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Address correspondence to M.A. Schwartz, Department of Vascular Biology, CVN228/VB4, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037. Tel.: (858) 784-7140. Fax: (858) 784-7360. E-mail: schwartz{at}scripps.edu
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Abstract |
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Key Words: mechanotransduction; mechanical stretch; lamellipodia; polarization; Rac
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Introduction |
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Classical studies of cellular responses to forces have demonstrated that neurites grow in response to mechanical tension in tissue culture (Bray, 1984). Fibroblasts were reported to be oriented along the axis of stretch (Haston et al., 1983), and electron microscopic studies indicated that mechanical tension, whether inherent in the cytoskeleton or imposed on the cell surface by exogenous force, can influence the alignment of tight-junction strands (Pitelka and Taggart, 1983). Rearward tension applied by a micropipette in platyfish epidermal cells dramatically suppressed protrusive activity at the leading edge and caused alignment of microfilaments parallel to the applied tension (Kolega, 1986).
Apart from static stretch, cyclic stretch applied to rat cardiac myocytes induces elongation and orientation perpendicular to the direction of the stretch (Terracio et al., 1988). In human umbilical vein endothelial cells, cyclic stretch stimulated stress fiber formation and alignment of cells which involved changes in adenylate cyclase activity (Shirinsky et al., 1989). Cyclic stretch-induced cell orientation was found to depend on calcium influx via stretch-activated cation channels (Naruse et al., 1998), and tyrosine phosphorylation of p130CAS, focal adhesion kinase (FAK)* and paxillin by c-src (Sai et al., 1999), as well as activation of mitogen-activated protein kinase (Wang et al., 2001b) and Rho (Yano et al., 1996).
Cells extend thin, sheet-like processes at their leading edge known as lamellipodia, which contain a dense meshwork of actin filaments. Some lamellipodia extend smoothly outward over the substratum, whereas others move back over the cell surface in a wavelike motion known as ruffling (Bray and White, 1988). These structures are regulated by the small GTPase Rac in response to a variety of stimuli, including growth factors and extracellular matrix (ECM) (Schwartz and Shattil, 2000). In the case of ECM, it is striking that cells plated on fibronectin or other adhesive proteins show a marked but transient activation of Rac (Price et al., 1998; del Pozo et al., 2000) that decreases to baseline levels once cells are fully spread. This observation prompted us to consider whether this downregulation of Rac might occur via mechanical effects.
In order to elucidate the effects of mechanical forces on Rac, we applied strain to vascular smooth muscle (VSM) cells and fibroblasts plated on elastic substrata coated with ECM protein. We found that increasing strain via a single step of equibiaxial stretch dramatically decreased lamellipodia due to inhibition of Rac. Conversely, decreasing cell-generated tension by inhibiting myosin phosphorylation increased lamellipodia through Rac activation, suggesting that endogenous tension plays a similar role to regulate Rac. Finally, uniaxial stretch suppressed lamellipodia in a directional fashion. Together with effects of myosin inhibitors, these results indicate that inhibition of Rac depends upon the direction of the tension and suggest that this effect contributes to the polarized morphology of migrating cells.
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Results |
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Effects of uniaxial stretch
Biaxial strain includes both a radial component (i.e., perpendicular to cell edges), and a tangential component (i.e., parallel to cell edges). Thus, biaxial strain does not address whether the direction of the applied strain is important. Therefore, we investigated the effects of uniaxial stretch on lamellipodia. VSM cells were spread on collagen-coated membranes in a device that applies stretch in one dimension as described in Materials and methods. Cells at 75 min after plating were well spread but remained largely circular and unpolarized, thus changes in polarity induced by strain could be easily assessed. Cells were then stretched to increase length by 8%. Strain was maintained for the duration of the experiment as before. Time-lapse images (Fig. 4 A) showed that cells immediately after stretch had increased their length in the direction of strain as expected. Quantitative analysis of the effects on cell size also revealed some decrease in size perpendicular to stretch (Fig. 4 C, 0 min). This change in area reflects the combination of tensile strain in the direction of stretch and a smaller amount of compression in the perpendicular direction, usually 15% of the increase (Sadoshima et al., 1992; Lee et al., 1999).
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In situ Rac assay
The above results suggest that Rac in different regions of a cell may be differentially affected by an asymmetric strain. To investigate this idea we turned to a recently developed fluorescence resonance energy transfer (FRET) assay for local Rac activity (Kraynov et al., 2000). The assay is based on the principle that upon activation, GFP-labeled Rac binds to Alexa-PBD, resulting in an increase in FRET that can be imaged in the microscope.
In these experiments, VSM cells containing GFP-Rac and Alexa-PBD were plated on collagen- coated membranes for 75 min as above. When FRET was determined, we observed that most of the cells had zones of high Rac activity near cell edges, though the cells were generally circular and nonpolarized (Fig. 6 A, top). Cells also showed a substantial level of perinuclear Rac activity. This pool was also observed by Kraynov et al. (2000) and did not noticeably change in response to extracellular signals. The nature of this compartment is presently unknown but does not correlate with lamellipodia. Examination of FRET near cell edges showed that uniaxial strain (8% increase in length) induced an increase in GTP-bound Rac at the ends and a decrease at the sides (Fig. 6 A, bottom). The total levels of FRET near cell edges did not change substantially until much later times when Rac activity diminished as it usually does at later times after cell spreading (Price et al., 1998; del Pozo et al., 2000).
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Effects of Y-27632 and ML-7 on lamellipodia
It is likely that the applied strain acts by increasing tension in specific elements within cells. Consequently, we investigated whether cell-generated tension functioned similarly to the externally applied strain in suppressing Rac activity. Therefore, inhibitors of myosin phosphorylation were examined. Blockade of Rho-kinase with Y-27632 or myosin light chain kinase with ML-7 is known to decrease cell-generated tension via distinct mechanisms (Zhong et al., 1997; Narumiya et al., 2000). Treatment of VSM cells for 60 min with either of these compounds moderately, but significantly increased Rac activity (Fig. 7, A and B). Staining of actin filaments in treated cells showed increased lamellipodia in cells treated with Y-27632 or low doses of ML-7 (Fig. 7 C). Y-27632 caused a noticeable but incomplete disassembly of stress fibers (Fig. 7 C). Higher doses of ML-7 (10 µM) caused disassembly of actin stress fibers and collapse of lamellipodia (unpublished data). Presumably, myosin phosphorylation is required for lamellipodia, which are therefore inhibited at higher doses of ML-7.
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Discussion |
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When cells are subjected to externally applied strain, there are two factors that could conceivably regulate signal transduction. The displacement of the cell edges could itself directly affect Rac, or alternatively, the resultant increase in tension within the cytoskeleton or membrane could be the critical factor. We found that treating cells with Y-27632 and ML-7 did not cause any significant displacement of cell edges that could account for the increase, whereas they did decrease cell tension applied to the substratum. Thus, altering tension under conditions where displacement is relatively constant also affects Rac activity, suggesting that tension within a cell transduces the signal to Rac.
The effect of tension on Rac may explain published observations from many systems. NIH3T3 cells plated on flexible substrates exert less tension and also show increased rates of lamellipodial activity and motility compared with cells on rigid substrates (Pelham and Wang, 1997). Adhesion of fibroblasts to surfaces coated with high concentrations of fibronectin inhibited Rac activation and decreased migration compared with moderate coating densities (Cox et al., 2001). In that study, Rac inhibition was linked to high Rho activity, which is a major determinant of myosin phosphorylation and contractility, again consistent with our findings. The well-known alignment of cells in response to stretch is also consistent with the effects reported here, as are developmental processes in which tractional fields govern cell migration and organization of cells into higher order structures (Davis and Camarillo, 1995; Ingber and Folkman, 1989).
Our results also demonstrated that with uniaxial strain, protrusive activity increased at the ends and decreased along the sides. The results of the in situ Rac assay also demonstrated that Rac is decreased at the edges that are subjected to increased tangential tension (i.e., the edges that actually lengthened). This is consistent with the previous finding that tension applied to the cell suppresses lateral protrusive activity (Kolega, 1986). The opposite edges that do not lengthen should experience increased radial tension, consequently this component must be less effective at inactivating Rac. Uniaxial stretch by 17% did not dramatically inhibit total protrusive activity, in sharp contrast with 15% equibiaxial stretch. These results demonstrated that direction of applied tension rather than increase in area is critical for the effects on Rac.
These observations also precisely match the morphology of cells under normal conditions and the effects of myosin inhibitors. Previous studies have shown that cell-generated tension is strongest along the frontrear axis of migrating cells (Galbraith and Sheetz, 1997; Pelham and Wang, 1999; Balaban et al., 2001). Cells under normal conditions extend lamellipodia primarily at edges perpendicular to actin stress fibers, whereas edges parallel to stress fibers that should experience tangential tension are inhibited. Therefore, one would predict that endogenous tension should inhibit Rac along the sides parallel to the direction of migration, which should help maintain the typical morphology of migrating cells. We observed that decreasing endogenous myosin-dependent force generation not only increased Rac activity, but specifically increased lamellipodia along the sides and tail to decrease polarity. These results demonstrate that cell-generated tension regulates Rac and suggest that this regulation contributes to cell polarity.
At later times, we observed redistribution of focal adhesion proteins. This observation is consistent with previous findings demonstrating that mechanical stretch induces focal adhesion assembly (Riveline et al., 2001; Wang et al., 2001a). The alignment of actin stress fibers in the direction of the applied stretch was seen at later time (3060 min). V12Rac completely blocks alignment of stress fiber after uniaxial stretch. This strongly suggests that early effects on Rac by uniaxial stretch is upstream of stress fiber alignments at later time. Further studies aiming to determine the signaling events that mediate regulation of Rac by tension are of particular interest in elucidating fundamental mechanisms of mechanotransduction.
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Materials and methods |
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Cell culture and DNA transfection
Rat aortic smooth muscle (VSM) cells, NIH3T3 cells and mouse embryonic fibroblasts were maintained in DME (Life Technologies) containing 10% FBS unless otherwise indicated. Cells were cultured at 37°C in a humidified incubator containing 7% carbon dioxide. Transient transfections were performed using Effectene reagents (QIAGEN) according to the manufacturer's instructions. pcDNAINeo-Tiam1 was provided by John G. Collard (The Netherlands Cancer Institute, Amsterdam, The Netherlands). GFP-V12Rac was described previously (del Pozo et al., 1999).
Stretch devices
The equibiaxial stretch device was previously described (Lee et al., 1996). Briefly, a silicone elastic membrane is attached to the membrane holder to form the bottom of the device. Indentation of the ring against the membrane results in a homogenous plane equibiaxial stretch of the membrane. Strains along circumferential and radial axes were equal in magnitude and homogenously distributed, and two-dimensional strains in the stretched cells were not significantly different from mean equibiaxial strains measured in the substrate for nominal strains up to 10% (Lee et al., 1996). The uniaxial stretch devices were similar to previously described instruments (Lee et al., 1999). In brief, a rectangular membrane is attached to rigid supports at either end, while the sides are free. Strain is applied by increasing the distance between the ends with a pair of stainless steel rods and screws. The displacement of markers during stretch was measured in microscopic digital images using a 4x objective, showing the strain in the direction parallel to the stretch axis has no significant difference at various points measured. Compression perpendicular to the axis of stretch did vary; for an 8% stretch it was 2.2% in the center of the membrane and decreased to 0.1% at the edges. Cells were examined close to the center for all experiments. For both types of devices, the strain was applied over 10 s, which is the time required to manually turn the ring or screws. Strain remained constant for the duration of the experiments. For VSM cells, membranes in the devices were coated with a solution of 40 µg/ml collagen type I for 2 h, and then sterilized by irradiation under the UV light in a laminar flow hood for 15 min. For fibroblasts membranes were coated with 20 µg/ml fibronectin using the same protocol.
GTPase assays
Rac assays were performed as described (del Pozo et al., 2000). Treated cells were washed with ice-cold PBS and lysed in 400 µl buffer containing 0.5% NP-40, 50 mM Tris, pH 7.0, 500 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 20 µg GSTPBD. Lysates were centrifuged to remove particulates and incubated with glutathione-agarose beads (Sigma-Aldrich) for 30 min at 4°C, washed three times, and eluted with SDS sample buffer. Rac protein was detected by Western blotting using a monoclonal antibody against Rac1 (Upstate Biotechnology). Rho assays were performed as described (Ren et al., 1999). Treated cells were washed with ice-cold phosphate-buffered saline and lysed in 400 µl RIPA buffer (50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycolate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2, 10 µg/ml each of aprotinin and leupeptin, 1 mM PMSF). Lysates were centrifuged as described above, equal volumes were incubated with the GST-Rho binding domain of Rhotekin (RBD) beads (20 µg protein/sample) for 45 min at 4°C, washed three times, and eluted with SDS sample buffer. Rho protein was detected by Western blotting using a polyclonal antibody against RhoA (Santa Cruz Biotechnology). Densitometry analysis was performed using Scion Image software (Scion Corporation).
Fluorescence microscopy
Cells were fixed for 20 min in 2% formaldehyde in PBS. For the cells on the stretch apparatus, the silicone membrane was adhered to slide glass with 732 multipurpose sealant (Dow Corning) at 4°C for 16 h. Cells were then permeabilized for 5 min with 0.2% Triton X-100/PBS, and were stained for 60 min in either 1:250 or 1:500 dilution of rhodamine-phalloidin (Molecular Probes). Images were recorded using a BioRad 1024 confocal microscope with a 60x SPlanApo 60 PL oil objective.
Quantification of lamellipodia
Cells stained with rhodamine-phalloidin were analyzed by either Inovision ISEE imaging software (Inovision) or Image Pro Plus software program (Media Cybernetics). Both the total cell periphery and the portion of the periphery occupied with lamellipodia were outlined. The percentage of lamellipodia was defined as: (the length of perimeter occupied by lamellipodia/total perimeter) x 100.
Real-time video phase-contrast microscopy
VSM cells were spread on collagen Icoated silicone membranes in either the equibiaxial or uniaxial stretch device. They were placed in an open chamber with atmospheric and temperature control (Schwartz, 1993) before and after the stretch. Cells were filmed from 10 min before to 70 min after the stretch at a 1-min interval with a Nikon DiaPhot microscope equipped with a SenSys cooled CCD video camera linked to a Silicon Graphics workstation running the Inovision ISEE software program (Kiosses et al., 1999). To calculate areas, cells were outlined manually in phase contrast images and areas within the boundary determined by the software.
Cells and microscopy for FRET assays
VSM cells were plated in 6-cm cell culture dishes and were transfected with 2 µg of pEGFP-C1 wild- type Rac with 3.3 mM thymidine to synchronize the cell cycle to enhance transfection efficiency (Goldstein et al., 1989). After the transfection, 5 mM of sodium butyrate was added to enhance expression (Goldstein et al., 1989). After 24 h, cells were trypsinized and were adjusted to 4 x 105 cells/ml. To one ml was added 3.75 µg of Alexa-PBD and cells were sheared through a 27-gauge needle as described (Clarke and McNeil, 1992). Cells were plated on collagen Icoated silicone membrane assembled in the uniaxial stretch device. At 75 min after plating, cells were stretched by 8% for the indicated time, and fixed with 2% formaldehyde in PBS.
FRET analysis
Imaging was performed using a BioRad 1024 Confocal Microscope where the filters were optimized to best acquire the GFP-, FRET-, and Alexa 546labeled images of cells (Del Pozo et al., 2002). These filters are: (1) GFP-Rac, excitation 488, emission 522; (2) Alexa 546, excitation 568, emission 598; and (3) FRET, excitation 488, emission 598. GFP and FRET images were acquired simultaneously, and Alexa 546 images were acquired separately, at the same plane and without any shift in the sample. GFP and FRET images of cells injected with GFP Rac only, and Alexa and FRET images of cells injected with Alexa PBD only were also obtained in order to determine bleed through and background levels to correct the initial FRET images (Chamberlain et al., 2000; Kraynov et al., 2000). These calculations were made using the ISEE software running on a UNIX workstation. Corrected FRET images on an 8-bit, 512 x 512 scale typically had a fluorescence intensity range of 092 in these experiments. This fluorescence intensity range was displayed using a color spectrum, where blue was closest to 0 and red was closest to 92. Positive FRET signals typically appeared between 46 and 92, in the green-yellow to red range. Below that was considered baseline.
Real-time video phase-contrast microscopy to quantify lamellipodia of living cells
To assay lamellipodia, VSM cells were plated on coverslips coated with collagen I. Dishes were prepared by cutting a hole in the bottom of the dish and attaching a coverslip to the outside of the dish with silicon grease. These were placed in an open chamber with temperature control. The medium was replaced by Hepes medium to keep a physiologic pH without CO2. Cells were viewed with an Olympus IX70 microscope equipped with a coolSNAP-Pro camera (Media Cybernetics), linked to a Gateway computer running the Image Pro Plus software program. Cell morphology was assayed by time-lapse imaging (one image per minute) 20 min before treatment and 6080 min after treatment. To quantify lamellipodia, the portion of the cell periphery with and without lamellipodia were outlined separately using the Image Pro Plus software program. The length of total lamellipodia and lamellipodia at the tail (defined as the region located behind the nucleus), and the length of the total perimeter of the cell were compiled for multiple cells. The percentage of lamellipodia was defined as: (the length of total or tail lamellipodia/total perimeter) x 100.
Measurements of traction and contractile force
Contractile forces were measured using flexible polyacrylamide substrates (5% acrylamide and 0.1% Bis-acrylamide) containing fluorescent beads as described (Wang et al., 2002). Cells were plated on collagen Icoated polyacrylamide and treated with ML-7 or Y-27632 for 1h. At the end of the experiment, cell-generated traction was eliminated by adding 1% SDS at final concentration. Calculations of traction forces were done as described (Butler et al., 2002).
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Footnotes |
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Acknowledgments |
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This work was supported by USPHS grant P01HL57900 (to M.A. Schwartz), GM57464 (to K.M. Hahn), Uehara Memorial Foundation (to A. Katsumi), Association pour la recherché contre Le cancer (to J. Milanini), EMBO (to M.A. del Pozo), Leukemia and Lymphoma Society Special Fellowship #3347-02 (to M.A. del Pozo), and American Heart Association fellowship 0120069Y (to W.B. Kiosses).
Submitted: 23 January 2002
Revised: 16 May 2002
Accepted: 17 May 2002
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