The actin crosslinking protein palladin modulates force generation and mechanosensitivity of tumor associated fibroblasts

Abstract

Cells organize actin filaments into higher-order structures by regulating the composition, distribution and concentration of actin crosslinkers. Palladin is an actin crosslinker found in the lamellar actin network and stress fibers, which are critical for mechanosensing of the environment. Palladin also serves as a molecular scaffold for α-actinin, another key actin crosslinker. By virtue of its close interactions with actomyosin structures in the cell, palladin may play an important role in cell mechanics. However, the role of palladin in cellular force generation and mechanosensing has not been studied. Here, we investigate the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. Traction force microscopy revealed that tumor-associated fibroblasts generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells and inhibited their ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in actin organization, adhesion dynamics and altered myosin organization in palladin knock-down cells. Our results suggest that actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

The actin crosslinking protein palladin modulates force generation and mechanosensitivity

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Mikheil Azatov, Silvia M. Goicoechea, Carol A. Otey & Arpita Upadhyaya,

and concentration of actin crosslinkers. Palladin is an actin crosslinker found in the lamellar actin

interactions with actomyosin structures in the cell, palladin may play an important role in cell studied. Here, we investigate the role of palladin in regulating the plasticity of the actin cytoskeleton down cells. Our results suggest that actin crosslinkers such as palladin and myosin motors coordinate

Many aspects of cell behavior are dependent on the physical properties of a cells environment1,2. Cell migration is susceptible to the mechanical properties of the environment such as substrate elasticity3. Stem cell dierentiation into dierent cell types is modulated by the elasticity of the microenvironment4. It is becoming increasingly clear that the mechanical interactions of cancer cells with their environment are essential components in tumor progression and metastasis5,6.

The molecular mechanisms that enable cells to sense and respond to the mechanical properties of their environment are being intensely studied7. The cytoskeleton and cell adhesions are key components that enable cells to sense their mechanical environment. Extensive work has shown that focal adhesions act as mechanosensors812.

In accord with this, the size, morphology and dynamics of focal adhesions depend on matrix stiness1315. The

coupling of focal adhesions to actin laments enables myosin motors to exert forces and transmit contractile tension to the substrate allowing the cell to sample the substrate stiness. Actin crosslinking proteins which link actin laments with developing adhesions and the extracellular matrix, and which organize actin laments into large-scale coherent structures are important for force generation8. However, their contribution to mechanotransduction is only now being understood1618.

Most mammalian cells express a diverse array of actin crosslinking proteins. The contribution of crosslinkers in organizing actin networks has been examined for crosslinkers such as -actinin and zyxin1923. -actinin is

involved in force transmission to the ECM via integrin binding21, while zyxin is important in maintenance of stress ber integrity under applied loads22. The actin-binding protein, palladin, occupies a unique molecular niche, functioning as a molecular scaold that directs the assembly and organization of actin networks24. Palladin directly binds actin filaments through its multiple Ig (Immunoglobulin-like) domains25, binds to the actin

Department of Cell Biology and Physiology and the Lineberger Institute

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Figure 1. Palladin associates with focal adhesions and modulates focal adhesion maturation. (A) TIRF images of EGFP-palladin cell spreading on glass showing palladin organization as it changes from a diuse localization into mature adhesions and stress bers. Scale bar: 15 m. (B) Le panel: Dual color image of a cell expressing EGFP-palladin (green) and mCherry-paxillin (red) showing focal adhesions and stress bers. Scale bar: 5 m. Right panel: Kymograph along the direction of growth of a focal adhesion (as indicated by white line in the le), showing accumulation of paxillin and palladin in a focal adhesion. Scale bars: 3 m horizontal, 2 min vertical. TIRF image of (C) EGFP-palladin cell and (D) Palladin knockdown (Palld4) cell transfected with mApple-paxillin (red) showing multiple focal adhesions along the cell periphery. Scale bar: 15 m. Plot of the mean uorescence intensity of a focal adhesion as a function of time during adhesion maturation in (E) an EGFP-palladin cell and (F) a palladin knockdown cell. (G) Beeswarm graphs showing comparison of the maturation times of focal adhesions in EGFP-palladin and Palld4 cells (p<0.0001, Wilcoxon ranksum test). (H) Comparison of focal adhesion length in EGFP-palladin and Palld4 cells (p < 0.01, Wilcoxon rank sum test). The box shows the interquartile range of the data, spanning from rst quartile to the third quartile of the data, red line indicates the median and whiskers denote 1.5x interquartile range.

crosslinker, -actinin, and colocalizes with -actinin along stress bers2628. In vitro assays show that palladin crosslinks actin into viscoelastic networks and synergistically combines with -actinin29. Palladin is up-regulated in pancreatic tumor-associated broblasts (TAFs) which have been shown to promote the progression of pancreatic tumors, metastasis, and resistance to therapy3032. Evidence suggests that the misregulation of actin reorganization resulting from altered palladin levels may contribute to aberrant cellular behavior. Given its localization in the cell, it is a likely candidate for force transmission. However, the role of palladin in focal adhesion maturation and actin organization for force transmission and cell response to ECM properties, such as stiness, is unclear.

Here, we use pancreatic TAFs to examine the role of palladin in actin organization, force generation and mechanosensing. As a model to study mechanosensing, TAFs are of particular interest because of their complex role in the assembly and dynamic remodeling of the tumor stroma33,34. We found that palladin plays a role in adhesion maturation, stress ber formation and actin ows, and has a signicant eect on cellular forces. Our experiments also suggest that palladin may have an eect on myosin activity and organization in cells. Taken together, our results demonstrate an important role for palladin in regulating cellular forces and mechanosensing.

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Methods

Cell culture, transfection and immunostaining. The palladin knockdown (KD) cell line (Palld4) in which palladin was silenced using shRNA sequence and the scrambled siRNA control (PGIPZ), were created as described previously35. Quantitative Western blots showed that palladin levels were reduced by 90% in the Palld4 line35. Wildtype, EGFP-palladin, KD and shRNA control cells were cultured in DMEM with 10% FBS, 1% PS and sodium pyruvate at 37 C. For spreading experiments, cells were plated at 15% conuence on bronectin (from bovine plasma, Sigma-Aldrich) coated glass coverslips. Coverslips were incubated with with 500l of 10 g/ml bronectin solution for 2 hours at room temperature. Imaging media L-15 (Life technologies, Grand Island, NY) was used for microscopy. For actin visualization, cells were xed using paraformaldehyde and stained with rhodamine-phalloidin. Transient transfections were done with mApple-paxillin, mCherry-actin, mCherry-MHC-IIA (myosin heavy chain) using Fugene HD tranfection reagent (Promega, Madison WI) and manufacturer protocol. For immunostaining, cells were xed with 4% paraformaldehyde (PFA) solution for 7minutes, washed with phosphate buered saline (PBS) and permeabilized with 0.2% solution of Triton-X for 2minutes. They were then washed with PBS and blocked (2% BSA in PBS) for 1h. Cells were incubated with primary antibody (Myosin IIa antibody), in blocking solution for 1h, washed in PBS and incubated in secondary antibody solution (Alexa Fluor 546 goat anti-rabbit, Invitrogen A11010) for 1h in the dark.

EGFP-palladin fragment was ligated into Z4-MSCV-mEos2-actin (a gi from Morgan Huse, Rockefeller University, New York, NY). Retroviruses were generated according to standard protocol36, with Phoenix Amphotropic cells and transduced into TAFs. The cells were then selected in 100g/ml zeocin for 2 weeks and sorted with uorescence-activated cell sorting to obtain uorescent cells. Palladin showed a 2X overexpression in EGFP-palladin cells compared to WT cells based on quantitative Western blot (data not shown).

Traction forces and preparation of PAA gels. For traction force experiments bronectin-coated polyacrylamide (PAA) gels with uorescent beads on the top layer were prepared as before37,38. The ratio of 40% acrylamide to 2% BIS (Bio-Rad, Hercules, CA) was varied (2:0.1, 3:0.1, 4:0.1, 5:0.1) to obtain gels of dierent stiness ranging from 1kPa60kPA. Glass coverslips were coated with 3-aminopropyl-trimethocysilane and glutaraldehyde (Sigma-Aldrich, St. Louis, MO) to allow the polymerizing gels to conjugate to the surface. A thin (5m) layer of gel with 200 nm diameter uorescent beads was attached to the top surface. 500 l of sulfo-SANPAH (ProteoChem, Loves Park, IL) solution was added to the top of the gel and incubated in the dark for 30 minutes. The sulfo-SANPAH was washed away with PBS and 500l of bronectin solution (10g/ml) was pipeted onto the gel and placed 2 inches below an 8 W UV lamp for 8minutes.

Aer obtaining images of multiple cells and corresponding beads on a gel, cells were detached by trypsinization to obtain a reference (or zero displacement) image for traction force analysis. Aer each imaging experiment, the gel height was determined using the microscopes z-focus mechanism and corrected for axial scaling. Typical gel heights were 6812m. The Youngs modulus of each gel was measured using the stainless steel ball indentation method39 rather than using a relation between gel stiness and BIS concentration since the measured modulus varied from gel to gel even with the same formulation.

Aer dri correction, the displacement of uorescent beads between two images (corresponding to the deformed and undeformed gel) was calculated using particle image velocimetry (PIV) (using the open-source MATLAB package MPIV, http://folk.uio.no/jks/matpiv/index2.html). The window size for the adaptive PIV algorithm ranged from 88 to 6464 pixels with an overlap of 50%, yielding a resolution of ~23m. Displacement vector maps were input into an unconstrained Fourier Transform Traction Cytometry (FTTC) algorithm implemented in MATLAB40 and extended to include nite thickness correction41. FTTC analysis was used to obtain the traction stress magnitude and vector maps. The total force exerted by the cell was calculated using F = T x y dxdy

( , ) , where T(x, y) is the stress at location x,y.

Live cell microscopy. Fluorescence and Interference Reection Microscopy images were collected at 37C using an inverted microscope (TE2000 PFS, Nikon, Melville, NY) with a cooled CCD camera (Coolsnap HQ2, Photometrics, Tucson, AZ). TIRF imaging was done using a 60x magnication,1.49 NA objective lens, a 491nm laser (100mW, Andor, South Windsor, CT) for EGFP excitation and a 561nm laser (75mW, Andor) for mApple and mCherry excitation.

For blebbistatin recovery experiments, cells were allowed to spread on bronectin-coated gels for 3hours and imaged. 15M blebbistatin was added to the imaging chamber and incubated for 30min. Blebbistatin was then washed out and replaced with imaging medium while cell recovery was monitored. Time-lapse imaging of cells and beads throughout the washout and recovery process enabled traction forces to be computed. Finally, cells were detached by trypsinization to obtain reference bead images.

Image Analysis. To quantify focal adhesions, polygons were drawn around maturing focal adhesions. Focal adhesion length was dened as the diagonal of the rectangle around a polygon. The focal adhesion maturation time was dened as the time taken for the mean uorescence intensity in the dening polygons to reach its maximum value from the onset of the rise. Radial Stress Fibers were marked by their location as in Oakes et al.20. RSF were identied by eye using rhodamine-phalloidin actin staining 4h aer spreading and dened as stress bers that are roughly perpendicular to the cell boundary.

Statistical analysis. Since many of the measured parameters were not Gaussian-distributed, we used the non-parametric Wilcoxons rank-sum test to determine the signicance with respect to WT. The resulting p-values are indicated on the gures and in the gure legends. Error bars denote SEM (standard error of the mean).

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Figure 2. Palladin knockdown impairs radial ber formation. (A) Snapshot of EGFP-palladin cell labeled with Rhodamine-phalloidin for actin showing strong radial bers (RSF). A typical RSF is indicated by the arrow. Scale bar: 15m. (B) Snapshot of EGFP-palladin cell showing localization of palladin in RSF. Scale bar: 15 m. (C) Merged image showing co-localization of actin and palladin in RSF. (D) Zoomed in image of the regionthe dotted boxed from (C) showing RSF in greater detail. (E) Snapshot of palladin KD cells (Palld4) labeled with Rhodamine-phalloidin showing a lack of radial stress bers in the cell. Scale bar: 15m. (F) Snapshot ofa control shRNA cell (PGIPZ) showing the presence of radial bers as indicated by the arrow for an example. Scale bar: 15m for all panels. (G) Bar graph showing comparison of the percentage of cells (EGFP-palladin, control sh-RNA (PGIPZ), and palladin KD (Palld4)) which displayed radial bers quantied at 4hours aer spreading initiation (N=30 for each condition).

Results

We rst examined the distribution of palladin in TAFs expressing green uorescent protein tagged palladin (EGFP-palladin) and visualized the cell morphology as it spread on bronectin-coated glass coverslips using total internal reection uorescence (TIRF) (Fig.1A). During early spreading, palladin appeared diusely in the contact zone or formed highly mobile puncta that assembled into nascent adhesions at the cell periphery. Aer 30 min of spreading, when the spread area was maximal, palladin puncta organized to form mature focal adhesions and palladin was recruited into assembling stress bers. Upon completion of spreading, palladin underwent a constant retrograde ow from the cell edge towards the interior along stress ber templates. Rhodamine-phalloidin staining to visualize lamentous actin (f-actin) simultaneously with EGFP-palladin showed that palladin colocalized with actin and formed punctate spots along stress bers, as observed before (Supplementary Fig. S1)24,28,42,43.

Mechanosensing is believed to arise from the tension-mediated maturation of focal adhesions1315,44. To exam

ine the eect of palladin expression on focal adhesion formation and dynamics, we constructed a cell line knocked down in palladin (Palld4) in which palladin expression levels were reduced by ~90% (see Methods)35. We used paxillin, a key component of adhesions in cells, as a focal adhesion marker. We transfected EGFP-palladin and Palld4 cells with mApple-paxillin and used dual-wavelength TIRF to visualize focal adhesion formation. Paxillin primarily localized to the tips of adhesions while palladin appeared in adhesions and stress bers (Fig.1B le). During early spreading, palladin localized to a thin region at the cell periphery, while paxillin appeared next to palladin towards the cell interior. Focal adhesions grew towards the interior as shown in the kymograph (Fig.1B right). To examine whether palladin expression aects focal adhesion maturation, we measured focal adhesion length and timescale of formation in EGFP-palladin and Palld4 cells (Fig.1C,D). The uorescence intensity of maturing adhesions rapidly increased as adhesions formed and grew (Fig.1E,F). Palladin knockdown resulted in shorter focal adhesions with a smaller maturation time (Fig.1G,H). This indicates a role for palladin in focal adhesion templating and growth.

We next examined whether palladin knockdown aected actin organization, specically stress ber formation. A wide variety of adhesive and contractile cells possess actin stress bers, which have been postulated to play an important role in the transmission of cellular forces45,46. Since focal adhesions are known to serve as templates for stress ber assembly19,20, we hypothesized that palladin is important for stress ber formation in TAFs. We allowed EGFP-palladin, Palld4 and cells with control sh-RNA (PGIPZ) to spread on FN coated coverslips, xed the cells aer 4hours of spreading, and stained with Rhodamine-phalloidin to visualize f-actin. EGFP-palladin cells displayed radial stress bers (RSF) enriched in both f-actin and EGFP-palladin (Fig.2AD), visible as bright structures roughly oriented radially with respect to the cell edge. Palld4 cells showed signicantly fewer RSF (Fig.2E). Further example images of both types of cells are shown in Supplementary Fig. S2.

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Figure 3. Tumor-associated broblasts are mechanosensitive. (A) Snapshot of an EGFP-palladin cell (green) on an elastic gel (in the 1030kPa stiness range) embedded with uorescent beads (red). Scale bar: 10m. (B) Snapshot of the traction stress map for the stresses generated by the same cell. Colors correspond to the stress values as indicated by the color scale. The contour of the cell is superimposed in black. (C) A map of the local traction force vectors superimposed on the cell contour. (D) Traction force per unit area for WT and EGFP-palladin cells on gels of dierent stiness ranges. (WT cells: grey: p<0.001 between so and intermediate; p<0.01 between intermediate and sti; Wilcoxon ranksum test) (EGFP-palladin cells: black: p<0.0001 between so and intermediate; p<0.0001 between intermediate and sti; Wilcoxon ranksum test). Numberof cells: N>20 from ~5 independent experiments for each condition (corresponding to a total of ~120 cells). Rhodamine-phalloidin staining of WT cell to visualize f-actin on (E) a so (2kPa) gel and (F) a stier (25kPa) gel. Scale bars: 10m.

Cells with control sh-RNA (PGIPZ) also displayed robust RSF formation (Fig.2F). We scored cells as having RSF if they had more than ve RSF. We found that a greater fraction of EGFP-Palladin cells have RSF as compared to palladin KD cells (Fig.2G), consistent with previous results with -actinin20,21. The average number of RSF per cell was measured to be 13.9 1.7 and 7.62.2 for EGFP-palladin and Palld4 respectively (N=30 cells of each type). We observed qualitatively that RSF have enhanced levels of EGFP-palladin uorescence, consistent with previous observations of increased F-actin in RSF20,21. These observations show that EGFP-palladin cells have a greater ability to form RSF conrming palladins role in RSF formation.

Actin crosslinkers have been implicated in the regulation of cellular forces1618. Our ndings that palladin is a regulator of focal adhesion maturation and RSF formation, and the postulated role of RSF in force transmission from cells to ECM via focal adhesions, led us to surmise that palladin may be critical for cellular force generation and mechanosensing. We therefore investigated palladins role in cellular forces.

We examined the response of TAFs to varying matrix stiness using traction force microscopy. Cells were allowed to spread on bronectin-coated polyacrylamide gels for 34 h and then imaged with wide-eld uorescence microscopy. Fluorescence images of EGFP-palladin cells in the green channel, or bright-eld images of wild-type TAF cells were obtained simultaneously with bead images in the red channel (Fig.3A). Traction forces were obtained from the displacement of beads on elastic gels deformed by cellular forces (see Methods for details). Figure3B shows a representative map of traction stresses generated by the cell in 3A. Typical peak tractions were on the order of a few hundred Pa, consistent with observations in other cells47. A representative vector map of traction forces superimposed on the cells uorescence image shows centripetal or inward directed tractions (Fig.3C). The majority of stress was exerted in the cell periphery with strong cell-substrate attachments.

To examine the eect of substrate stiness on cellular force generation, we modulated the gel stiness (from 460kPa) by varying the ratio of acrylamide to the crosslinker, bis, but maintaining the same FN concentration and measured the forces exerted by wild-type TAF and EGFP-palladin cells. Wild-type cells exerted higher forces on gels of increasing stiness, saturating at very high stiness, indicating mechanosensitivity over this range of substrate compliance (Fig.3D). We veried that EGFP-palladin expression did not aect the overall mechano-sensitivity of TAF cells. The traction forces exerted by EGFP-palladin cells were similar to those of WT cells and showed a similar increase with gel stiness (Fig.3D). Cell shape and actin organization were also modulated by the gel stiness. On the soest gels (Fig.3E shows an example of a cell on a 2kPa gel), the cell shape was rounded

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Figure 4. Palladin modulates cellular traction forces and mechanosensitivity. (A) Snapshot of a Palld4cell on elastic gel embedded with beads. Scale bar: 10m. (B) Snapshot of traction stress map with colorvalues corresponding to dierent stress values. Cell contour is superimposed in black. (C) Total traction force per unit area of Palld4 cells shown in the same plot with EGFP-palladin cells for comparison. (p<0.01 for 410kPa, p<0.001 for 1030kPa, Wilcoxon ranksum test). Each bar represents an average of data obtained from N=3040 cells from ~6 independent experiments per condition. (D) Bar graph for comparison between traction stresses exerted by dierent cell types on intermediate stiness (1030kPa) gels, showing that shRNA control cells (PGIPZ) exert similar stresses as WT and EGFP palladin cells. (p<0.001, Wilcoxon ranksum test for comparison between EGFP-palladin and Palld4 cells). Each bar represents an average of data obtained from N>20 cells from ~5 independent experiments. Rhodamine-phalloidin staining of a Palld4 cell on (E) a so (2kPa) gel and (F) a sti (25kPa) gel. Scale bar: 10m (G) Bar graphs comparing the spread areas of EGFP-palladin (black) and Palld4 (gray) cells as a function of gel stiness. (p<0.001, Wilcoxon ranksum test, N>20 cells in each bar for comparison between 14kPa and 410kPa, 1030kPa, 3060kPa gels; only rst comparison shown in graph; p<0.05, Wilcoxon ranksum test, N>20 cells for comparison between 1030kPa and 3060kPa for Palld4 cells).

with a smaller area, fewer actin stress bers and more disorganized actin structures. On stier gels, cells spread to a greater extent and formed robust stress bers (Fig.3F shows an example of a cell on a 25kPa gel). This qualitatively underscores the fact that actin organization is correlated with cellular force generation.

Palladin modulates cellular traction forces and mechanosensitivity. To examine how absence of palladin aects cell-generated forces, we imaged Palld4 cells expressing cytoplasmic GFP as a marker of stable transfection35 (Fig.4A). The traction stress map (Fig.4B), shows that Palld4 cells exert higher stresses than EGFP-palladin cells for a given stiness range, as indicated by higher stress values. We further found that Palld4 cells also exhibited sensitivity to substrate stiness as they generated larger forces on stier surfaces. However, the nature of this mechanosensitivity was dierent from EGFP-palladin and WT cells. The traction stresses exerted by Palld4 cells increased from so to intermediate stiness gels, but showed no further increase on the stiest gels. The average stress (force per unit area) exerted by Palld4 cells was signicantly higher (almost double) than those exerted by EGFP-palladin cells in the intermediate stiness range 1030 kPa (Fig.4C). These data indicate that Palld4 cells are more sensitive to substrate stiness in a lower range of stiness as they exert larger forces and the exerted force plateaus at a lower stiness than for control cells.

We conrmed that the eect of palladin knockdown was specic as control shRNA cells (PGIPZ) exerted similar forces as WT and EGFP-Palladin cells with no signicant dierence in the forces between these cells (Fig.4D). As with WT cells, we found that on very so gels cells spread less and lacked actin stress bers (Fig.4E shows an example Palld4 cell on 2kPa gel), while on stier gels (25kPa) cells had a larger spread area and numerous stress bers (Fig.4F shows an example Palld4 cell on a 25 kPa gel). The spread area of Palld4 cells (grey bars) was significantly smaller for the soest gels (14kPa) compared to all stier gels (410kPa, 1030kPa, 3060kPa), similar to the results with EGFP-palladin cells (black bars) (Fig.4G). However, for the dierent cell types, we measured similar areas in all the ranges of substrate stiness (ranging from 4kPa60kPa) where tractions were measured, and cells formed stress bers in this range of stiness. These results indicate that the dierence in traction stress between EGFP-palladin and Palld4 cells on so and intermediate stiness gels is not merely due to a dierence in spread area but rather due to a change in intrinsic force generation capacity of the cell.

Actomyosin networks are known to be important for cellular traction force generation [51]. Myosin localizes to stress bers in a striated pattern resembling sarcomeres in striated muscle, which suggests a role for myosin contractility in force transmission across the cell. To examine the relative localization of myosin and palladin, we transfected EGFP-palladin cells with

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Figure 5. Palladin and myosin localize in alternate bands across actin stress bers. (A) Wideeld uorescence image of EGFP-palladin cell labeled with mCherry-myosin-IIA showing localization of palladin (green) and myosin (red) in a spread cell. Scale bar: 15m. (B) Magnied image of the square highlighted in (A) showing alternating bands of palladin (green) and myosin (red) along actin stress bers. Scale bar: 5m.(C) Intensity prole of the line highlighted in (B) showing alternating intensity peaks of palladin (green, lower prole) and myosin (red, upper prole) uorescence. (D) Bar graph comparing the band spacing for palladin and myosin.

mCherry-myosin-IIA and obtained uorescence images of EGFP-palladin (green) and mCherry-myosin-IIA (red) (Fig.5A). Palladin localized in a striated pattern on stress bers (Fig.5B) similar to myosin. Intensity proles along stress bers showed that myosin and palladin localized to alternate bands (Fig.5C). For selected regions of stress bers, we calculated the correlation coefficient between the intensities of the two line proles. While it varied throughout the cell, in regions with dense stress bers the correlation coefficient was signicantly negative (C=0.39, p=0.02), indicating that palladin and myosin puncta are largely anti-correlated. For comparison, in areas away from stress bers, C=0. The band spacing, measured as the distance between peaks in the intensity proles, was 1.10.3m for myosin, similar to the spacing 1.210.05m, of palladin bands (Fig.5D). Cells on gels of intermediate stiness exhibited smaller band spacing in EGFP-Palladin cells compared to Palld4 cells (Supplementary Fig. S3.) These observations are consistent with previous studies showing the localization of myosin and -actinin in alternate bands across stress bers47, since -actinin and palladin co-localize. This periodic appearance and close proximity of myosin and palladin as well as changes in band spacing in Palld4 cells led us to hypothesize that palladin expression may have an eect on myosins ability to generate contractile stresses on actin networks.

To examine the role of palladin in the generation of contractile stresses, we dynamically perturbed myosin contractility using blebbistatin, a small molecule inhibitor of myosin II. Blebbistatin treatment results in loss of stress bers, focal adhesions and traction force generation in a reversible manner47. We used blebbistatin to inhibit myosin II activity and quantied the recovery of force upon removal of blebbistatin (see Methods for details). We used gels of the intermediate stiness range (1030 kPa) as this stiness yielded the greatest dierence in forces between Palld4 and EGFP-palladin cells. Upon treatment with blebbistatin, stress bers disassembled and cells drastically changed their shape, shrinking and leaving behind retraction bers as shown in a representative Palld4 cell (Fig.6A,B). Aer washout of blebbistatin, most cells recovered their shape (Fig.6C) and partially recovered their stress bers. Traction stresses dropped almost entirely upon incubation with blebbistatin for 30min (Fig.6D before and 6E aer inhibition) and then largely recovered 60 min aer washout (Fig.6F). As seen from the plots of force recovery versus time, a large fraction of the force recovered during the rst 20minutes aer blebbistatin washout (Fig.6G).

Subsequently, the forces recovered by palladin KD cells continued to increase, while those of EGFP-palladin cells plateaued. The absolute forces recovered by KD cells were larger than those by EGFP-palladin cells, as expected since KD cells exerted higher forces before inhibition. To quantify the relative values of forces recovered, we calculated the ratio of recovered force to the initial force (before blebbistatin treatment) for each cell to obtain the percentage recovery with respect to the initial force. While the reduced value of force aer blebbistatin treatment was higher for KD cells than for EGFP-palladin cells, the percentage drop in force for KD cells was greater (~15% of the original) than for EGFP-palladin cells (~30% of the original). Palladin KD cells showed a faster force recovery at early times (rst 20 min during which most of the force builds up) as compared to EGFP-palladin cells indicated by a larger slope (Fig.6H). We quantied the increase in force aer blebbistatin washout as the dierence (D) between the force 60min post-recovery and at the time of blebbistatin removal as a

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Figure 6. Palladin knockdown cells show more efficient recovery from Blebbistatin treatment. (A) DIC image of a palladin KD (Palld4) cell on a gel of intermediate stiness (1030kPa range). Scale bar: 10m. (B) DIC imageof the same cell as in A, 30minutes aer incubation in 15M blebbistatin. (C) DIC image of the cell 1hour aer washout from blebbistatin, showing recovery of cell morphology. (D) Traction force map of the cell in A showing robust generation of traction forces. (E) Traction force map of the cell in B, showing disappearance of traction forces upon blebbistatin addition. (F) Traction force map of the cell in C, showing recovery of traction forces 1hour aer Blebbistatin washout. (G) Total force as a function of time aer removal of blebbistatin for GFP-palladin (black) and Palld4 (grey) cells. Each data point is an average of forces from N=10 cells from independent experiments for each condition. The rst data point represents the initial (pre-blebbistatin) force. The graphs show the increase in cellular traction forces as the cell recovers from blebbistatin washout, subsequent to 30min incubation in Blebbistatin. (H) The percentage force (with respect to original forces) during recovery from blebbistatin aer washout plotted as a function of time for EGFP-palladin cells (black) and Palld4 cells (grey). (I) The percentage increase of stress aer washout of blebbistatin quantied as the dierence between force recovered 1hour aer washout, Frecov, and the force aer incubation in blebbistatin for 30minutes, Fblebb (with respect to the initial force prior to blebbistatin addition). The data represents an average for 2030 cells of each type. (p<0.05, Wilcoxon ranksum test).

percentage of the original force. The extent of force recovery was higher for Palld4 cells than EGFP-palladin cells, again indicating more efficient force recovery in Palld4 cells (Fig.6I). These results indicate that lower expression of palladin facilitates recovery from the eects of myosin inhibition and that lower palladin levels are correlated with higher force generation.

The coordination of actin dynamics and myosin II activity in lamellar and lamellipodial networks results in a continuous retrograde ow of actin, myosin, and other crosslinkers from the cell periphery towards the center48. Retrograde ow speed is determined by actin assembly and disassembly kinetics and myosin motor activity, and may be sensitively related to the forces generated49.

Previous studies have shown that traction stresses are correlated with retrograde ow speeds in lamellipodia50.

We examined the eect of palladin expression on retrograde ow in TAFs by quantifying the ow speed for EGFP-palladin and Palld4 cells spread on gels of dierent stiness. Cells were transfected with mCherry-myosin,

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Figure 7. Palladin involvement in retrograde ow. (A) Fluorescence image of a Palld4 cell expressing mCherry-myosin. Scale bar 10m. (B) Kymograph generated along the line drawn in A showing retrograde ow of myosin which appears as red linear streaks. (C) Comparison of the retrograde ow speed for EGFP-palladin and Palld4 cells for dierent gel stiness. Each bar represents the average of ~100200 tracked lines along kymographs similar to the one in B (p<0.01, t-test).

plated on bronectin-coated gels, and imaged for 30min to observe the centripetal ow of myosin and palladin (Fig.7A). Kymographs along radial lines parallel to the ow show uorescent streaks corresponding to the movement of myosin structures (Fig.7B). The slopes of these streaks yield the ow speed (Fig.7C). The retrograde ow speed was smaller for Palld4 cells than for EGFP-palladin cells for both stiness ranges examined. The retrograde ow rate also varied with substrate stiness. For both cell types, ow rates were higher on intermediate stiness gels, while the forces exerted on these gels were smaller. This suggests an inverse dependence between traction force and retrograde ow. This is in agreement with the previously reported biphasic dependence50 of retrograde ow on traction forces since our observed speeds (1530 nm/s) are in the higher ow phase of the biphasic dependence curve.

Discussion

Here we examined the role of the actin crosslinking protein, palladin, in cell mechanics. Our results show that palladin plays a critical role in cellular force generation and mechanosensing. Palladin is essential for the efficient formation of radial stress bers consistent with prior results in osteosarcoma cells20. We have previously shown that palladin knockdown cells have decreased Rac activity51. Conversely, increased Rac activity was shown to result in an increased number of radial stress bers52, which correlates with our result of increased RSF in EGFP-palladin cells (cells with higher Rac activity) as compared to cells lacking palladin. We also found that reduced expression of palladin aects focal adhesion maturation leading to smaller adhesions which turnover more rapidly. Importantly, we found that palladin knockdown increases the force generating capacity of cells, facilitates the rapid buildup of tension within the lamellar actin network but impairs the ability to sense substrate rigidity for sti gels. We found that cells had slower retrograde ow rates on stier surfaces and consistently that palladin KD cells exhibit slower ows as compared to EGFP-palladin cells. These indicate that slower ows are associated with greater traction forces implying that palladin enhances myosin-mediated actin ows on so substrates, which result in smaller traction forces. Overall, our ndings indicate that the relationship between local changes in cell response e.g. actin ow, focal adhesion dynamics and actin organization can enable the cell to sense and adapt globally to material parameters of the environment such as substrate stiness.

Our nding that palladin knockdown cells exert forces that are almost twice as large as those exerted by wild type cells is consistent with previous studies showing that -actinin knockdown also results in higher forces20,21.

We found that the loss of radial stress bers, decreased retrograde ow and altered focal adhesion lifetimes that accompany the loss of palladin, reduces the sensitivity of cells to sense substrate stiness, suggesting that palladin plays a role in cellular mechanosensing. To obtain deeper insight into the mechanisms involved, we dynamically inhibited myosin activity and examined the subsequent recovery of forces aer removal of inhibitor in cells with normal and reduced levels of palladin. Palladin knockdown cells showed a greater rate of force recovery,

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Palladin

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Figure 8. Model showing the putative role of palladin in stress ber assembly and force generation. (A) Schematic representation of the proposed model. For actin laments to move past each other, actin crosslinkers, here indicated as palladin, need to detach from the laments. (B) Prediction for the dependence of force on cross-linker density.

indicating that lower expression of palladin facilitates more efficient force generation by myosin. The modulation of traction stresses and kinetics of force recovery suggest that palladin expression may modulate the behavior of the actomyosin network in cells. Experiments have shown that knockdown of palladin is correlated with higher activation levels of Rho in cells (Goicoechea, unpublished). Since activated Rho is a positive regulator of myosin activity in cells, this may provide a potential link between palladin expression and myosin-based force generation.

Based on previous studies and our observations, we propose the following qualitative model of stress ber contraction and force generation. Typically, three types of stress bers are observed in adherent cells including TAFs ventral stress bers, which span the entire cell and lie along the base of the cell, radial stress bers which are attached at one end to focal adhesions, and transverse bers, with a sarcomeric structure, which are not attached to focal adhesions53,54. Ventral stress bers are attached to focal adhesions at each end and show a graded polarity of actin laments, and hence are most likely associated with contractile force generation in cells5356. For

bers with graded polarity to contract, they should be able to displace -actinin and palladin relative to each other and along the laments57. In a proposed model53,54, actin laments are able to contract because of the rapid association/dissociation rate of -actinin.

In our representation of the proposed model, (Fig.8A), the displacement of actin crosslinkers (e.g. palladin), which is modulated by their association/dissociation kinetics, is required for actin laments to contract. High concentrations of crosslinkers (such as those in WT or EGFP-Palladin cells) would restrict laments from sliding past each other and stien the stress ber, resulting in decreased contraction and force generation. Conversely, lower expression levels of cross-linkers (i.e. palladin knockdown cells) would lead to greater force generation as observed. Such a mechanism may enable cells to regulate force generation by adjusting the overall concentrations of actin crosslinkers. However, a very low concentration of crosslinkers is not optimal58, as in this situation laments can slide past each other without signicant exertion of forces. This model, therefore, predicts a biphasic dependence of contractility on crosslinker density (Fig.8B). The right branch of the curve represents the crosslinker levels in the WT or the EGFP-Palladin cells whereas the middle branch would represent the crosslinker levels in the Palld4 cells.

Active matter theory predicts the general sigmoidal form of the force v/s stiness relationship59. In this model, the relevant parameters are the force capacity of the cell, which likely corresponds to the expression level and activity of myosin motors and the internal stiness of the cellular cytoskeleton, relative to the substrate stiness. In this model, lowering the level of cross-linkers would correspond to a steeper transition to the saturating force. Thus, the dierence between WT and KD cells would be most apparent at intermediate stiness.

Actin crosslinkers and myosin may be present in an optimum concentration for proper force generation and mechanical response. Exertion of forces that are too large may hinder cells ability to discriminate between different mechanical properties of substrates, as forces need to be tuned closely to match the cell surroundings. In summary, the dierences in cellular contractility arising from palladin expression levels suggest that palladin is involved in many aspects of cell mechanics. Its interaction with myosin motors may serve as a foundation for traction force regulation. Understanding the molecular mechanisms underlying palladins involvement in cellular forces and mechanical sensing, in particular the role of its interactions with other actin crosslinkers such as alpha-actinin, will be a topic of our future studies.

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Acknowledgements

This work was supported by the National Science Foundation grants 1121710 to AU and 1121365 to CO. The authors thank Dr. King Lam Hui for help with construction of the EGFP-palladin cell line.

M.A., C.O. and A.U. designed the research; S.G. constructed the knockdown and contributed cell lines; M.A. performed the experiments and analyzed the data; M.A. and A.U. wrote the manuscript; C.O. reviewed the manuscript. All authors gave nal approval for publication.

Additional Information

Supplementary information accompanies this paper at http://www.nature.com/srep

Competing nancial interests: The authors declare no competing nancial interests.

How to cite this article: Azatov, M. et al. The actin crosslinking protein palladin modulates force generation and mechanosensitivity of tumor associated broblasts. Sci. Rep. 6, 28805; doi: 10.1038/srep28805 (2016).

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