Fig 2: Lateral and vertical segregation of proteins within cornerstone FA. a–c Interferometric photo-activated localisation microscopy (iPALM) images of Eos-tagged integrin ß5 (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 ß5 and active integrin ß1 (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 aV (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 ß5 (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

Fig 3: Focal adhesions in MDA-MB-231 cells. The gene expression of ITGB1 (A), PTK2 mRNA expression (B), PXN mRNA expression (C) of MDA-MB-231 cells exposed to the RPM. In addition, the gene expression of TLN1 (D) and analysis of the fluorescence intensity of talin (E), IFS of 1g control cells (F) and RPM-exposed MDA-MB-231 cells [AD cells: (G) and MCS: (H)] is given. Moreover, the VCL mRNA (I) and analysis of the fluorescence intensity of vinculin (J), the IFS of vinculin are shown in 1g control cells (K) and RPM-exposed AD cells (L) and MCS (M). *p < 0.05 1g vs. AD and/or MCS and #p < 0.05 AD vs. MCS.

Fig 4: Vinculin distribution and orientation within cornerstone adhesions. 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 force transduction layer containing vinculin and talin is emphasised over other layers. b iPALM analysis of the Zcentre for N-terminally (Talin-1-N, n = 54 biologically independent ROI) and C-terminally (Talin-1-C, n = 8 biologically independent ROI) tagged (Eos) talin-1 in hPSC cornerstone adhesions. c iPALM analysis of the Zcentre of N-terminally (Vinculin-N, n = 55 biologically independent ROI) and C-terminally (Vinculin-C, n = 27 biologically independent ROI) tagged (Eos) vinculin in hPSC cornerstone adhesions. d Two-colour iPALM images of Eos-tagged vinculin (Vinculin-C) and endogenous paxillin in a cornerstone FA. Individual cornerstone FA are displayed. Where localisation of vinculin and paxillin are displayed separately, top-view and side-view images are colour-coded as a function of the z-position of the indicated protein. Where fluorescence channels are merged (paxillin, red; vinculin, 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. e 3D scatter plots displaying the individual iPALM localisations (grey dots) of endogenous paxillin and Eos-tagged Vinculin-N and Vinculin-C within a single cornerstone adhesion. Surface plots present the fit of those localisations using a two-dimensional polynomial equation. Note that the paxillin localisations are homogeneously flat while localisations of both vinculin constructs form a solid paraboloid. f iPALM xy images of Eos-vinculin-C at selected z-layers in an individual cornerstone FA. Scale bar 1 µm. 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

Fig 5: Localization of non-degraded KIND2 in mitotic adhesions.a,b, Stills of live-cell recordings generated by TIRF microscopy showing a representative interphase and mitotic (30 min after RO3306 release) HeLa cell stably expressing EGFP–KIND2 (a) or YPet–talin-1 (b), and Lifeact–mRuby and H2B–CFP. c,d, SIM images (c) of EGFP–KIND2 (green), the ß1 integrin activation-associated 12G10 epitope (red) and phalloidin (magenta) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Co-localization of EGFP–KIND2 with the 12G10 epitope is shown in FAs (arrowheads) of interphase cells and in puncta in retraction fibres and round cell bodies of mitotic cells. To visualize EGFP–KIND2 signals in retraction fibres, the fluorescence was enhanced by increasing the exposure time. Boxes indicate areas of retraction fibres shown at high magnifications. The arrow indicates the direction of line profile analysis (d) of EGFP–KIND2 and 12G10 epitope (red). Each line-profile assessment was done three times. AU, arbitrary unit. e, SIM images of immunostained endogenous KIND2 (green), phalloidin (red) and pH3 (blue) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Arrowheads indicate KIND2 in FAs. KIND2 in mitotic retraction fibres was visualized by increasing the exposure time. Boxes indicate retraction fibre areas shown at high magnifications. f, Schematic of the procedure, depicting the Z positions of round mitotic (30 min after RO3306 release) HeLa cell bodies: region 1, bottom stack; region 3, stack capturing the cortical end of retraction fibres; region 2, intermediate stack between region 1 and 3. Region 1 was recorded by TIRF microscopy and regions 2 and 3 by confocal microscopy. g, Representative image sections at different regions as depicted in f of round mitotic (30 min after RO3306 release) HeLa cell bodies immunostained for the 12G10 epitope or stably expressing EGFP–KIND2, YPet–talin-1 or EGFP. h, Quantification of adhesion assays of untreated and STLC-arrested HeLa cells on FN, VN, fetal bovine serum or BSA coatings. Mean ± s.d.; n = 9 independent experiments. P values calculated by unpaired Student’s t-test (two-tailed). Scale bars, 1 µm (magnifications in c and e) or 10 µm (a–c,e,g).Source data