Mechanical Stimulation of Piezo1 Receptors ... - ACS Publications

Feb 6, 2017 - In mammals, they play important roles in basic physiological functions including proprioception, sensation of touch, and vascular develo...
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Mechanical Stimulation of Piezo1 Receptors Depends on Extracellular Matrix Proteins and Directionality of Force Benjamin M. Gaub* and Daniel J. Müller* Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland S Supporting Information *

ABSTRACT: Piezo receptors convert mechanical forces into electrical signals. In mammals, they play important roles in basic physiological functions including proprioception, sensation of touch, and vascular development. However, basic receptor properties like the gating mechanism, the interaction with extracellular matrix (ECM) proteins, and the response to mechanical stimulation, remain poorly understood. Here, we establish an atomic force microscopy (AFM)-based assay to mechanically stimulate Piezo1 receptors in living animal cells, while monitoring receptor activation in real-time using functional calcium imaging. Our experiments show that in the absence of ECM proteins Piezo1 receptors are relatively insensitive to mechanical forces pushing the cellular membrane, whereas they can hardly be activated by mechanically pulling the membrane. Yet, if conjugated with Matrigel, a mix of ECM proteins, the receptors become sensitized. Thereby, forces pulling the cellular membrane activate the receptor much more efficiently compared to pushing forces. Finally, we found that collagen IV, a component of the basal lamina, which forms a cohesive network and mechanical connection between cells, sensitizes Piezo1 receptors to mechanical pulling. KEYWORDS: Mechanosensation, Piezo receptors, atomic force microscopy, extracellular matrix, single-cell force spectroscopy

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through lipid model”. However, membrane blebs, membrane patches, and lipid bilayers are artificial systems in which the Piezo1 receptors could not interact with ECM or/and cytoskeleton, both of which have been shown to influence mechanosensitive properties for a number of mechanosensitive receptors including ENaC, TRPV1, TRPV4, TRPP1, TRPA1, and NMDA.14 In context of Piezo receptors, it has been demonstrated that the ECM plays an important role in Piezo receptor localization and function,12 arguing in favor of the “tether model”. Piezo receptors have been shown to primarily localize at the cell substrate interface where they show significantly lower activation threshold to mechanical stimulation compared to the freely exposed cell surface.12,22 Additionally, in primary sensory neurons mechanosensitive properties depend on protein tethers coupling the cells to the ECM.23 Despite these detailed insights, it is still controversial which model is best suited to describe the force sensing mechanism of Piezo receptors. To elucidate the activation mechanism, recent functional studies stimulated Piezo receptors by pushing membranes using blunt glass pipettes7,8,24,25 or pulling membrane patches using patch pipettes.9,10,18,19,21 However, whereas blunt glass pipettes have a low spatial resolution (μm range) and lack force sensitivity, patch formation displaces the membrane and thus the Piezo receptor from its native cellular environment including the cytoskeleton and ECM.9,14 Accordingly, new

echanosensation is essential for many organisms. In bacteria, mechanosensitive ion channels balance osmotic stress,1 while in vertebrates they are needed for tactile,2 pain3,4 and auditory5 sensation. A number of mechanosensitive ion channels have been described in bacteria (MsC), Drosophila (NOMPC), C. elegans (DEG/ENaC family), and vertebrates (K2P family).6 Recently, Piezo receptors, a new class of mechanosensitive ion channels, have been identified in eukaryotes.7−9 As basic physiological processes including touch sensation, cardiovascular function and proprioception critically depend on Piezo receptor function,2,10,11 Piezo depletion in lower vertebrates is lethal and mutations altering their function can cause numerous diseases in humans.12 However, despite the physiological importance of Piezo receptors little is known how mechanical forces activate the receptor. Two models have been proposed to explain the coupling of macroscopic mechanical forces to open the ion channel of the Piezo receptor.13,14 In the “tether model”, mechanosensitive receptors couple to auxiliary proteins, which transmit macroscopic forces to induce ion channel opening.13,15 In the “membrane tension” or “force through lipid model”, the ion channel is gated by the surrounding lipid bilayer without the need of auxiliary proteins.16,17 A number of studies have shown that mechanical perturbations of the lipid bilayer alone are sufficient to gate Piezo1 receptors.18−21 Mechanosensitive properties of Piezo1 receptors are preserved in membrane blebs,18 membrane patches,9,19 and in reconstituted lipid bilayer systems,20 demonstrating that Piezo1 receptors are intrinsically mechanosensitive. These findings argue in favor of the “force © 2017 American Chemical Society

Received: January 13, 2017 Revised: February 3, 2017 Published: February 6, 2017 2064

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Figure 1. Assay for mechanical manipulation and functional readout of Piezo receptor activity. (a) Schematic of the experimental assay combining AFM with inverted confocal microscopy. The basic elements of the AFM (top) consist of a laser, which is reflected from the cantilever on to a photodiode to detect the cantilever deflection and force. The free end of the cantilever carries a bead (5 μm diameter), which can be functionalized with ECM proteins to facilitate specific interactions of bead and cell. The cellular response is read out via functional calcium imaging by confocal microscopy, while the cellular morphology is monitored using differential interference contrast (DIC) microscopy. (b) Cultured neuroblastoma (N2A) cells transiently transfected with the DNA encoding for the mechanosensitive channel Piezo1 IRES GFP (green) and the genetically encoded calcium indicator jRCaMP1a (red). (c) Single channel and overlaid DIC and fluorescent images of an AFM cantilever (see insert for the bead) with a neuroblastoma cell expressing Piezo1 IRES GFP (green) and jRCaMP1a (red). The red channel shows the baseline fluorescence of the calcium indicator before stimulation. All scale bars, 20 μm.

opening of the receptors. We find that in the absence of ECM proteins Piezo1 receptors are relatively insensitive to mechanical pushing of the cellular membrane. However, when conjugated to ECM proteins Piezo1 receptors are significantly sensitized and can be stimulated by much lower mechanical forces pulling the cellular membrane. Particularly, we find that collagen IV sensitizes the Piezo1 receptor to gate at much smaller mechanical pulling forces. Results and Discussion. Combining AFM and Confocal Microscopy To Characterize Piezo1 Receptor Activation. To systematically investigate how Piezo1 receptors couple mechanical forces to channel opening in live cells, we mechanically stimulated Piezo1 receptors by AFM-based force spectroscopy and monitored receptor activity by real-time fluorescent calcium imaging (Figure 1a). To define the contact area in which mechanical forces were applied to a cell, we glued a 5 μm diameter bead to the free end of the AFM cantilever. Neuroblastoma (N2A) cells and human embryonic kidney (HEK) cells were transiently transfected with the mechanosensitive receptor Piezo1 coexpressing GFP (green) and the calcium indicator jRCaMP1a (red) (Figure 1b,c and Supporting Information Figure 1). Expression of both was confirmed by confocal microscopy. Cells transfected with jRCaMP1a showed varying levels of baseline fluorescence (Figure 1b,c) before mechanical stimulation was applied (see below). High Pushing Forces Activate Piezo1 in the Absence of the ECM. For mechanical stimulation, the bead attached to the cantilever was centered above a single HEK or N2A cell

approaches are needed to quantitatively probe Piezo receptor activation in a cellular context with delicate force control. Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been established to characterize membrane proteins of isolated membranes at high spatial resolution and force sensitivity in the physiological relevant environment.26,27 Complementary, AFM-based single-cell force spectroscopy (SCFS) has been established to mechanically manipulate single cells and to measure the adhesion facilitated by cell membrane receptors.28,29 Both AFM-based approaches reached a precision where they can mechanically detect the binding strength of ligands to single membrane receptors in vitro and in living animal cells.30−33 Particularly when combined with optical microscopy and providing cell culture conditions, AFM has proven helpful to characterize the binding strength of ligands to cell surface receptors while morphologically characterizing the cell state.30,34 SMFS and SCFS are routinely applied to characterize mechanical forces at which single cell adhesion receptors unbind from ECM proteins (20− 80 pN), cell membrane receptors unbind from ligands (50−150 pN), and integral membrane proteins are unfolded and extracted from membranes (100−300 pN).27,31,35 Here we thus apply AFM-based force spectroscopy to investigate whether there are better modes toward mechanically activating Piezo1 receptors in living mammalian cells. The force spectroscopy experiments are conducted with time-lapse fluorescent calcium imaging to directly monitor and quantify the cellular response to the mechanically induced channel 2065

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Figure 2. Piezo1 receptor response to vertically pushing the cell with a micrometer-sized bead. (a) Representative example of jRCaMP1a fluorescence in a Piezo1-N2A cell pre (baseline) and post (peak) stimulation by mechanical pushing (250 ms). The GFP signal indicating the coexpression of Piezo1 receptors (Figure 1) is not shown. The time points are in reference to panel B. Scale bars, 20 μm. (b) Representative fluorescence trace of a stimulated Piezo1-N2A cell. The blue arrow indicates the time point of mechanical push stimulation (220 nN, 250 ms). (c) Normalized calcium signal of control N2A cells (n = 6), Piezo1-N2A cells (n = 21), and Piezo1-HEK cells (n = 11) in response to mechanical pushing. Calcium signals were calculated as described (Materials and Methods) and the noise level is shown in gray. (d) Threshold forces for control N2A cells (n = 6), Piezo1-N2A cells (n = 21), and Piezo1-HEK cells (n = 11). (e) Percentage of Piezo1-N2A cells responding to mechanical pushing pre (83%, n = 6) and post (7%, n = 15) application of the calcium chelator BAPTA (1 mM in 1% DMSO, 99% culture medium). Dots represent single cells. Boxes show median, first and third quartiles. Two tailed Mann−Whitney test, ****, P < 0.0001; ***, P < 0.001; ns, P > 0.05.

(1.48 ± 0.35 I/I0; n = 11) (Figure 2c). Whereas most cells exhibited an “all or none” type response to mechanical stimulation, very few cells (5.8%, n = 2/34) showed graded responses that increased with increasing mechanical stimulus (data not shown). To test if the calcium signals were due to the overexpressed Piezo1 receptors, we transfected cells with jRCaMP1a but not with Piezo1 and repeated the experiments (Figure 2c,d). We did not observe any calcium signals in response to force stimulation in the range of 100−400 nN. However, a small fraction of N2A control cells (26.1%, n = 6/ 23, Supporting Information Table 1) responded to forces >400 nN (Figure 2d). Because N2A cells natively express Piezo1 receptors,7 we assumed that these endogenous receptors responded to mechanical stimulation. However, the higher force thresholds at which the calcium signals were induced (432.5 ± 10.3 nN; n = 6) showed that the mechanical responses of N2A cells expressing endogenous and overexpressed Piezo1 were clearly separated. In contrast to N2A control cells, HEK control cells overexpressing jRCaMP1a but not overexpressing Piezo1 receptors did not respond to mechanical stimulation >500 nN (0%, n = 0/20, Supporting Information Table 1). The latter control experiments also showed that the cell morphology did not change considerably and that the cell membrane did not rupture upon mechanical stimulation as this would have caused calcium influx and increased fluorescence (Supporting Information Figure 3). To identify the source of calcium ions causing the fluorescent signal of mechanically stimulated Piezo1-N2A cells, we removed calcium from the extracellular space using the calcium chelator BAPTA and mechanically stimulated the cells. The percentage of Piezo1-N2A cells responding to the mechanical

coexpressing the Piezo1 receptor and calcium indicator (Figure 2a). Piezo1 receptors inactivate very quickly (τ ≈ 15 ms).7 Hence, we approached and retracted the cantilever to and from the cell surface at the maximum possible speed of ∼100 μm s−1, allowing only a short interaction time of ∼250 ms with the cell (Supporting Information Figure 2a). The force applied to the cell was measured by the deflection of the cantilever and in this case reached 220 nN. As observed by fluorescence microscopy, the application of mechanical force was followed by a strong increase of fluorescence signal of the calcium indicator jRCaMP1a to which we refer to as the calcium signal (Figure 2a). The Piezo1 channel thus opened in response to the mechanical perturbation, which allowed the calcium ions to flow along the concentration gradient into the cell. We optically tracked the functional activation of Piezo1 receptors at a time resolution of ∼100 ms, which allowed to precisely follow the activation and decay profile of the fluorescence signal (Figure 2b). Whereas the activation profile increased quickly in response to the mechanical stimulus, the decay of the cellular calcium signal was long lasting (Figure 2b; Supporting Information Movie 1). Next, we used our assay to characterize the forces at which Piezo1 overexpressing N2A (Piezo1-N2A) and HEK (Piezo1HEK) cells were mechanically stimulated. We stepwise increased the force pushing the bead on to Piezo1-N2A and Piezo1-HEK cells until observing calcium signals (Figure 2c,d and Supporting Information Figure 2). We found that the Piezo1 force thresholds were similar in Piezo1-N2A (219.9 ± 8.9 nN; n = 21) and Piezo1-HEK (184.5 ± 15.1 nN; n = 11) cells (Figure 2d) as were the normalized calcium signals for Piezo1-N2A (2.21 ± 0.28 I/I0; n = 21) and Piezo1-HEK cells 2066

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Figure 3. Piezo1 receptor response depends on mechanical stimulus and substrate. (a) Schematic of a Matrigel-functionalized AFM cantilever with bead stimulating Piezo1 receptors by mechanical pulling. (b) Representative fluorescence trace of a Piezo1-N2A cell responding to mechanical pulling with a Matrigel-coated bead. The mechanical stimulation has three phases as indicated by the gray shaded background. In phase I, the cantilever pushes the bead onto the cell (∼20 nN) as indicated by the left force arrow; in phase II, the force pressing the bead to the cell surface is kept constant for 60 s, resulting in the formation of adhesive forces between bead and cell surface; in phase III, the cantilever is retracted to apply a pulling force as indicated by the right force arrow. (c,d) Normalized calcium signals (c) and forces (d) resulting from mechanical stimulation by either pushing uncoated beads to Piezo1-N2A cells (n = 21), Matrigel-coated beads to Piezo1-N2A cells (n = 12) or mechanically pulling the membrane of Piezo1-N2A cells with Matrigel-coated beads (n = 11). Normalized calcium signals were calculated as described (Materials and Methods) and the noise level is shown in gray. Dots represent single cells. Boxes show median and first and third quartiles. Nonparametric one-way ANOVA (Kruskal−Wallis) test. ****, P < 0.0001; ns, P > 0.05.

Figure 4. Collagen IV sensitizes Piezo1-N2A cells to mechanical pulling. (a,b) Normalized calcium signals (a) and forces (b) of Piezo1-N2A cells in response to mechanical pulling with cantilevers functionalized with Matrigel (n = 11), collagen IV (Col IV) (n = 11), Laminin EHS (n = 30), or Laminin 551 (n = 20). Normalized calcium signals were calculated as described (Materials and Methods) and the noise level is shown in gray. Dashed lines show Ca2+(I/I0) = 0 and F = 0 nN. Dots represent single cells. Boxes show median and first and third quartiles. Nonparametric one-way ANOVA (Kruskal−Wallis) test. ****, P < 0.0001; ns, P > 0.05.

beads and cantilevers with Matrigel, a mix of different ECM proteins (Figure 3a).36 The functionalization of the beads was sufficient to last several rounds of mechanical stimulation experiments (Supporting Information Figure 4). To prevent wear or contamination effects, each ECM-functionalized bead and cantilever was exchanged after five stimulation experiments. Initially, Matrigel-functionalized beads had no effect on Piezo1 receptor activation when mechanically pushing the cellular membrane (Figure 3c,d). Threshold forces for mechanically

stimulus decreased from 83% before calcium depletion to 7% after calcium depletion (Figure 2e). Therefore, the mechanically induced calcium signal is linked to calcium influx across the membrane. ECM Sensitizes Piezo1 Receptor Response to Mechanical Pulling. In their natural environment, cells are surrounded by neighboring cells and ECM. To test the role of the ECM in our mechanical stimulation assay, we wanted to provide a range of ECM proteins to the cells. Therefore, we first functionalized 2067

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Figure 5. Model for ECM-mediated sensitization of Piezo1 receptors. (a−d) Summary of four different modes to mechanically stimulate Piezo1. (a) Pushing a nonfunctionalized micrometer sized bead onto a Piezo1-N2A cell mechanically activates Piezo1 receptors. Upon activation of the Piezo1 receptors, the cell shows a calcium response. This mechanical activation of the Piezo1 receptor through pushing the cell membrane requires high forces of ∼220 nN. (b) Pushing a micrometer-sized bead that has been functionalized with ECM proteins (Matrigel) onto a Piezo1-N2A cell requires equally high forces to mechanically stimulate Piezo1 receptors as observed for nonfunctionalized beads. (c) Functionalizing the bead with non-ECM adhesive proteins like concanavalin A (ConA) and allowing the bead to adhere to Piezo1-N2A cells enables pulling the cellular membrane. Cells stressed by nonspecific mechanical pulling forces show no calcium response and Piezo1 receptors are thus not activated. (d) Functionalizing the bead with ECM proteins such as Matrigel or collagen IV allows the bead to adhere to Piezo1-N2A cells and enables pulling the cellular membrane as well. In this mode, Piezo1 receptors are activated and the cell shows a strong calcium response. Piezo1 receptor activation by mechanical pulling occurs at much lower forces of ∼33 nN compared to activation via mechanical pushing. Forces are indicated by blue arrows and calcium signals by the coloring of the cell (red, response; gray, no response). Piezo1 receptors are shown in orange, ECM proteins (Matrigel and collagen IV) in blue, and non-ECM adhesive proteins in green.

(pushing versus pulling) or due to the conjugation of the cellular membrane with ECM proteins. To test this, we replaced Matrigel with concanavalin A (ConA), a carbohydratebinding protein that strongly adheres to sugar residues exposed by cellular membranes.38 Using ConA-functionalized beads, we could apply comparable pulling forces to membranes of Piezo1N2A cells. However, we could not observe any increased calcium signal in response to mechanical stimulation (Supporting Information Figure 5). Similarly, nonfunctionalized beads failed to induce calcium signal response and control N2A cells did not respond to stimulation with Matrigel either. Taken together, the data suggest that the force sensitivity of Piezo1 receptors depends on the directionality of the mechanical stimulus and the presence of ECM proteins. Collagen IV Sensitizes Piezo1 Receptors. Matrigel is composed of several different ECM proteins including laminins, collagens, and proteoglycans.39 This prompted us to test whether ECM proteins individually or a combination of ECM proteins mediates the observed increase in sensitivity to mechanical stimulation. First, we tested laminins that are the major constituent of Matrigel (∼60%).39 However, functionalizing the bead with a mix of different laminins (EHS laminin) or a single subtype of laminin (laminin 551) had no effect on mechanical sensitivity of Piezo1-N2A cells (Figure 4a), despite reaching pulling forces similar to Matrigel-functionalized beads

pushing Piezo1-N2A cells with Matrigel-functionalized cantilevers 220.8 ± 11.4 nN (n = 12) were the same as for uncoated beads 219.9 ± 8.9 nN (n = 21). Next, we reversed the direction of the mechanical stimulus and pulled the cellular membrane (Figure 3a,b). To pull the cell membrane, we lowered the ECM-functionalized bead onto the cell until reaching a contact force of ∼20 nN, which was well below the pushing force required to mechanically activate Piezo1 receptors (Figure 2). The cantilever was then kept at this force for 60 s to allow the cell to adhere to the ECM-functionalized bead.37 Finally, the cantilever was rapidly retracted (≈ 100 μm s−1) to locally stretch the cell membrane adhering to the bead (Figure 3b, Supporting Information Figure 2b). Retraction of the cantilever caused a strong calcium signal in Piezo1-N2A cells (Figure 3c,d and Figure 4, Supporting Information Movie 2). Notably, when pulling with Matrigel-functionalized beads, Piezo1 receptors were activated by pulling forces of 33.0 ± 3.7 nN (Figure 3d), which were much smaller compared to Piezo1 activation by pushing forces of 219.9 ± 8.9 nN (Figure 3d). Despite the considerable differences in forces required to stimulate the cells by pushing and pulling their membrane with functionalized and nonfunctionalized beads, all mechanically stimulated cells showed similarly strong calcium signals (Figure 3c,d). We next asked if the differences in activation forces were due to the opposing direction of the applied mechanical stimulus 2068

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Collagen IV Sensitizes Piezo1 Receptors to Mechanical Pulling. We found that the sensitivity of Piezo1 receptor overexpressing cells to mechanical pushing forces could not be further increased by functionalizing the bead with Matrigel, which contains several ECM proteins. We hence tested other mechanical stimuli. We observed that by reversing the direction of the stimulus and mechanically pulling instead of pushing the membrane the force thresholds reduced by nearly 1 order of magnitude to ∼33 nN. However, this effect could only be observed when the cells adhered to beads functionalized with Matrigel (Figure 5). Cells adhering to beads functionalized with non-ECM proteins did not show enhanced calcium signals in response to mechanical pulling. We tested various ECM proteins and found that collagen IV alone can sensitize Piezo1 receptors to mechanical stimulation. As this mechanical sensitization through collagen IV is as efficient as that observed for Matrigel, we conclude that collagen IV, which as a component of the basal lamina, forms a cohesive network and mechanical connection between cells, sensitizes Piezo1 receptors to mechanical pulling. Currently, we do not know whether collagen IV modulates Piezo1 receptor sensitivity by binding to it directly or indirectly by signaling through neighboring membrane bound proteins. Our observation that auxiliary proteins can sensitize Piezo1 receptors to mechanical stimulation is in agreement with recent studies reporting that the membrane bound scaffold protein stomatin-like protein 3 (STOML3) can sensitize Piezo receptors22 and that laminin-binding protein filaments are required for mechanosensation in primary sensory neurons.23 Nevertheless, the gating mechanism of Piezo receptors is still a matter of debate.12,14,17 The tether model16,22,23,42 and the force through lipid model18,19,21 have been proposed to explain gating of Piezo1 receptors. To date studies have provided evidence for one or the other model. Here, we provide evidence for the coexistence of both models. In our study, we identify two modes of Piezo1 receptor gating, which, dependent on the environmental context, operate at different force regimes and respond to opposite directions of forces. We find that Piezo1 receptors can be gated by an unspecific mechanism when pushing the cellular membrane in absence of the ECM. This mode of Piezo1 activation can be explained by the “force through lipid” model and high pushing forces of ≈220 nN are needed to gate the Piezo1 receptor in this context. However, we also find that Piezo1 receptors are gated by another mechanism when adhering to ECM proteins, particularly collagen IV, and stimulated by pulling on the cellular membrane. In this context, Piezo1 receptors are activated by much smaller and ligand specific pulling forces of ∼35 nN, which can be explained by the tether model. On the basis of our insights, we conclude that in the absence of ECM proteins Piezo1 receptors are gated by forces transmitted through pushing the cellular membrane, while in the presence of ECM proteins Piezo1 receptors are modulated by collagen IV and thus become sensitive to much smaller mechanical pulling forces (Figure 5). It will be interesting to test whether ECM proteins enhance force sensitivity by binding directly to Piezo1 receptors or indirectly via associated intracellular protein or signaling pathways. Materials and Methods. Cell Culture. Human embryonic kidney HEK cells (HEK-293T, ATCC #11268) and mouse neuroblastoma N2A cells (ATCC #131) were seeded on glassbottom Petri dishes (FluoroDish; WPI) in DMEM (highglucose, GlutaMAX supplement, pyruvate; Life Technologies),

(Figure 4b). In contrast, we found that functionalizing the beads with collagen IV, another constituent of Matrigel (∼30%),39 induced strong calcium signals in response to pulling the membrane (Figure 4a). The cellular calcium responses (1.3 ± 0.2 I/I0; n = 11) were induced at low pulling forces of 31.3 ± 3.6 nN (n = 11). Both the responses and the pulling forces were very similar to that observed using Matrigelfunctionalized beads (Figure 4). For control N2A cells expressing only endogenous Piezo1 we did not observe any responses to the mechanical pulling of the cellular membrane using collagen IV-functionalized beads (Supporting Information Figure 5). Taken together, the similarity of Matrigel- and collagen IV-mediated Piezo1 receptor sensitization and the absence of laminin-mediated effects on Piezo1 receptors suggests that collagen IV sensitizes Piezo1 to pulling forces. Conclusion. AFM-Based Mechanical Stimulation and Optical Readout of Piezo1 Activity in Live Cells. The structural and electrophysiological characterization of Piezo1 receptors has majorly progressed in recent years.2,11,18,24 However, the methods to characterize force sensing and gating mechanisms of Piezo receptors applied mechanical pushing or pulling with limited force and positional control and were mostly restricted to lipid membranes, membrane patches, or membrane blebs. These highly artificial systems lack cellular components including cytoskeleton and ECM and thus remove Piezo receptors from their native environment. To overcome these limitations, we developed an AFM-based assay with nanonewton force control and nanometer positional accuracy that can be applied to live cells. By functionalizing a bead mounted to an AFM cantilever with ECM proteins, our assay could stimulate Piezo1 receptors biochemically and mechanically thereby mimicking the physiochemical environment of a cell embedded in the ECM. With this setup, we applied pushing and pulling forces to cells while monitoring the functional response of Piezo1 receptors in real-time using calcium imaging. Gating Piezo1 Receptors by Mechanical Pushing Requires Physiologically Relevant Forces. Our experiments show that HEK cells not overexpressing Piezo1 receptors do not respond to mechanical pushing forces up to 500 nN. The mechanical load applied in our experiments does not damage or rupture the cell membrane as this would lead to a change in fluorescence or morphology, which was not observed (Supporting Information Figure 3). N2A cells in contrast show clear calcium signals in response to pushing forces above 400 nN. This mechanosensitive response is likely due to endogenous Piezo1 receptors expressed in N2A cells.7 Overexpression of Piezo1 receptors caused HEK and N2A cells to respond to pushing forces of ∼180−220 nN. Applying a force of ∼220 nN to push a bead having a diameter of 5 μm onto a cell surface corresponds to a pressure of ∼41.85 mmHg (Supporting Information Note 1). This pressure lies within the range of physiological pressures encountered in tissues having high mRNA levels of Piezo1.7 In the human bladder, for instance, a recent study measured pressures ranging from 1−22 cm H2O, which correspond to 0.73−16.88 mmHg (Supporting Information Note 2).40 For the human lung, a study found maximal expiratory pressure values of 143 ± 10 cm H20, which amounts to 97.83−112.54 mmHg (Supporting Information Note 2).41 Lung pressures are thus ∼2−3 times higher than the pressure at which Piezo1 receptors gated in our AFM experiments. This leads to the conclusion that the mechanical stimuli applied in our experiments are physiologically relevant. 2069

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CellHesion software. For the push stimulus, the AFM lowered the bead on the cantilever onto the cell with a speed of 10 μm s−1 until the force reached the set point, held the set point force at constant force for 250 ms, and then retracted with a speed of 100 μm s−1. Set point forces were applied in intervals from 100−400 nN with 50 nN increments and time between intervals ranged from 10−25 s. For the pull stimulus, the ECMcoated cantilever with bead was lowered onto the cell until reaching a force of 20−25 nN at which it was kept for 60 s and then retracted at a speed of 100 μm s−1. Separation of bead and cell resulted in rupture forces as measured by the deflection of the cantilever. Detailed experimental parameters during push and pull stimulation are shown in Supporting Information Figure 2. Functionalized cantilevers were changed after stimulation of five cells to avoid cell debris masking the functionalization of the beads.44 Nonfunctionalized cantilevers were not changed during the experiment. Data Analysis. To classify responding and nonresponding cells, we mechanically stimulated N2A cells expressing GFP only (Supporting Information Figure 1), recorded the fluorescent signal, and analyzed the data according to eq 1 (Supporting Information Figure 6). Mechanical stimulation of cells caused cytosolic fluorophores to move in or out of the confocal imaging plane, creating fluorescence artifacts. We used these fluorescence artifacts to determine the experimental noise level for the assay (Supporting Information Figure 6). All cells having normalized fluorescence greater than the noise threshold of I/I0 > 0.18 for pushing stimulus and I/I0 > 0.25 for pulling stimulus were defined as “responding”; all cells that fell below the threshold were defined as “non-responding”. Statistical Analysis. Data was plotted using Profit software (Quantum Soft). Statistical analysis was performed with the Prism 6 (GraphPad). The nonparametric two-tailed Mann− Whitney test was used in Figure 2 and the nonparametric oneway ANOVA (Kruskal−Wallis) test was used for Figures 3 and 4.

supplemented with 10% (v/v) fetal bovine serum (FBS, life Technologies), 100 μg mL−1 penicillin and 100 μg mL−1 streptomycin (Life Technologies), and cultured at 37 °C and 5% CO2 until further use (24−48 h). Cells were transiently transfected with a CMV-jRCaMP1a plasmid (500 ng) alone, a CMV-GFP plasmid (500 ng) alone or cotransfected with the CMV-jRCaMP1a plasmid and a CMV-mousePiezo1-IRES-GFP plasmid (250 ng) using polyethylenimine (PEI, Chemie Brunschwig AG) or lipofectamine 2000 for N2A and HEK cells, respectively, 24−48 h before recording. Piezo DNA constructs were kindly provided by A. Patapoutian (San Diego), and jRCaMP1a DNA was ordered from Addgene (# 61562). Confocal Microscopy and Calcium Imaging. Confocal imaging was performed using an inverted laser-scanning microscope (LSM 700, Zeiss) equipped with a 25×/0.8 LCI PlanApo water immersion objective (Zeiss). Lasers and channels were assigned using the “best signal” option to ensure there was no spectral overlap between them. Transfected cells were located and regions of interest around probed cells were assigned to maximize the acquisition speed. Time-lapse images were acquired with 100−300 ms time resolution. To minimize cell-to-cell variability, a Z-stack scan was performed to determine the center of the cell before starting mechanical stimulation. Time-lapse imaging was initiated >10 s before the onset of the mechanical stimulus. Time-lapse images of calcium responses were analyzed using the built in ZEN blue software. The calcium signal was calculated as follows ⎛I⎞ |I(peak) − I(baseline)| Ca 2 +⎜ ⎟ = I(baseline) ⎝ I0 ⎠

(1)

With Ipeak being the maximum fluorescent intensity, and IBaseline being the average fluorescent intensity of 5 s preceding the stimulus. The calcium chelator BAPTA (Enzo Life Sciences) was applied at 1 mM concentration in 1% DMSO, 99% culture medium. Cells were incubated for 15 min with BAPTA before stimulation. Cantilever and Bead Functionalization. Five micrometer diameter silica beads (Kisker Biotech) were glued to the tip of the cantilever using UV glue (Dymax) and cured under UV light for 20 min. Cantilevers with beads were plasma treated for 5 min using a plasma cleaner (Harrick Plasma) to ensure a clean surface. After this, ECM proteins were physisorbed to bead and cantilever by dipping the plasma-cleaned cantilever into a 25 μL drop containing the protein at concentrations and incubation times given as the following: Matrigel (BD biosciences) was diluted to 50 μg mL−1 in PBS and incubated for 1 h at room temperature. Concanavalin A (Sigma-Aldrich) was diluted to 2 mg mL−1 in PBS and laminin EHS (SigmaAldrich), laminin 551 (Biolamina), and collagen IV (Advanced BioMatrix) were diluted to 50 μg mL−1 in PBS and incubated for 2 h at 37 °C. Functionalized cantilevers were gently rinsed with PBS before mounting to the AFM. Atomic Force Microscopy. An AFM (CellHesion 200, JPK Instruments) was mounted on an inverted confocal microscope (Observer Z1, LSM 700, Zeiss). Cells were kept at 37 °C using a Petri dish heater (JPK Instruments). Tipless microcantilevers (CSC-37, Micromash HQ) with 5 μm beads glued to the free end of the cantilever were mounted on a standard glass cantilever holder (JPK Instruments) of the AFM. Cantilevers were calibrated using the thermal noise method.43 Mechanical stimulation protocols were programmed using the JPK

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.7b00177. Comparison of Piezo1 sensitivity values from literature (Supporting Information Note 1). Physiological pressures encountered in human tissue expressing native Piezo1 receptors (Supporting Information Note 2). Transient Piezo1, jRCaMP1a, and GFP expression in HEK and N2A cells (Supporting Information Figure 1). Experimental parameters for push and pull protocols (Supporting Information Figure 2). Experiments mechanically indenting control HEK cells without Piezo1 (Supporting Information Figure 3). Lifetime of fluorescently labeled ECM proteins on cantilever (Supporting Information Figure 4). Piezo1 receptors do not respond to pulling with unspecific adhesive proteins (Supporting Information Figure 5). Fluorescence artifacts from mechanical pushing and pulling (Supporting Information Figure 6). Summary of the number of stimulated cells and number of responding cells for various experimental conditions (Supporting Information Table 1) (PDF) 2070

DOI: 10.1021/acs.nanolett.7b00177 Nano Lett. 2017, 17, 2064−2072

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Nano Letters

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Representative example of Piezo1-N2A response to push stimulus (AVI) Representative example of Piezo1-N2A response to pull stimulus with collagen IV functionalized cantilever (AVI)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: (B.M.G) [email protected]. *E-mail: (D.J.M.) [email protected]. ORCID

Daniel J. Müller: 0000-0003-3075-0665 Author Contributions

B.M.G. and D.J.M. designed the experiment. B.M.G. performed the experiments. B.M.G. and D.J.M. wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank A. Patapoutian for kindly providing DNA constructs of the Piezo1 receptor and the Addgene repository for DNA constructs of genetically encoded calcium indicators. We thank N. Strohmeyer, T. Strittmatter, C. Cattin, G. Flaeschner, D. Martinez-Martin, A. Lomakin and R. Schubert for constructive ideas and discussion. We thank EMBO for support in form of a long-term fellowship (ALTF 424-2016) provided to B.G. This work was supported by the NCCR Molecular Systems Engineering.

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