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Am J Physiol Cell Physiol 295: C1579-C1589, 2008. First published October 15, 2008; doi:10.1152/ajpcell.00223.2008
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EXTRACELLULAR MATRIX, CELL INTERACTIONS

Mechanosensing machinery for cells under low substratum rigidity

Wei-Chun Wei,1 Hsi-Hui Lin,2 Meng-Ru Shen,1,3 and Ming-Jer Tang1,2,3

1Institute of Basic Medical Sciences, 2Department of Physiology, and 3Center of Gene Regulation and Signal Transduction, National Cheng Kung University Medical College, Tainan, Taiwan

Submitted 23 April 2008 ; accepted in final form 4 October 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mechanical stimuli are essential during development and tumorigenesis. However, how cells sense their physical environment under low rigidity is still unknown. Here we show that low rigidity of collagen gel downregulates β1-integrin activation, clustering, and focal adhesion kinase (FAK) Y397 phosphorylation, which is mediated by delayed raft formation. Moreover, overexpression of autoclustered β1-integrin (V737N), but not constitutively active β1-integrin (G429N), rescues FAKY397 phosphorylation level suppressed by low substratum rigidity. Using fluorescence resonance energy transfer to assess β1-integrin clustering, we have found that substratum rigidity between 58 and 386 Pa triggers β1-integrin clustering in a dose-dependent manner, which is highly dependent on actin filaments but not microtubules. Furthermore, augmentation of β1-integrin clustering enhances the interaction between β1-integrin, FAK, and talin. Our results indicate that contact with collagen fibrils is not sufficient for integrin activation. However, substratum rigidity is required for integrin clustering and activation. Together, our findings provide new insight into the mechanosensing machinery and the mode of action for epithelial cells in response to their physical environment under low rigidity.

focal adhesion kinase; β1-integrin activation; β1-integrin clustering; lipid raft; actin cytoskeleton

EXTRACELLULAR MATRIX (ECM) has an active and complex role in regulating the behavior of the cells that contact it, influencing their survival, adhesion, spreading, migration, proliferation, apoptosis, and differentiation (32). The major receptors of ECM are integrins, which are transmembrane receptors composed of two subunits, {alpha} and β, that form connections between the cytoskeleton and ECM. Each {alpha}β heterodimer has its own binding and signal specificity (25). The initiation of integrin-mediated activities involves two steps, namely, integrin activation and clustering. Integrins are locked in the inactive form without stimulation of extracellular ligands. After binding to its ligand with the extracellular domain (outside-in) (19) or stimulation by intracellular signals (inside-out) (38), an integrin is activated and its extracellular domain undergoes conformational changes, which lead to exposure of several ligand-induced binding sites (LIBS) (49). In addition to conformation changes, ligand binding also induces elevation of lateral mobility and clustering of integrins in the plasma membrane (31). Both ligand binding and clustering of integrins are critically important to activate intracellular signals. However, the regulatory mechanisms of controlling integrin activation and clustering are still unclear.

Activation of integrin leads to the recruitment and organization of a number of different cytoskeletal proteins ({alpha}-actinin, talin, paxillin, etc.) and signaling molecules [focal adhesion kinase (FAK), Src, etc.] into focal adhesion sites (9, 18, 30, 59, 61). Among numerous focal adhesion complex proteins, FAK is a key regulator (26). It transmits information from integrin to the various signaling pathways, including those that regulate cellular events such as cell shape, migration, growth, differentiation, and apoptosis (22, 23, 44, 52). FAK is a 125-kDa cytoplasmic tyrosine kinase and is autophosphorylated at Tyr397 immediately after integrin clustering. This creates a binding site for the src homology 2 (SH2) domain of Src (46, 58). The recruited, active Src phosphorylates several other tyrosine residues in FAK and creates phosphotyrosine binding sites for other signaling molecules, such as Grb2, p130cas, and phosphatidylinositol 3-kinase (6, 24). There are five major sites phosphorylated by Src: Y407, Y576/577, Y861, and Y925. Phosphorylation at Y576/577 and Y861 enhances FAK kinase activity (3), and Y925 is a binding site for the SH2 domain protein Grb2 (47).

Current understanding of how ECM affects cell behaviors has been taken primarily from in vitro studies in which cells were cultured on rigid dishes coated with a thin layer of ECM. However, resent studies showed that sensing mechanical stimuli is also crucial for cells to detect several useful signals from the external environment during development and tumorigenesis (14, 15, 27, 42, 51). The three-dimensional collagen gel culture system has been used as a model to study ECM signaling because it provides not only biochemical signals but also biophysical properties (52, 53). The elastic modulus of soft tissue is in the few hundreds to several thousands of pascals range that can be made by collagen gel (37, 46, 57). Previous studies showed that low substratum rigidity downregulates focal adhesion complex protein expression and phosphorylation (13, 54). However, the mechanosensing machinery as well as its mode of action are unclear.

In this study, we attempted to examine whether and how collagen gel affects ECM signaling via its biophysical property. Our data indicate that cells sense external mechanical stimulation by using a set of mechanical sensing machinery that is composed of lipid rafts, β1-integrin, FAK, and the actin cytoskeleton. We also delineated the mode of action of this mechanical sensing machinery.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells and cell culture. Madin-Darby canine kidney (MDCK) and HEK293T transfectants were maintained in DMEM (from GIBCO) supplemented with 10% fetal bovine serum under 5% CO2 at 37°C (51). These cells were seeded on different substrata at a density of 106 cells per 100-mm dish. In some experiments, cells were pretreated with different inhibitors such as cytochalasin D, colcemide (from Biosource), and methyl-β-cyclodextrin (MβCD; from Sigma).

Preparation of collagen gel and polyacrylamide gel. For preparation of collagen gel, type I collagen was prepared according to the established procedure described previously (29) from tendons of rat tails provided by the National Cheng Kung University Medical College animal center. To prepare 0.3% collagen gel, collagen stock was mixed with 5.7x DMEM, 2.5% NaHCO3, 0.1 M HEPES, 0.17 M CaCl2, 1 N NaOH, and culture medium. The mixtures were dispensed in the culture dish (4 ml/100 mm dish) and allowed to gelate. To prepare the collagen gel-coated dish, 0.3% collagen gel solution was added to culture dishes to cover the surface. The culture dish was then tilted, and the excess amount of collagen gel was aspirated. The collagen gel-coated dishes were air-dried and washed twice with normal culture medium before use. Polyacrylamide gel was prepared according to a previously described method (55) with the following modifications. Gels were prepared with 5% acrylamide and bisacrylamide ranging from 0.02% to 0.085%. The elastic modulus of the gels was detected by rheometer (Thermo, HAAK Rheometer). The polyacrylamide gel was coated with 200 µg/ml type I collagen (from BD) by using a photoactivatable sulfonated cross-linker, sulfo-SANPAH (from Pierce), before addition of cells.

Collection of cell lysate and Western blot. Cell lysates were harvested in modified RIPA buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris pH 7.4, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail), and the lysates were analyzed by Western blot with antibodies against FAK (from Transduction Lab), FAKY397P, FAKY407P, FAKY577P, FAKY861P, and FAKY925P (from Biosource), β1-integrin, activated β1-integrin (clone B44) (from Chemicon), Myc, hemagglutinin, discoidin domain receptor (DDR)-1 (from Santa Cruz Biotechnology), and β-actin (from Amersham) and an enhanced chemiluminescence (ECL) system (from Amersham-Pharmacia).

Immunofluorescence and confocal study. Cells cultured on collagen gel-coated dishes or collagen gel for 30 min were washed three times with ice-cold phosphate-buffered saline (PBS) and fixed and permeabilized with 4% paraformaldehyde and 0.5% Triton X-100 prepared in PBS for 15 min at room temperature. Fixed cells were incubated with anti-FAK or anti-phosphorylated FAKY397 antibody overnight at 4°C. Cells were then washed and incubated with the proper Alexa Fluor 488-conjugated secondary antibody (from Molecular Probes) and phalloidin-tetramethylrhodamine isothiocyanate (from Sigma) for 1 h at room temperature. The immunofluorescence images were taken by confocal microscopy (Olympus, FV-1000).

Raft fractionation and staining. Cells were cultured under different substratum rigidities for 30 min and were lysed in ice-cold lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl) containing 1% Triton X-100, 1 mM sodium orthovanadate, and protease inhibitor cocktail. Equal amounts of these cell lysates were brought to a volume of 2 ml with 40% OptiPrep and then overlaid with 4 ml of 30% OptiPrep and 2 ml of sucrose 5% OptiPrep. Lysates were then ultracentrifuged in a SW40.1Ti rotor (Beckman, Palo Alto, CA) at 40,000 rpm for 16 h to separate lipid rafts from the cytosol. Aliquots of 1 ml of gradient fractions were then collected to yield a total of eight fractions. Proteins were concentrated in each fraction by TCA precipitation. The resulting pellets of each fraction were dissolved in lysis buffer and stored at –80°C before being tested.

Fluorescence resonance energy transfer measurement. Fluorescence resonance energy transfer (FRET) efficiency was measured as described previously (34). In brief, 293T cells were transiently transfected with wild-type, constitutively activated, or autoclustering β1-integrin for 48 h and were suspended or cultured on different substratum rigidities for 30 min. Cells exhibiting relatively similar intensity levels of monomeric cyan fluorescent protein (mCFP) and yellow fluorescent protein (mYFP) under confocal microscope (Olympus, FV-1000) were selected for experiments. Ten to fifteen regions of interest (ROIs) at adhesion points of the mCFP image were randomly chosen and analyzed. Each data set contains ~100 individual ROIs from 10–15 individual cells in at least 3 separate experiments. ROIs exhibiting saturation in either the CFP or YFP channel were eliminated from the analysis. FRET efficiency (E, %) was calculated as E = {1 – [FmCFP(Pre)/FmCFP(Post)]} x 100, where FmCFP(Pre) and FmCFP(Post) are intensity of mCFP emission before and after mYFP photobleaching, respectively. Data were fit to Lineweaver-Burk plots (1/E = 1/Emax+ K/Emax x 1/F) with Prism software. K was calculated from the slope of Lineweaver-Burk plots (K/Emax) and 1/Emax at the y-intercept. The curves shown in Figs. 5 and 6 were drawn by using the K and Emax values with Prism software.


Figure 5
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  		Fig. 5. Substratum rigidity controls β1-integrin clustering. A and B: HEK293T cells transfected with wild-type, constitutively active (G429N), or autoclustered (V737N) β1-integrin-conjugated monomeric cyan fluorescent protein (mCFP) and yellow fluorescent protein (mYFP) were suspended or cultured on collagen gel-coated dish or collagen gel for 30 min. β1-Integrin clustering was assessed by fluorescence resonance energy transfer (FRET) under confocal microscopy (Olympus, FV-1000). A: representative image of FRET. B: quantitative results of FRET. C and D: HEK293T cells were transfected with wild-type β1-integrin-conjugated mCFP and mYFP. Cells were cultured on polyacrylamide gel with different moduli of rigidity for 30 min. β1-Integrin clustering was assessed by FRET under confocal microscopy (Olympus, FV-1000). C: representative image of FRET. D: quantitative results of FRET. E: plot of substratum rigidity vs. FRET constant K.

 

Figure 6
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  		Fig. 6. Substratum rigidity-controlled β1-integrin clustering is regulated by actin cytoskeleton. HEK293T cells transfected with wild-type β1-integrin-conjugated mCFP and mYFP were cultured on a collagen gel-coated dish for 4 h and treated with cytochalasin D (10 µg/ml), ML-7 (50 µg/ml), Y27632 (10 µg/ml), or colcemide (10 µg/ml) for 30 min. A: cells were fixed and stained for actin (red), Y397-phosphorylated FAK (green), and nucleus (blue). Images were taken by confocal microscopy (Olympus, FV-1000). Bar, 10 µm. B and C: β1-integrin clustering was assessed by FRET under confocal microscopy (Olympus, FV-1000). B: representative image of FRET. C: quantitative results of FRET. D: cell lysates were analyzed by Western blot using antibodies to detect activated β1-integrin (LIBS), β1-integrin, Y397-phosphorylated FAK, and FAK.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Low substratum rigidity downregulates FAKY397 phosphorylation and β1-integrin activation. To investigate whether low substratum rigidity affects focal adhesion signals, we examined FAK phosphorylation levels of MDCK cells cultured on dish, collagen gel-coated dish, or collagen gel. By rheometer measurement, the rigidity of the collagen gel-coated dish and the collagen gel are 109 Pa and 20 Pa, respectively (57). When cells were seeded and allowed to spread on collagen gel for 30 min, only the level of FAKY397 phosphorylation was suppressed, while phosphorylation of FAK at other tyrosine sites (407, 577, 861, and 925) remained elevated similar to those of the control (Fig. 1A). We further observed the localization of FAK and Y397-phosphorylated FAK in cells under different rigidities. When MDCK cells were cultured on the collagen gel-coated dish, they exhibited well-organized actin stress fibers with a significant level of FAK and Y397-phosphorylated FAK being localized at the end of actin fibers, which are characteristics of focal adhesions. In contrast, when MDCK cells were cultured on collagen gel, they displayed little spreading and few actin stress fibers and focal adhesions (Fig. 1B). More importantly, MDCK cells exhibited little FAKY397 phosphorylation on collagen gel, consistent with the observation from Western blotting (Fig. 1A). A time course study was performed to monitor the FAKY397 phosphorylation and upstream β1-integrin activation levels within 4 h in cultures. The LIBS was considered as a marker for β1-integrin activation. Activation of FAKY397 and β1-integrin was diminished when cells were suspended. After cells were plated on the normal-cultured dish or the collagen gel-coated dish, the level of FAKY397 phosphorylation and β1-integrin activation was elevated. Low rigidity of collagen gel downregulated the expression level of FAK but not β1-integrin after 1 h, as in our previous report (54). However, the level of phosphorylated FAKY397 and β1-integrin activation remained low in cells cultured on collagen gel throughout the 4 h (Fig. 1C).


Figure 1
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  		Fig. 1. Regulation of focal adhesion kinase (FAK) Y397 phosphorylation by substratum rigidity. A: Madin-Darby canine kidney (MDCK) cells were seeded and allowed to spread on dish (C), collagen gel-coated dish (Co), or collagen gel (G) for 30 min. Cell lysates were analyzed by Western blot with antibodies for detecting FAK protein or specific FAK phosphorylation sites on Y397, Y407, Y577, Y861, or Y925. B: cells were immunostained and show actin (red), FAK, or Y397-phosphorylated FAK (green). Immunofluorescence images were taken by confocal microscope (Olympus, FV-1000). Bar, 10 µm. C: MDCK cells were suspended (S) or cultured on dish, collagen gel-coated dish, or collagen gel for indicated times. Cell lysates were analyzed by Western blot with antibodies for detecting activated β1-integrin [ligand-induced binding sites (LIBS)], β1-integrin, Y397-phosphorylated FAK, and FAK.

 
To examine whether reduced activation of β1-integrin was induced by inhibition of externalization of β1-integrin under low substratum rigidity, we detected the amount of membrane-bound β1-integrin by FACScan. No significant difference in membrane-bound β1-integrin was observed in cells cultured under different conditions (Fig. 2A). The requirement of substratum rigidity for β1-integrin activation was further examined. MDCK cells were suspended and treated with soluble collagen (200 µg/ml) for 30 min. As expected, soluble collagen could not activate β1-integrin in suspended cells (Fig. 2B). To further investigate whether substratum rigidity is correlated with FAKY397 phosphorylation and β1-integrin activation, cells were seeded and allowed to spread on collagen-coated polyacrylamide gel with different moduli of rigidity for 30 min. The levels of FAKY397 phosphorylation as well as β1-integrin activation depended on substratum rigidity, particularly at rigidities lower than 386 Pa, where they react in a dose-responsive manner (Fig. 2C). The responses of β1-integrin activation and FAKY397 phosphorylation fit a simple hyperbola in elastic modulus. The KD (half-saturation) values for β1-integrin and FAKY397 are 346.3 Pa and 277.1 Pa, respectively (Fig. 2D). These data indicate that low substratum rigidity fails to fully activate FAKY397 and β1-integrin.


Figure 2
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  		Fig. 2. Regulation of β1-integrin activation by substratum rigidity. A: after being cultured for 30 min, living cells were incubated with β1-integrin antibody and the membrane-bound β1-integrin was detected by FACScan analysis. B: suspended MDCK cells were treated with soluble collagen (200 µg/ml) or denatured collagen gelatin (200 µg/ml) for 30 min. Cell lysates were harvested and analyzed by Western blot using antibodies to detect activated β1-integrin (LIBS) and β1-integrin. C: MDCK cells were cultured on polyacrylamide gel with different moduli of rigidity for 30 min. Cell lysates were analyzed by Western blot using antibodies to detect activated β1-integrin (LIBS), β1-integrin, Y397-phosphorylated FAK, and FAK protein. D: quantitative densitometry of relative β1-integrin and FAKY397 activation level. Fitting curves were calculated by Prism software. Solid and dashed lines represent the curve fit for β1-integrin activation and FAKY397 phosphorylation, respectively.

 
Lipid raft plays crucial role in activating β1-integrin. Since β1-integrin activation is the upstream signal of FAKY397 phosphorylation, we addressed how low substratum rigidity regulates β1-integrin activation. Previous studies showed that FAK regulates the "inside-out" signal of integrin activation (16). However, neither FAK nor another collagen receptor, DDR-1, which inhibits {alpha}2β1-integrin-induced signal (51), was involved in low-rigidity-regulated β1-integrin inactivation (data not shown). Recent studies suggested that the membrane microdomain, lipid raft, participated in regulation of integrin function (36). We employed live cell imaging to observe the distribution of lipid rafts in cells seeded and allowed to spread on a collagen-coated dish and collagen gel within 4 h. Cells were stained with Alexa Fluor 488-conjugated cholera toxin (CTX) B as a lipid raft marker. We found that low rigidity delayed the formation of lipid rafts (Fig. 3, A and B). A disperse distribution of lipid raft on the cell membrane was observed when cells were seeded and allowed to spread on collagen gel-coated dishes for 30 min. In contrast, little rafts were seen when cells were seeded on collagen gels for the same time. An immunofluorescence study was performed in order to delineate the localization of β1-integrins and rafts in the cell membrane. We found that active β1-integrins were present and colocalized with lipid rafts when cells were cultured on the collagen gel-coated dish but not the collagen gel for 30 min (Fig. 3C). Furthermore, we employed a sucrose gradient to separate raft and nonraft fractions of cells. β1-Integrin was not present in raft fractions when cells were cultured on collagen gel (Fig. 3D). Thus our data show that the activated β1-integrin is associated with markers of lipid rafts. To further confirm this, cells were cultured on a collagen gel-coated dish with treatment with MβCD, an inhibitor of lipid raft formation by depletion of membrane cholesterol. We found that MβCD inhibited β1-integrin activation as well as FAKY397P, while repletion of membrane cholesterol by exposing cells to MβCD saturated with cholesterol (39) reversed both β1-integrin activation and FAKY397 phosphorylation (Fig. 3E). These results indicate that under rigid substratum the activation of β1-integrin requires lipid rafts.


Figure 3
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  		Fig. 3. Lipid raft is required for β1-integrin activation and FAKY397 phosphorylation. A: MDCK cells were cultured on collagen gel-coated or collagen gel for indicated times. Living cells were stained with cholera toxin (CTX) B-conjugated Alexa Fluor 488. Immunofluorescence images were taken by confocal microscopy (Olympus, FV-1000). B: quantification of CTX B-positive area on the cell membrane with Image Pro Plus software. C: MDCK cells were culture on collagen gel-coated dish or collagen gel for 30 min. Cells were fixed but not permeabilized and then stained with mouse anti-active β1-integrin (Huts-4) or anti-β1-integrin antibodies followed by Alexa Fluor 594-conjugated secondary goat IgG (red) and Alexa Fluor 488-labeled CTX B (green). Immunofluorescence images were taken by confocal microscopy (Olympus, FV-1000). Bar, 10 µm. D: MDCK cells were cultured on collagen gel-coated dish or collagen gel for 30 min. Lysates were fractionated by OptiPrep and analyzed by Western blot using antibodies to detect β1-integrin, caveolin-1, Na+/K+ pump. E: MDCK cells were pretreated with methyl-β-cyclodextrin (MβCD, 20 mM) or repletion of cholesterol for 1 h, followed by culture on collagen gel-coated dish for 30 min. Cell lysates were harvested and analyzed by Western blot using antibodies to detect activated β1-integrin (LIBS), β1-integrin, Y397-phosphorylated FAK, and FAK.

 
β1-Integrin clustering is essential for FAKY397 phosphorylation. To delineate whether low substratum rigidity-induced downregulation of FAKY397 phosphorylation is mediated by reduction of β1-integrin activation, MDCK cells stably overexpressing wild-type, constitutively active (G429N) β1-integrin (40) were employed. The results showed that constitutively active β1-integrin failed to rescue FAKY397 phosphorylation when cells were cultured under low substratum rigidity (Fig. 4A). To specify whether β1-integrin clustering is involved in regulating FAKY397 phosphorylation, green fluorescent protein (GFP)-conjugated wild-type, constitutively active, or autoclustered (V737N) β1-integrin (46) was transiently transfected into HEK293T cells. Autoclustered β1-integrin formed aggregations under low substratum rigidity (Fig. 4B). We found that autoclustered but not wild-type or constitutively active β1-integrin rescued FAKY397 phosphorylation when cells were cultured under low rigidity (Fig. 4C). To further investigate whether autoclustered β1-integrin regulates focal adhesion formation, actin organization, and cell spreading, GFP-conjugated wild-type, constitutively active, or autoclustered β1-integrin was transiently transfected into HEK293T cells. These β1-integrin transfectants were seeded and allowed to spread on a collagen gel-coated dish or collagen for 30 min and were immunostained with FAK (marker of focal adhesion) or actin. The focal adhesion sizes and numbers were then measured from the FAK-positive area with Image Pro Plus software under confocal microscopy (Fig. 4, D and E). In addition, the staining of actin filament that revealed the cell shape and the cell area was measured with the same software. The results are shown in Fig. 4, F and G. Autoclustered but not wild-type or constitutively active β1-integrin increased the sizes and number of focal adhesion formation, actin organization, and cell spreading even in cells cultured under low rigidity. These data indicate that β1-integrin clustering but not activation is prerequisite for FAKY397 phosphorylation, regardless of substratum stiffness.


Figure 4
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  		Fig. 4. β1-Integrin clustering is indispensable for FAKY397 phosphorylation. A: MDCK cells transfected with constitutively active β1-integrin (G429N) were suspended or cultured on dish, collagen gel-coated dish, or collagen gel for 30 min. Cell lysates were analyzed by Western blot using antibodies to detect activated β1-integrin (LIBS), β1-integrin, Y397-phosphorylated FAK, and FAK. B–G: HEK293T cells were transfected with green fluorescent protein (GFP)-conjugated wild-type, constitutively active (G429N), or autoclustered (V737N) β1-integrin. Cells were suspended or cultured on dish, collagen gel-coated dish, or collagen gel for 30 min. B: cells were fixed, and fluorescence images were taken by confocal microscopy (Olympus, FV-1000). Arrowhead represents aggregated β1-integrin. Bar, 10 µm. C: cell lysates were analyzed by Western blot using antibodies to detect activated β1-integrin (LIBS), β1-integrin, FAK, and Y397-phosphorylated FAK. D and E: cells were immunostained with anti-FAK antibody as a marker of focal adhesion. Images were taken under confocal microscope. The sizes and numbers of FAK-positive area were counted with Image Pro Plus software. F: cells were immunostained and showed actin (red). Immunofluorescence images were taken by confocal microscope (Olympus, FV-1000). Bar, 10 µm. G: quantitative results of cell areas in F were assessed with Image Pro Plus software. *P < 0.05, Co vs. G.

 
Low substratum rigidity downregulates level of β1-integrin clustering. To delineate how substratum rigidity regulates β1-integrin clustering, we employed FRET. HEK293T cells transiently transfected with wild-type, constitutively active, or autoclustered β1-integrin-conjugated mCFP or mYFP were established for the measurement of FRET. Cells were suspended or seeded and allowed to spread either on a collagen gel-coated dish or collagen gel for 30 min and subjected to FRET examination. The FRET efficiency was assessed based on methods previously described (34, 60). Results showed that when cells were suspended β1-integrin was not clustered at all. Clustering of β1-integrin was markedly increased when cells were cultured on the collagen gel-coated dish. However, β1-integrin clustering remained at a low level when cells were cultured on collagen gel (Fig. 5, A and B). To delineate the correlation between substratum rigidity and β1-integrin clustering, HEK293T cells transiently cotransfected with wild-type β1-integrin-conjugated mCFP or mYFP were cultured on collagen-coated polyacrylamide gel with different moduli of rigidity. At a rigidity of >386 Pa, β1-integrin was highly clustered. The level of β1-integrin clustering was highly correlated with substratum rigidity in the range between 58 and 386 Pa. β1-Integrin clustering was diminished under substratum rigidity of 58 Pa (Fig. 5, C and D). The responses of value of FRET constant K fit a simple hyperbola in elastic modulus. The KD (half-saturation) was 251.2 Pa (Fig. 5E). These data show that β1-integrin clustering depends on an external force provided by substratum rigidity.

Actin cytoskeleton contributes to β1-integrin clustering. We also explored whether internal forces provided by the cytoskeleton are also involved in regulating β1-integrin clustering. Cells were cultured on a collagen gel-coated dish and then treated with cytochalasin D, myosin light chain kinase (MLCK) inhibitor ML-7, Rho kinase (ROCK) inhibitor Y27632, or colcemide to disrupt actin filaments or microtubules. Cells still spread out when microtubules were disrupted but spread very little after the disruption of actin filaments (Fig. 6A). Although β1-integrin activation was not affected by disruption of the actin filaments or microtubules, β1-integrin clustering and FAKY397 phosphorylation level were downregulated by cytochalasin D, ROCK inhibitor Y27632, and MLCK inhibitor ML-7 but not by colcemide (Fig. 6, B–D). These data indicate that β1-integrin clustering but not activation depends on internal force provided by the actin cytoskeleton.

Augmentation of β1-integrin clustering enhances interaction of β1-integrin, FAK, and talin. We further investigated the molecular mechanism by which β1-integrin clustering leads to FAKY397 phosphorylation. Recent evidence revealed that FAK activity is regulated through the interaction of its FERM-like NH2-terminal domain with the cytoplasmic domain of β1-integrin to release an autoinhibitory interaction of FERM domain with its kinase domain (11, 12). Other evidence also showed that talin is important in regulating integrin signaling (22). Therefore, whether β1-integrin clustering affects the interaction between β1-integrin, FAK, and talin was determined. HEK293T cells transiently transfected with GFP-conjugated wild-type β1-integrin were cultured on polyacrylamide gel with different substratum rigidities, and the interactions between exogenous β1-integrin, FAK, and talin were examined. With the substratum rigidity increase, the interaction between exogenous β1-integrin, FAK, and talin increases (Fig. 7A). The response of the amount of β1-integrin associate protein fits a simple hyperbola in elastic modulus (Fig. 7B). Transient transfection of equal amounts of GFP-conjugated wild-type, constitutively active (G429N), or autoclustered (V737N) β1-integrin into HEK293T cells was performed (Fig. 7C). Cells were seeded and allowed to spread on a collagen gel-coated dish, collagen gel, or polyacrylamide gel with different moduli of rigidity for 30 min, and the interactions between exogenous β1-integrin, FAK, and talin were examined. When cells harboring wild-type or constitutively active β1-integrin were cultured on collagen gel, little interaction between β1-integrin, FAK, and talin was observed. Only cells harboring autoclustered β1-integrin displayed higher interaction between exogenous β1-integrin, FAK, and talin under low substratum rigidity (Fig. 7D). Together, these data suggest that β1-integrin clustering, but not activation, leads to FAKY397 phosphorylation by augmentation of the interaction between β1-integrin, FAK, and talin.


Figure 7
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  		Fig. 7. Autoclustered β1-integrin increases the interaction between β1-integrin, FAK, and talin in cells cultured under low substratum rigidity. A and B: transient transfection of equal amount of wild-type (WT), constitutively active (G429N), or autoclustered (V737N) β1-integrin into HEK293T cells was performed. A: cells were cultured on dish for 24 h, and lysates were analyzed by Western blot (IB) with antibodies against talin, activated β1-integrin (LIBS), β1-integrin, Y397-phosphorylated FAK, or FAK protein. IP, immunoprecipitation. B: cells were cultured on collagen gel-coated dish or collagen gel for 30 min. Cell lysates were immunoprecipitated with control bead (–), anti-GFP antibody and then immunoblotted with anti-talin, -Y397 phosphorylated FAK, -FAK, or -GFP antibodies. C: HEK293T cells transiently transfected with wild-type β1-integrin were cultured on polyacrylamide gel with different moduli of rigidity for 30 min. Cell lysates were immunoprecipitated with control bead (–), anti-GFP antibody and then immunoblotted with anti-talin, -Y397-phosphorylated FAK, -FAK, or -GFP antibodies. D: quantitative densitometry of relative β1-integrin associate protein level. Fitting curves were calculated with Prism software.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we provide data to characterize the components and molecular functions of the mechanical sensing machinery in detecting substratum rigidity around the 100-Pa range. The mode of action in this set of mechanical sensing machinery is associated with lipid rafts and controlled by the actin cytoskeleton with a positive feedback loop (Fig. 8). Our data indicate that the physical environment affects β1-integrin clustering, which is regulated by the actin cytoskeleton. Above a rigidity of 386 Pa, through the integration of external and internal forces, the cells are able to construct well-organized actin filaments that lead to a full activation of FAKY397. On the other hand, below a substratum rigidity of 386 Pa, the reduction of integrin clustering leads to a downregulation of FAKY397 activation and actin organization. Since β1-integrin clustering does not occur below a rigidity of 58 Pa, the feasible range of this mechanical sensing machinery lies within the substratum rigidity range of 58 to 386 Pa. Thus the mechanical sensing machinery provided here is involved in regulating cell behavior of soft tissue, since previous studies showed that the elastic modulus of soft tissues, such as brain, liver, and mammary gland, falls within this range.


Figure 8
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  		Fig. 8. Schematic figure depicting the mechanosensing machinery and the mode of action of cells in response to low rigidity. Substratum rigidity-induced β1-integrin activation is mediated by lipid rafts. Substratum rigidity also regulates β1-integrin clustering and FAKY397 phosphorylation through actin organization.

 
Integrin signaling has been studied for many decades. However, currently the sequential order of β1-integrin activation and clustering is still unknown. In this study, we pointed out the importance of substratum rigidity in controlling β1-integrin activation and clustering. Our data showed that substratum rigidity-related lipid raft externalization controls β1-integrin activation. We also showed that substratum rigidity regulates the level of β1-integrin clustering by well-organized actin filaments. However, constitutive activated β1-integrin could not enhance β1-integrin clustering under low substratum rigidity. Moreover, downregulation of β1-integrin clustering by disruption of actin organization had no effect on β1-integrin activation. Thus we reasoned that β1-integrin activation is prior to clustering. Another important finding in this work is that we provide evidence showing that integrin clustering and actin organization are controlled by a positive feedback loop. Clustering of β1-integrin promotes actin organization, since cells overexpressed with autoclustered β1-integrin but not constitutively active β1-integrin enhanced actin organization when cells were cultured on collagen gel. On the other hand, integrated actin structures also control β1-integrin clustering, since disruption of actin organization also downregulates the level of β1-integrin clustering. However, FAKY397 phosphorylation is not involved in actin organization. Disruption of actin organization abolished FAKY397 phosphorylation. Cells overexpressing autophosphorylation site mutant FAK (FAKY397F) retain well-constructed actin filaments when they are cultured on collagen gel (unpublished data). Thus we deduce that the feedback control loop leads to downstream FAKY397 phosphorylation.

We showed that lipid raft externalization to the membrane is associated with cell spreading. A recent study showed that adhesion-induced lipid raft externalization depends on microtubules (2). To test whether the cytoskeleton is also involved in regulating raft transport in our model, HEK293T cells were cultured on a collagen gel-coated dish for 4 h and treated with or without drugs to disrupt actin filaments or microtubules. However, our data showed that disruption of actin filament or microtubules does not affect the raft transport (data not shown). Although the cell spreading process is impeded by low substratum rigidity, we found that disruption of lipid rafts by MβCD reduced β1-integrin activation, FAK phosphorylation (Fig. 3E), the level of β1-integrin clustering, and cell spreading as well (data not shown). Thus we deduced that raft formation is prior to β1-integrin signaling. Recent work also suggested that line tension, the interfacial energy at the raft domain edge, is a key parameter determining the distribution of rafts (17). Applying lateral tension on the membrane increases raft formation through increasing line tension (1). Thus it is also possible that substratum rigidity regulates raft formation by controlling the physical property of the cell membrane. The details of this mechanism will be investigated in the future.

Talin is a cytoplasmic protein with a globular head domain and an elongated rod domain that acts as an essential linkage between integrins and the actin cytoskeleton. Previous studies showed that talin is also crucial in regulating integrin signaling. The NH2-terminal globular head domain of talin is responsible for promoting integrin activation and clustering (4, 10, 44, 56). Since force-induced exposure of vinculin-binding sites on talin through a conformational change leads to the reinforcement of the focal adhesions, talin is also considered as a key mechanotransduced molecule in focal adhesions (5, 20, 28, 35). Furthermore, previous evidence suggested that talin is functionally associated with the lipid rafts (33, 41, 45). Together, the retardation of the lipid rafts as well as talin externalization may lead to decreased β1-integrin activation under low substratum rigidity. However, the exact role of talin in this mechanosensing machinery still needs further investigation.

The mechanosensing machinery responsible for sensing the rigidity beyond the range of 58–386 Pa could be more comprehensive. A recent study showed that substratum rigidity controls mesenchymal stem cell lineage specification (15). Stem cells differentiate into myogenic and osteogenic lineages under rigidities of 11 kPa and 34 kPa, respectively. Moreover, our recent study showed that low substratum rigidity-induced apoptosis is inversely correlated with substratum rigidity. Nevertheless, a significant difference of apoptosis ratio still exists when cells are cultured under substratum rigidity between 20 and 103 Pa (52), in which range β1-integrin is neither activated nor clustered. We noted that other stretch-activated ion channels or capacitative calcium entries may be involved in detecting matrix rigidity at this range (7, 8). In light of these findings, it is feasible that various tissues may possess different sets of mechanical sensing machinery to detect different substratum rigidities.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Health Research Institute grant NHRI-EX95-9430SI and National Science Council, ROC Grant 95-2120-M-006-007 to M.-J. Tang.


   ACKNOWLEDGMENTS
 
We thank Tsu-Ling Chen for her excellent technical assistance; Dr. Timothy A. Springer for the gift of β1-integrin (G429N) plasmids; Dr. Valerie M. Weaver for the gift of β1-integrin (wild type, G429N, V737N)-conjugated GFP plasmids; Dr. Roger Y. Tsien for the gift of monomeric CFP and YFP plasmids; and Dr. Hong-Chen Chen for the gift of MDCK 3B5 cells harboring FAK and FRNK.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M.-J. Tang, No. 1, University Rd., Dept. of Physiology, National Cheng Kung Univ. Medical College, Tainan 701, Taiwan (e-mail: mjtang1{at}mail.ncku.edu.tw)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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