Mechanosensor in integrin signaling: The emerging role of EGFR

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1 Mechanosensor in integrin signaling: Cynthia Hajal, Columbia University The emerging role of EGFR Professor James C. Hone, Mentor Shuaimin Liu, Columbia University Abstract Although the epidermal growth factor receptor (EGFR) is known to interact with integrins in the processes of cellular spreading and motility, little is known about the actual role of EGFR. Previous studies from our laboratory have shown that in the early interactions of cells with rigid Arg-Gly-Asp (RGD) ligands, EGFR activity is needed for normal cell spreading and for the assembly of local contraction units that sense rigidity. EGFR inhibitors blocked local contractions and normal spreading in media lacking serum and soluble EGF. Here, we test the hypothesis that EGFR is mechanically activated by substrate stiffness by selectively modulating stiffness of submicron pillar arrays and we analyze the correlation between local contraction force and EGFR phosphorylation. Introduction An experimentally fast approach to assess how modifications in the substrate stiffness affect cells relies on the use of elastomeric submicron pillars. These submicron structures (500 nm in diameter, 1 to 5 aspect ratio of diameter to height) are used as a cellular substrate to test the mechanical force exerted by cells. Given their ability to mimic a continuous substrate on which cells usually grow in tissue environment, submicron pillars allow scientists to study how cells respond to the stiffness of the extracellular matrix (ECM) in vivo. Previous studies from our lab reveal that cells sense substrate rigidity through local contractile units by force analysis on submicron pillars 1. The ability to measure these contraction forces is fundamental to understand the mechanism of how cells sense the substrate stiffness and respond to it at a molecular level and nanometer scale. In fact, mechanisms of force production with respect to various stiffnesses are different on submicron and micron pillars. While submicron pillars have a constant displacement of ~60 nm regardless of the pillar stiffness, micron pillars are moved by a constant force, leading to different pillar displacements as their stiffness varies 1. Hence, the mechanism cells use to probe rigidity by pulling pillars to a constant distance is established. These local contractions of constant displacement act on pairs of pillars by pulling them towards each other and the elements responsible for these forces are thus described as contractile pairs or units 2. Micron and submicron pillars have distinct force patterns: while micron pillars have balanced, inward centripetal forces located at the periphery of the cell, submicron pillars are deflected towards one another from the presence of contractile units bridging them together (Fig. 1). Contractile units are located in the lamellipodium area of the cell and are usually between 2 and 4µm in length. They constitute about 30 percent of the forces displacing these submicron pillars but they are not involved in the displacement of micron pillars since they occur on their edges. Delving deeper into the force parameters involved in these different mechanisms and patterns, studies in Michael Sheetz s lab with assistance of super resolution microscopy technique have shown that the local contractile force is generated by a sarcomere-like complex comprised by myosin, α-actinin, Tm and tropomodulin3 2. On the other hand, cells on micron pillars have adhesion complexes forming on the edges of the pillar tops which can be attributed to rearward actin flow. The differences in the parameters involved in these forces are responsible for the different patterns observed in submicron and micron pillars. 1

2 is shown to be co-localized with phosphorylated EGFR (pegfr). Experimental Methods Double Stiffness Submicron Pillar Fabrication Figure 1. Cell plated on a submicron pillar substrate with force vectors shown around its active leading edge. The yellow force vectors refer to all the forces displacing the pillars which are not contractile units. Contractile pairs are shown as red vectors and pull the pillars towards each other. Silicon structures with holes of 0.5µm in diameter were used as molds for the fabrication of elastomer submicron pillars using PDMS. The PDMS was formed by mixing its base with the curing agent (Slygard 184; Dow Corning) with a ratio of 10:1 in order to achieve a Young s modulus of ~ 2MPa. The equation below was used to calculate pillar bending stiffness based on Euler-Bernouilli theory: EGFR has long been known as a hallmark for cancer and the ligand binding activation of EGFR is well understood. However, more and more evidence shows that EGFR can be activated ligand-free. It is known that certain cancer cells, compared to normal ones, are able to ignore signals from the matrix and grow under anchorage-independent conditions. Since their rigidity sensing ability is hampered, we were interested in understanding whether EGFR plays any role in a cell s rigidity sensing machinery. Previous studies suggest that EGFR can be mechanically activated at the cell-cell contact 3. We delved deeper using pillar substrates and force analysis to show that EGFR is associated with local contractility and its inhibition led to abnormal cell spreading and less contractility force. Furthermore, by selectively modifying the stiffness of pillars, we observed a correlation between substrate stiffness and EGFR phosphorylation. To mimic differences in cellular substrate rigidities, double stiffness pillars with different Young modulus values were used to observe cells straddled between the stiff and soft pillar regions. Finally, paxillin, a protein responsible for cellular adhesions to extracellular matrices, where D, L and E are the diameter, length and Young modulus of the pillar, respectively. Using this equation, the 1.8µm-high and 1.3µmhigh pillars, before UV treatment, had a stiffness of 3.2 nn/µm and 8.4 nn/µm respectively. The PDMS was then centrifuged at a speed of 3000 RPM and a temperature of 24ºC for 3 minutes. To be able to observe differences in the soft and stiff pillars, Coumarin 343 (393029; Sigma Aldrich), a dye sensitive to light, was mixed into the PDMS. For 2g of PDMS, 40 µl of dye were used and this procedure was done under minimum bright lighting to avoid damaging the dye. The final product was placed in the vacuum chamber for ~ 10 minutes. A droplet of the mixed PDMS and dye was placed on each silicon mold and the molds were vacuumed again for ~ 10 minutes to ensure a uniform pillar size. The samples were then placed on a spinner for 9 seconds at 2800 RPM with an acceleration level of The PDMS mixed with dye was cured at 65ºC for 13 hours. After the samples had cooled down, the pillars were demoded carefully in air and were placed upside down in individual small Petri dishes 2

3 previously cleaned in the O2 plasma machine for 1 minute. The pillar arrays obtained had a center-to-center spacing set to 1µm, twice the pillar diameter to maintain a pillar density favorable for cell growth 1, Mesh Annealed square masks with a pitch of 62µm (hole of 40µm and bar of 22µm) were then placed on the appropriate pillar areas and the dishes were shined with UV lighting for an hour and a half in order to increase the pillar stiffness of exposed regions 12 fold. The samples were then ready to be plated with cells. Cell Culture and Plating To promote cellular adhesion, the pillar substrates were coated with fibronectin (50µg/L; Roche). Mouse Embryonic Fibroblasts (MEFs) were cultured in Dulbecco s Modified Eagle Medium (DMEM, 1X; Gibco) and phosphate buffered saline (PBS, 1X; Corning cellgro) and then incubated for 30 minutes. In our experiment, two dishes with different pillar stiffnesses did not contain any Fetal Bovine Serum (FBS), one dish contained 1% FBS as an EGFR growth factor. Since the concentration is less than 2% FBS, we assume that all three dishes are serum-depleted. With cells placed on the substrates, we were able to immunostain for specific proteins and hormones of interest. Immunostaining We immunostained the cells for both paxillin and pegfr by first fixing them with 4% (vol/vol) paraformaldehyde (PFA) in PBS for 10 minutes at room temperature. The cells were then rinsed with PBS and permeabilized with 0.2% (vol/vol) Triton X-100 in PBS for 5 minutes at room temperature. We again rinsed them with PBS and blocked all the samples with 1% (vol/vol) bovine serum albumin (BSA) in PBS for 30 minutes at room temperature on a shaker. The primary antibodies were then added to the dishes: goat antibody for pegfr (p- EGFR Antibody (Tyr 1173): sc-12351; Santa Cruz Biotechnology) and rabbit antibody for paxillin (paxillin Antibody (H-114): sc-5574; Santa Cruz Biotechnology). After having incubated the samples overnight at 4ºC, the cells were washed 3 times with PBS for 5 minutes. They were incubated for 1 hour on the shaker with the corresponding secondary antibodies: chicken anti-goat 647 for pegfr (Chicken anti- Goat IgG (H+L), Alexa Fluor 647; Life Technologies) and donkey anti-rabbit 555 for paxillin (Donkey anti-rabbit IgG (H+L), Alexa Fluor 555; Life Technologies). Cells were finally washed 3 times with PBS for 5 minutes to conclude the immunostaining process. Confocal Microscopy Cells were transported to the confocal laserscanning microscope (LSM 700 with microscope axio observer.z1; Zeiss) for confocal microscopy using an objective of 63X. The cells of interest had to be straddled between soft and stiff pillar regions in order to observe any differences in pegfr and paxillin concentrations between the two areas with different rigidities. The software Zen (Zen B SP1; Zeiss) was used to observe the cell images from the confocal microscope. In addition to the two channels for paxillin and pegfr in green and red respectively (Alexa Fluor 555 and 488), a blue channel Coumarin 343 was used to distinguish soft from stiff pillars. Live Imaging Microscopy To compare wild-type MEFs and MEFs treated with EGFR-inhibitor, time-lapse imaging of pillars with bright-field microscopy was accomplished for both groups using an objective of 60X. The setup maintained at 37ºC consisted of an inverted microscope (Olympus IX-81) with a CoolSNAP HQ (Photometrics) attached to it. Image filtering For the wild-type MEFs compared to the ones treated with EGFR-inhibitor, the position of each pillar in each frame was tracked using the NanoTrack plugin for ImageJ (National Institutes of Health). In order to find contractile units, pillars within 2 to 4µm of the cell s leading edge were analyzed. This is due to the fact that contractile units are usually between 2 and 4µm in size. Pillars were tracked in these areas before the cell spread over them in order to have an initial position with no force acting 3

4 on them. The position of the pillars at each point in time (in each frame) was then calculated by subtracting this reference position with no force from the position at a given frame. To account for stage drift, a set of reference pillars far from any cell was analyzed in order to subtract their average displacement from the displacement of the pillars of interest at the leading edge 1. A customized program on Matlab (MathWorks) was used to analyze force vectors and count the number of contractile Results and Discussion Using pillar substrates with a stiffness of 8.4 nn/µm and a diameter of 0.5µm (this stiffness corresponds to 1.3µm-high pillars based on the equation shown in the Experimental Methods section), we plated 5 wild-type MEFs on a set of dishes and 5 MEFs with EGFR-inhibitor on another set. Both groups of cells were plated in serum-depleted media. We then analyzed the number of local contractile units in the two groups of cells to evaluate the relation between EGFR and contractile forces in the early spreading phase. For different time intervals and for each cell, we counted the numbers of contractile pairs per second and averaged them for the time period chosen (for example, 10 minutes). The error bars in the graph represent the standard error of the mean (SEM) for a number of different cells. Organizing the data by contractile pairs per second allows for a more accurate comparison between the two groups of cells. The results obtained are shown in Fig. 2 below. Figure 2. Graph representing the contractile pair number per second averaged over each time units in each cell to obtain real-time force measurements. ImageJ was also used in the second part of the experiment, after having obtained the images from the software Zen, in order to apply appropriate filters, adjust the contrast and brightness, as well as superimpose the paxillin and pegfr observations to determine whether the two are colocalized. period chosen for both wild-type (WT) and EGFR-inhibitor (inhibitor) cells. The error bars represent the SEM for a number of different cells. We observe that cells in a serum containing EGFR-inhibitor have less contractile pair numbers than the wild-type ones. This assumption is valid over time for most measurements shown in Fig. 2. We could minimize the size of the error bars and reduce the overlap in error bars between the WT group and the inhibitor one by increasing the number of cells analyzed in each set. Although some overlap exists in the error bars, referring to the variations in terms of cells in each group, the overall result suggests that EGFR is associated with local contractility in the early spreading phase and that blocking it with inhibitors would lead to less contractile units observed. Having assessed that EGFR plays an important role in rigidity sensing by comparing the number of contractile units for wild-type cells and cells treated with EGFR-inhibitor, we went further to determine whether any correlation exists between substrate stiffness and EGFR phosphorylation. In order to do so, poly(dimethysiloxane) (PDMS) double stiffness pillars were prepared on 5x5mm square arrays from silicon molds with diameter 0.5µm in order to obtain a stiffness of 3.2 nn/µm (this stiffness corresponds to 1.8µm-high pillars, respectively, based on the equation shown in the Experimental Methods section). The submicron pillar fabrication steps are shown in Fig. 3. For a UV treatment of 1 and a half hours, the stiffness of the exposed pillar regions increased 12 fold 4

5 (from 3.2 to 38.4 nn/µm). Double stiffness submicron pillar fabrication is discussed in detail in the Experimental Methods section. Once the pillar substrates were shined with UV lighting, they were coated with fibronectin and MEFs were plated on them. In this experiment, we were interested in determining whether the concentration of pegfr is higher on stiffer substrates compared to softer ones. A higher level of EGFR phosphorylation on stiff pillars would suggest that EGFR is mechanically activated by the rigidity of the substrate. We performed the immunostaining of the cells for paxillin, a focal adhesion protein, and pegfr during the spreading process. We then examined their localization by fixing cells after 30 minutes of spreading. This immunostaining process involved the permeabilization of the cellular membrane followed by the addition of primary antibodies for both paxillin and pegfr. The secondary corresponding antibodies were then added to the cellular medium: anti-rabbit 555 for paxillin and anti-goat 647 for pegfr. More details on the immunostaining process are outlined in the Experimental Methods section. We were interested in cells straddled between the stiff and soft pillars to be able to determine whether a difference in substrate rigidity can influence the concentration of both paxillin and pegfr. During the confocal microscopy procedure, the Coumarin 343 channel was used to observe the pillar substrate, represented in blue in our images. Moreover, the Alexa Fluor 555 and 639 channels were used respectively for paxillin and pegfr and they were represented respectively in green and red in our images. The results are shown in Fig. 4 and 5. Figures 4 and 5. MEFs incubated on 3.2 and 38.4 nn/µm soft and stiff pillars in serumdepleted medium for 30 minutes in (a). In (b) and (c), the cell is immunostained for paxillin and pegfr respectively. Part (d) represents the colocalization of paxillin and pegfr As it is shown in Fig. 4, paxillin is concentrated on the edges of the cell and is colocalized with pegfr. By comparing the stiff and soft areas of Fig. 4a with the signal localizations for paxillin and pegfr in Fig. 4b and 4c, we observe that the concentration of these two molecules is considerably more prominent on the stiffer pillar 5

6 areas than on the softer ones. This suggests that the mechanism for EGFR phosphorylation is influenced by the rigidity of the substrate. pegfr is more uniformly mechanically activated around the cell, as opposed to the observed behavior in Fig. 4 and 5. Fig. 5 reinforces this claim since we can observe that paxillin is concentrated where the substrate is stiffer as shown in Fig 5b. This paxillin presence is again colocalized with pegfr implying on the one hand, that EGFR activation is emphasized on rigid substrates and on the other hand, that there is a correlation between EGFR and focal adhesion proteins, such as paxillin. Another interesting point shown in Fig. 5 concerns the spreading of the cell s filopodia. The cell appears to be reaching for the stiffer substrate areas during its spreading with filopodia located at the border of the stiff and soft areas. This non-isotropic spreading behavior combined with the increased concentration of paxillin and pegfr at the ends of the filopodia suggests that cellular spreading is emphasized on stiffer substrates. At these soft-stiff borders, cells stretch towards rigid pillars where EGFR is mechanically activated. In order to evaluate the influence of the original substrate stiffness on the concentrations of paxillin and pegfr, cells were also plated on another set of submicron pillars with a stiffness of 8.4 nn/µm before UV treatment. This set of pillars was made using the same protocol outlined in the Experimental Methods section. The stiffness of the area exposed to UV lighting increased 12 fold to nn/µm. The other conditions of the extracellular matrix were kept identical. From Fig. 6, we observed that paxillin and pegfr have higher concentrations on the stiff regions than on the soft ones, as was shown in Fig. 4 and 5. However, the pegfr and paxillin signals are more evenly distributed on the edge of the cell in this case, compared with the signals from softer pillars in Fig. 4 and 5. This suggests that the unexposed pillars in Fig. 6 are already sensed as stiff by the cells. As a result, Figure 6. MEFs incubated on 8.4 and nn/µm soft and stiff pillars in serum-depleted medium for 30 minutes in (a). In (b) and (c), the cell is immunostained for paxillin and pegfr respectively. Their signals are evenly distributed on the edge of the cell. Part (d) represents the colocalization of paxillin and pegfr Conclusions Observing that the ability of cancer cells to sense substrate stiffness is hindered, we were interested in determining whether EGFR is correlated with rigidity sensing. Using wild-type and EGFR-inhibitor MEFs, we were able to determine that EGFR plays an important role in recognizing different substrate stiffnesses, with an increased amount of contractile units in wild-type cells. Moreover, the experiment on double stiffness pillars with cells straddled in the two regions allowed us to depict accurately how cells react on substrates with different rigidities by studying the activation of EGFR. This double stiffness pillar 6

7 experiment allowed us to understand how a single cell locally responds to different rigidities. Our results suggested that regions of the cells on stiffer pillars have higher concentrations of paxillin and pegfr, indicating that EGFR is a mechano-sensor with an increased activity on rigid substrates. Moreover, paxillin and EGFR were colocalized in all the cells observed, implying that cellular adhesions to external matrices and cellular spreading are correlated. Finally, we observed that cells have a nonisotropic expansion suggesting that they prefer stiffer substrates. Submicron pillars have then provided the mechanical tool needed to study the effect of certain proteins and hormones on cellular growth, leading to the conclusion that the activation of EGFR is mechanically influenced by the rigidity of the cellular substrate. References 1. Ghassemi S, Meacci G, Liu S, Gondarenko AA, Mathur A, Roca-Cusachs P, Sheetz MP, Hone J. Cells test substrate rigidity by local contractions on submicrometer pillars. Proc Natl Acad Sci. 2012, Vol. 109, Wolfenson H, Meacci G, Liu S, Stachowiak MR, Iskratsch T, Ghassemi S, Roca-Cusachs P, O Shaughnessy B, Hone J, Sheetz MP. Tropomyosin Controls Nanometre Steps in Sarcomere-like Contractions for Rigidity Sensing. Manuscript submitted. 3. Yu X, Miyamoto S, Mekada E. Integrin α2β1-dependent EGF receptor activation at cell-cell contact sites. Journal of Cell Science. 2000, Vol. 113, Ghassemi S, Biais N, Maniiura K, Wind SJ, Sheets MP, Hone J. Fabrication of elastomer Pillar Arrays with Modulated Stiffness for Cellular Force Measurements. J Vac Sci Technol B Microelectron Nanometer Struct Process Meas Phenom. 2008, Vol 26(6), Iskratch T, Yu CH, Marthur A, Liu S, Stevenin V, Dwyer J, Hone J, Ehler E, Sheetz M. FHOD1 is needed for directed Forces and Adhesion Maturation during Cell Spreading and Migration. Dev Cell. 2013, Vol 27(5), Acknowledgments I would like to acknowledge support from Shuaimin Liu 1, Junqiang Hu 1, Professor James C. Hone 1, the members of the Sheetz Lab 2, as well as the REU program funded by the NSF for the opportunity to conduct this research. Funds for this research were provided by the National Institutes of Health through Analyses of 120nm Local Contractions Linked to Rigidity Sensing under grant 1R01GM Partial support was provided by the Center for Precision Assembly of Superstratic and Superatomic Solids: an NSF MRSEC under award number DMR Department of Mechanical Engineering, 2 Department of Biological Sciences, Columbia University After graduating with a B.S. in Mechanical Engineering from the School of Engineering and Applied Sciences at Columbia University, I plan to pursue a thesis M.S. in Mechanical Engineering. Through research and graduate level courses, my goal is to familiarize myself with new innovations and techniques to prepare myself better for a career in engineering. 7