Figure 2. Piezo1 does not cluster in red blood cells (RBCs).

(A) Representative 2D stimulated emission depletion (STED) image of an unroofed RBC immunostained with an anti-HA tag antibody for Piezo1 (green) and labeled with phalloidin STAR 580 for F-actin (magenta). Scale bar, 1 μm. (B) 3D reconstruction from SIM imaging of immunostained RBCs, where Piezo1 spots are detected by the spot detector function in Imaris and are colored yellow. The distance from each Piezo1 spot to the nearest Piezo1 spot in this cell was calculated in Imaris and is shown on the right. Scale bar, 1 μm. (C) The same analysis as in (B) was carried out on a 2D STED image of an unroofed RBC, but here Piezo1 spots at the edge of the membrane were masked out from spot detection to prevent edge-based artifacts in estimating nearest neighbor distances. Scale bar, 1 μm. (D) The mean nearest spot distance was calculated for SIM and STED analyses and plotted. For SIM the mean is 540 ± 37 nm (n = 19 cells) and for STED the mean is 544 ± 106 nm (n = 12 cells). (E) (Left) Huygens deconvolution of a Piezo1 puncta, imaged by 2D STED, resolving into a triplet after deconvolution, scale bar is 50 nm. (Bottom right) A top-down view of the Piezo1 channel structure with red asterisks indicating the positions of antibody binding. (Top right) An intensity profile of fluorescent signal from a line-scan of two adjacent bright pixels from the bottom left image, showing a distance of ~25 nm. (F) Negative stain electron microscopy of an unroofed RBC immunostained with an anti-HA tag primary antibody and a secondary antibody conjugated to 18-nm gold. Low magnification image of an unroofed RBC (left) with a region highlighted in red imaged at medium magnification (right). 18-nm gold particles corresponding to labeled Piezo1 channels are highlighted by red circles. As in fluorescence microscopy images, Piezo1 channels do not appear to cluster. (G) Negative stain electron microscopy image at high magnification of a triple-labeled Piezo1 channel.

Figure 2—figure supplement 1. Cluster analysis of fluorescent Piezo1 spots in a 2D stimulated emission depletion (STED) image of a labeled unroofed red blood cell (RBC) membrane.

(A) Representative 2D STED image of an unroofed RBC membrane immunostained with an anti-HA tag antibody for Piezo1 (green) and labeled with phalloidin STAR 580 for F-actin (magnta). Scal bar, 1 μm. (B) Binarized mask of the RBC membrane using thresholding of the rhodamine–phalloidin (left) and binarized thresholding of fluorescent Piezo1 spots (right). (C) Cluster analysis using the spatial statistics 2D/3D ImageJ plugin. The cumulative distribution frequency of the distance between a typical position within the masked reference structure and its nearest point in the pattern is plotted on the y-axis against the distance, x-axis, for a simulated random spatial distribution (black) with 95% confidence limits (gray) compared to the observed experimental distribution (green). There is no statistical difference between the simulated randomly distributed data and observed data. N = 1 cell.

Figure 2—figure supplement 2. 2D STED and Huygens deconvolution analysis of immunolabelled Piezo1 in unroofed RBCs.

(A) Two representative sets of images of Piezo1 fluorescent spots after stimulated emission depletion (STED) data collection (top panels) followed by Huygens deconvolution (bottom panels) Piezo1 spots resolve into singlets, doublets, or triplets, with the bright pixels separated by approximately 25 nm. Scale bar, 50 nm. (B) After selecting Piezo1 fluorescent puncta with a max intensity above 20,000 arbitrary units we counted the putative number of antibodies bound based on resolved bright pixels where n = 33 fluorescent puncta from 3 unroofed cells. Here, we show a relative frequency distribution for 1, 2, or 3 antibodies bound. Assuming a binomial distribution for antibody binding, our data are consistent with approximately 60% antibody binding.