophysiologic factor in glaucoma.

methods. Trabecular meshwork cells derived from donor cornea rings and passaged three to seven times were plated on collagen-coated tissue culture plastic or polyacrylamide gels of different rigidity. Cell spreading and focal adhesions were assessed by immunofluorescence microscopy. Expression of focal adhesion kinase (FAK), α-smooth muscle actin (α-SMA), tubulin, α-B-crystallin and GAPDH, as well as phosphorylation of FAK and serum-induced activation of ERK, were studied by Western blot. The subcellular distributions of α-SMA and fibronectin were examined by confocal immunofluorescence microscopy.

results. ECM rigidity modulated cell spreading and focal adhesion size. FAK activation and serum-induced ERK phosphorylation increased with rising substrate rigidity. Expression of α-SMA and recruitment of α-SMA to stress fibers were enhanced on rigid substrates, whereas myocilin and α-B-crystallin expression increased on soft substrates. The structure of fibronectin deposits differed on stiff and soft matrices.

conclusions. Extracellular matrix rigidity modulates cytoskeletal structures, protein expression patterns, signal transduction, and fibronectin deposition in HTM cells. ECM changes altering trabecular meshwork resiliency may therefore have significant effects on ocular outflow tract functions with implications in glaucoma.

The trabecular meshwork is critically involved in the regulation of aqueous humor outflow. 1 2 It is a mechanosensitive structure under dynamic mechanical stress exerted by the ciliary muscle, intrinsic contractile elements, and intraocular pressure. 3 4 5 6 Primary open-angle glaucoma is associated with structural changes in the trabecular meshwork, which likely affect tissue rigidity and biomechanics. 7 In particular, an increase in extracellular matrix (ECM) material in the cribriform meshwork adjacent to Schlemm’s canal and a loss of cells in the uveoscleral meshwork have been observed. 2 8 Similar, but less pronounced, changes occur with age, 9 another risk factor for glaucoma. The extent of ECM alterations correlates with the severity of glaucomatous damage to the optic nerve. 10 The driving force for these ECM changes is not entirely clear, but the growth factor TGF-β, whose aqueous humor levels are increased in glaucoma, 11 may have an important role. It enhances trabecular meshwork cell contractility and actin stress fibers 12 and promotes ECM deposition to increase outflow resistance. 13 TGF-β also induces cross-linking of ECM proteins by tissue transglutaminase, which may have additional detrimental effects in glaucoma. 14 All these observations suggest an essential role of cell–ECM interactions in IOP homeostasis and glaucoma. 7  
On a cellular level, the interactions of cells and surrounding ECM have a decisive impact on cell fate and govern various cell functions. 15 While cells deposit and remodel ECM, they are themselves subject to regulatory signals elicited by ECM components in an interdependent signaling network. 16 Cells bind to ECM molecules using integrins, a family of dimeric transmembrane receptors. Integrins serve as mechanical anchors connecting ECM molecules to the actin cytoskeleton. At the same time, integrins form the core of signaling molecule clusters (e.g., focal adhesions), which modulate cytoskeletal rearrangements, growth factor signaling, cytokinesis, and cell survival. 15 17 Cells respond to ECM-mediated mechanical stimulation by integrin-dependent cytoskeletal adaptation and changes in protein expression and phosphorylation patterns. 18 19 20 Furthermore, the rigidity of the ECM modulates cell migration and survival. 21 22 23 A recent study revealed that adjusting ECM rigidity suffices to determine mesenchymal stem cell differentiation into neuronal cells, myotubes, or osteoblasts. 24 Thus, changes in ECM composition or its mechanical properties can affect a range of adhesion-dependent cellular functions. We therefore studied the effects of ECM rigidity on human trabecular meshwork (HTM) cells. Here we show that ECM rigidity modulates cytoskeletal configuration, protein expression, and signal transduction in this cell type and may contribute to changes in trabecular meshwork function related to glaucoma. 
Materials and Methods
Reagents
Antibodies raised against the following proteins were used: α-smooth muscle actin (α-SMA), vinculin, tubulin, and fibronectin (Sigma, Taufkirchen, Germany), focal adhesion kinase (FAK; Cell Signaling Technology/NEB, Frankfurt, Germany), pY397FAK (Biosource, Nivelles, Belgium), extracellular regulated kinase (ERK; Santa Cruz Biotechnology, Santa Cruz, CA), pERK (Promega, Mannheim, Germany), GAPDH, myocilin (affinity-purified goat anti-human; R&D Systems, Wiesbaden, Germany), α-B-crystallin (Stressgen/Biomol, Hamburg, Germany), fluorescent dye (Alexa 488)-conjugated goat anti-mouse (Molecular Probes, Eugene, OR), and horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson/Dianova, Hamburg, Germany). Phalloidin-TRITC (Sigma, Taufkirchen, Germany) was used to stain filamentous actin. 
Cell Culture
HTM tissue was derived from donor cornea rings, and cells were cultivated according to methods published earlier 25 26 with slight modifications. The tenets of the Declaration of Helsinki were followed in all procedures. In brief, donor rings were transferred from the storage medium and kept in Dulbecco modified Eagle medium (DMEM; PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% heat-inactivated fetal calf serum (Biochrom, Berlin, Germany) and with 100 U/mL penicillin and 100 μg/mL streptomycin (both from PAA) for 24 hours. Under microscopic guidance, anterior and posterior incisions were placed to isolate the trabecular meshwork, which was then removed using forceps and was cut into smaller sections. The tissue sections were placed in 24-well plates, covered with a glass coverslip to avoid floating, and incubated in growth medium. Confluent cell layers were passaged by trypsinization. Cells were characterized by assessing baseline α-B-crystallin expression and increased myocilin expression after 7 days of dexamethasone treatment. The myocilin response to dexamethasone treatment has been reported as specific for trabecular meshwork cells. 27 28 Cells from three different donors were used from passages 3 to 7. All experiments were performed at least three times with similar results. 
Flexible Substrates
Polyacrylamide substrates were prepared as described. 29 30 The gels were composed (final concentrations) of 7% acrylamide, 3% acrylamidopropyl trimethyl ammonium chloride, 0.1% to 0.8% bis-acrylamide to control rigidity, 0.01% ammonium persulfate and 0.003% N,N,N′,N′-tetramethyl methylene diamine (all Sigma) in PBS. The charged trimethyl ammonium chloride compound allows for ECM binding. In this study, gels containing 0.1% and 0.2% bis-acrylamide were termed soft gels, and gels containing 0.4% or 0.8% bis-acrylamide were termed stiff gels. For immunofluorescent stains, 24-mm round coverslips were coated with amino-silane (Sigma) for 2 minutes, washed in water, activated with 0.5% glutaraldehyde (Roth, Karlsruhe, Germany) for 30 minutes, washed again, and air dried. Polyacrylamide solution (14 μL) was pipetted onto the coverslip and covered with an 18-mm round coverslip. After polymerization for 30 minutes, the smaller coverslip was removed, and the gel was washed in PBS and coated with collagen (15 μg/mL PBS; Inamed Biomaterials, Fremont, CA) for 1 hour in a cell culture incubator, washed again extensively in PBS, and subsequently incubated in DMEM. To cultivate cells for Western blots, gels 0.75-mm thick were cast in a regular minigel casting stand (BioRad, Munich, Germany), removed after 60-minute polymerization, and washed in PBS. A 6-cm cell culture plate was used to punch out round gels, which were transferred to dishes of respective size and coated with collagen as described. 
Spreading Assay
HTM cells were trypsinized, kept in suspension in a cell culture incubator for 1 hour to allow for equal retraction of all cells, and subsequently plated on collagen-coated glass coverslips or polyacrylamide gels. At 20, 40, or 60 minutes after plating, the cells were fixed in 2% paraformaldehyde (Merck, Mannheim, Germany) and permeabilized with 0.1% Triton X-100, and F-actin was stained with phalloidin-TRITC (Sigma). After washing in PBS, the stained samples were mounted (Vectashield; Vector, Burlingame, CA) and viewed under a fluorescence microscope (Axiophot; Zeiss, Oberkochen, Germany). To assess cell spreading, slide labels were blinded, and cells were counted in randomly chosen fields until at least 100 cells were counted. Spreading cells were identified by their formation of lamellipodia and were scored as spreading; all other cells were scored as nonspreading. 
Western Blot
Cells plated on collagen-coated tissue culture plastic or polyacrylamide gels were used for Western blot analysis. Cells were rinsed with ice-cold PBS, and total cell protein extracts were prepared using a RIPA lysis buffer (20 mM Tris, 150 mM NaCl, 0.1 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS) containing phosphatase and protease inhibitors (Phosphatase Inhibitor Cocktail III, Calbiochem/Merck, Bad Soden, Germany; Complete Protease Inhibitor, Roche, Mannheim, Germany). Protein concentrations were measured using a BCA assay (KMF, Lohmar, Germany). Ten micrograms protein extract was boiled in Laemmli sample buffer and subjected to SDS polyacrylamide gel electrophoresis. Proteins were transferred onto a polyvinylidene membrane (Amersham, Braunschweig, Germany) using a BioRad gel blotting apparatus. Membranes were blocked in 3% BSA in TBST (10 mM Tris HCl, 150 mM NaCl, 0.1% Tween 20) for 1 hour. Membranes were incubated with primary antibody overnight at 4°C and with a peroxidase-conjugated secondary antibody for 45 minutes at room temperature. After each incubation step, membranes were washed in TBST for 30 minutes. Peroxidase was visualized by enhanced chemiluminescence (ECL) and exposure to ECL films (Amersham, Braunschweig, Germany) for appropriate times. 
Immunofluorescence Confocal Microscopy
Cells plated on collagen-coated glass coverslips or polyacrylamide gels were fixed in 2% paraformaldehyde, permeabilized in 0.2% Triton X-100, blocked in 2% normal goat serum (Jackson-Immuno, Hamburg, Germany), labeled with primary antibodies against vinculin (1:400), α-SMA (1:500), or fibronectin (1:500) in blocking buffer at 4°C overnight, and washed in PBS. Fluorescent dye (Alexa 488)-conjugated secondary antibodies against mouse or rabbit (for fibronectin stains) IgG were used at a dilution of 1:500 for 1 hour at room temperature in blocking buffer. Phalloidin-TRITC (Sigma) was used to counterstain the F-actin cytoskeleton (1 hour, room temperature), and after washing in PBS the samples were mounted in medium (Vectashield; Vector Laboratories) containing DAPI. Cells were viewed with a laser scanning confocal microscope (TCS SP-2; Leica Microsystems, Bensheim, Germany). 
Results
Substrate Rigidity Modulates Cell Spreading
Interactions of cells with ECM mediate the spreading of newly plated cells in vitro. Cell spreading involves integrin binding to ECM molecules with initiation of integrin signaling, RhoGTPase activation, and subsequent actin reorganization. 31 We studied cell spreading to assess the influence of substrate rigidity on cell–matrix interactions in a functional assay. Cells were trypsinized, kept in suspension for 1 hour, and replated on collagen-coated polyacrylamide gels of different rigidity. Spreading cells were scored 20, 40, or 60 minutes after plating. Cell spreading was rigidity dependent and was initiated more rapidly on rigid substrates in a dose-dependent manner (Fig. 1) , indicating an influence of substrate rigidity on cell–ECM interactions. 
Substrate Rigidity Affects Focal Adhesion Formation
To allow for cell spreading, integrin-mediated cell–ECM interactions induce early adhesive sites and the subsequent formation of focal complexes and focal adhesions. 32 Focal adhesions serve as signaling centers and anchor the actin cytoskeleton to the ECM. To further elucidate the effect of substrate rigidity on the formation of cell–ECM interaction sites, we used immunofluorescence microscopy to examine focal adhesions in spread cells. In these experiments, cells were plated at a low density to avoid possible confounding effects of cell–cell adhesions and were fixed and stained 2 days after plating. HTM cells showed strong actin stress fibers (Figs. 2A 2G)and elongated focal adhesions (Figs. 2D 2G)on collagen-coated coverslips. On polyacrylamide gels, actin stress fibers were thinner and less intensely stained. This effect was dependent on the rigidity of the polyacrylamide gel (Figs. 2B 2C) . The loss of prominent stress fibers was associated with a redistribution of vinculin to the perinuclear region (Figs. 2D 2E 2F) . On 0.8% bis-acrylamide-containing gels (Figs. 2B 2E 2H) , adhesions at the ends of stress fibers appeared shorter and stained less intensely for vinculin than on tissue culture plastic. On soft gels containing 0.2% bis-acrylamide, vinculin prevailed in a punctate pattern with no apparent elongated focal adhesions (Figs. 2C 2F 2I)
Substrate Rigidity Modulates Growth Factor Signaling
The activation of mitogen-activated protein kinases (MAPKs) by growth factors requires integrin-mediated cell adhesion signaling. 33 Given that the previous experiments suggested an influence of substrate rigidity on cell adhesion sites, we next explored possible rigidity-dependent alterations in MAPK activation. Cells were plated on substrates of different rigidity, serum starved overnight in 3% FCS, and stimulated with 10% FCS for 30 minutes. Activation of ERK was determined by Western blot using a phosphospecific antibody. Baseline ERK phosphorylation was not detected on soft substrates (Fig. 3)but was present in cells on tissue culture plastic. Stimulation by 10% FCS induced strong ERK phosphorylation in cells on tissue culture plastic and elicited weaker effects on soft substrates in a rigidity-dependent manner (Fig. 3)
Substrate Rigidity Influences Focal Adhesion Kinase Phosphorylation and Expression of Cytoskeletal Proteins
Cell–ECM interactions modulate gene expression in an integrin-dependent fashion. 15 Recent studies indicate an influence of ECM rigidity on protein expression patterns. 24 To further explore the effects of substrate rigidity on focal adhesions and cytoskeletal proteins, we studied the phosphorylation of focal adhesion kinase (FAK) and the expression of α-smooth muscle actin (α-SMA) and tubulin. HTM cells were plated on substrates of different rigidity, allowed to adjust for 7 days in 3% FCS, and harvested for Western blot analysis. FAK phosphorylation was dependent on substrate rigidity showing decreased activation on softer substrates, whereas total FAK protein expression was unaffected by substrate rigidity (Fig. 4) . The differences in FAK phosphorylation suggest different levels of cell–ECM interaction-induced focal adhesion signaling. GAPDH-corrected OD ratios for pFAK/FAK were 0.97, 0.52, and 0.53 for plastic, 0.8% gels, and 0.2% gels, respectively. Expression of the contractility-related protein α-SMA declined substantially with decreasing substrate rigidity (Figs. 4A 4B) . In contrast, tubulin showed a biphasic response with diminished expression at intermediate rigidity and increased expression on soft polyacrylamide gels. These data suggest that distinct substrate rigidity levels may induce specific protein expression patterns. 
Substrate Rigidity Modulates α-Smooth Muscle Actin Localization
Expression of α-SMA is associated with increased cell contractility 34 and has been reported in the trabecular meshwork of young healthy eyes. 2 α-SMA is incorporated into the actin cytoskeleton to enhance cellular contractile strength. 34 To address the localization of α-SMA, HTM cells were plated on substrates of different rigidity, kept in 10% FCS for 10 days, and fixed and stained for F-actin and α-SMA. Expression of α-SMA and its incorporation into actin stress fibers were both dependent on ECM rigidity (Fig. 5) . α-SMA expression was most pronounced on glass coverslips. In cells on 0.8% bis-acrylamide gels, α-SMA was diffusely distributed throughout the cytoplasm, and few cells presented α-SMA in stress fibers. Cells on soft 0.2% bis-acrylamide gels showed a weak, diffuse cytoplasmic α-SMA signal (see also Fig. 4 ) and no detectable incorporation into stress fibers. 
Substrate Rigidity Modulates Myocilin and α-B-Crystallin Expression
Increased expression of myocilin and the chaperone α-B-crystallin has been detected in the trabecular meshwork of glaucomatous eyes. 35 Because ECM changes associated with glaucoma 2 may induce alterations in ECM rigidity, 7 we were compelled to study the influence of ECM rigidity on the expression of these proteins. Both proteins appeared to be increasingly expressed on polyacrylamide gels compared with rigid tissue culture plastic (Fig. 6)
Substrate Rigidity Modulates Fibronectin Deposition
HTM cells deposit and remodel ECM. Integrin-mediated cell–ECM interactions are essential in these processes 36 as cells exert mechanical force on ECM molecules to expose cryptic sites and assemble matrix components in a structured manner. 37 38 39 To investigate the effect of substrate rigidity on ECM deposition, HTM cells were plated on collagen-coated substrates of different rigidity, kept in 10% FCS for 10 days, fixed, and stained for fibronectin and F-actin. 
The morphology of fibronectin deposits differed with substrate rigidity (Fig. 7) . On rigid collagen-coated glass coverslips, the cells deposited extensive fibronectin networks with abundant nodular connections (Figs. 7A 7D) . On 0.8% bis-acrylamide gels, the fibronectin network appeared less elaborate with fewer nodes (Fig. 7B 7E) . On soft 0.2% bis-acrylamide gels, fibronectin was deposited in short, singular strips lacking a network architecture (Figs. 7C 7F) . Fibronectin mRNA expression was not significantly affected by substrate rigidity (data not shown). 
Discussion
Primary open-angle glaucoma is associated with distinct morphologic changes in the ECM of the trabecular meshwork. 2 It is not entirely clear how these changes affect ocular outflow resistance. Recent studies indicate that cell–ECM interactions and the actin cytoskeleton of trabecular meshwork cells have essential regulatory roles in aqueous humor dynamics. 1 7 It has also been suggested that increased ECM deposition and protein cross-linking observed in glaucoma may alter trabecular meshwork rigidity. 1 ECM rigidity was shown to have a major impact on cell differentiation and function in general. 22 23 29 It modulates the activation of adhesion proteins and alterations of the actin cytoskeleton in fibroblasts 29 and is sufficient to guide stem cell lineage specification. 24 Here we show that ECM rigidity has a substantial influence on cell–ECM interactions, protein expression, and matrix deposition in HTM cells. 
Unlike regular cell culture dishes, polyacrylamide gel substrates allow reproduction of the rigidity of natural tissues such as brain, muscle, or bone by varying the amount of bis-acrylamide cross-linker. 24 29 40 Using this technique in a functional cell spreading assay, we observed that HTM cells indeed sense different levels of substrate rigidity (Fig. 1) . The cells spread most rapidly on the stiffest gel tested (0.8% bis-acrylamide) and showed a gradual rigidity-dependent decline in cell spreading on softer substrates (Fig. 1) . These findings are supported by other reports. To spread, newly plated fibroblasts require cell–ECM binding, integrin activation, Rho GTPase signaling, and subsequent actin polymerization. 31 Spreading cells exert contractile force on the ECM substrate to allow for focal adhesion formation 41 and locomotion, and sufficient ECM resiliency is required to balance cellular tension force and to support the spreading process. 41 42 Thus, cells spread more readily on stiff substrates. 42 Our data also indicate that the polyacrylamide substrates used do have functionally different rigidity characteristics. 
Cell–ECM interactions shape the actin cytoskeleton and focal adhesions. 16 To explore a possible influence of substrate rigidity on these structures in HTM cells, we examined the localization of filamentous actin and the focal adhesion protein vinculin 41 in cells plated on substrates of different stiffness. We used collagen-coated glass as a reference because rigid glass or plastic is the current standard substrate in cell biology. When cells were plated on collagen-coated glass, vinculin localized mainly to elongated focal adhesions at the ends of strong actin stress fibers (Figs. 2D 2G) , similar to previous observations. 43 On less rigid polyacrylamide gels, actin stress fibers were more delicate and less intensely stained (Figs. 2B 2C) , whereas vinculin was more diffusely distributed and was enhanced in a perinuclear compartment (Figs. 2E 2F)as rigidity decreased. Cells plated on soft gels (0.2% bis-acrylamide) were devoid of typical vinculin-positive focal adhesions, suggesting that substrate rigidity regulates focal adhesion size in HTM cells. It has been shown previously that adhesion size and structure depend on the mechanical load applied to the adhesion site. 44 45 Because external forces and cell-generated tension are in a continuous equilibrium, 42 rigid substrates allow for larger mechanical loads and thus support larger cell adhesions. 29  
Focal adhesions not only anchor the actin cytoskeleton to the ECM, they have important signaling functions. 15 16 33 To assess whether the rigidity-dependent changes of the cytoskeleton and cell adhesions are associated with altered signal transduction, we studied growth factor-induced ERK activation in cells plated on different substrates. After starvation in 2% FCS overnight, the cells were stimulated with 10% FCS or control medium for 30 minutes. Baseline ERK activation was negligible on polyacrylamide gels but present on tissue culture plastic (Fig. 3) . Growth factor stimulation elicited an increase in ERK phosphorylation that was strongly rigidity dependent (Fig. 3) . Thus, ECM rigidity modulates growth factor signaling in HTM cells. 
To further elucidate the influence of ECM rigidity on protein expression and phosphorylation, we studied the expression levels of FAK, α-SMA, tubulin, and GAPDH as a loading control. All these proteins were strongly expressed in cells on tissue culture plastic (Fig. 4A) . FAK protein expression was largely independent of substrate rigidity, but FAK phosphorylation decreased in a rigidity-dependent manner (Figs. 4A 4B) . This finding is consistent with our previous observations and suggests diminished adhesion-dependent signaling in cells plated on soft substrates. Cells plated on relatively stiff polyacrylamide gels (0.8% bis-acrylamide) expressed less tubulin than cells plated on either tissue culture plastic or soft polyacrylamide gels (0.2% bis-acrylamide; Figs. 4A 4B ). Increased tubulin expression on very soft substrates seems unexpected, but a lack of cell–ECM binding has been reported to delay tubulin monomer degradation 46 and could explain this result. α-SMA is expressed throughout the trabecular meshwork in younger human eyes, and its expression declines with age. 47 In our experiments, α-SMA expression strongly declined with decreasing substrate rigidity (Figs. 4A 4B) . Interestingly, α-SMA has been characterized as a mechanosensitive protein 48 whose expression is upregulated by the application of mechanical force on cell adhesions. We also studied α-SMA localization because its incorporation into actin stress fibers enhances cell contractility. 34 48 In line with our results, α-SMA incorporation into stress fibers was rigidity dependent (Fig. 5) . On stiff polyacrylamide gels, diffuse staining for α-SMA was observed, but few cells had α-SMA-positive stress fibers; almost no staining for α-SMA was observed on soft polyacrylamide gels. Our results are therefore consistent with the concept that cells are able to develop stronger mechanical forces on rigid substrates. 
Expression of myocilin and the chaperone α-B-crystallin is increased in the trabecular meshwork of glaucomatous eyes. 35 We detected enhanced expression of both proteins on soft substrates (Figs. 6A 6B) . It has been shown that myocilin expression is induced by dynamic mechanical stimulation and declines in trabecular meshwork cells cultivated on standard rigid substrates. 49 Cells are able to probe substrate rigidity, 21 and ensuing molecular changes at cell adhesion sites may be more dynamic on soft substrates, which could serve to enhance myocilin expression. α-B-crystallin protein levels also respond to mechanical stimulation 50 with an initial increase in proteolysis followed by increased transcription. However, the molecular mechanisms of rigidity-dependent regulation of myocilin or α-B-crystallin expression remain to be elucidated. The pattern of decreased α-SMA and increased myocilin and α-B crystallin expression we detected on soft substrates is reminiscent of findings in the trabecular meshwork of glaucomatous eyes. 
ECM deposition and remodelling are cell-mediated processes that require integrin binding of matrix molecules with subsequent cytoskeletal contraction to unfold ECM monomers and to allow for fibrillogenesis and ECM network assembly. 16 51 Because the amount and composition of ECM are altered in glaucoma, we studied the influence of ECM rigidity on further ECM deposition by trabecular meshwork cells. Cells were plated at confluent density on collagen-coated substrates of different rigidity, incubated for 10 days, and stained for F-actin and fibronectin. On collagen-coated glass, trabecular meshwork cells deposited elaborate multinodular fibronectin networks, indicating extensive remodelling activities (Figs. 7A 7D) . On stiff collagen-coated acrylamide gels, the deposits were less extensive and had fewer nodes and shorter fibrils (Figs. 7B 7E) . On soft polyacrylamide gels, the deposits appeared dotted with some short fibrils and only occasional nodes. These experiments reveal that ECM deposition and remodelling differ depending on substrate rigidity. ECM assembly appears most elaborate on rigid substrates. 
In summary, our data indicate that substrate rigidity modulates trabecular meshwork cell functions on several levels: cell–matrix interactions, adhesion-dependent signaling, and protein expression patterns. In light of these findings and the fact that trabecular meshwork resiliency is likely affected by the ECM changes associated with glaucoma, trabecular meshwork cell substrate rigidity may have an impact on glaucoma pathophysiology. 
 
Figure 1.
 
Substrate rigidity modulates cell spreading. HTM cells were plated on polyacrylamide gels of different stiffness (softest gel contained 0.1%, stiffest gel contained 0.8% bis-acrylamide), were allowed to spread for the indicated times, were fixed and stained for F-actin with phalloidin-TRITC, and were counted and scored as spreading or nonspreading depending on the presence of lamellipodia. Quantitation of the spreading cells at the indicated times (mean ± SEM, A) and cell morphology 20 minutes after plating (BE) are shown. Arrowheads indicate cells that formed lamellipodia and were scored as spreading (BE).
Figure 1.
 
Substrate rigidity modulates cell spreading. HTM cells were plated on polyacrylamide gels of different stiffness (softest gel contained 0.1%, stiffest gel contained 0.8% bis-acrylamide), were allowed to spread for the indicated times, were fixed and stained for F-actin with phalloidin-TRITC, and were counted and scored as spreading or nonspreading depending on the presence of lamellipodia. Quantitation of the spreading cells at the indicated times (mean ± SEM, A) and cell morphology 20 minutes after plating (BE) are shown. Arrowheads indicate cells that formed lamellipodia and were scored as spreading (BE).
Figure 2.
 
Effects of substrate rigidity on the actin cytoskeleton and focal adhesions. HTM cells were plated on collagen-coated glass coverslips (A, D, G) and stiff (B, E, H) or soft (C, F, I) collagen-coated polyacrylamide gels. F-actin and vinculin were stained with phalloidin-TRITC and a monoclonal antibody, respectively. Composite images (GI) depict F-actin (red), vinculin (green), and the DAPI-stained nucleus (blue). On rigid substrates (D, less in E) focal adhesions show as elongated foci of vinculin staining (DF, insets). Identical acquisition settings were used for all images. Scale bar, 20 μm.
Figure 2.
 
Effects of substrate rigidity on the actin cytoskeleton and focal adhesions. HTM cells were plated on collagen-coated glass coverslips (A, D, G) and stiff (B, E, H) or soft (C, F, I) collagen-coated polyacrylamide gels. F-actin and vinculin were stained with phalloidin-TRITC and a monoclonal antibody, respectively. Composite images (GI) depict F-actin (red), vinculin (green), and the DAPI-stained nucleus (blue). On rigid substrates (D, less in E) focal adhesions show as elongated foci of vinculin staining (DF, insets). Identical acquisition settings were used for all images. Scale bar, 20 μm.
Figure 3.
 
Substrate rigidity modulates serum-induced ERK activation. HTM cells were plated on collagen-coated tissue culture plastic or collagen-coated soft (0.1% bis-acrylamide) or stiff (0.4% bis-acrylamide) polyacrylamide gels, starved overnight, and stimulated by 10% fetal calf serum for 30 minutes. (A) Expression of ERK protein and its phosphorylation on substrates of different rigidity is shown. The Western blot membrane was reprobed for GAPDH to control loading. (B) Relative signal intensity of the bands shown in (A) normalized for GAPDH.
Figure 3.
 
Substrate rigidity modulates serum-induced ERK activation. HTM cells were plated on collagen-coated tissue culture plastic or collagen-coated soft (0.1% bis-acrylamide) or stiff (0.4% bis-acrylamide) polyacrylamide gels, starved overnight, and stimulated by 10% fetal calf serum for 30 minutes. (A) Expression of ERK protein and its phosphorylation on substrates of different rigidity is shown. The Western blot membrane was reprobed for GAPDH to control loading. (B) Relative signal intensity of the bands shown in (A) normalized for GAPDH.
Figure 4.
 
Effects of substrate rigidity on protein expression and phosphorylation patterns. HTM cells were plated onto collagen-coated tissue culture plastic or collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels and were harvested for Western blot 7 days after plating. (A) The blot was repeatedly probed to assess FAK, pY397FAK, α-SMA, and tubulin. GAPDH served as a loading control. (B) Relative signal intensity of the bands shown in (A) normalized for GAPDH.
Figure 4.
 
Effects of substrate rigidity on protein expression and phosphorylation patterns. HTM cells were plated onto collagen-coated tissue culture plastic or collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels and were harvested for Western blot 7 days after plating. (A) The blot was repeatedly probed to assess FAK, pY397FAK, α-SMA, and tubulin. GAPDH served as a loading control. (B) Relative signal intensity of the bands shown in (A) normalized for GAPDH.
Figure 5.
 
Substrate rigidity modulates α-SMA localization. HTM cells were grown on collagen-coated coverslips and collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels for 10 days. (AC) Composite image depicts F-actin (red), α-SMA (green), and cell nuclei (blue). (D-F) Grayscale view of α-SMA staining. Few cells demonstrated α-SMA-positive stress fibers on stiff polyacrylamide gels (B, E), whereas intense staining is observed on glass coverslips (A, D). Identical acquisition settings were used for all images. Scale bar, 40 μm.
Figure 5.
 
Substrate rigidity modulates α-SMA localization. HTM cells were grown on collagen-coated coverslips and collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels for 10 days. (AC) Composite image depicts F-actin (red), α-SMA (green), and cell nuclei (blue). (D-F) Grayscale view of α-SMA staining. Few cells demonstrated α-SMA-positive stress fibers on stiff polyacrylamide gels (B, E), whereas intense staining is observed on glass coverslips (A, D). Identical acquisition settings were used for all images. Scale bar, 40 μm.
Figure 6.
 
Effects of substrate rigidity on myocilin and α-B-crystallin expression. HTM cells were plated onto collagen-coated tissue culture plastic or collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels and were harvested for Western blot 7 days after plating. (A) The blot was reprobed for myocilin and α-B-crystallin. GAPDH served as a loading control. (B) Relative signal intensity of the bands shown in (A) normalized for GAPDH.
Figure 6.
 
Effects of substrate rigidity on myocilin and α-B-crystallin expression. HTM cells were plated onto collagen-coated tissue culture plastic or collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels and were harvested for Western blot 7 days after plating. (A) The blot was reprobed for myocilin and α-B-crystallin. GAPDH served as a loading control. (B) Relative signal intensity of the bands shown in (A) normalized for GAPDH.
Figure 7.
 
Effect of substrate rigidity on fibronectin deposition. HTM cells were plated onto collagen-coated coverslips or collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels and were allowed to grow for 10 days. Images show F-actin (red), fibronectin (green), and the DAPI-stained nucleus (blue). (DF) Close-ups as marked in (AC). Arrowheads indicate fibronectin nodules. Scale bar: (AC) 40 μm; (DF) 10 μm.
Figure 7.
 
Effect of substrate rigidity on fibronectin deposition. HTM cells were plated onto collagen-coated coverslips or collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels and were allowed to grow for 10 days. Images show F-actin (red), fibronectin (green), and the DAPI-stained nucleus (blue). (DF) Close-ups as marked in (AC). Arrowheads indicate fibronectin nodules. Scale bar: (AC) 40 μm; (DF) 10 μm.
The authors thank Michael Sendtner and his coworkers at the Institut für Klinische Neurobiologie Würzburg for generously sharing equipment. They also thank Martin A. Schwartz and Johan de Rooij for their advice on the preparation of ECM-coatable polyacrylamide gels. 
GabeltBT, KaufmanPL. Changes in aqueous humor dynamics with age and glaucoma. Progr Retin Eye Res. 2005;24:612–637. [CrossRef]
Lütjen-DrecollE. Functional morphology of the trabecular meshwork in primate eyes. Progr Retin Eye Res. 1998;18:91–119.
BradleyJM, KelleyMJ, ZhuX, AnderssohnAM, AlexanderJP, AcottTS. Effects of mechanical stretching on trabecular matrix metalloproteinases. Invest Ophthalmol Vis Sci. 2001;42:1505–1513. [PubMed]
TumminiaSJ, MittonKP, AroraJ, ZelenkaP, EpsteinDL, RussellP. Mechanical stretch alters the actin cytoskeletal network and signal transduction in human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 1998;39:1361–1371. [PubMed]
WiederholtM, ThiemeH, StumpffF. The regulation of trabecular meshwork and ciliary muscle contractility. Progr Retin Eye Res. 2000;19:271–295. [CrossRef]
VittalV, RoseA, GregoryKE, KelleyMJ, AcottTS. Changes in gene expression by trabecular meshwork cells in response to mechanical stretching. Invest Ophthalmol Vis Sci. 2005;46:2857–2868. [CrossRef] [PubMed]
TanJC, PetersDM, KaufmanPL. Recent developments in understanding the pathophysiology of elevated intraocular pressure. Curr Opin Ophthalmol. 2006;17:168–174. [PubMed]
RohenJW, Lutjen-DrecollE, FlugelC, MeyerM, GriersonI. Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma (POAG). Exp Eye Res. 1993;56:683–692. [CrossRef] [PubMed]
GabeltBT, GottankaJ, Lutjen-DrecollE, KaufmanPL. Aqueous humor dynamics and trabecular meshwork and anterior ciliary muscle morphologic changes with age in rhesus monkeys. Invest Ophthalmol Vis Sci. 2003;44:2118–2125. [CrossRef] [PubMed]
GottankaJ, JohnsonDH, MartusP, Lutjen-DrecollE. Severity of optic nerve damage in eyes with POAG is correlated with changes in the trabecular meshwork. J Glaucoma. 1997;6:123–132. [PubMed]
TripathiRC, LiJ, ChanWF, TripathiBJ. Aqueous humor in glaucomatous eyes contains an increased level of TGF-beta 2. Exp Eye Res. 1994;59:723–727. [CrossRef] [PubMed]
TammER, SiegnerA, BaurA, Lütjen-DrecollE. Transforming growth factor-beta 1 induces alpha-smooth muscle-actin expression in cultured human and monkey trabecular meshwork. Exp Eye Res. 1996;62:389–397. [CrossRef] [PubMed]
GottankaJ, ChanD, EichhornM, Lutjen-DrecollE, EthierCR. Effects of TGF-β2 in perfused human eyes. Invest Ophthalmol Vis Sci. 2004;45:153–158. [CrossRef] [PubMed]
Welge-LüssenU, MayCA, Lütjen-DrecollE. Induction of tissue transglutaminase in the trabecular meshwork by TGF-β1 and TGF-β2. Invest Ophthalmol Vis Sci. 2000;41:2229–2238. [PubMed]
GiancottiFG, RuoslahtiE. Integrin signaling. Science. 1999;285:1028–1032. [CrossRef] [PubMed]
GeigerB, BershadskyA, PankovR, YamadaKM. Transmembrane extracellular matrix-cytoskeleton crosstalk. Nat Rev Mol Cell Biol. 2001;2:793–805. [CrossRef] [PubMed]
SchwartzMA, SchallerMD, GinsbergMH. Integrins: emerging paradigms of signal transduction. Annu Rev Cell Dev Biol. 1995;11:549–599. [CrossRef] [PubMed]
WangN, ButlerJP, IngberDE. Mechanotransduction across the cell surface and through the cytoskeleton. Science. 1993;260:1124–1127. [CrossRef] [PubMed]
BershadskyAD, BalabanNQ, GeigerB. Adhesion-dependent cell mechanosensitivity. Annu Rev Cell Dev Biol. 2003;19:677–695. [CrossRef] [PubMed]
KatsumiA, OrrAW, TzimaE, SchwartzMA. Integrins in mechanotransduction. J Biol Chem. 2004;279:12001–12004. [CrossRef] [PubMed]
LoCM, WangHB, DemboM, WangYL. Cell movement is guided by the rigidity of the substrate. Biophys J. 2000;79:144–152. [CrossRef] [PubMed]
WangHB, DemboM, WangYL. Substrate flexibility regulates growth and apoptosis of normal but not transformed cells. Am J Physiol Cell Physiol. 2000;279:C1345–C1350. [PubMed]
DischerDE, JanmeyP, WangYL. Tissue cells feel and respond to the stiffness of their substrate. Science. 2005;310:1139–1143. [CrossRef] [PubMed]
EnglerAJ, SenS, SweeneyHL, DischerDE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126:677–689. [CrossRef] [PubMed]
PolanskyJR, WeinrebRN, BaxterJD, AlvaradoJA. Human trabecular cells, I: establishment in tissue culture and growth characteristics. Invest Ophthalmol Vis Sci. 1979;18:1043–1049. [PubMed]
RheeDJ, TammER, RussellP. Donor corneoscleral buttons: a new source of trabecular meshwork for research. Exp Eye Res. 2003;77:749–756. [CrossRef] [PubMed]
LoWR, RowletteLL, CaballeroM, YangP, HernandezMR, BorrasT. Tissue differential microarray analysis of dexamethasone induction reveals potential mechanisms of steroid glaucoma. Invest Ophthalmol Vis Sci. 2003;44:473–485. [CrossRef] [PubMed]
StamerWD, RobertsBC, HowellDN, EpsteinDL. Isolation, culture, and characterization of endothelial cells from Schlemm’s canal. Invest Ophthalmol Vis Sci. 1998;39:1804–1812. [PubMed]
PelhamRJ, Jr, WangY. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci USA. 1997;94:13661–13665. [CrossRef] [PubMed]
de RooijJ, KerstensA, DanuserG, SchwartzMA, Waterman-StorerCM. Integrin-dependent actomyosin contraction regulates epithelial cell scattering. J Cell Biol. 2005;171:153–164. [CrossRef] [PubMed]
PriceLS, LengJ, SchwartzMA, BokochGM. Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol Biol Cell. 1998;9:1863–1871. [CrossRef] [PubMed]
ZimermanB, VolbergT, GeigerB. Early molecular events in the assembly of the focal adhesion-stress fiber complex during fibroblast spreading. Cell Motil Cytoskeleton. 2004;58:143–159. [CrossRef] [PubMed]
RenshawMW, PriceLS, SchwartzMA. Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase. J Cell Biol. 1999;147:611–618. [CrossRef] [PubMed]
HinzB, CelettaG, TomasekJJ, GabbianiG, ChaponnierC. Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell. 2001;12:2730–2741. [CrossRef] [PubMed]
Lütjen-DrecollE, MayCA, PolanskyJR, JohnsonDH, BloemendalH, NguyenTD. Localization of the stress proteins alpha B-crystallin and trabecular meshwork inducible glucocorticoid response protein in normal and glaucomatous trabecular meshwork. Invest Ophthalmol Vis Sci. 1998;39:517–525. [PubMed]
WuC, KeivensVM, O’TooleTE, McDonaldJA, GinsbergMH. Integrin activation and cytoskeletal interaction are essential for the assembly of a fibronectin matrix. Cell. 1995;83:715–724. [CrossRef] [PubMed]
IlicD, KovacicB, JohkuraK, et al. FAK promotes organization of fibronectin matrix and fibrillar adhesions. J Cell Sci. 2004;117:177–187. [CrossRef] [PubMed]
ZhongC, Chrzanowska-WodnickaM, BrownJ, ShaubA, BelkinAM, BurridgeK. Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly. J Cell Biol. 1998;141:539–551. [CrossRef] [PubMed]
MeshelAS, WeiQ, AdelsteinRS, SheetzMP. Basic mechanism of three-dimensional collagen fibre transport by fibroblasts. Nat Cell Biol. 2005;7:157–164. [CrossRef] [PubMed]
YeungT, GeorgesPC, FlanaganLA, et al. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil Cytoskeleton. 2005;60:24–34. [CrossRef] [PubMed]
EzzellRM, GoldmannWH, WangN, ParasharamaN, IngberDE. Vinculin promotes cell spreading by mechanically coupling integrins to the cytoskeleton. Exp Cell Res. 1997;231:14–26. [CrossRef] [PubMed]
ChicurelME, ChenCS, IngberDE. Cellular control lies in the balance of forces. Curr Opin Cell Biol. 1998;10:232–239. [CrossRef] [PubMed]
GroshevaI, VittitowJL, GoichbergP, et al. Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells. Exp Eye Res. 2006;82:945–958. [CrossRef] [PubMed]
GalbraithCG, YamadaKM, SheetzMP. The relationship between force and focal complex development. J Cell Biol. 2002;159:695–705. [CrossRef] [PubMed]
NicolasA, GeigerB, SafranSA. Cell mechanosensitivity controls the anisotropy of focal adhesions. Proc Natl Acad Sci USA. 2004;101:12510–12525.
MooneyDJ, HansenLK, LangerR, VacantiJP, IngberDE. Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover. Mol Biol Cell. 1994;5:1281–1288. [CrossRef] [PubMed]
FlügelC, TammER, Lütjen-DrecollE, StefaniF. Age-related loss of a smooth muscle actin in normal and glaucomatous human trabecular meshwork of different age groups. J Glaucoma. 1992;1:165–173. [CrossRef]
WangJ, ZoharR, McCullochCA. Multiple roles of alpha-smooth muscle actin in mechanotransduction. Exp Cell Res. 2005;312:205–214. [PubMed]
TammER, RussellP, EpsteinDL, JohnsonDH, PiatigorskyJ. Modulation of myocilin/TIGR expression in human trabecular meshwork. Invest Ophthalmol Vis Sci. 1999;40:2577–2582. [PubMed]
MittonKP, TumminiaSJ, AroraJ, ZelenkaP, EpsteinDL, RussellP. Transient loss of αB-crystallin: an early cellular response to mechanical stretch. Biochem Biophys Res Commun. 1997;235:69–73. [CrossRef] [PubMed]
Wierzbicka-PatynowskiI, SchwarzbauerJE. The ins and outs of fibronectin matrix assembly. J Cell Sci. 2003;116:3269–3276. [CrossRef] [PubMed]
Figure 1.
 
Substrate rigidity modulates cell spreading. HTM cells were plated on polyacrylamide gels of different stiffness (softest gel contained 0.1%, stiffest gel contained 0.8% bis-acrylamide), were allowed to spread for the indicated times, were fixed and stained for F-actin with phalloidin-TRITC, and were counted and scored as spreading or nonspreading depending on the presence of lamellipodia. Quantitation of the spreading cells at the indicated times (mean ± SEM, A) and cell morphology 20 minutes after plating (BE) are shown. Arrowheads indicate cells that formed lamellipodia and were scored as spreading (BE).
Figure 1.
 
Substrate rigidity modulates cell spreading. HTM cells were plated on polyacrylamide gels of different stiffness (softest gel contained 0.1%, stiffest gel contained 0.8% bis-acrylamide), were allowed to spread for the indicated times, were fixed and stained for F-actin with phalloidin-TRITC, and were counted and scored as spreading or nonspreading depending on the presence of lamellipodia. Quantitation of the spreading cells at the indicated times (mean ± SEM, A) and cell morphology 20 minutes after plating (BE) are shown. Arrowheads indicate cells that formed lamellipodia and were scored as spreading (BE).
Figure 2.
 
Effects of substrate rigidity on the actin cytoskeleton and focal adhesions. HTM cells were plated on collagen-coated glass coverslips (A, D, G) and stiff (B, E, H) or soft (C, F, I) collagen-coated polyacrylamide gels. F-actin and vinculin were stained with phalloidin-TRITC and a monoclonal antibody, respectively. Composite images (GI) depict F-actin (red), vinculin (green), and the DAPI-stained nucleus (blue). On rigid substrates (D, less in E) focal adhesions show as elongated foci of vinculin staining (DF, insets). Identical acquisition settings were used for all images. Scale bar, 20 μm.
Figure 2.
 
Effects of substrate rigidity on the actin cytoskeleton and focal adhesions. HTM cells were plated on collagen-coated glass coverslips (A, D, G) and stiff (B, E, H) or soft (C, F, I) collagen-coated polyacrylamide gels. F-actin and vinculin were stained with phalloidin-TRITC and a monoclonal antibody, respectively. Composite images (GI) depict F-actin (red), vinculin (green), and the DAPI-stained nucleus (blue). On rigid substrates (D, less in E) focal adhesions show as elongated foci of vinculin staining (DF, insets). Identical acquisition settings were used for all images. Scale bar, 20 μm.
Figure 3.
 
Substrate rigidity modulates serum-induced ERK activation. HTM cells were plated on collagen-coated tissue culture plastic or collagen-coated soft (0.1% bis-acrylamide) or stiff (0.4% bis-acrylamide) polyacrylamide gels, starved overnight, and stimulated by 10% fetal calf serum for 30 minutes. (A) Expression of ERK protein and its phosphorylation on substrates of different rigidity is shown. The Western blot membrane was reprobed for GAPDH to control loading. (B) Relative signal intensity of the bands shown in (A) normalized for GAPDH.
Figure 3.
 
Substrate rigidity modulates serum-induced ERK activation. HTM cells were plated on collagen-coated tissue culture plastic or collagen-coated soft (0.1% bis-acrylamide) or stiff (0.4% bis-acrylamide) polyacrylamide gels, starved overnight, and stimulated by 10% fetal calf serum for 30 minutes. (A) Expression of ERK protein and its phosphorylation on substrates of different rigidity is shown. The Western blot membrane was reprobed for GAPDH to control loading. (B) Relative signal intensity of the bands shown in (A) normalized for GAPDH.
Figure 4.
 
Effects of substrate rigidity on protein expression and phosphorylation patterns. HTM cells were plated onto collagen-coated tissue culture plastic or collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels and were harvested for Western blot 7 days after plating. (A) The blot was repeatedly probed to assess FAK, pY397FAK, α-SMA, and tubulin. GAPDH served as a loading control. (B) Relative signal intensity of the bands shown in (A) normalized for GAPDH.
Figure 4.
 
Effects of substrate rigidity on protein expression and phosphorylation patterns. HTM cells were plated onto collagen-coated tissue culture plastic or collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels and were harvested for Western blot 7 days after plating. (A) The blot was repeatedly probed to assess FAK, pY397FAK, α-SMA, and tubulin. GAPDH served as a loading control. (B) Relative signal intensity of the bands shown in (A) normalized for GAPDH.
Figure 5.
 
Substrate rigidity modulates α-SMA localization. HTM cells were grown on collagen-coated coverslips and collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels for 10 days. (AC) Composite image depicts F-actin (red), α-SMA (green), and cell nuclei (blue). (D-F) Grayscale view of α-SMA staining. Few cells demonstrated α-SMA-positive stress fibers on stiff polyacrylamide gels (B, E), whereas intense staining is observed on glass coverslips (A, D). Identical acquisition settings were used for all images. Scale bar, 40 μm.
Figure 5.
 
Substrate rigidity modulates α-SMA localization. HTM cells were grown on collagen-coated coverslips and collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels for 10 days. (AC) Composite image depicts F-actin (red), α-SMA (green), and cell nuclei (blue). (D-F) Grayscale view of α-SMA staining. Few cells demonstrated α-SMA-positive stress fibers on stiff polyacrylamide gels (B, E), whereas intense staining is observed on glass coverslips (A, D). Identical acquisition settings were used for all images. Scale bar, 40 μm.
Figure 6.
 
Effects of substrate rigidity on myocilin and α-B-crystallin expression. HTM cells were plated onto collagen-coated tissue culture plastic or collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels and were harvested for Western blot 7 days after plating. (A) The blot was reprobed for myocilin and α-B-crystallin. GAPDH served as a loading control. (B) Relative signal intensity of the bands shown in (A) normalized for GAPDH.
Figure 6.
 
Effects of substrate rigidity on myocilin and α-B-crystallin expression. HTM cells were plated onto collagen-coated tissue culture plastic or collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels and were harvested for Western blot 7 days after plating. (A) The blot was reprobed for myocilin and α-B-crystallin. GAPDH served as a loading control. (B) Relative signal intensity of the bands shown in (A) normalized for GAPDH.
Figure 7.
 
Effect of substrate rigidity on fibronectin deposition. HTM cells were plated onto collagen-coated coverslips or collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels and were allowed to grow for 10 days. Images show F-actin (red), fibronectin (green), and the DAPI-stained nucleus (blue). (DF) Close-ups as marked in (AC). Arrowheads indicate fibronectin nodules. Scale bar: (AC) 40 μm; (DF) 10 μm.
Figure 7.
 
Effect of substrate rigidity on fibronectin deposition. HTM cells were plated onto collagen-coated coverslips or collagen-coated stiff (0.8% bis-acrylamide) or soft (0.2% bis-acrylamide) polyacrylamide gels and were allowed to grow for 10 days. Images show F-actin (red), fibronectin (green), and the DAPI-stained nucleus (blue). (DF) Close-ups as marked in (AC). Arrowheads indicate fibronectin nodules. Scale bar: (AC) 40 μm; (DF) 10 μm.
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