Fig. 3 | Nature Communications

Fig. 3

From: Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells

Fig. 3

Lateral and vertical segregation of proteins within cornerstone FA. ac Interferometric photo-activated localisation microscopy (iPALM) images of Eos-tagged integrin β5 (a), paxillin (b), and talin-1-N (N-terminally tagged talin-1) (c) in cornerstone FA. Individual cornerstone FA are displayed. Both top-view (xy) and side-view (xz) images are displayed. White boxes indicate the area used to generate the side-view images (in the absence of a white box, the entire image was used). For all images, individual localisations are colour-coded as a function of their z-positioning (distance from the coverslip). In addition, normalised line intensity profiles of the region of interest, in both z- and x-axis, are shown in red lines. d hPSC plated for 24 h on VTN were stained for endogenous integrin β5 and active integrin β1 (mAb 12G10) and imaged using an Airyscan confocal microscope. The yellow line highlights the area used to measure the intensity profiles displayed on the side. Scale bars (a) 1 μm, (b, c) 2 μm, (d) 5 μm. e Schematic representation of the different FA layers previously identified using iPALM in U2OS cells13, colour bars highlight the z range for each of the three layers and the integrin signalling layer is emphasised over other layers. f iPALM analysis of the z position (distance from the coverslip, Zcentre) of some of the integrin signalling layer components, Eos-tagged integrins αV (n = 17 biologically independent ROI. Given that cornerstone FAs are clusters of FAs, one ROI may contain multiple FAs. For details on the number of localisations, and HPSC colonies see supplementary Table 1) and β5 (n = 22 biologically ROI), and Eos-tagged paxillin (n = 23 biologically independent ROI), in hPSC cornerstone adhesions. Boxes display the median, plus the 1st and 3rd quartiles (IQR: 25th– 75th percentile). Whiskers correspond to the median ± 1.5 × IQR. Source data are provided as a Source Data file

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