Fig. 5

Actin and α-actinin-1 lateral distribution. a Schematic representation of the different FA layers previously identified using iPALM in U2OS cells, colour bars highlight the z range for each of the three layers and the actin-regulatory layer containing actin and α-actinin-1 is emphasised over other layers. b Two-colour iPALM images of Eos-tagged actin and endogenous paxillin in a cornerstone FA. One individual cornerstone FA is displayed. Where localisation of actin is displayed separately, top-view and side-view images are colour-coded as a function of the z-position of the actin molecules. Where fluorescence channels are merged (paxillin, red; actin, green), the z position is only displayed in the side view and the colours represent the fluorescence signal for each protein. Scale bar 1 μm. c Z density profile of paxillin (red) and actin (green) displaying the number of localisations as a function of the z position in an individual cornerstone adhesion. Dotted lines correspond to the experimental data, while solid lines correspond to the fitted data obtained using either a single Gaussian distribution (paxillin) or a sum of two Gaussian distributions (actin). Dashed black lines highlight these two Gaussian distributions. d iPALM image of Eos-tagged α-actinin-1 in an individual hPSC cornerstone FA. Top-view and side-view images are colour-coded as a function of the z-position of the α-actinin-1 molecules. Scale bar 1 μm. e Z-density profile of α-actinin-1 (purple) showing the number of localisations as a function of the z position. Dotted line corresponds to the experimental data while the solid line corresponds to the fitted data obtained using a sum of two Gaussian distributions (dashed black lines). f iPALM analysis of the Z_centre of actin (actin low n = 28 biologically independent ROIs, actin high n = 47 biologically independent ROIs) and α-actinin-1 (α-actinin high n = 31 biologically independent ROIs, α-actinin low n = 27 biologically independent ROIs) in hPSC cornerstone adhesions. “Low” and “High” denote separate peaks in the distribution of the same protein. Source data are provided as a Source Data file. Boxes display the median, plus the 1st and 3rd quartiles (IQR: 25th–75th percentile). Whiskers correspond to the median ± 1.5 × IQR