Mechanical stimulation of Piezo1 receptors depends on extracellular matrix proteins and directionality of force. Supporting Information

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1 Mechanical stimulation of Piezo1 receptors depends on extracellular matrix proteins and directionality of force Benjamin M. Gaub and Daniel J. Müller Supporting Information Supporting Information Note 1. Comparison of Piezo1 force threshold values with pressure values from literature. In order to compare our Piezo1 force threshold values with published Piezo1 pressure values we calculated the pressure resulting from push stimulations using the AFM cantilever with 5 µm diameter bead. The contact area A between bead and cell is given as half sphere surface of the bead: =2 Supporting Equation 1 with r = 2.5 µm, the area A is: =2 (2.5 μm) =39.26 The pressure P is given as: = Supporting Equation 2 with F being the force. To mechanically stimulate Piezo1- cells by a pushing bead, the threshold force is nn (Figure 2d). Therefore, the threshold pressure is: 1 kpa=7.5 mm Hg, therefore: = kg m s = kg m s = kpa=41.85 mm Hg Coste et al. 1 reported Piezo1 P 50 pressure sensitivity values in cells in cell-attached patches of: P 50 = 28.1 ± 2.8 mm Hg; Lewis et al. 2 reported Piezo1 P 50 pressure sensitivity values in cell-attached patches of: P 50 = 19.3 ± 1.2 mm Hg; Cox et al. 3 reported Piezo1 P 50 pressure sensitivity values in cell-attached patches of: P 50 = 45 ± 3 mm Hg;

2 Supporting Information Note 2: Physiological pressures encountered in human tissue with native Piezo1. The mrna expression profiles for Piezo1 show the highest levels in bladder and lung. 1 Urinal bladder pressures have been determined in patients with indwelling transurinal catheters. 4 The pressure values reported in this study ranged from 1 22 cm H 2 O. 1 cm H O =0.73 mm Hg, therefore: 1 cm H O =0.73 mm Hg, and 22 cm H O =16.88 mm Hg. Thus, urinal bladder pressures range from mm Hg. In our experiments we had to mechanically push the Piezo1- cells by 220 nn to induce Piezo1 gating. This force applied via a 5 µm bead corresponds to mm Hg (Supporting Information Note 1). Maximal expiratory pressure values which can be taken as proxy for lung pressure were determined in human subjects with normal respiratory function and amounted to 143 ± 10 cm H 2 0 while standing upright. 5 1 cm H O =0.73 mm Hg, therefore: 133 cm H O =97.83 mm Hg, and 153 cm H O = mm Hg. Maximal expiratory pressure values range from mm Hg, which is 2 3 times the pressure we had to apply to mechanically gate Piezo1 by pushing the bead onto Piezo1- cells (Supporting Information Note 1).

3 Supporting Information Figures Supporting Information Figure 1. Transient Piezo1, jrcamp1a and GFP expression in HEK and cells. a-d) Piezo IRES GFP (green) and jrcamp1a (red) expression in HEK-293T cells 48 h after transfection. (b,c) Single channel and (a,d) overlaid DIC and fluorescent images. The red channel shows the baseline fluorescence of the calcium indicator before stimulation. A differential interference contrast (DIC) image was simultaneously acquired. Yellow cells are expressing both Piezo IRES GFP and jrcamp1a. e) GFP (green) expression in cells 48 h post transfection used for fluorescent assay calibration (Methods). All scale bars, 20 µm.

4 Supporting Information Figure 2. Experimental parameters for push and pull protocols. a,b) Experimental parameters for push (a) and pull (b) stimulation. The three vertical panels show parameters from one representative recording per condition. Calcium indicator fluorescence signal as measured by confocal microscopy are shown in the top panel, the mechanical force exerted via the AFM cantilever is shown in the middle panel and the position of the cantilever is shown in the bottom panel. a) For the push stimulus, 250 ms bouts of positive pressure were applied to the cell with increasing intensity (middle). The cantilever was lowered onto the cell with a speed of 10 µm s 1 until the force reached the set point and the cantilever was withdrawn with a speed of 100 µm s 1 (bottom). Calcium signals were recorded in real time and the stimulation was terminated as soon as the cell responded (top). b) For the pull stimulus, the ECM coated cantilever was lowered onto the cell up to a contact force of nn. The cantilever was held at constant contact force for 60 s to allow ligandreceptor interactions to form. Finally, the cantilever was retracted (100 µm s 1 ) to stretch the cells locally and the resulting rupture forces were readout using the deflection of the cantilever (middle).

5 Supporting Information Figure 3. Control recordings from HEK cells without Piezo1. a-c) Representative images of HEK cells expressing jrcamp1a (red) but not Piezo1, before (a), during (b) and after (c) mechanical push by AFM. The time points in the upper right corner refer to (d). Scale bars, 10 µm. d) Fluorescence of the cell depicted in (a-c) during stimulation with pushing forces from nn. The cell does not respond to mechanical pushing in the absence of Piezo1 and cell morphology is conserved throughout the experiment. For a summary of all control HEK cells refer to Supporting Information Table 1.

6 Supporting Information Figure 4. Lifetime of fluorescently labeled ECM proteins on cantilever. a) Fluorescent signal of a bead functionalized with rhodamine conjugated laminin EHS used several times to mechanically stimulate cells. Fluorescent images of the bead were recorded after each mechanical stimulation (pulling) and the fluorescence intensity was calculated using the Zeiss ZEN blue software. The two experiments shown were done with two different beads. Data points 1 and 2 show fluorescence after calibration measurements without cell contact. The later data points (3 7) show fluorescence after stimulation of cells (20 nn contact force for 60 s). b) Representative overlay of fluorescence and DIC images showing the bead functionalized with rhodamine conjugated laminin EHS and the AFM cantilever before (left) and after (right) the experiment. The numbers refer to experiment 2 in panel (a). Scale bars, 20 µm.

7 Supporting Information Figure 5. Piezo1 receptors do not respond to mechanical pulling when adhering unspecific to substrates. a,b) Summary of calcium signals (a) and forces (b) of cells with or without Piezo1 receptor overexpression in response to mechanical pulling with various adhesive proteins attached to the cantilever. a) Normalized calcium signals from left to right: without Piezo1 overexpression, cantilever with Matrigel (0.05 ± 0.02 I/I 0 ; n = 13), without Piezo1 overexpression, cantilever with collagen IV (Col IV) ( 0.11 ± 0.02 I/I 0 ; n = 16), with Piezo1 overexpression, cantilever without functionalization (blank lever) (0.04 ± 0.03 I/I 0 ; n = 15), with Piezo1 overexpression, cantilever with concanavalin A (ConA) (0.02 ± 0.02 I/I 0 ; n = 24), without Piezo1 overexpression, cantilever with concanavalin A (0.05 ± 0.02 I/I 0 ; n = 13). Noise level in gray. b) Rupture forces from left to right: without Piezo1 overexpression, cantilever with Matrigel (40.16 ± 5.30 nn; n = 13), without Piezo1 overexpression, cantilever with collagen IV (28.44 ± 3.21 nn; n = 16), with Piezo1 overexpression, cantilever without functionalization (22.53 ± 1.96 nn; n = 15), with Piezo1 overexpression, cantilever with concanavalin A (34.1 ± 3.04 nn; n = 24), without Piezo1 overexpression, cantilever with concanavalin A (38.8 ± 3.97 nn; n = 13). Normalized calicum signals were calculated as described (Methods). Dots represent single cells. The dotted line indicates the baseline (I/I 0 = 0). Values in legend are given as mean ± SEM. Data in Figure are shown by the box (median, first and third quartiles).

8 Supporting Information Figure 6. Fluorescence artifacts from mechanical pushing and pulling single cells with the AFM cantilever. a) Summary of normalized fluorescence intensity for push (0.08 ± 0.01 I/I 0 ; n = 15) and pull (0.11 ± 0.02 I/I 0 ; n = 15) stimulation of control cells expressing GFP only. These values reflect the noise level (0.25 I/I 0 ) of the assay. Artifacts result from compression of the cells due to mechanical stimulus and thus transient increase or decrease in fluorescence of GFP. These values were used to set the cutoff for responsive vs. non-responsive cells (Methods). Dots represent single cells. Data are given as mean ± SEM. Additionally, median and first and third quartiles are shown by the box. b,c) Representative traces showing GFP fluorescence for push (b) and pull (c) stimulation of GFP expressing cells.

9 Supporting Information Table 1 Cells Cantilever Stimulus Number of Number of Success Figure cells tested responders* rate (%) Control HEK Push Supporting Information Figure 6 Piezo1- Push Figure 2c,d HEK Control Push Figure 2c,d Piezo1- Push Figure 2c,d Piezo1- Matrigel Push Figure 3c,d Piezo1- Matrigel Pull Figure 3c,d Figure 4 Piezo1- Pull Figure 3c,d Piezo1- Collagen Pull Figure 4 IV Piezo1- Laminin Pull Figure 4 EHS Piezo1- Laminin 551 Pull Figure 4 Supporting Information Table 1. Summary of the number of stimulated cells and number of responding cells for various experimental conditions. *Responding cells were classified as described in Methods. All cells analyzed overexpressed jrcamp1a.

10 Supporting Information Movie 1. Representative example of Piezo1- response to push stimulus. Piezo1- cell stimulated by mechanical pushing using a non-functionalized cantilever and bead. The cell responded to 205 nn pushing force with strong influx of calcium as seen by the increasing fluorescence of the calcium reporter jrcamp1a (red) at t = 30 s. Time (s) is shown in top left. Scale bar, 10 µm. Supporting Information Movie 2. Representative example of Piezo1- response to pull stimulus with collagen IV functionalized cantilever. Piezo1- cell stimulated by pulling using a collagen IV functionalized cantilever and bead. The cell responded to 10 nn pulling force with strong influx of calcium as seen by the increasing fluorescence of the calcium reporter jrcamp1a (red) at t = 71 s. Time (s) is shown in top left. Scale bar, 10 µm. Supporting Information References (1) Coste, B.; Mathur, J.; Schmidt, M.; Earley, T. J.; Ranade, S.; Petrus, M. J.; Dubin, A. E.; Patapoutian, A. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 2010, 330, (2) Lewis, A. H.; Grandl, J. Mechanical sensitivity of Piezo1 ion channels can be tuned by cellular membrane tension. elife 2015, 4, e (3) Cox, C. D.; Bae, C.; Ziegler, L.; Hartley, S.; Nikolova-Krstevski, V.; Rohde, P. R.; Ng, C. A.; Sachs, F.; Gottlieb, P. A.; Martinac, B. Removal of the mechanoprotective influence of the cytoskeleton reveals PIEZO1 is gated by bilayer tension. Nat. Commun. 2016, 7, (4) Chionh, J. J.; Wei, B. P.; Martin, J. A.; Opdam, H. I. Determining normal values for intraabdominal pressure. ANZ J. Surg. 2006, 76, (5) Badr, C.; Elkins, M. R.; Ellis, E. R. The effect of body position on maximal expiratory pressure and flow. Aust. J. Physiother. 2002, 48,