Fig 2: KANK1-talin interaction is required for recruiting CMSCs to FAs.(A) Widefield fluorescence images of HeLa cells depleted of KANK1 and KANK2 and expressing the indicated siRNA-resistant GFP-KANK1 fusions (rescue), stained for the FA marker phospho-tyrosine (pY). (B) Widefield fluorescence images of HeLa cells transfected with the control siRNA or siRNAs against KANK1 and KANK2, expressing GFP alone or the indicated siRNA-resistant GFP-KANK1 fusions and stained for LL5ß or KIF21A. (C) Quantification of peripheral clustering of LL5ß and KIF21A in cells treated as in panel (B) (n=12, 5–6 cells per condition). (D) TIRFM images of live HeLa cells depleted of KANK1 and KANK2 and co-expressing the indicated siRNA-resistant GFP-KANK1 fusions and mCherry-CLASP2. (E) Quantification of peripheral clustering of mCherry-CLASP2 in cells treated as in panel (D) (n=20, 8 cells per condition). (F) Widefield fluorescence images of HeLa cells transfected with the indicated GFP-KANK1 fusions and stained for endogenous LL5ß. (G) Quantification of peripheral clustering of LL5ß in cells treated as in panel (F) (n=12, 6 cells per condition). (H) Widefield fluorescence images of HeLa cells transfected with GFP-tagged KANK1 or its CC1 mutant and stained for LL5ß. (I) Quantification of peripheral clustering of LL5ß cells treated as in panel (H) (n=12, 6 cells per condition). Error bars, SEM; ns, non-significant; **p<0.005; ***p<0.001, Mann-Whitney U test.DOI: http://dx.doi.org/10.7554/eLife.18124.01610.7554/eLife.18124.017Figure 4—source data 1.An Excel sheet with numerical data on the quantification of peripheral clustering of different markers represented as plots in Figure 4C,E,G,I.DOI: http://dx.doi.org/10.7554/eLife.18124.017

Fig 3: The KN motif of KANK1 interacts with the R7 domain of talin1.(A) Schematic representation of KANK1 and the deletion mutants used in this study, and the summary of their interactions and localization. N.d., not determined in this study. (B) TIRFM images of live HeLa cells transiently expressing the indicated GFP-tagged KANK1 deletion mutants together with the focal adhesion marker mCherry-paxillin. In these experiments, endogenous KANK1 and KANK2 were also expressed. (C) Identification of the binding partners of Bio-GFP-tagged KANK1 and its indicated deletion mutants by using streptavidin pull down assays from HEK293T cells combined with mass spectrometry. (D) Streptavidin pull down assays with the BioGFP-tagged KANK1 or the indicated KANK1 mutants, co-expressed with GFP-talin1 in HEK293T cells, analyzed by Western blotting with the indicated antibodies. (E) Sequence alignment of KANK1 and KANK2 with the known talin-binding sites of DLC1, RIAM and Paxillin. The LD-motif and the interacting hydrophobic residues are highlighted green and blue respectively. (F) Schematic representation of talin1 and the deletion mutants used in this study, and their interaction with KANK1. (G) Streptavidin pull down assays with the BioGFP-tagged talin1 or the indicated talin1 mutants, co-expressed with HA-KANK1 in HEK293T cells, analyzed by Western blotting with the indicated antibodies.DOI: http://dx.doi.org/10.7554/eLife.18124.003

Fig 4: Overall vertical stratification of cornerstone FA. a Z positioning (Zcentre) of aV and ß5 integrins, paxillin, vinculin, talin-1, actin, a-actinin-1, kank1 and kank2 in cornerstone adhesions. Letters –N and –C denote the location of the tag while “low” and “high” denote separate peaks in the distribution of the same protein. Boxes display the median, plus the 1st and 3rd quartiles (IQR: 25th– 75th percentile). Whiskers correspond to the median ± 1.5 × IQR. b Schematic model of the 3D architecture of hPSC cornerstone adhesions. The lateral and vertical positioning of each protein are based on the data presented here. This model does not depict protein stoichiometry. Source data are provided as a Source Data file

Fig 5: The role of talin-KANK1 interaction in regulating microtubule plus end dynamics around FAs.(A) Schematic representation of the pattern of microtubule growth in control HeLa cells and in cells with perturbed CMSCs, based on (van der Vaart et al., 2013). (B) TIRFM images of live HeLa cells depleted of KANK1 and KANK2 and co-expressing the indicated siRNA-resistant GFP-KANK1 fusions and EB3-mRFP. Images are maximum intensity projection of 241 images from time lapse recording of both fluorescence channels. (C) Distributions of microtubule growth rates at the 3 µm broad cell area adjacent to the cell edge, and in the central area of the ventral cortex for the cells treated as described in (B) (n=87–153, 7–8 cells). (D) Ratio of microtubule growth rate in the cell center and at the cell edge for the cells treated as described in B (n=7–8 cells). (E) Angles of microtubule growth relative to the cell margin for the cells treated as described in B. Box plots indicate the 25th percentile (bottom boundary), median (middle line), mean (red dots), 75th percentile (top boundary), nearest observations within 1.5 times the interquartile range (whiskers) and outliers (black dots) (n=93–114, 7–8 cells). (F) TIRFM images of live HeLa cells depleted of talin1 and talin 2 and co-expressing the indicated GFP-talin1 fusions and EB3-mRFP. Images are maximum intensity projection of 241 images from time lapse recordings of both fluorescence channels. (G) Distributions of microtubule growth rates at the 3 µm broad cell area adjacent to the cell edge, and in the central area of the ventral cortex for the cells treated as described in F (n=88–154, 7 cells). (H) The ratio of microtubule growth rate in the cell center and at the cell edge for the cells treated as described in panel (F) (n=7 cells). (I) Angles of microtubule growth relative to the cell margin for the cells treated as described in F. Box plots as in (E) (n=155–166, 10 cells). In all plots: error bars, SEM; ns, non-significant; **p<0.01; **p<0.005; ***p<0.001, Mann-Whitney U test.DOI: http://dx.doi.org/10.7554/eLife.18124.01810.7554/eLife.18124.019Figure 5—source data 1.An Excel sheet with numerical data on the quantification of different aspects of microtubule organization and dynamics represented as plots in Figure 5C–E,G–I.DOI: http://dx.doi.org/10.7554/eLife.18124.019