External mechanical strain regulates membrane targeting of Rho GTPases by controlling microtubule assembly

Andrew J. Putnam1, James J. Cunningham1, Brendan B. L. Pillemer2, and David J. Mooney1,3,4

Departments of 1 Chemical Engineering, 2 Microbiology, 3 Biomedical Engineering, and 4 Biologic and Materials Sciences, University of Michigan, Ann Arbor, Michigan 48109-1078


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transmission of externally applied mechanical forces to the interior of a cell requires coordination of biochemical signaling pathways with changes in cytoskeletal assembly and organization. In this study, we addressed one potential mechanism for this signal integration by applying uniform single external mechanical strains to aortic smooth muscle cells (SMCs) via their adhesion substrate. A tensile strain applied to the substrate for 15 min significantly increased microtubule (MT) assembly by 32 ± 7%, with no apparent effect on the cells' focal adhesions as revealed by immunofluorescence and quantitative analysis of Triton X-100-insoluble vinculin levels. A compressive strain decreased MT mass by 24 ± 9% but did not influence the level of vinculin in focal adhesions. To understand the decoupling of these two cell responses to mechanical strain, we examined a redistribution of the small GTPases RhoA and Rac. Tensile strain was found to decrease the amount of membrane-associated RhoA and Rac by 70 ± 9% and 45 ± 11%, respectively, compared with static controls. In contrast, compressive strain increased membrane-associated RhoA and Rac levels by 74 ± 17% and 36 ± 13%, respectively. Disruption of the MT network by prolonged treatments with low doses of either nocodazole or paclitaxel before the application of strain abolished the redistribution of RhoA and Rac in response to the applied forces. Combined, these results indicate that the effects of externally applied mechanical strain on the distribution and activation of the Rho family GTPases require changes in the state of MT polymerization.

cytoskeleton; mechanotransduction; focal adhesions


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MECHANOTRANSDUCTION, THE PROCESS by which cells and tissues sense and respond to mechanical changes in their environment, involves a cascade of events that eventually converts a mechanical signal into a biochemical response (13, 57). The response can be vastly different, depending on the type of signal and the context in which it is applied. Externally applied mechanical signals were shown to alter cell proliferation (66, 67), influence production of extracellular matrix (ECM) proteins (8, 11, 14), and induce alterations in gene expression (44, 57, 65), often in an ECM-dependent manner (31, 36, 40, 67). The mechanisms utilized by cells to transduce these mechanical inputs into phenotypic changes are the focus of increasing numbers of studies, but they remain poorly understood.

Two paradigms have been proposed to explain the responses of cells to changes in the mechanical microenvironment. In the first paradigm, mechanical stimuli applied to sites of cell adhesion trigger a cascade of soluble biochemical signals, likely derived from the cluster of signaling molecules at focal adhesion sites (12, 57) or from ion fluxes via mechanosensitive ion channels (4, 23, 62). The second paradigm, based on models of tensegrity architecture first put forth by Buckminster Fuller (26, 29, 46, 63), suggests that externally applied mechanical forces may be directly transduced to the underlying cytoskeleton via integrins, altering a preexisting cellular force balance and thereby changing the assembly and organization of cytoskeletal elements. These changes in filament assembly may then directly or indirectly alter cellular gene expression by influencing a variety of signaling pathways (13, 28). Although these two paradigms have largely been regarded as distinct, clearly the integration of biochemical signals and the cytoskeleton is required to yield an appropriate cellular response to a mechanical input.

The Rho family GTPases represent one potential target involved in the integration of these cellular responses to mechanical signals. Widely studied for their role in regulating the assembly and organization of actin cytoskeletal structures (16, 50, 51, 60), the Rho family GTPases have also been implicated in integrin-mediated signaling (17, 49, 56) and in the cellular response to applied mechanical forces (1, 2, 32, 43). RhoA plays a critical role in the assembly of actin stress fibers in response to various soluble stimuli, including serum, growth factors, and lysophosphatidic acid (LPA) (15, 16, 42), and to insoluble adhesion ligands such as fibronectin (5, 49).

A definitive connection between the microtubule (MT) cytoskeleton and cell contractility mediated by the Rho family GTPases has been elucidated over the past decade. Increased contractility, characterized by an increased formation of actin stress fibers and focal adhesions, was first shown to occur after depolymerization of MTs with various pharmacological agents (e.g., nocodazole and colchicine) in 1989 (20). This increased contractility was later shown to be the result of greater myosin light chain phosphorylation (37), now known to be mediated via activation of RhoA (24, 39). Other studies demonstrated that Rho is required for the selective stabilization of MTs (18) via a mechanism that involves the Rho effector mDia (45). Thus there is clear evidence that the signaling capabilities of the Rho GTPases and MT assembly and/or organization are linked.

In this study we have directly tested the hypothesis that changes in MT assembly and/or disassembly driven by mechanical strain can modulate the distribution of Rho GTPases between cytoplasmic and membrane-associated subcellular compartments. This hypothesis stems from recent findings that externally applied mechanical strain, typical of that experienced by smooth muscle cells (SMCs) in blood vessels, leads to predictable alterations in MT assembly (47, 48). Single step changes in mechanical strain were applied to cultured SMCs to alter the assembly of MTs, and the subcellular localization of two Rho GTPases, RhoA and Rac, was quantified. The results presented here demonstrate that single step changes in strain applied to SMCs alter the quantity of membrane-associated RhoA and Rac. In parallel experiments, the size and vinculin content of focal adhesions remained relatively constant in response to both compressive and tensile single applied strains. Prolonged treatments with low doses of either nocodazole or paclitaxel before the application of strain significantly altered the amount of membrane-associated RhoA and Rac after the applied strain, suggesting that the force-mediated regulation of Rho GTPases requires force-induced changes in MT polymerization. Combined, these results suggest that changes in MT assembly may be able to regulate the targeting of RhoA and Rac to the membrane in response to externally applied mechanical forces. We speculate that this may provide a means of spatially regulating the balance of activity of RhoA vs. Rac.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation and culture of rat aortic SMCs. Vascular SMCs were isolated from thoracic rat aortas with an adaptation of a previously published technique (47, 53). Routine SMC culture was performed with medium 199 supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all from GIBCO/Life Technologies, Gaithersburg, MD). SMCs between passages 4 and 15 were used in all studies.

Application of external step changes in strain to cultured SMCs. Cells were plated out at densities between 20,000 and 30,000 cells/cm2 on six-well culture dishes made of silicon rubber (BioFlex plates; Flexcell Intl., Hillsborough, NC). These plates, initially untreated, were coated with a theoretical density of 1 µg/cm2 (accounting for the additional surface area created by the volume of the liquid) of fibronectin (human plasma fibronectin; GIBCO). Fibronectin was adsorbed with a carbonate-bicarbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.4) (19). Cells cultured on these substrates were allowed to attach and spread for ~24 h. Cells were subsequently serum starved by culturing in medium 199 alone for a period of 18-24 h before experimentation to eliminate the effects of serum on Rho signaling activity. Dishes were exposed to positive step changes in strain with a custom-built device described in earlier publications (47, 48) by clamping the plate down over a series of Teflon posts, effectively imparting a tensile force to the adherent cells. Cells were then held in this position for a specific period of time before being analyzed. By contrast, for the negative step change experiments, the plates were clamped down over the Teflon posts for a period of 24 h, allowed to equilibrate, and then subsequently released to result in a compressive, or negative, strain to the cell culture surface.

Extraction and quantification of MT and total tubulin fractions from cultured cells. MTs and total tubulin were differentially extracted from cultured SMCs according to methods described in detail elsewhere (10, 41, 47). A competitive ELISA technique (61) was used to quantify the cellular tubulin distribution as previously described (47).

Extraction and quantification of Triton X-100-insoluble vinculin. Control and stretched SMCs were rinsed once with cold PBS and then permeabilized for 10 min with ice-cold permeabilization buffer (in mM: 10 HEPES, pH 6.9, 50 NaCl, 3 MgCl2, and 300 sucrose plus 1 EGTA; all reagents from Sigma, St. Louis, MO) containing 0.1% Triton X-100, followed by a brief rinse with the same buffer. The remaining detergent-insoluble proteins were solubilized in lysis buffer (25 mM Tris Cl, pH 7.4, 0.4 M NaCl, 0.5% SDS; all reagents from Sigma) for 5 min with mechanical scraping. Protease and phosphatase inhibitors were added to both buffers to prevent protein degradation [10 µg/ml aprotinin, 10 µg/ml leupeptin, 5 mg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium fluoride, and 1 mM sodium orthovanadate; all from Sigma]. Lysates were stored at -70°C until analysis. Total protein was measured with the bicinchoninic acid (BCA) assay (Pierce Chemical, Rockford, IL). Equal amounts of total protein were subjected to SDS-PAGE, and vinculin was quantified by standard Western blotting techniques with enhanced chemiluminescence. Vinculin was detected with a monoclonal anti-vinculin antibody (MAB1624; Chemicon, Temecula, CA), followed by a 1:1,000 dilution of a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad). Densitometry of scanned films was performed with NIH Image software (National Institutes of Health, Bethesda, MD).

Immunofluorescent localization of focal adhesions. Control and stretched SMCs were rinsed with PBS, followed by a 1-min permeabilization in permeabilization buffer (see Extraction and quantification of Triton X-100-insoluble vinculin) containing 0.5% Triton X-100. Cells were then fixed for 20 min with 4% formaldehyde in cytoskeleton buffer (CBS; in mM: 10 MES, pH 6.1, 138 KCl, 3 MgCl2, and 2 EGTA with 0.32 M sucrose; all reagents from Sigma). Nonspecific binding was blocked with 2% BSA (GIBCO), and monoclonal anti-vinculin (Chemicon) was followed by rhodamine anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). Cells were visualized on a Nikon E800 microscope equipped for epifluorescence with a charge-coupled device (CCD) camera (Optronics) and a frame-grabber board (Scion). Images were compiled with Adobe Photoshop 5.0.

Cell fractionation and detection of membrane-associated RhoA and Rac. Whole cell lysates were prepared from SMCs with an adaptation of a previously published technique (2, 7, 25, 43). Cells cultured on six-well dishes (either tissue culture polystyrene or flexible BioFlex plates) were washed two times in PBS before being lysed. Cells were lysed for 5 min by adding 1 ml of a lysis buffer containing 20 mM Tris (pH 8.0), 250 mM sucrose, and protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 5 µg/ml pepstatin A, and 0.5 mM PMSF). Samples were scraped from the dish and collected into 1.5-ml microcentrifuge tubes. These tubes were then flash-frozen in liquid nitrogen and subsequently subjected to three cycles of freezing and thawing to completely lyse the cells. Samples were analyzed for total protein content with the Pierce BCA protein assay kit. Parallel samples from each condition (1 from each well of the 6-well dish) were pooled into a single sample containing 200-300 µg of total protein. Each sample was prepared by aliquoting an appropriate volume of each sample into a Beckman ultracentrifuge tube to yield equal amounts of protein across all conditions. With this approach, equal amounts of protein were then subjected to centrifugation at 100,000 g and 4°C for 1 h with a Beckman Preparative Ultracentrifuge with a Ti65.2 rotor. After centrifugation, the supernatant was aspirated off and discarded. The remaining insoluble pellet was resuspended in 200 µl of Laemmli sample buffer and allowed to sit on ice for 1 h to solubilize all of the protein. At that point, 50 µl of each sample was boiled for 3 min and separated via electrophoresis on 10-20% Tris-glycine polyacrylamide gels (Bio-Rad). Western blots were obtained by transferring the proteins to a polyvinylidene difluoride (PVDF) membrane for 90 min with a Bio-Rad Mini-Protean II transfer apparatus (Bio-Rad Laboratories).

Blots were blocked overnight at 4°C in Tris-buffered saline with 0.1% Tween-20 (TBS-T) with 5% nonfat dried milk (NFDM). RhoA was detected with a mouse monoclonal anti-RhoA antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1,000 in TBS-T + 5% NFDM followed by an HRP-conjugated goat anti-mouse IgG secondary antibody (Santa Cruz) diluted 1:2,000 in TBS-T + 5% NFDM. Rac was detected with a rabbit polyclonal anti-Rac antibody (Cytoskeleton, Denver, CO) diluted 1:1,000 in TBS-T + 5% NFDM followed by an HRP-conjugated goat anti-rabbit IgG secondary antibody (Santa Cruz) diluted 1:2,000 in TBS-T + 5% NFDM. Bands were detected by adding an enhanced chemiluminescent substrate (ECL Plus, Amersham-Pharmacia, Arlington Heights, IL) and exposing the blots to autoradiography film (Hyperfilm ECL, Amersham-Pharmacia). Developed films were scanned and quantified by densitometry with NIH Image software.

Generation of whole cell lysates for total cellular RhoA/Rac. Whole cell lysates were prepared from SMCs as in Cell fractionation and detection of membrane-associated RhoA and Rac. After three cycles of freezing and thawing to completely lyse the cells, samples were analyzed for total protein content with the Pierce BCA protein assay kit. Specific volumes of these lysates were then subsequently mixed with Laemmli sample buffer to yield a final mass of 1 µg of total protein to load onto a 10-20% polyacrylamide gel. Western blots for total cellular RhoA and Rac were then performed as described for membrane-associated RhoA and Rac.

Detection of activated (GTP-bound) Rac in cell lysates. Affinity precipitation of activated Rac was performed with a commercially available kit (Cytoskeleton) according to the manufacturer's instructions. The protocol followed was essentially that described in several other studies (6, 49). Control and stretched SMCs were washed with ice-cold PBS and lysed in buffer containing 50 mM Tris, pH 7.4, 10 mM MgCl2, 0.2 M NaCl, 2% NP-40, 10% sucrose, and protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 5 µg/ml pepstatin A, and 0.5 mM PMSF). After clarification of the lysates by centrifugation, equal amounts of protein were incubated with 10 µg of recombinant PAK-binding domain-GST fusion protein (PBD-GST) for 30 min at 4°C and then incubated with glutathione-agarose beads for an additional 30 min at 4°C. Collected beads were washed several times with lysis buffer, and proteins were eluted with Laemmli sample buffer. Bound Rac was analyzed by Western blotting with a polyclonal Rac antibody (Cytoskeleton) as described in Cell fractionation and detection of membrane-associated RhoA and Rac. Whole cell lysates were also analyzed to provide an indirect measure of equal loading.

Alteration of MT network with paclitaxel or nocodazole. The assembly of the MT network was altered with paclitaxel (Sigma) or nocodazole (Sigma) before the application of external strain. These agents were solubilized in DMSO and diluted into cell culture medium. Because drug-induced alteration of the MT network can directly influence RhoA and Rac activity (39, 64), we adopted a procedure whereby cells were initially treated with standard doses of nocodazole (10 µg/ml) or paclitaxel (15 µM) for a short period (90 min), followed by serum starvation and reduced drug concentration (1 µg/ml nocodazole or 1 µM paclitaxel) for 18-24 h to allow the activity of RhoA and Rac to attain a steady state before applying strain. These conditions were verified to maintain the expected disruptions in the MT network without inducing any perceptible changes in cell adhesion or the actin cytoskeleton.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MT assembly is modulated by externally applied strain. We demonstrated previously (47, 48) that forces applied to SMCs via their adhesion substrate trigger alterations in MT assembly and/or disassembly in a manner that depends on the duration, direction, and magnitude of the applied strain, and these effects appear to be a direct result of the applied force. These strain-induced effects on MTs depend on ECM ligand density but appear to be independent of ECM ligand identity (48). In this study, we investigated what influence these mechanical effects on the cytoskeleton might have on other signaling events within the cell, focusing on the two Rho family GTPases, RhoA and Rac. Because serum by itself can influence the activity and subcellular localization of Rho GTPases, all experiments described in this study were performed with serum-starved cells. To confirm that MTs in serum-starved SMCs behave in the same fashion as those in our earlier studies, cells were cultured for 24 h in 10% serum-containing medium on silicon rubber substrates precoated with fibronectin and subsequently serum-starved for another 18-24 h. Single externally applied step changes in strain were subsequently applied with a custom-built strain device that was characterized and described previously (47). Triton X-100-insoluble (n = 6) and total (n = 6) cellular lysates from cells held in a strained position were quantified for tubulin content. In agreement with our earlier studies, a tensile strain applied to serum-starved SMCs drove increased MT assembly by 32 ± 7% over nonstrained controls (Fig. 1A), whereas a compressive strain decreased MT assembly by 24 ± 9% (Fig. 1B). In all experiments conducted in this study, quantification of total cellular tubulin levels revealed no change (data not shown), suggesting that strain-induced alterations in MT assembly occur in a reciprocal fashion with changes in the preexisting cytoplasmic pool of alpha - and beta -tubulin monomers.


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Fig. 1.   Microtubule (MT) assembly is regulated via externally applied step changes in strain. A: smooth muscle cells (SMCs) subjected to a single (+)10% step change in strain, which imparts a tensile force to the substrate, have an increased polymeric tubulin mass (and hence net MT assembly) after 15-min exposure to the strain. B: by contrast, SMCs subjected to a single (-)10% step change in strain, which imparts a compressive force to the substrate, have a decreased polymeric tubulin content representing net MT disassembly. Values represent means ± SD from a single representative experiment. * Statistical significance (P < 0.05 compared with t = 0; n = 6).

Focal adhesions remain stable when subjected to single applications of strain. Parallel experiments were performed to determine whether either tensile or compressive strains influenced the composition or stability of focal adhesions in SMCs, because these structures are likely required to convey the extracellular strain to the cytoskeleton. Several recent reports suggested a role for focal adhesions in transducing local mechanical forces but did not address the response when mechanical strains are applied uniformly to the cell adhesion substrate (3, 27, 52). Qualitative immunofluorescent localization of vinculin, one of several abundant structural focal adhesion proteins, revealed no obvious change in the relative size or number of focal adhesions in response to either a 10% tensile strain or a 10% compressive strain (Fig. 2A). These images were supported by Western blot quantification of the amounts of Triton X-100-insoluble vinculin in cells exposed to both tensile and compressive strain (Fig. 2B). No significant changes were observed in the levels of focal adhesion-associated paxillin, focal adhesion kinase (FAK), or alpha -actinin, either (data not shown).


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Fig. 2.   Focal adhesion size and vinculin content remain unchanged after application of strain. A: relative size and number of focal adhesions in SMCs subjected to a compressive strain (left), held static (center), or subjected to a tensile strain (right) remain qualitatively unchanged as revealed by immunofluorescent localization of vinculin in the focal adhesions. Representative images are shown in the top images, with the boxed regions blown up in the bottom images. B: quantification of Western blots for Triton X-100-insoluble cell lysates supports the immunofluorescent staining, demonstrating no significant differences in the level of focal adhesion-associated vinculin in SMCs exposed to either compressive or tensile strain. Values provided are means ± SD from a single representative experiment, with 5 Triton X-100-insoluble lysates analyzed for vinculin content (n = 5).

Membrane targeting of both RhoA and Rac is influenced by application of single step changes in strain. We next determined whether strain influenced the activation of the Rho family GTPases RhoA and Rac. RhoA and Rac have been widely studied for their roles in the assembly and/or organization of the actin cytoskeleton and focal adhesions and are appropriately situated to transduce an extracellular mechanical signal into biochemical signals within cells. To indirectly measure the levels of the active GTP-bound form of RhoA, an assay was used that takes advantage of the fact that these small GTPases translocate to a membrane-bound fraction on activation (2, 7, 25, 43). To confirm the validity of these methods for our studies, serum-starved SMCs on static six-well tissue culture dishes were exposed to 2 µg/ml LPA, which has been well documented to activate RhoA. Equal protein lysates from LPA-treated cells were subjected to ultracentrifugation at 100,000 g for 1 h at 4°C. The crude pellets containing insoluble cell lysate material were solubilized in Laemmli sample buffer and analyzed via SDS-PAGE and Western blotting as described in MATERIALS AND METHODS. Treatment of the cells with LPA induced a net increase in the amount of membrane-associated RhoA in the particulate fraction compared with untreated controls, indicating an altered distribution of RhoA in SMCs correlating with an increased activity level (Fig. 3). A similar membrane targeting of RhoA occurred after short-term treatment with nocodazole, which was also shown previously to trigger activation of RhoA (data not shown).


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Fig. 3.   Activation of RhoA by lysophosphatidic acid (LPA) triggers a redistribution of RhoA to a membrane-associated pool. A: SMCs exposed to LPA (2 µg/ml), which was previously shown to activate RhoA, demonstrate a redistribution of RhoA from a cytoplasmic to a particulate membrane-associated fraction as measured with a translocation-cell fractionation assay. B: 15-min treatment with LPA increased the amount of RhoA in the membrane-associated fraction compared with control cells as detected by Western blotting followed by quantitative densitometry. Images shown are representative of 2 independent experiments.

Using the amount of membrane-associated Rho as an indirect measure of Rho GTPase activity, we monitored the distribution of RhoA and Rac in SMCs exposed to single step changes in applied strain. A 10% step tensile strain, which induces net MT assembly (Fig. 1A), induced a redistribution of RhoA from the membrane fraction to the soluble fraction, decreasing the membrane fraction to 30 ± 9% of the static controls (a decrease of 70%; Fig. 4A). By contrast, a 10% step compressive strain led to an increase of 74 ± 17% in the amount of membrane-associated RhoA over static controls (Fig. 4B). The level of total cellular RhoA remained constant throughout the course of the experiments (not shown). Recall that this same mechanical stimulus triggered net MT disassembly (Fig. 1B). Rac localization was assayed with the same methodologies used for RhoA. A 10% step tensile strain induced a net decrease in the amount of membrane-associated Rac in the high-speed pellet to a value of 55 ± 11% of the static controls (a decrease of 45%; Fig. 5A). That these fractionation techniques were a valid indirect measure of Rac activity was confirmed by use of a commercially available affinity precipitation, or pull-down, assay to directly measure GTP-bound Rac in cells subjected to a 10% step tensile strain. This assay, repeated several times, clearly demonstrated a decrease in the activity of Rac in cells subjected to a tensile strain for 15 min (Fig. 5B), paralleling our cell fractionation assay data. By contrast, a 10% step compressive strain resulted in an increase of 36 ± 13% in the amount of membrane-associated Rac over static controls (Fig. 5C). Similar to the results for RhoA, the level of total cellular Rac remained unchanged both before and after the applied strain (Fig. 5B). These findings clearly demonstrate a redistribution of RhoA and Rac, which correlates to their activity state, to an insoluble membrane-bound compartment in response to the application of mechanical forces applied to the substrate.


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Fig. 4.   Membrane association of RhoA is modulated by single step changes in strain applied to SMCs. A: SMCs exposed to a 10% step change tensile strain have a significantly reduced amount of membrane-associated RhoA compared with static controls. B: by contrast, a 10% step change compressive strain significantly increased the amount of membrane-bound RhoA over static controls. Representative Western blots from a single experiment with a sample size of 3 for each condition are shown, and the results shown are typical of 3 separate experiments that were performed (each with n = 3/condition). Values provided are means ± SD from a representative experiment. * Statistical significance (P < 0.05 compared with t = 0; n = 3).



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Fig. 5.   Membrane targeting of Rac is also modulated by single step changes in strain. A: SMCs exposed to a 10% step change tensile strain show a reduction in the amount of membrane-associated Rac compared with static controls. B: these changes in the distribution of Rac between membrane-associated and cytoplasmic pools correlated with a decreased activity of Rac as measured by the interaction of GTP-bound Rac in cell lysates to a PAK-binding domain-GST fusion protein (PBD-GST). Western blots from a representative experiment show a clear decrease in the levels of active Rac after application of a tensile strain. The levels of total Rac remained constant throughout these experiments. C: by contrast, a 10% step change compressive strain increased the amount of Rac in the particulate fraction compared with static controls. Western blots from a single experiment with a sample size of 2 or 3 for each condition are shown. Values provided are means ± SD from a representative experiment. * Statistical significance (P < 0.05 compared with t = 0; n = 3).

Strain-induced alterations in distribution of RhoA and Rac are dependent on presence of dynamic MT cytoskeleton. To test whether the strain-induced changes in MT assembly and the signaling activity of RhoA and Rac are related, experiments were performed in which the MT cytoskeleton was compromised before the application of mechanical strain. Because short-term drug-induced alteration of the MT network alone was previously shown to alter RhoA and Rac activity (39, 64), a procedure was used in which cells were subjected to standard doses of nocodazole (10 µg/ml) or paclitaxel (15 µM) for short periods of time (90 min) before subsequent exposure to much lower drug concentrations (1 µg/ml nocodazole and 1 µM paclitaxel) for an extended time (18-24 h) to allow the cells to reequilibrate. To ensure that these protocols led to the expected disruptions of the MT network without long-term influences on RhoA and Rac activity, untreated and drug-treated cells were fixed and stained to visualize the MT and actin networks. The long-term nocodazole treatment regimen led to a complete ablation of the MT network but appeared to have little effect on the ability of the cells to organize actin, suggesting that the influence on RhoA and Rac was negligible (Fig. 6). Similarly, the long-term paclitaxel treatment regimen clearly stabilized the MTs but had no apparent adverse effects on the actin network (Fig. 6).


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Fig. 6.   Disruption of MTs with prolonged low doses of nocodazole or paclitaxel. SMCs in static 6-well dishes were treated with either nocodazole (10 µg/ml) or paclitaxel (15 µM) for 90 min in serum-containing medium and subsequently serum-starved in the presence of a reduced drug concentration (1 µg/ml for nocodazole, 1 µM for paclitaxel) for 24 h. These drug treatments induced the expected alterations in MTs without adversely affecting the actin cytoskeleton. Assessment of cells treated with the vehicle used to solubulize these drugs (DMSO; control) verified the presence of normal MTs in the absence of either paclitaxel or nocodazole. MFs, microfilaments.

After these chronic drug treatment regimens, cells were subjected to a tensile strain. SMCs lacking a MT network did not demonstrate any significant change in the levels of membrane-associated RhoA in response to the strain (Fig. 7A). This finding is in contrast to the response of untreated cells, which show a significant redistribution of RhoA away from membrane pools in response to the same mechanical stimulus (Fig. 4A). Similarly, cells treated with nocodazole before the application of a 10% compressive strain did not significantly redistribute their pools of RhoA in response to the applied strain (Fig. 7B), in contrast to the response of untreated cells subjected to the same mechanical stimulus (Fig. 4B). Importantly, this regimen of drug treatment did not alter the baseline distribution of RhoA in the SMCs (compare t = 0 conditions in Fig. 7B with and without nocodazole). Interestingly, elimination of the MT network with nocodazole pretreatment amplified the reduction in membrane-associated Rac in response to a 10% tensile strain (Fig. 7C) compared with untreated cells (Fig. 5A). By contrast, pretreatment with nocodazole before application of a 10% compressive strain blocked the increase in membrane-associated Rac that was induced by this same mechanical stimulus in control SMCs (Fig. 7D). That long-term pretreatment of the SMCs with nocodazole did not disrupt the baseline distribution of Rac before the application of strain is further confirmed by the absence of any differences between the t = 0 time points for control and nocodazole-treated cells (Fig. 7D). In light of these results, mechanical strain-induced alterations in the subcellular distribution of both RhoA and Rac appear to be dependent on the presence of MTs in the cells.


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Fig. 7.   Strain-induced alterations in Rho and Rac distribution are eliminated by removal of the MT cytoskeleton. A: SMCs exposed to prolonged pretreatment with a low dose of nocodazole (1 µg/ml) were subjected to a 10% step change tensile strain. The amount of membrane-associated RhoA was unchanged compared with unstretched controls. B: similarly, nocodazole-stimulated SMCs subjected to a 10% step change compressive strain did not significantly alter the levels of RhoA in the particulate fraction in response to the applied strain compared with static controls. C: by contrast, a 10% step change tensile strain applied to nocodazole-pretreated cells reduced the amount of membrane-associated Rac to an even greater extent than that observed with untreated cells (compared with Fig. 4A). D: however, a 10% step change compressive strain did not significantly alter the levels of membrane-associated Rac compared with static controls, in contrast to the increase in the amount of membrane-targeted Rac observed after the same mechanical stimulus in untreated cells (Fig. 4B). Values represent means ± SD from a representative experiment. * Statistical significance (P < 0.05 compared with t = 0; n = 3).

To determine whether the mere presence of MTs is sufficient to permit a strain-induced redistribution of Rho GTPases, the same positive and negative step change experiments were performed with cells that had been pretreated with paclitaxel following the regimen described above. In the presence of a paclitaxel-stabilized MT network, strain-induced alterations in the localization of Rho were not evident (Fig. 8). Whereas control cells responded to the tensile strain by diminishing their level of membrane-associated RhoA (Fig. 4A), paclitaxel-treated cells did not show any significant changes in the levels of RhoA in the membrane fraction after exposure to strain (Fig. 8A). Likewise, the increased proportion of RhoA in the membrane-associated fraction in response to a compressive strain (Fig. 4B) was significantly reduced by treatment with paclitaxel before application of the strain (Fig. 8B). As for nocodazole, long-term pretreatment of the SMCs with paclitaxel before the application of strain did not result in any differences between the t = 0 time points for control and paclitaxel-treated cells (data not shown).


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Fig. 8.   Strain-induced alterations in membrane targeting of RhoA depend on dynamic MTs. A: SMCs exposed to prolonged pretreatment with a low dose of paclitaxel (1 µM) were subjected to a 10% step change tensile strain. No significant change in the levels of membrane-associated RhoA was observed compared with unstretched controls, in striking contrast to the statistically significant decrease in the amount of membrane-associated RhoA in untreated cells exposed to the same stimulus (Fig. 4A). B: similarly, paclitaxel-pretreated SMCs exposed to a 10% step compressive strain showed no significant change in the distribution of RhoA, in contrast to the significant increase in membrane-associated RhoA induced by the same mechanical stimulus in untreated cells. Values represent means ± SD (n = 3). No statistically significant differences were observed between t = 0 and t = 15 min conditions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These studies demonstrate that tensile and compressive strains applied to the substrate are capable of altering the subcellular distribution of RhoA and Rac in SMCs. The strain-induced targeting of these Rho GTPases to a membrane fraction was dependent on the presence of a dynamic MT cytoskeleton, supporting a linkage between MT assembly and Rho GTPase localization. Given the evidence that RhoA and Rac can translocate to membranes on GTP loading (7, 25), these data suggest that MTs may be required to spatially regulate the activity of these signaling proteins by controlling their distribution between subcellular compartments. Combined with our previous demonstrations (47, 48) that the state of MT polymerization in smooth muscle cells depends on the duration, direction, and amplitude of externally applied strains, these new data suggest that changes in MT assembly in response to externally applied strain regulate RhoA and Rac distribution.

Although previous studies support a role for the Rho family GTPases in response to mechanical forces (1, 32, 43), the findings described here explicitly reveal a direct link between these proteins and the MT cytoskeleton in the context of mechanotransduction. This convergence between MT assembly and Rho signaling in response to applied force parallels similar relationships between MTs and Rho GTPases described in studies using pharmacological agents (24, 39, 64). Depolymerization of MTs after transient exposure to nocodazole was shown previously to trigger activation of RhoA and a subsequent increase in contractility (24, 39). By analogy, a compressive (negative) strain applied to the substrate in our studies triggered net MT depolymerization and induced a redistribution of RhoA from cytoplasmic to membrane-associated pools, indicative of an increase in activity. Similarly, a tensile (positive) strain applied to the substrate, which induced net MT polymerization, reduced the amount of membrane-associated RhoA, indicative of a decrease in activity.

Changes in the distribution of the small GTPase Rac occurred in response to externally applied strain as well. Our finding that tensile stress triggers a decrease in Rac activity agrees well with recent data from the literature (32). However, the MT dependence of these alterations in Rac is not quite as clear as for RhoA. It was shown previously that MT growth activates Rac to promote lamellipodial protrusion in fibroblasts (64). Although MTs influence membrane targeting of Rac in our system, a direct correlation between strain-induced MT growth and membrane targeting of Rac is not supported by our results. Although drug-induced MT growth may trigger local Rac activation in lamellipodial ruffles, our findings suggest that MT assembly driven by a single application of global tensile strain in fact correlates with decreased Rac activation and association with the membrane. One possibility is that the GTP loading of Rac may require MT assembly at the leading edge of migrating cells, but MT assembly may not regulate Rac localization in the same fashion in response to mechanical strain applied to the entire cell. Regardless of this, in light of our findings, MTs clearly play an important role in permitting mechanical forces to influence both RhoA and Rac and may be important for regulating other cellular signaling events outside mechanotransduction.

Translocation of the Rho GTPases to a membrane-associated fraction has been used widely as an indirect measure of their activity (2, 7, 25, 43). However, it was shown recently that the active form of Rac is targeted to the membrane only when decoupled from a guanine nucleotide dissociation inhibitor, Rho-GDI (21, 22). Hence, although our use of a cell fractionation assay as an indirect measure of the activity of RhoA and Rac has its limitations, it appears to correlate well with activity based on parallel affinity-precipitation pull-down experiments (Fig. 5B). Regardless of this, the results from these studies clearly demonstrate that the application of single mechanical strain to cultured SMCs results in MT-dependent changes in the distribution of RhoA and Rac between soluble and membrane-associated compartments. Combined with a recent study demonstrating the capacity of a Rho exchange factor (GEF-H1) to bind to MTs (38), our findings suggest that MTs may spatially regulate the active forms of the Rho GTPases, possibly by controlling subcellular trafficking of these proteins themselves or some of their regulators. Future studies will require quantification of both the total cellular activity of Rho GTPases and the spatial regulation of that activity in cells subjected to an applied strain.

Interestingly, single applications of externally applied strain resulted in no significant changes in the levels of focal adhesion-associated vinculin. First, this finding indicates that changes in MT assembly and disassembly are not tied directly to stress-induced changes in focal adhesion vinculin content. Second, this finding suggests that the net changes in the distribution of RhoA and Rac do not drive large-scale changes in focal adhesion assembly in response to applied strain in this system. In light of these findings and our earlier work, we propose a model (Fig. 9) in which mechanical control of MT assembly regulates the local relative balance of RhoA vs. Rac activity by controlling their subcellular distributions. Maintaining an appropriate balance of RhoA vs. Rac activity may therefore provide a means to stabilize focal adhesions in the face of a single mechanical input, providing a stable bridge between the ECM and the cytoskeleton across which mechanical forces can travel and be transduced into biochemical signals inside the cell. In contrast to our findings, other reports have demonstrated that focal adhesions increase in size and number in response to an increase in contractility via a pathway that involves the activation of RhoA (16, 50). In addition, externally applied strain has been shown to trigger a number of integrin-mediated signaling events that can subsequently influence focal adhesion assembly (55, 57-59). One possible explanation for these apparently contradictory findings is that the SMCs used in these studies, despite being deprived of serum for 18-24 h before application of strain, never lose large, stable focal adhesion structures and actin stress fibers as has been noted with various types of 3T3 fibroblasts under similar conditions (16, 39, 50, 52). Two additional possibilities are that vinculin may not be found within a subset of focal adhesions that remodel in response to applied mechanical forces (69) or that vinculin is part of a subset of focal adhesion proteins that are not altered by stretch (54).


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Fig. 9.   Proposed model: regulation of MT assembly and disassembly via external mechanical strain influences membrane targeting of RhoA and Rac GTPases. The findings in this study suggest that regulation of MT assembly by mechanical strain is necessary for changes in the association of RhoA and Rac GTPases with a particulate membrane-associated fraction in SMCs subjected to a single mechanical input. Regulating the local relative activity of RhoA vs. Rac by controlling membrane targeting in response to the applied strain may represent a feedback mechanism by which focal adhesions are stabilized in the face of the mechanical signal. ECM, extracellular matrix.

Our earlier studies (47, 48) describe the dependence of MT assembly on the direction, duration, and magnitude of an applied stress and have recently been corroborated by other evidence in the literature (33). These findings support a model for the cytoskeleton in which MTs are partly responsible for supporting compressive loads (9, 30). According to this model, a tensile strain applied to the substrate presumably relieves compressive loads on the MTs and a net increase in polymerization occurs. By contrast, a compressive strain applied to the substrate would increase the compressive load on MTs, inducing their net depolymerization. We did not observe any reciprocal changes in the actin cytoskeleton (data not shown) or the structure of cell-ECM adhesions in response to tensile or compressive strain. However, the absence of actin/focal adhesion remodeling does not refute a tensegrity-based model for the cytoskeleton. Because MTs and cell-ECM interactions are presumably both involved in resisting tensional forces generated by the actin network in a tensegrity model (30, 47), the absence of focal adhesion remodeling in response to the external force may be due to a portion of the load being transferred from the ECM to the underlying MT network. This transfer of load then directly alters the assembly and disassembly of MTs, driving coordinated changes in RhoA and Rac activation to serve as means for cells to stabilize these other structures in the face of a changing mechanical environment.

Although the precise mechanisms by which MTs and Rho GTPases communicate, particularly in response to applied mechanical forces, remain unknown, several potential mechanisms for MT and Rho GTPase integration in cell motility were recently reviewed (68). One hypothesis is that MTs deliver a limiting signal or a factor to focal adhesion sites that allows for communication with the Rho GTPases. This hypothesis is further supported by evidence that MTs repeatedly target focal adhesions, possibly as a mechanism to deliver signals to relax local contractility as a cell migrates across its substrate (34, 35). Similar mechanisms may be at work in mechanotransduction as well. We speculate that alterations in MT assembly alone do not control the activity of Rho and Rac but may be required for the precise spatial regulation of that activity.

In summary, the results presented in these studies demonstrate that externally applied mechanical forces alter both MT assembly and the targeting of the small GTPases RhoA and Rac to a membrane-associated compartment in primary SMCs. Furthermore, membrane targeting of RhoA and Rac is dependent on changes in the state of MT assembly. Together these data suggest that stresses transferred between the ECM and cytoskeletal filaments alter biochemical signal transduction by modulating MT polymerization and support tensegrity models that predict stress-dependent control of cytoskeletal assembly.


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge financial support from the National Institutes of Health (NIH) (R01-DE-I3349 issued to D. J. Mooney). A. J. Putnam was supported by a fellowship from the NIH Organogenesis Training Grant (5T32-HD-07505-02) during the course of this work.


    FOOTNOTES

Present address of A. J. Putnam: Department of Chemical Engineering and Materials Science and Department of Biomedical Engineering, University of California, Irvine, CA 92697.

Address for reprint requests and other correspondence: D. J. Mooney, Dept. of Biologic and Materials Sciences, School of Dentistry, Univ. of Michigan, Ann Arbor, MI 48109-1078 (E-mail: mooneyd{at}umich.edu).

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.

First published October 30, 2002;10.1152/ajpcell.00137.2002

Received 25 March 2002; accepted in final form 28 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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