1Department of Mechanical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, Cambridge 02139; and 2Department of Medicine, Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts 02115
Submitted 12 December 2003 ; accepted in final form 3 June 2004
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ABSTRACT |
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mechanotransduction; endothelial cell; paxillin; viscoelastic model
Fluid shear stress studies have used cell morphology, gene expression, and intracellular calcium concentration changes to show that cells respond differently to time-varying stress. Cells exposed to rapidly fluctuating shear stress environments, generated with turbulent flow, do not align with the direction of flow as do cells exposed to laminar fluid shear stress (10), whereas oscillating fluid shear stress with a low mean positive force does not induce the same gene expression as flow with a high mean positive force (15, 18, 44). Furthermore, ramped levels of laminar fluid shear stress result in graded nitric oxide (29) and intracellular calcium (38) responses. These studies show that the biological response of a cell to mechanical force depends on both the magnitude and time course of applied external force but present little information regarding the physical basis for these observations.
Focal adhesion site remodeling has also been used as a marker of mechanotransduction, which has the advantage of being rapid (occurring in minutes after stimulation) and site specific (14, 30, 37). Integrin signaling is critical in forming and developing focal adhesion sites as well as in recognizing external force by orchestrating mitogen-activated protein kinase activity and stress fiber formation (42). Both external and internal mechanical stress leads to focal adhesion reinforcement via the recruitment and binding of focal adhesion proteins (14). Src family kinase activation and tyrosine phosphorylation both have roles in early focal adhesion-associated mechanosensing (17). Tyrosine phosphorylation of focal adhesion kinase (FAK) assists in the recruitment and binding of focal adhesion proteins by regulating protein-protein interactions in proteins that contain the Src homology 2 (SH2) (32), whereas Src family kinase activation and interaction with FAK initiates tyrosine phosphorylation of paxillin and p130Cas, leading to Rac-dependent focal adhesion turnover and cell migration (12, 34).
Single cells respond to external forces with focal adhesion translocation and protein recruitment, as shown by fluid shear stress acting as a mechanotaxis stimulus to preferentially translocate focal adhesion sites in the direction of shear (30) and entire cell stretch resulting in increased focal adhesion protein recruitment (37). Cells also respond to locally applied mechanical loads, suggesting that individual focal adhesions sense local levels of force. Pico-Newton level forces applied to the apical cell surface result in focal adhesion protein recruitment to the point of load application (14). Conversely, concentrated apical surface loads on the order of 10 nN per focal adhesion site initiate complex formation and protein recruitment along the basal cell surface in regions local to the concentrated load (35). Understanding how cells transmit mechanical forces from the cell apex to basal adhesions is critical for further characterization of focal adhesion mechanosensing.
In the present study, we examine force magnitude and frequency thresholds for transducing local mechanical loads into biological signals through focal adhesion sites. Using a single pole magnetic trap, we delivered a highly controllable local shear force to the apical cell surface and quantified basal surface focal adhesion translocation. Focal adhesion translocation was monitored with GFP-paxillin, a focal adhesion protein that binds to the focal adhesion targeting (FAT) region of FAK. Translocation exhibited a dependence on both the magnitude and frequency of loading, as well as Src family kinase activity and tyrosine phosphorylation, suggesting that focal adhesion mechanotransduction depends on a balance between the local mechanical stress and specific biochemical signals.
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MATERIALS AND METHODS |
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On reaching 70% confluence, the BAEC were transiently transfected with the GFP-paxillin vector (gift of K. Yamada, National Institutes of Health, Bethesda, MD) using FuGene6 (Roche, Indianapolis, IN) with a 3:1 transfection reagent (µl)-to-DNA (µg) ratio. Western blot analysis with anti-paxillin (RDI, Flanders, NJ) and anti-GFP (Sigma, St. Louis, MO) monoclonals verified the expression of GFP-paxillin 24 h posttransfection. After incubating at 37°C for 24 h, the medium was exchanged for fresh DMEM, containing 10% FCS, antibiotics, and a suspension of 4.5-µm diameter fibronectin-coated magnetic beads (Dynabeads M-450; Dynal Biotech, Lake Success, NY). The final bead concentration was
1.6 x 106 beads/dish. BAEC were given a 60-min incubation period at 37°C to allow bead attachment to the cell surface. Src family kinase and tyrosine kinase inhibition was achieved by applying 10 µM PP2 (Calbiochem, La Jolla, CA) or 100 µM genistein (Calbiochem) during the 60 min magnetic bead attachment incubation period at 37°C.
Fluorescent microscopy. Cells were imaged at x60 magnification with an inverted light microscope (model IX-70; Olympus, Melville, NY) equipped with a water-immersion objective and temperature control plate and recorded with a digital camera (CoolSNAP; Roper Scientific MASD, San Diego, CA). Subconfluent cells expressing GFP-paxillin and binding a single magnetic bead were selected for experimentation. After locating a cell that met the experimental criteria, the magnetic trap was positioned 75 µm from the magnetic bead and lowered to the bead plane. A control sequence of fluorescent images was first obtained by imaging at t = 0, 1, 3, and 5 min. After the control sequence, an external forcing function was applied and the cells were again imaged at t = 1, 3, and 5 min. The control image at t = 5 min was used for the t = 0 min forcing sequence image. To ensure basal surface imaging, both the control and forcing function images were recorded as an image stack by scanning vertically 5 µm with 0.25-µm step sizes.
Image analysis.
The image that best captured the basal surface focal adhesion plane was chosen for analysis after deconvolving the image stacks (VayTek, Fairfield, IA). Custom image analysis algorithms for segmenting individual focal adhesion sites and tracking the corresponding translocation vectors were written for MATLAB (Math Works, Natick, MA). Translocation vectors were calculated with respect to the initial bead position, which typically did not displace more than 1 µm.
Magnetic trap force application.
The magnetic trap generated both a steady and nonreversing sinusoidal shear force of varying frequency. The steady load was applied at 0.90, 1.45, and 2.25 nN force levels, whereas the nonreversing sinusoidal forcing functions maintained a mean of 1.45 with a 2.25 nN maximum force and were applied at frequencies of 0.1, 1.0, 10, and 50 Hz. The magnetic trap force levels were calibrated by suspending the experimental magnetic beads in dimethylpolysiloxane (12 M; Sigma) and monitoring the local bead velocity as the bead moved toward the magnetic trap. With the use of Stokes Law for viscous drag force (F) around a sphere, F = 6 µrV, where µ is the fluid viscosity, r is the sphere radius, and V is the sphere velocity, the magnetic trap force generation was calibrated. For more information regarding the magnetic trap design and use refer to Huang et al. (25).
Computational simulations.
Our viscoelastic, finite element model of the cell was extended to incorporate variable basal surface contact conditions mimicking the typical focal adhesion plane topology observed in our experiments. The simulation assumed a continuum, incompressible, homogeneous, isotropic Maxwell viscoelastic material with a shear modulus (G) of 100 Pa and viscosity (µ) of 100 Pa·s (characteristic time constant, = 1 s) (28). A continuum approach effectively models large-scale cellular deformation distributions that exceed the length scales of individual microstructural cytoskeleton constituents without specifically assigning material properties for actin filaments and microtubules or accounting for the inhomogeneity observed by others (24). This continuum-like behavior has been verified experimentally by Karcher et al. (28). Geometrically the cell was modeled as a 20-µm radius half cylinder, either 5 or 3 µm high. A ramp force reaching 1.125 nN, half of the 2.25 nN experimental load, was applied to the apical cell surface over a period of 0.2 s. Initial simulations used a 5-µm height and constrained the entire basal cell surface in all three translational directions. Subsequent simulations were modified to better represent experimental adhesion conditions by correlating specific sets of model nodes on the basal cell surface to experimental regions of focal contact, fixing the focal adhesion site-associated nodes, and reducing the model height to 3 µm. The remaining basal surface nodes were free to translate. For simulation purposes, it was assumed that focal adhesion sites undergo negligible translocation during the 0.2-s simulation.
Statistical analysis. All data reported were collected from at least three separate experiments and reported as means ± SE. Differences among experimental parameters were assessed by using one- and two-way ANOVA. Post hoc paired comparisons were also performed with the Bonferroni test (Prism 4.0; GraphPad Software, San Diego, CA). Differences were accepted as significant for P values < 0.05. Absence of a comparison bar in data figures indicates a significant data comparison with baseline.
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RESULTS |
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Translocation values for the control group were measured from the same cells used in forcing experiments before applying the external load. Each cell contained a single fibronectin-coated magnetic bead bound to its apical surface. The translocation magnitudes calculated for this control group were compared with the force-induced translocations. A second control group tested the potential effects of the magnetic field and local temperature elevation on focal adhesion dynamics. These control cells had single fibronectin-coated polystyrene beads bound to their apical cell surface to maintain consistency with the unforced control group. Comparison of the unperturbed and magnetic field exposed control cells confirmed that the magnetic trap had negligible effects on focal adhesion translocation (Fig. 3).
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Comparison of local and global translocation magnitudes, as defined previously by a 7.5-µm radius, revealed that none of the sinusoidal forcing functions, regardless of frequency, resulted in local region translocations that exceeded global translocations. Furthermore, the local translocation values resulting from the steady 1.45-nN load significantly exceeded the local values for 1.0 and 0.1 Hz loading (Fig. 7). The global translocations associated with 10 and 50 Hz forcing functions were comparable in magnitude to the steady load global translocation, whereas the 0.1 Hz forcing function produced slightly smaller translocations. Translocations associated with 1.0 Hz forcing were not significantly different than baseline translocations. Comparing the high frequency (10 and 50 Hz) forcing results to the steady load results suggests that cells transmit and transduce oscillating forces differently compared with steady forces.
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After 5 min of loading, tyrosine kinase inhibition differentially affected focal adhesion translocation depending on the inhibitor, forcing frequency, and focal adhesion site location with respect to the point load. Genistein reduced both the local and global response to steady loading and the local response to high frequency loading compared with unforced baseline levels of translocation (Fig. 8, A and B). PP2, on the other hand, effectively impaired translocation of global focal adhesion sites without inhibiting local focal adhesion sites from translocating significantly greater than baseline. The global translocation after PP2 inhibition, observed for both high frequency and steady loading, was significantly less than the local translocation levels (Fig. 8, C and D). The altered levels of focal adhesion translocation resulting from tyrosine kinase and specific Src family kinase inhibition imply that force-induced focal adhesion translocation depends on force-activated kinase activity and tyrosine phosphorylation, as well as local stress distributions.
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DISCUSSION |
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The threshold reported in this study is comparable to the total integrated shear force required to activate endothelial cells with fluid shear stress. Mechanotransduction studies with fluid shear stress typically apply stresses on the order of 110 dynes/cm2 (0.11.0 Pa) and observe changes in intracellular calcium and nitric oxide levels (22, 27, 29, 38), ion channel activity (2, 33), G protein activation (20), gene expression (15), and focal adhesion translocation (30). This level of stress, applied over a typical endothelial cell area on the order of 1,000 µm2, results in nN-level shear forces. Similarly, nN-level lifting forces applied normal to the apical cell surface via multiple integrin linked magnetic beads induces F-actin accumulation and tyrosine phosphorylation, increasing local membrane stiffness at the bead-membrane interface as measured with atomic force microscopy (19).
Fluid shear stress studies (38) that monitor intracellular calcium concentration changes report a stress threshold of 0.1 dynes/cm2 for cell activation or roughly 10 pN per cell with a graded response up to 4.0 dynes/cm2. Our results differ in both the force-level threshold and graded response findings. Focal adhesion translocation and calcium signaling likely result from two different mechanically activated biological pathways (7) and might therefore have two different activation thresholds. Furthermore, by using single bead magnetic trap loading similar to this focal adhesion translocation study did not induce a significant change in intracellular calcium compared with unforced control cells (unpublished data). These findings are consistent with previous studies indicating that calcium signaling is not an essential component to focal adhesion dynamics (13) and with data from Sawada and Sheetz (37) showing that substrate strain activates focal adhesion formation in Triton-X cytoskeletons, even in the absence of cell membranes, cytosolic-free ions, and other intracellular biomolecules.
The increased focal adhesion translocation observed at a steady load of 1.45 nN or greater did not correlate with the direction of force application after 5 min. This finding is consistent with the study of Li et al. (30), who found random focal adhesion movement during the first 10 min of fluid shear stress application. Only after 1 h did endothelial cells subjected to fluid shear stress begin to migrate in the direction of shear force application. Furthermore, it has been shown that endothelial cells align, and actin filaments orient, with the direction of shear stress within hours of the onset of stress (3, 11). These observations suggest that the random direction of elevated focal adhesion translocation observed after 5 min of loading resulted from an unaligned actin filament organization characteristic of short loading times.
An insignificant change in focal adhesion area was observed after applying a steady mechanical load via fibronectin-coated beads, which would have presumably corresponded to the recruitment of focal adhesion proteins. Other studies have shown that external forces, applied locally with a micropipette tip (35) and optical trap (14) or globally with fluid shear stress (30) and substrate strain (37), as well as internal myosin II-driven contractile forces, modulate focal adhesion protein recruitment, which leads to focal adhesion growth (16, 45). The estimated forces required for basal surface focal adhesion growth, however, range from 3 (14) to 10 nN (35) per focal adhesion site in experiments with fibroblasts. Cells in the present study contained at least 10 focal adhesion sites per cell, which would correspond to forces per focal adhesion site one to two orders of magnitude smaller than the forces reported to induce focal adhesion recruitment and growth. These force-level comparisons suggest that focal adhesion translocation is activated with externally applied forces at significantly lower levels than required for focal adhesion reinforcement.
The details of shear force transmission from the apical cell surface to the basal surface and mechanotransduction through focal adhesions are not fully understood. It is clear, however, that externally applied forces act directly on fibronectin-bound integrins and transmit through the cytoskeleton to analogous basal surface attachments. The observed distribution of these forces among the numerous focal adhesions is quite complex and can apparently give rise to locally elevated stress levels even at locations relatively far removed from the site of forcing (24). In contrast, a continuum, viscoelastic description of the cell predicts that force levels decay with distance from a point load. Finite element analysis of magnetocytometry estimates a radial decay of force transmission through the cell to the basal surface for continuous basal surface constraints (28). The simulations performed for this study, with constrained basal surface regions corresponding to experimental focal adhesion sites, estimated that shear forces transmit nonuniformly to basal surface focal adhesion sites. The local focal adhesion sites with concentrated shear stress were shown experimentally to translocate significantly greater compared with global regions under steady loading conditions. Viscoelastic characterization of cellular force transmission would interpret this result as a direct consequence of focal adhesion sites sensing local levels of stress. However, focal adhesion sites in the global region still experienced translocations significantly greater in forced cells compared with nonforced cells, suggesting that soluble mechanosensing biomolecules, such as tyrosine kinases, direct focal adhesion dynamics in peripheral cell regions.
Observations that local cell stiffness increases within minutes of applying nN-level magnetic bead forces to integrin linkages (4, 19) and Pascal-level fluid shear stress (5), provide a possible biological response mechanism for local force concentration. The formation of stress fibers could increase the local cell stiffness, elevate local force transmission, and lead to more force being supported by focal adhesion sites close to the magnetic bead. Visualization of GFP-actin, however, did not show an observable increase in local stress fiber formation after 5 min of loading (unpublished data). Consequently, the elevated focal adhesion translocation in local regions most likely did not result from the rapid formation of local stress fibers and rapid cell stiffening in response to a concentrated external force.
Focal adhesion sites appear to function as individual mechanosensors responding to local levels of external force, as supported by comparing local and global region translocation results. Other investigators report a similar sensing capability of focal adhesions in response to locally applied external forces (14, 35) as well as in response to local mechanical substrate properties (45). Internally generated actin-myosin II contraction also leads to focal adhesion activation (14, 16, 45). Two theories attempt to explain how focal adhesion sites sense local levels of force. The first recognizes that external forces locally perturb structural elements, making new binding partners more readily available. For example, tension induced integrin density increases at focal adhesion sites in coordination with focal adhesion site development and growth (1, 42). The second theory hypothesizes that external forces applied directly to structural proteins alter the protein conformational state, which transform the protein from an inactive state to an active state by exposing new binding sites (16). Both theories suggest an altered state of molecular-level equilibrium induces protein binding changes and subsequently initiates a cascade of local biological responses.
Frequency dependence of focal adhesion translocation. The experiments performed with 0.1, 1.0, 10, and 50 Hz sine wave forcing functions oscillated around a mean positive force of 1.45 nN and exhibited a biphasic cellular response with a minimum at 1.0 Hz. Frequencies greater than the maximum level were limited by the magnetic trap control capabilities. Others have reported inhibited responses of cells exposed to a near-physiological 1.0 Hz oscillating force with a positive mean compared with steady force responses (9). This frequency dependence offers a more complex view of the cellular response to external forces. For the oscillatory forcing functions, the net time during which force levels exceeded the mechanotransduction threshold did not vary with frequency nor did the minimum and maximum force levels. However, focal adhesion translocation varied with respect to the loading rate, implying that the shear force transmission distributions at the basal surface and mechanosensing signal pathways depend on the frequency of loading.
Force transmission pathways have different relaxation time constants depending on the properties of individual structural elements, and as a result, different relative strains with frequency variations. Consider the simple viscoelastic example of a parallel, two branch system where the first branch contains a single spring and the second a spring and a dashpot in series, and acknowledge extension continuity between the two branches. In the present case of constant force amplitude, extension of the isolated spring will be greatest under a steady load or at low frequencies and smallest at high frequencies. Conversely, the extension experienced by the spring in series with the dashpot will be smallest under a steady load and will increase to a constant, nonzero value at high frequencies. The transition between branch responses depends on the characteristic relaxation time of the model. If each of the two springs represents a different force transducing element within the cell, and if the system is excited near the threshold level of extension for both of these elements, then the character of the response could vary in the manner observed in our experiments. In physical terms, the two branches of this network might be on the scale of the entire cell (e.g., the cytoskeleton and the cell membrane) or on the scale of individual molecules. Others have accounted for the differential response of cellular components by modeling the viscoelastic cellular response with multiple base units in series and parallel, where each unit consists of a single spring and a spring-dashpot branch. These models show force transmission increases in branches as stiffness increases and that overall deformation decreases with increasing forcing frequency (31). The biphasic response to sinusoidal forcing functions of varying frequency may have also resulted from a combination of viscoelastic force transmission and tyrosine kinase activity, which has been shown to affect focal adhesion protein recruitment and site turnover (17, 25, 34). Although local force-induced structural changes mediated by talin 1 binding occur independently of tyrosine phosphorylation, the role of tyrosine kinases in focal adhesion mechanotransduction is not fully understood and could be partially responsible for the biphasic response observed with variable forcing frequency.
Tyrosine kinase inhibition differentially affects focal adhesion mechanotransduction. To investigate the parallel effects on focal adhesion translocation of direct force transmission and kinase signaling, tyrosine kinase activity was pharmacologically inhibited during steady and high frequency loading. Inhibition had both local and global effects that varied with the forcing function (see Fig. 8).
Applying genistein, an ATP-competitive tyrosine kinase inhibitor, impaired force-induced focal adhesion translocation both locally and globally in response to steady force application and local translocation in response to high frequency (50 Hz) loading. Although others have shown that genistein selectively impairs directed endothelial cell migration under fluid shear stress depending on the temporal variation in shear stress delivery (23), the precise effects of genistein on focal adhesion mechanotransduction remain unknown. Results from tyrosine kinase inhibition with genistein, however, do suggest that tyrosine kinase activity and tyrosine phosphorylation can differentially affect focal adhesion translocation with respect to the loading frequency.
PP2 is a more specific protein kinase inhibitor compared with genistein that inhibits Src family kinase activation and phosphorylation of FAK (36), a known upstream pathway of Rac-dependent cell migration (34). Application of PP2 reduced global focal adhesion translocation to baseline during both steady and high frequency loading but did not adversely affect local force-induced translocation. PP2 has previously been shown to affect cell motility by blocking biochemically induced focal adhesion translocation (40). Furthermore, it is known that Src family kinase recruitment to FAK leads to paxillin phosphorylation and cell migration (21). Therefore, the observation that PP2 impairs global focal adhesion translocation in response to steady and high frequency loading suggests that inhibition of Src family kinase activity impairs global signaling pathways that are otherwise initiated by local force application to integrin receptors. Furthermore, in local regions of high basal shear stress where PP2 inhibition did not significantly affect translocation, the elevated focal adhesion translocation may have resulted directly from force transmission and independently of Src family kinase activity.
In summary, focal adhesion translocation resulting from mechanical stimulation has a threshold between 0.90 and 1.45 nN. The cellular response to external force depends both on the magnitude and frequency of force application. Steady load application promotes elevated local force transmission and focal adhesion translocation. At frequencies greater than the 1.0 Hz, however, loading induces focal adhesion translocation of similar magnitude in both local and global regions. Furthermore, the mechanotransduction differences between loading patterns may depend on a balanced response to mechanical force transmission and tyrosine kinase signaling. Overall these findings suggest that focal adhesion sites function as individual mechanosensors responding to local levels of force and further emphasize the intricate balance of force transmission and biochemical signaling events involved with focal adhesion mechanotransduction.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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