The PDMS substrates of varying stiffness were made from Sylgard 184 and Sylgard 527 (Dow Corning). The stiffness of the substrates was modified by varying ratios of elastomer base to curing agent targeting three regions, hard: ∼1 MPa, medium: ∼50 kPa (Balaban et al., 2001), soft: <5 kPa (Moraes et al., 2015). Glass coverslips (∼1 GPa) were used for comparison (Supplementary Table S1). For the experiments, two-well (3 mm diameter) chips were made by bonding a PDMS compartment on coverslip. PDMS mixtures were placed into the wells and cured for 24 h at room temperature. To separate the effect of surface chemical variation from mechanical properties, all substrates were treated with fibronectin (50 μg/ml) in phosphate-buffered saline (PBS) and incubated for 45 min prior to experiments.

The spreading area of each cell was quantified using ImageJ (NIH) with MATLAB plugin. Briefly, brightfield images were imported to MATLAB and converted to binary images, and individual cells were identified using built-in “adaptive threshold.” The images were processed with several erosions and dilations to clearly define the cell boundary. The binary images were then transferred back to ImageJ and the cell areas were measured. F-actin thickness was measured using Plot Profile in ImageJ. Focal adhesions were selected manually and analyzed using ImageJ. Multiple cells were selected from each image and multiple images were used. A minimum of 4 experiments were performed under each condition.

Madin-Darby Canine Kidney (MDCK) cells (ATCC) were grown to confluence in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 1% penicillin and streptomycin. The cells were trypsinized and seeded in the wells and allowing to grow for 30 min in the incubator. The cells were then washed with media that removed unattached cells and grown for additional 2 h. Live cell imaging were conducted in a stage-top incubator (INUB-ZILCSD-F1-LU, Tokai Hit Co., Ltd., Japan), maintained at 37°C and 5% CO₂. No-phenol Red DMEM media with 10% fetal bovine serum (Gibco, TX) was used for fluorescence imaging to reduce the background illumination. Isotonic saline solution was used for experiments with GsMTx4 and Gd³⁺, as it was known that culture media reduced the efficiency of the peptide (Bae et al., 2011).

Piezo1 knockdown was performed using previously validated Piezo1 miRNA targeting Piezo1 and co-expressed with EGFP to verify transfection (Jetta et al., 2019). Cells were cultured to ∼60% confluence and transfected with plasmid DNA miRNA (0.4 µg) using Effectene (Qiagen, Valencia, CA) at 1:50 DNA to effectene ratio. Cells were incubated in the transfection media for another 48 h prior to experiments. The transfection efficiency was ∼25%.

Cells were fixed with 4% paraformaldehyde for 15 min, and permeabilized with Triton X-100 (0.1% in PBS) for 15 min, followed by blocking with 5% Goat Serum (Sigma-Aldrich) for 1 h at room temperature. For staining paxillin, blocking buffer, made with 5% Goat Serum and 0.01% Triton in PBS was used for diluting primary and secondary antibodies. Cells were incubated with primary antibody, Anti-Paxillin (BDB610052, Fischer Scientific), at 1:50 dilution overnight at 4°C and secondary antibody, Alexa fluor 488 (A11001-Thermo Fischer) at 1:200 for 1 h at room temperature. F-actin was stained with Phalloidin Alexa Flour 568 (A12380, Fischer Scientific) at 1:100 in PBS and was incubated at room temperature for 1 h. After every step, cells were washed three times with PBS. Imaging was done in slow fade Gold Anti-fade Reagent (1:100, Invitrogen) in PBS to protect from photobleaching. Images were acquired using an inverted microscope (Axiovert 200M, Zeiss) with a CCD camera (AxioCam, Zeiss). Fluorescence images were obtained with two filter sets: (Ex: 470/40 nm; Em: 525/50 nm) and (Ex: 550/25, Em: 605/70), and a ×63 oil immersion objective. The B/W images were obtained using a ×20 objective.

Cytosolic Ca²⁺ was measured using Ca²⁺ sensitive dye, Fluo-4 AM (5 μM, Invitrogen). The normalized Ca²⁺ intensity was calculated using ∆ F F 0 = F − F 0 F 0 , where F and F ₀ are the mean fluorescence intensities of individual cells at time t and t = 0, respectively.

GsMTx4 was purified and diluted in saline to a final concentration of 5 μM. Yoda1 (Tocris Bioscience) was dissolved in DMSO as stock solution (48 mM), then diluted in saline to a final concentration of 25 µM. Gadolinium chloride (Sigma-Aldrich, St. Louis, MO) was freshly prepared and diluted to 20 µM in saline.

The data is shown as the mean ± standard error of the means (s.e.m.). Statistical analysis was performed using two sample t-test, and value of p < 0.05 was considered statistically significant.

To investigate the effect of local mechanical properties, we cultured MDCK cells on four different substrates, glass, hard, medium-soft and soft with stiffness ranging from ∼1 GPa to ∼5 Pa (Supplementary Table S1). Cells spread to a full extent on all substrates within 3 h, and showed hexagonal/polygonal shape, typical for epithelial cells (Figure 1A). The spreading area of individual cells varies slightly depending on the substrate stiffness. Cells spread to larger area (∼20%) on glass and hard substrates compared with the soft substrates (Figures 1A, C). However, the organization and amount of actin fibers vary drastically on different substrates. On stiffer substrates, glass and hard, cells developed high density actin stress fibers across the cell body that extends to cell protrusions (yellow arrows, Figure 1B). On soft substrates, the actin fibers arranged along the cell periphery to form a ring-shape boundary and there was no stress fiber like structure in the cell interior (blue arrows, Figure 1B). The amount of F-actin was quantified by the mean fluorescence intensity of cell interior and normalized with the mean intensity of whole cell, showing that interior F-actin intensity is greater for the hard substrates compared with soft ones (Figure 1E). The actin fibers were quantified by the thickness and number of fibers in individual cells, which shows that both thickness and number of fibers are significantly greater on stiffer substrates (Figures 1F, G, *p < 0.001). The percentage of attached cells was lower on soft substrates (Figure 1D, *p < 0.001), and only attached cells were used for analysis. This result indicates actin cytoskeleton are sensitive and responsive to the stiffness of the substrates.

MDCK cells endogenously express mechanosensitive Piezo1, that can be activated by changes in membrane tension or fluid shear stress (Gudipaty et al., 2017; Jetta et al., 2019). To assess whether Piezo1 is responsible for detection of substrate stiffness, we knockdown Piezo1 with miRNA, and observed the cell shape and actin reorganization with varying substrate stiffness. Cells were transfected with Piezo1 miRNA and co-expressed with EGFP to verify transfection (green, SM Supplementary Figure S1). Piezo1 knockdown (P1KD) cells showed significantly low intensity of actin fibers in the cell interior on all substrates (Figure 2A). These cells developed F-actin rings along the cell periphery on hard and glass substrates. In contrast, the nearby control cells (non-transfected), showed strong actin stress fibers (red arrows, Figure 2A). Piezo1 knockdown appeared to have subtle effect on cells on soft substrates (Figure 2A). The thickness of actin fibers in P1KD cells was measured and compared with the control cells, which shows Piezo1 knockdown reduced the fiber thickness significantly on stiffer substrates (Figure 2B, *p < 0.001, **p < 0.05). The stress fibers in P1KD cells were diminished (Figure 2C, *p < 0.001, **p < 0.05). The F-actin intensity in interior of the P1KD cells is also reduced significantly (Figure 2D, n = 30 for all, *p < 0.001). However, the cell shape was not altered, nor the spreading area (Figure 2E, n = 50 for glass and hard, n = 40 for soft, **p < 0.05). This result indicates that Piezo1 along with actin cytoskeleton forms a vital force transduction apparatus in epithelial cells.

To assess if Piezo1 mediated cation current is needed for cell remodeling on hard substrates, we blocked the channels using Piezo1 specific inhibitor, GsMTx4. On hard substrates, inhibiting Piezo1 significantly reduced the actin stress fibers in the cell interior, but it has little effect (<10%) on cell spreading (Figures 3A, C). Some F-actin appeared in punctuated form in the cell interior after treatment. On soft substrates, neither actin organization nor the cell area were altered by Piezo1 inhibition (Figures 3A, C). We quantified the thickness and number of actin fibers in cells and found that inhibiting Piezo1 channels reduced the fiber thickness and the number of stress fibers significantly (Figures 3D, E, *p < 0.001). As controls, we used non-specific mechanosensitive channel (MSC) inhibitor Gd³⁺ to inhibit all MSCs. The interior actin stress fibers were eliminated in all cells. The cell spreading area was also moderately reduced in the presence of Gd³⁺, it is because some cells started to shrink under the treatment of the drug for 2.5 h. Thus, Piezo1 mediated Ca²⁺ current is necessary in stiffness triggered F-actin reorganization. We have previously shown that cell expansion requires opening of Piezo1 channels with Ca²⁺ influx that leads to activation of Rho-ROCK pathway (Jetta et al., 2021). This result is consistent with previous findings, and further shows that Piezo1 mediated Ca²⁺ signal is involved in cell sensing of stiffness.

We evaluated the effect of stiffness on focal adhesions by immunostaining Paxillin, a protein located in the focal adhesion complex. We found that FA enlargement was strictly modified by the stiffness. Cells developed large mature FAs on stiffer substrates, that were aligned with stress fibers at the ends (yellow arrows, Figure 4A, Control panel). Some short FAs also exist, however, those FAs were located in the middle of the cell or dissociated with the stress fibers (not shown). The FAs are also present in Piezo1 knockdown cells, although the size of the FAs is much smaller (Supplementary Figure S2). P1KD only reduced the number of FAs slightly (Supplementary Figure S2). On soft substrates, the FAs were much smaller and shorter compared with those on hard substrates, and they were distributed along the cell periphery. Interestingly, these FAs are mostly radial although they are not associated with stress fibers (blue arrows, Figure 4A). We measured the size of FAs and found that the mean FA size on glass and hard substrates are significantly larger than that on soft substrate (Figure 4E, *p < 0.001). The stiffness had minimal effect on the number of FAs in individual cells (Figure 4F). These results show that cell spreading was facilitated by two stage of FAs, mature FAs that couple with stress fibers to transmit traction forces, and nascent FAs that are linked to actin mesh providing guidance for spreading. Small nascent FAs located at the lamellipodium edge can facilitate cell spreading without stress fibers facilitated traction forces.

We then evaluated the role of Piezo1 on F-actin and FAs with Piezo1 agonist, Yoda1. Yoda1 was added to the culture media at 1 h after seeding the cells, and cells were fixed and co-stained at 2.5 h. Yoda1 treated cells showed thicker actin fibers and larger mature FAs compared with control cells, while the organization of actin and FAs remain the same (Figure 4A). Statistical analysis shows that fiber thickness increased on all substrates after Yoda1 treatment (Figure 4B, *p < 0.001, **p < 0.05), while the number of fibers did not show significant change (Figure 4C). The enhancement of actin fibers and FAs did not result in a significant increase in cell area (Figure 4D).

FA sizes increased moderately on stiffer substrates after Yoda1 treatment (Figure 4E). On soft substrates, the effect of Yoda1 on size and number of FAs per cell is negligible (Figure 4F). The existence of nascent FAs at cell protrusions provided the guidance for cells expansion. These FAs were never grown to mature even in the fully spreading cells on soft substrate.