Focal adhesion elongation occurs along an actin bundle template

U2OS human osteosarcoma cells were transfected with GFP-actin and mApple-Paxillin and plated on fibronectin-coated traction force substrates compatible with high resolution imaging (Video S1). Consistent with previous reports [4], [7], nascent focal adhesions underwent elongation into mature focal adhesions concomitantly with the assembly of a radial stress fiber (Fig. 1a+b). During this process, a constant retrograde flow of actin persisted across the lamella at a rate of 0.3 µm/min (Fig. 1c), constituting a “slipping clutch” between lamellar actin and the focal adhesion plaque [5], [12], [13]. During these changes to focal adhesion size and actin organization, the traction stress exerted on the underlying substratum increased from background levels to nearly 100 Pa.

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Figure 1. Focal Adhesion Maturation in Wild Type Cells.

(a) Time-lapse images of a U2OS cell expressing GFP-actin and mApple-paxillin, with images of traction stress magnitude and overlaid traction stress vectors. Time is in min:sec. (b) Plot of focal adhesion length (black squares) and traction stress (blue circles) at the focal adhesion (FA) indicated in (a) as a function of time. (c) Region of a U2OS cell with overlaid actin retrograde flow vectors. (d) Histogram of FA lifetimes in U2OS cells. (e) Histogram of FA elongation rate, measured by the rate of change in the length during FA elongation. (f) Histogram of focal adhesion loading rate, measured by the rate of change of the traction stress during FA elongation.

https://doi.org/10.1371/journal.pone.0070652.g001

Time-lapse imaging of GFP-paxillin over 30–60 min enabled measurements of focal adhesion dynamics. Focal adhesions which elongated into mature focal adhesions persisted for an average of approximately 55 minutes before disassembling, characterizing a focal adhesion lifetime (Fig. 1d). The rate of FA growth during maturation, or its “elongation rate” was peaked around 0.15 µm/min (Fig. 1e). The rate at which tension increased at the focal adhesion plaque, or “loading rate”, was broadly peaked around a mean of 25 Pa/min (Fig. 1f). Thus, consistent with previous measurements we observed a correlation between tension and focal adhesion size during assembly [14], [15], which has previously been interpreted as evidence for focal adhesion mechanosensitivity [8].

Adhesion maturation persists when cellular tension is reduced by 75%

In order to assess the role of tension in the focal adhesion elongation process, we modulated myosin activity by incubating the cells with varying concentrations (1–10 µM) of the Rho-associated protein kinase (ROCK) inhibitor Y-27632 for 1 hour prior to fixing and staining for actin and paxillin. As expected, at high concentrations (10 µM) of Y-27632, lamellar actin bundles were completely abrogated and focal adhesions appeared as near diffraction-limited puncta near the cell edge (Fig. 2a+b). However, at low concentrations of the inhibitor (2 µM), transverse arcs and radial stress fibers terminating in elongated focal adhesions were still observed (Fig. 2a+b). As the concentration was increased to 5 µM, the appearance of radial stress fibers was markedly reduced but some elongated focal adhesions terminating in radial stress fibers still remained (Fig. 2a+b). Thus, elongated focal adhesions persisted at intermediate concentrations of Y-27632 (Fig. 2b).

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Figure 2. Focal Adhesion Morphology and Traction Forces under Treatment with ROCK inhibitor Y-27632.

(a) Images of F-actin visualized by fluorescent phalloidin (top row) and paxillin immunofluorescence (middle row) in U2OS cells treated with the indicated concentrations of the rho-kinase inhibitor, Y-27632. (b) Box plots of FA lengths in U2OS cells as a function of Y-27632 concentration (n = 101 (WT), 78 (1 µM), 134 (2 µM), 117 (5 µM), 124 (10 µM) FAs). (c) Representative GFP-actin images (top row) and traction stress maps (bottom row) and for U2OS cells treated with 2, 5 and 10 µM Y-27632. (d) Plot of the total traction force exerted by U2OS cells as a function of Y-27632 concentration (n = 23 (WT), 13 (1 µM), 8 (2 µM), 14 (5 µM), 6 (10 µM) cells).

https://doi.org/10.1371/journal.pone.0070652.g002

To assess the cellular tension at each of these drug concentrations, we summed the magnitude of the traction stress over the entire cell area to obtain the total traction force. Control cells exerted approximately 250 nN of traction force (Fig. 2c+d). Upon treatment with 1 µM Y-27632, the traction force was reduced by ∼50% and continued to decrease further as the concentration was increased (Fig. 2c+d). In 10 µM Y-27632, the traction force exerted was reduced to 25 nN, approximately 10% that of control cells and consistent with previously reported values of the myosin-independent force exerted by adherent cells (Fig. 2c+d) [16], [17]. Thus, despite reductions in cellular tension by 50–80% in intermediate concentrations of Y-27632 (Fig. 2b+c), elongated focal adhesions persisted.

Reduced myosin activity shortens FA lifetime but does not affect their growth rate

To assess changes in focal adhesion dynamics at intermediate levels of Y-27632 where elongated focal adhesions were observed despite significantly reduced cellular tension, we imaged GFP-actin and mApple-paxillin in drug-treated cells plated on traction force substrata (Video S2–S4). For cells treated with 2 and 5 µM Y-27632, focal adhesion assembly appeared remarkably similar to that of control cells. Nascent focal adhesion puncta appeared at the cell edge and elongated as traction stress increased (Fig. 3a+b). Surprisingly, the FA elongation rate was unaffected (Fig. 3c) despite a dramatic decrease in the loading rate (Fig. 3d). However, an enhanced disassembly rate (Fig. 3e) resulted in a significantly reduced focal adhesion lifetime (Fig. 3f).

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Figure 3. Focal Adhesion Maturation under Treatment with ROCK inhibitor Y-27632.

(a) Time-lapse images of GFP-actin and mApple-paxillin in U2OS cell treated with 2 µM Y-27632, with images of traction stress magnitude. Time is in min:sec. (b) Focal adhesion length (black squares) and traction stress (blue circles) at the focal adhesion indicated in (a) as a function of time. Focal adhesion assembly characteristics in varied Y-27632 concentrations: (c) FA elongation rate, (d) FA loading rate, (e) FA disassembly rate, (f) FA lifetime under indicated conditions (n = 10 FA for each condition for FA elongation, loading rate, disassembly rate, and lifetime). (g) Lamellar actin retrograde flow speeds in varied conditions (n = 2 cells per condition, 5 to 7 regions per cell). (h) Box plot of peak traction stress reached during focal adhesion lifetimes in cells treated with Y-27632. “Stable FA” indicates FA that had lifetime greater than 30 minutes, while “Unstable FA” indicates FA with lifetime less than 30 minutes.

https://doi.org/10.1371/journal.pone.0070652.g003

The constant focal adhesion elongation rate was surprising because of the dramatically reduced loading rate. If tension were an important parameter in focal adhesion elongation, we would have expected a corresponding decrease with reduced cellular tension. Two other roles of myosin activity that may persist at these intermediate tensions are myosin-driven retrograde flow and cross-linking. Indeed, we found that actin retrograde flow persisted at a constant rate in cells treated with 2 and 5 µM Y-27632 and was only abrogated at the highest concentration of 10 µM Y-27632 (Fig. 3g). Interestingly the speed of retrograde flow (0.3 µm/min) was approximately twice that of FA elongation (0.15 µm/min), suggesting that constant FA retrograde flow dynamics control the elongation rate of FA over a broad range of cellular tension.

At these reduced levels of myosin activity, focal adhesion stability was severely impacted, with lifetimes being reduced by nearly 50% (Fig. 3f). Interestingly, under partial myosin inhibition, we observed a fraction of adhesions which elongated but which were unstable, having a lifetime of less than 25 min. The maximal traction stress generated at these unstable focal adhesions remained low, approximately 60 Pa (1.8 nN) on average. By contrast, the population of stable FAs that elongated and then persisted for at least 30 minutes typically built up a tension of 100 Pa (3 nN) (Fig. 3h).

Over-expression of α-actinin or Dia1 stabilizes FAs under low tension

Our previous work showed that a stress fiber template is necessary for focal adhesion maturation (Oakes et al., 2012). In order to assess the role of radial stress fibers in stabilizing and elongating focal adhesions at low tensions, we chose to explore the roles of two proteins known to play important roles in the formation of RSFs: the actin cross-linker α-actinin 1, and the actin nucleator Dia1 [7], [9], [17]. Constructs of mApple α-actinin or constitutively active Dia1 were over-expressed in U2OS cells treated with 5 µM Y-27632, and traction forces and adhesion dynamics were obtained as previously described (Fig. 4a–d; Video S5+S6). For both constructs, cells exerted a total average force of between 50 to 75 nN (Fig. 4b), a level comparable with control cells treated with 5 µM Y-27632 (Fig. 2d).

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Figure 4. Focal Adhesion Stability is Rescued at Low Tension by Over-expression of α-actinin and Dia1.

(a) Representative GFP-paxillin image and traction stress map of a U2OS cell treated with 5 µM Y-27632 and over-expressing mApple-α-actinin. (b) Total traction force generated by cells treated with 5 µM Y-27632 and over-expressing either mApple-α-actinin or mCherry-Dia1 (n = 9 cells per condition). (c) Time-lapse GFP-paxillin and traction stress map images of a U2OS cell treated with 5 µM Y-27632 and over-expressing mApple-α-actinin. Time is in min:sec. (d) Focal adhesion length (black squares) and traction stress (blue circles) at the focal adhesion as a function of time for red oval indicated in (c). Focal adhesion assembly characteristics for cells treated with 5 µM Y-27532 and over-expressing either mApple-α-actinin or mCherry-CA-Dia1: (e) FA elongation rate, (f) FA loading rate, (g) FA disassembly rate, (h) FA lifetime and (i) Maximum FA stress attained by FAs (n = 12 FA for each condition for FA elongation, loading rate, disassembly rate, and lifetime).

https://doi.org/10.1371/journal.pone.0070652.g004

The reduced level of cellular tension resulted in a loading rate during focal adhesion growth of 5 Pa/min (Fig. 4f), comparable to control cells treated with 5 µM Y-27632 (Fig. 3c,d) but at a similar elongation rate to all previous conditions (Fig. 4e). Over-expression of either α-actinin or CA-Dia1, however, resulted in a reduced disassembly rate (Fig. 4g) and an increased focal adhesion lifetime (Fig. 4h) comparable to control cells at normal levels of intracellular tension (Fig. 3e,f). Thus, promotion of RSF assembly was sufficient to stabilize focal adhesions at low levels of intracellular tension (Fig. 4i). This result suggests that an important role of myosin II activity in the stabilization of focal adhesions is in its function to assemble RSFs at focal adhesion sites.

Cell culture

U2OS human osteosarcoma cells (ATCC) were cultured in McCoy's 5A media (Sigma) supplemented with 10% fetal bovine serum (HyClone), 2 mM L-glutamine (Gibco) and penicillin-streptomycin (Gibco). Cells were transfected with plasmid DNA constructs encoding for GFP-actin (gift of the Gary Borisy Lab, Northwestern University), mApple-paxillin and mApple-α-actinin, EGFP-paxillin [21], GFP-Dia1 (gift of the Henry Higgs lab) using the transfection reagent FuGENE 6 (Roche). Y-27632 was purchased from Calbiochem and used at concentrations of between 1 and 10 µM as indicated. Cells were plated for 24 hours and then incubated for 1 hour in Y-27632 at the concentration indicated for all experiments.

Polyacrylamide substrates for traction force microscopy

Polyacrylamide (PAA) substrates containing far-red 40 nm fluorescent microbeads (Invitrogen) were prepared on glass coverslips using previously published methods [22], [23]. Briefly, PAA gels with 7.5%/0.1% weight percentage of acrylamide/bis-acrylamide were used to create a gel with a shear elastic moduli of 2.8 kPa [22], [24]. Fibronectin (Millipore) was coupled to the surface of the PAA gels by means of hydrazine hydrate [15], [22], [25], as previously described. Briefly, PAA gels were incubated for at least 2 hours in undiluted hydrazine, followed by a 1 hour incubation in 5% acetic acid and then washed. A 10 µg/mL fibronectin solution was prepared in sodium acetate buffer (pH 4.5), and oxidized by addition of 40 µg/mL sodium meta-periodate prior to a 30 min incubation on the PAA gel at room temperature. The PAA gels were then rinsed repeatedly and plated with cells.

Microscopy

Live cell traction force measurements were performed on an inverted Nikon Ti-E microscope with a CSU-X confocal scanhead (Yokogawa), laser merge module containing 491, 561 and 642 laser lines (Spectral Applied Research) and an HQ2 cooled CCD camera (Roper Scientific). All hardware was controlled via Metamorph acquisition software (MDS Analytical Technologies). Traction force data was obtained at 37°C in a perfusion chamber (Warner Instruments) using a 60×1.2 NA Plan Apo WI objective (Nikon). Cells were maintained in culture media supplemented with 10 mM HEPES and 30 µl/ml Oxyrase (Oxyrase, Inc.).

Actin flow speed measurements

Cells were transfected with GFP-actin and plated on a 2.8 kPa PAA gel for live imaging. Images were collected at 30 second intervals for 30 minutes for each cell. Fiducial marks within the lamellar actin were identified and tracked over time using custom software developed by the Danuser Lab (Harvard University) as previously described [26]. Briefly, local intensity maxima were identified and tracked using time-integrated cross-correlations. Templates for the object size for the correlations were adaptively chosen as 1.6×1.6 µm. Approximately 1000–3000 flow vectors were generated for each image. Flow vectors were chosen from regions exhibiting coherent flow, and the means were calculated for each condition by averaging the magnitudes of the flow vectors from 5–7 suitable regions for each cell, for two cells per condition.

Displacement analysis and force reconstruction

Methods for traction force microscopy have been previously described [22], [23], [25]. Briefly, images of fluorescent beads embedded in the PAA gel were aligned to compensate for experimental drift and the bead displacement field was calculated between pairs of images by comparing the unstrained bead images obtained after the cell had been removed to images obtained with an attached cell. Displacement fields were calculated using Particle Imaging Velocimetry (PIV) software in MATLAB (available at http://www.oceanwave.jp/softwares/mpiv/), using the Minimum Quadratic Differences (MQD) algorithm which calculates the shift necessary to produce the minimum cross-correlation coefficient between a small region of the experiment image and the reference image. Displacement vectors were filtered and interpolated using the kriging interpolation method. We used a displacement grid size of 0.86 µm for these measurements. From the displacement data, Fourier transform traction cytometry (FTTC) [27] was then used to estimate traction stress and force at focal adhesions [23]. Traction stresses were reconstructed with zeroth-order regularization, which has been shown to yield traction force measurements consistent with the boundary element method [23]. Regularization parameters remained constant for all data sets directly compared.

The total traction force exerted by cells was computed by summing the magnitudes of the traction stress vectors under and near the cell of interest, subtracting the background value (taken from a region outside of the cell – typically around 20 Pa) and multiplying by the area covered by the chosen traction stress vectors.

Focal Adhesion Dynamics

Focal adhesion lengths and lifetimes were found by analyzing time-lapse images of focal adhesions by hand in order to measure the focal adhesion length as a function of time. Focal adhesion lifetimes were determined as the total amount of time a focal adhesion was present in the time-lapse movie. Focal adhesion elongation and disassembly rate were computed by dividing the change in focal adhesion length during assembly and disassembly. Forces at individual focal adhesions were measured by computing the stress at the focal adhesion by interpolating traction stress vectors using a Gaussian weight function and converting the peak stress measured in the adhesion into force by multiplying by the area of the stress footprint at the adhesion [25].