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Kank2 activates talin, reduces force transduction across integrins and induces central adhesion formation

Nature Cell Biology volume 18, pages 941–953 (2016)Cite this article

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Abstract

Integrin-based adhesions play critical roles in cell migration. Talin activates integrins and flexibly connects integrins to the actomyosin cytoskeleton, thereby serving as a ‘molecular clutch’ that transmits forces to the extracellular matrix to drive cell migration. Here we identify the evolutionarily conserved Kank protein family as novel components of focal adhesions (FAs). Kank proteins accumulate at the lateral border of FAs, which we term the FA belt, and in central sliding adhesions, where they directly bind the talin rod domain through the Kank amino-terminal (KN) motif and induce talin and integrin activation. In addition, Kank proteins diminish the talin–actomyosin linkage, which curbs force transmission across integrins, leading to reduced integrin–ligand bond strength, slippage between integrin and ligand, central adhesion formation and sliding, and reduced cell migration speed. Our data identify Kank proteins as talin activators that decrease the grip between the integrin–talin complex and actomyosin to regulate cell migration velocity.

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Figure 1: Kank2 is a novel FA protein.
Figure 2: Kank2 is targeted to FAs through the KN motif.
Figure 3: Kank2 curbs cell migration by inducing adhesion sliding.
Figure 4: Adhesion sliding occurs at the interface between integrin and ligand.
Figure 5: Kank2 impairs force transmission across integrins.
Figure 6: Kank2 directly binds the talin rod.
Figure 7: Kank2 induces talin and integrin activation.
Figure 8: Kank2 decreases F-actin binding to talin-ABS2.

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Acknowledgements

We thank N. Nagaraj for technical support, and C. Grashoff, O. Rosslier, G. Giannone and R. Böttcher for discussions and reading of the manuscript. The work was supported by the National Institutes of Health R01GM112998 (to A.R.D.), Stanford Graduate Fellowship (to S.T.) and ERC, DFG and Max Planck Society (to R.F.).

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Authors and Affiliations

  1. Max Planck Institute of Biochemistry, 82152 Martinsried, Germany

    Zhiqi Sun, Hui-Yuan Tseng, Dirk Dedden, Naoko Mizuno, Anita A. Wasik & Reinhard Fässler

  2. Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA

    Steven Tan & Alexander R. Dunn

  3. CytoMorpho Lab, Biosciences & Biotechnology Institute of Grenoble, UMR5168, CEA/INRA/CNRS/Université, 38054 Grenoble-Alpes, Grenoble, France

    Fabrice Senger, Laetitia Kurzawa & Manuel Thery

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Contributions

Z.S. and R.F. initiated the project, designed the experiments and wrote the paper; Z.S. performed the vast majority of the experiments; H.-Y.T., S.T., F.S., L.K., D.D. and A.A.W. performed experiments; Z.S., S.T., N.M., M.T., A.R.D. and R.F. analysed data; all authors read and approved the manuscript.

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Integrated supplementary information

Supplementary Figure 1

(a) SILAC ratio plot showing specific β1wt tail interactors with high β1 wt/β1 scr and low β1 scr/β1 wt SILAC ratios in reverse SILAC labeling experiments and with high MS intensities in the FA-enriched proteome of FN-seeded fibroblasts in red. Dab-2 (green) and SNX17 (grey) show a high β1 wt/β1 scr SILAC ration but were absent from the FA-enriched proteome. (b) Split channels of Fig. 1b. Mouse fibroblasts seeded on FN for 3h and immunostained for Kank2 (green), Kindlin-2 (red) and DAPI (blue). (c) Mouse fibroblasts seeded on FN for 3h and immunostained for Kank2 (green), Talin (red) and DAPI (blue). (d) Confocal image of immunostained (Kank2, green; Kindlin-2, red; DAPI, blue) wild-type fibroblasts seeded for 3h on a FN-coated micropattern. The boxed areas show Kank2 puncta around paxillin-positive FAs. (e) Mouse fibroblasts seeded on FN for 5h and immunostained for β1 integrin (Itgb1, green), β3 integrin (Itgb3, red), Paxillin (Pxn, blue) and DAPI (grey). (f,g) Mouse fibroblasts expressing Kank2-GFP seeded for 5h on FN and immunostained for β1 integrin (Itgb1, red) or active β1 integrin (9EG7, red), Paxillin (Pxn, blue) and DAPI (grey). Scale bar in b-g, 10 μm.

Supplementary Figure 2

(a) MS intensities of Kank1-4 in the FA-enriched sub-proteome and total proteome of wild-type mouse kidney fibroblasts (mean ± s.d.; n = 3 independent MS measurements). (b) Western blot analysis of Kank2 and β1 integrin (Itgb1) in cells expressing scrambled (shScr) and Kank2-specific shRNAs. Actin served to control protein loading. (c) Western blot analysis of Kank2 before and after shRNA-mediated knockdown using polyclonal anti-Kank2, and of indicated GFP-tagged Kank2 constructs using anti-GFP antibodies, respectively. Vinculin is used as loading control.

Supplementary Figure 3

(a,b) Immunofluorescence of GFP-tagged Kank2 constructs (green) co-stained for Liprin-β1 (a, red) or ELKS (b, red), Paxillin (Pxn; blue) and DAPI (grey). (c) Line profile analysis of GFP-tagged FL-Kank2 co-stained with Liprin-β1 and ELKS. Full arrowheads indicate the position of FA belt and open arrowheads indicate Liprin-β1- and ELKS-enriched regions outside the FA belt. Scale bar, 2 μm. (d,e) Western blot testing GFP-tagged Kank-KN (d), FL-Kank2 and Kank2ΔKN (e) binding to biotinylated β1 integrin tails. FL and mutant Kank2 proteins were detected with the anti-GFP antibody, and Kindlin-2 was used to control the β1 tail pull-down assay. (f) GFP-tagged Kank1, Kank3 and Kank4 expressed in Kank2-depleted fibroblasts and co-stained for Paxillin (Pxn; red), F-actin (blue) and DAPI (grey). Scale bars in a,b,f, 10 μm.

Supplementary Figure 4

(a) Time lapse images of cells stably expressing GFP-tagged FL-Kank2 (green) and Paxillin-TagRFP (Pxn-TagRFP; red) during isotropic spreading on FN. Arrow heads indicate Kank2 recruitment to the proximal FA border. (b) Normalized Pxn-TagRFP intensities during FA disassembly behind lamella in cells expressing FL-Kank2 (red) or Kank2ΔKN (blue) (mean ± s.d.; n = 8 cells for FL-Kank2, and n = 10 cells for Kank2ΔKN pooled from 4 independent experiments). (c) FA disassembly rates calculated from (b) as rate constants in a one-phase exponential decay (mean ± s.d.). (d) Ten overlaid, sequential time lapse images of Vinculin-mCherry in the cell centre temporally colour-coded using spectrum look up tables (LUT). Corresponding time lapse images of GFP are shown as projection of average intensity. Scale bar, 5 μm. (e) Percentage of adhesion sites with sliding velocity above 0.3 μm min−1 in indicated cell lines (mean ± s.d., n = 5 cells from 3 independent experiments; P values were calculated using one-way ANOVA Tukey test). (f) 2-D random migration of indicated cells on FN (box plot with median, 1st–3rd quantile box, minimal to maximal whiskers; shScr, n = 52 cells; shKank2#1, n = 58 cells; shKank2#2, n = 60 cells; data aggregated from 4 independent experiments; P values were calculated using Student t-test). (g) Cell spreading area of indicated cells at different time points (n ≥ 50 cells for each condition). (h) Percentage of adhesion sites with sliding velocity above 0.3 μm min−1 in Kank2-depleted fibroblasts stably expressing Paxillin-TagRFP and Kank2-GFP on FN treated with or without 50 μM broad spectrum MMP inhibitor Gm6001 for 5h (mean ± s.d., n = 5 cells from 3 independent experiments).

Supplementary Figure 5

(a) Western blot analysis of Kank2 and Liprin-β1 in wild-type cells expressing FL-Kank2-GFP together with scrambled (shScr) and two Liprin-β1-specific shRNAs. Vinculin served to control protein loading. (b) 2D correlation coefficient between GFP and inverted FRET signals in FL-Kank2-GFP expressing cells before and after Liprin-β1 depletion (dot plot and box plot; FL-Kank2 shScr, n = 30 cells; FL-Kank2 shLiprin-β1#1, n = 31 cells; FL-Kank2 shLiprin-β1#2, n = 33 cells; data aggregated from 3 independent experiments for each condition; P values were calculated using the Wilcoxon Rank Sum test). (c) Ratio between adhesion area with high tension (≥250 pN μm−2) and adhesion area with low tension (<250 pN μm−2) were calculated in cells expressing indicated constructs (dot plot and box plot; FL-Kank2, n = 24 cells; Kank2ΔCoil, n = 45 cells; FL-Kank2 shScr, n = 30 cells; FL-Kank2 shLiprin-β1#1, n = 31 cells; FL-Kank2 shLiprin-β1#2, n = 33 cells; data aggregated from 3 independent experiments for each condition; P values were calculated using the Wilcoxon Rank Sum test).

Supplementary Figure 6

(a) SDS-PAGE analysis of bacterially expressed and purified FL-Talin-1, Talin head domain (THD), Venus-His-Sumo-tagged Talin R4-R8 domain, GST-KN fusion protein and GST. (b) HEK293 cells expressing GFP-tagged Talin rod truncations were lysed, incubated with recombinant GST-KN, immunoprecipitated with anti-GFP antibody and analyzed with Western blot. Red asterisk marks the recombinant GST-KN protein.

Supplementary Figure 7

(a) Western blot and (b) densitometry of Talin binding to β1 integrin tails in cells treated with increasing doses of dexamethasone (Dox) to induce expression of either FL-Kank2 or Kank2ΔKN (mean ± s.d.; n = 3 independent experiments; P values were calculated using Student t-test). Asterisk marks additional Kank2 bands after transient induction of protein expression, which is likely due to post-translational modification. (c,d) Representative Western blot (c) and densitometry (d) of endogenous Kank2, Talin and Kindlin-2 binding to β1 integrin tails in the presence of increasing concentrations of recombinant KN-GST. (e) Western blot of endogenous Kank2, Talin and Kindlin-2 binding to β1 integrin tails in the presence of increasing concentrations of chemically synthesized KN peptide. (f) SDS-PAGE analysis of FL-Kank2 expressed and purified from HEK293 cells. (g) Western blot analysis of recombinant Talin-1 binding to β1 integrin tail in the presence of the KN peptide and increasing concentration of recombinant THD. (h) Western blot analysis of β1 integrin tail pull downs of endogenous Talin detected with an antibody recognizing THD in cells overexpressing the GFP-tagged Talin rod domain (Talin-Rod-GFP) in the presence or absence of KN peptide. Source data for b can be found in Supplementary Table 2.

Supplementary Figure 8

(a) Immunofluorescence of FL-Kank2-GFP (green) co-stained with endogenous RhoGDIα (red) and DAPI (blue). Scale bar, 10 μm. (b) Western blot (left) and densitometric (right) analysis of RhoA activation in Kank2-depleted cells expressing indicated Kank2 constructs seeded on FN for 1h (mean ± s.d.; n = 3 independent experiments; P values were calculated using one-way ANOVA Tukey test; N.S., no significance). (c) Western blot (left) and densitometric (right) analysis of myosin light chain (MLC) phosphorylation at serine-19 (pMLC-Ser19) in Kank2-depleted cells expressing indicated Kank2 constructs seeded on FN for 1h. Actin was used to control protein loading (mean ± s.d.; n = 4 independent experiments; P values were calculated using one-way ANOVA Tukey test; N.S., no significance). (d) Still images from a representative time lapse FRAP experiment of Talin-1-GFP shown in rainbow LUT. Talin-1-GFP was co-expressed with Cherry-tagged FL-Kank2 or Kank2ΔKN, respectively, in Kank2-deplted fibroblasts. Co-localization of Kank2-mCherry and Talin-1-GFP in the region of interest (ROI, red circle) was confirmed in pre-bleached images. Scale bar, 2 μm. Source data for b,c can be found in Supplementary Table 2.

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Supplementary Table 1

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Supplementary Table 2

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FL-Kank2-GFP (green) and Paxillin-TagRFP (red) were stably expressed in Kank2-depleted mouse fibroblasts.

Confocal live cell imaging during isotropic cell spreading 5 min after plating on FN in both fluorescence channels and phase contrast. Scale bar, 10 μm. (AVI 1586 kb)

FL-Kank2-GFP (green) and Paxillin-TagRFP (red) were stably expressed in Kank2-depleted mouse kidney fibroblast.

Confocal live cell imaging of polarized cells 45 min after plating on FN in both fluorescence channels and phase contrast. Scale bar, 10 μm. (AVI 9783 kb)

FL-Kank2-GFP (green) and Paxillin-TagRFP (red) were stably expressed in Kank2-depleted mouse kidney fibroblast.

Confocal live cell imaging of migrating cells 4 h after plating on FN in both fluorescence channels and phase contrast. Scale bar, 10 μm. (AVI 6606 kb)

Kank2ΔKN-GFP (green) and Paxillin-TagRFP (red) were stably expressed in Kank2-depleted mouse kidney fibroblast.

Confocal live cell imaging of polarized cells 45 min after plating on FN in both fluorescence channels and in phase contrast. Scale bar, 10 μm. (AVI 7998 kb)

FL-Kank2-GFP (green) and Vinculin-mCherry (red) were expressed in Kank2-depleted mouse kidney fibroblast.

Confocal live cell imaging of migrating cells 4 h after plating on FN in both fluorescence channels and phase contrast. Scale bar, 10 μm. (AVI 6758 kb)

Kank2ΔKN-GFP (green) and Vinculin-mCherry (red) were expressed in Kank2-depleted mouse kidney fibroblast.

Confocal live cell imaging of migrating cells 4 h after plating on FN in both fluorescence channels and phase contrast. Scale bar, 10 μm. (AVI 3070 kb)

Kank2ΔCoil-GFP (green) and Vinculin-mCherry (red) were expressed in Kank2-depleted mouse kidney fibroblast.

Confocal live cell imaging of migrating cells 4h after plating on FN in both fluorescence channels and phase contrast. Scale bar, 10 μm. (AVI 5110 kb)

Kank2(1–670)-GFP (green) and Vinculin-mCherry (red) were expressed in Kank2-depleted mouse kidney fibroblast.

Confocal live cell imaging of migrating cells 4h after plating on FN in both fluorescence channels and phase contrast. Scale bar, 10 μm. (AVI 5302 kb)

GFP-tagged Kank2-KN (green) and Vinculin-mCherry (red) were expressed in Kank2-depleted mouse kidney fibroblast.

Confocal live cell imaging of migrating cells 4h after plating on FN in both fluorescence channels and phase contrast. Scale bar, 10 μm. (AVI 3059 kb)

GFP control (green) and Vinculin-mCherry (red) were expressed in Kank2-depleted mouse kidney fibroblast.

Confocal live cell imaging of migrating cells 4h after plating on FN in both fluorescence channels and in phase contrast. Scale bar, 10 μm. (AVI 0 kb)

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Sun, Z., Tseng, HY., Tan, S. et al. Kank2 activates talin, reduces force transduction across integrins and induces central adhesion formation. Nat Cell Biol 18, 941–953 (2016). https://doi.org/10.1038/ncb3402

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