Force-induced conformational changes in PIEZO1

Abstract

PIEZO1 is a mechanosensitive channel that converts applied force into electrical signals. Partial molecular structures show that PIEZO1 is a bowl-shaped trimer with extended arms. Here we use cryo-electron microscopy to show that PIEZO1 adopts different degrees of curvature in lipid vesicles of different sizes. We also use high-speed atomic force microscopy to analyse the deformability of PIEZO1 under force in membranes on a mica surface, and show that PIEZO1 can be flattened reversibly into the membrane plane. By approximating the absolute force applied, we estimate a range of values for the mechanical spring constant of PIEZO1. Both methods of microscopy demonstrate that PIEZO1 can deform its shape towards a planar structure. This deformation could explain how lateral membrane tension can be converted into a conformation-dependent change in free energy to gate the PIEZO1 channel in response to mechanical perturbations.

Conflict of interest statement

The authors declare no competing interests.

Figures

Extended Data Fig. 1.. Architecture and topology of mechano-sensitive channel Piezo1.

(a) Top, bottom and side views of Piezo1 (PDB 6B3R) in cartoon representation (top) and embedded in the micelle density map (EMD-7042) contoured at 6σ. CED: C-terminal extracellular domain, CTD: C-terminal domain. (b) Top: Topology of Piezo1, rainbow-colored with N-terminus in blue and C-terminus in red, except for structurally unsolved TM 1–12 regions in grey. Helices are represented as cylinders, loops as solid lines, and unresolved regions as dotted lines. Bottom: Top view of TMs, labeled as in the topology. Red squares outline 4-TM units that constitute the arm. TM 21–24 is at the ‘elbow’ of the arm. The hypothetical position of the unresolved units TM 1–4, TM 5–8 and TM 9–12 are indicated (dashed outline).

Extended Data Fig. 2.. Image processing procedure to determine the radius of curvature of side-view Piezo1 channels and the intrinsic radius of curvature of the vesicles in which they are embedded.

(a) The input 2D averaged image of one side of the vesicle (here Piezo1 occupied side). (b) Edge detection output using the Canny method. (c) Edge detection output overlaid with the selection polygon. (d) Edges in the selected polygon region annotated with numbers in different colors for easier identification. (e) and (f) Measured (blue circle) and fitted (red dotted line) circles of the edges corresponding to the outer (e) and inner (f) boundaries of the vesicle membrane. Center coordinates, radius and its 95% confidence interval are shown. The unit of all values is in pixels. (g) Fitted circles (red dashed lines) overlaid onto the edge detection output. Radii with the confidence interval of outer and inner boundaries are shown in units of nm. (h) Fitted circles (red dashed lines) overlaid onto the input 2D averaged image.

Extended Data Fig. 3.. Estimation of tip-sample interaction force (Fts) in HS-AFM at various Aratio(=Aset/Afree) based on experimental tip motion analysis and numerical simulation.

(a) and (b) Average tip motions observed at different Aratio in buffer solution during HS-AFM imaging on mica (a) and a supported lipid bilayer (DOPC/DOPS; 4:1) (b), respectively. (c) and (d) Forces caused by the elastic response of the cantilever (top), the hydrodynamic damping with the medium (middle) and the total force that governs the tip motion (bottom). (e) and (f) Sum of the drive force and tip-sample interaction force. (g) and (h) Reconstructed Fts trajectories during a single oscillation cycle based on the point mass model (Equations 7 and 8, Methods). (i) Comparison between peak forces obtained from reconstructed Fts trajectories on mica (blue dashed line) and membrane (red dashed line), and peak forces simulated using different surface stiffness using VEDA: Virtual Environment for Dynamic AFM (https://nanohub.org/resources/veda) (dashed lines and grey shadowed area). The VEDA simulation is performed by using the amplitude modulation approach curve tool with the following settings: discrete approach steps within a defined z-range, acoustic excitation, Afree=2 nm, Hertz contact model (Etip=130 GPa, νtip=0.3, νsample=0.5, ν is the Poisson’s ratio) with a tip radius of 1 nm, and other HS-AFM experimental parameters, e.g. k, Q, and ω0. (j) Comparison between average forces obtained from reconstructed Fts trajectories on mica (blue dashed line) and membrane (red dashed line) and values calculated through Equation 1 (thick black dashed line). (k) Comparison between peak forces obtained from experimental Fts trajectories on membrane at different Afree values. Using second order polynomial fitting, the peak force reconstructed in the condition of Afree=1.5 nm can be well described by y = −688.7x2 + 633.4x + 55 (black dashed line) with R2 = 0.99. This fitting allows us to estimate the upper bound of force, peak force, applied to Piezo1 channels at any given Aratio. Tip trajectories are representative of ≥5 independent experiments using ≥3 different HS-AFM cantilevers.

Extended Data Fig. 4.. Classification of Piezo1 channels using cross-correlation analysis on characteristic dimensions measured upon force application.

(a) and (b) Cross-correlation density maps of smallest halo radius (Rmin) at low applied force versus largest halo radius (Rmax) at highest applied force (a) and of smallest halo radius (Rmin) versus maximum central height (Hmax) at lowest applied force. Along the diagonal direction of the cross-correlation density maps, the molecules separate into two peaks that suggests the existence of two Piezo1 subtypes with different size in HS-AFM force-sweep movies. Therefore, we assigned the molecules in the peaks as Type-1 (~70%, white-dashed circle) and Type-2 (~30%, yellow-dashed circle) Piezo1, respectively. The same molecules populate in the same subtype according to all analyzed criteria. The total number of analyzed Piezo1 particles is 143 from 11 HS-AFM movies acquired on ≥5 different samples, days and HS-AFM tips.

Extended Data Fig. 5.. Mechanical response of Type-2 Piezo1 to applied force.

(a) HS-AFM image at ‹FHS-AFM› ~52 pN of Type-2 Piezo1 viewed from the extracellular face. (b) Height section profile (top) and radial height profile (bottom) of the topography displayed in (a). (c) Dimensional analysis of single Type-2 Piezo1 particle in a force-sweep HS-AFM experiment. Like the Type-1 Piezo1 reported in the main text, each single molecule (left) is 360-fold symmetry averaged (center) and a kymograph (right) across the center profile (dashed line in 360-fold image) calculated. The kymograph highlights the halo expansion (dashed line) as a function of force. Bottom: Force as function of frame acquisition or time during the force-sweep HS-AFM movie acquisition. The yellow colored area corresponds to the image acquisition of the particle shown (left). (d) Normalized probability density maps of halo radius (R) as a function of force. Type-2 Piezo1 also show the structural reversibility. The total number of analyzed Type-2 Piezo1 particles are 43 from 11 HS-AFM movies acquired on ≥5 different samples, days and HS-AFM tips.

Extended Data Fig. 6.. Piezo1 has an exceptional low 2D-density of TM helices.

Transmembrane (TM) helix 2D-density analysis for channels and transporters of known 3D structure. The TM helix density is estimated by the total TM helices number divided by the occupied membrane area of each known structure. A total of 201 channels and transporters structures have been analyzed. A general trend seems that channels are somewhat less densely packed than transporters, possibly a signature of the existence of a protein-free pore region. The Piezo1 structure is an outlier: It is of exceptional size and has an exceptionally low TM helices density, 2. This unique feature might be a signature and prerequisite for Piezo1 mechano-sensing.

Fig 1.. Proposed activation mechanisms of Piezo1.

(a) Lateral membrane tension model: Changes in membrane properties, e.g. tension or curvature, lead to a gating force applied onto Piezo1. (b) Tethered spring model: Piezo1 channel is activated through interactions with the cytoskeleton or the extracellular matrix. Red arrows indicate force application.

Fig 2.. Reconstitution of Piezo1 in vesicles exhibit various orientations in cryo-EM micrographs.

(a) Piezo1 channels reconstituted in POPC/DOPS/CHOL (8:1:1) vesicles (≥1000 images). Top/bottom-view and side-view particles are highlighted by white and yellow arrowheads, respectively. Inset: magnified, filtered and contrast adjusted view containing a top-view Piezo1 channel with left-handed curved arms in projection (red dashed circle and arrowheads). (b) Averages of the top-view (n=322) and bottom-view (n=120) Piezo1 compared to the structural model (PDB 6B3R). The handedness of the three arms in projection permits the determination of Piezo1 orientation. Scale bars: 20 nm.

Fig 3.. Piezo1 channels become flatter in large vesicles.

(a) Cryo-EM image of a vesicle with a Piezo1 channel in side-view. Membrane curvatures for the region centered on Piezo1 and on the opposite pole (1166 particles). (b) Comparison of the average membrane densities at the opposite pole (top) and at Piezo1 (bottom). Vesicles with 13 nm (n=19), 19 nm (n=25) and 31 nm (n=19) curvature radius (opposite pole) are shown. (c) Circles defining the radius of curvature for outer (red) and inner (blue) membrane leaflets at the opposite pole (top) and at Piezo1 (bottom). (d) The midplane radius of curvature for Piezo1 is graphed against the midplane radius of curvature at the opposite pole (circles and dashed curve). The straight dotted line shows the relationship for spherical vesicles. Data are mean ±95% confidence intervals of the fitted radii (n ≥ 15).

Fig 4.. HS-AFM experiments of Piezo1.

(a) Schematic diagram of force-controlled HS-AFM imaging of membrane-embedded Piezo1: The ratio Aset/Afree defines the average applied force ‹FHS-AFM›. (b) Top: Simulated topographies of Piezo1 in the detergent micelle viewed from the extracellular (left) and the intracellular (right) faces. The membrane was set as a uniform height level extending from the most peripheral resolved TM helices. The three black arrowheads indicate the positions of the three arms. Bottom: Section profiles of the simulated topographies. (c, d) HS-AFM images at specific ‹FHS-AFM› of Piezo1 viewed from the extracellular ((c), ~20 pN and ~50 pN) and intracellular ((d), ~30 pN) faces. Right: Section profiles (red traces) of the topographies. Extracellular face: a central plug surrounded by a bowl with ‘negative height’, i.e. at a level below the surrounding bilayer. The three arms of Piezo1 are observed within the deep ring area when imaged at ~50 pN, as highlighted by the radial profile with ~120° periodicity (green trace). The intracellular face is contoured as a featureless dome. HS-AFM images are representative of 100 particles from ≥5 different samples.

Fig 5.. Mechanical response of Piezo1 to applied force.

(a) Top: HS-AFM images from a force-sweep movie of Piezo1 in a POPE/POPG = 85:15 (w/w) bilayer. Each image is an average over 10 frames acquired at a specific loading force. Bottom: Force (red), Aset/Afree ratio (blue) and Z-piezo displacement (green) as function of frame acquisition time. The 5 yellow colored areas correspond to the image acquisition periods of the frames shown above. Afree~1.5 nm. Representative of ≥11 independent experiments from ≥5 different Piezo1 samples. (b) Example of lateral expansion analysis of a single Piezo1 particle. Each single molecule (left) is 360-fold symmetry averaged (middle) and a kymograph (right) across the center of Piezo1 calculated (white dashed line). The kymograph highlights the outer radius (halo) expansion (black dashed line) as a function of force. Kymograph is representative of ≥100 particles. (c) Normalized probability density map of outer ring radius (R) as a function of force. 100 Piezo1 particles from 11 movies acquired on ≥5 different samples, days and HS-AFM tips. The symmetric distribution of R shows the structural reversibility upon force increase and decrease. A critical force Fc~18 pN is required during HS-AFM operation. (d) Applied force as a function of the sample displacement towards the AFM tip. The linear regression (red dashed line), performed by disregarding the zero-force and negative Δz data points, provides the stiffness constant K~7.0 pN/nm for stressing a spring-like piezo1 based on our proposed dome-flattening model. The green-colored area indicates the work exerted by the HS-AFM measurement for dome-flattening of Piezo1. Data are mean ± s.d. with n≥10.