Integrin-containing focal adhesions transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, the potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell–ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin–ECM downstream signalling, localizes with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage independence and metastasis.
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We thank J. Siivonen and P. Laasola for excellent technical assistance, M. Georgiadou, D. Schlaepfer and J. Heino for insightful comments on the manuscript and H. Hamidi for scientific writing and editing of the manuscript. S. Corvera is acknowledged for the GFP–EEA1 plasmid (Addgene), D. Schlaepfer for the FAK wt and −/− MEFs and the GFP–FAK plasmids, T. Näreoja and S. Koho for assistance with the STED instrument, and Cell Imaging Core facility, University of Turku Centre for Biotechnology, and the Nikon Imaging Centre at Institut Curie-CNRS for help with the imaging. Turku Centre for Disease Modelling is acknowledged for help with the metastasis assays. This study has been supported by the Academy of Finland, ERC Starting Grant, ERC Consolidator Grant, the Sigrid Juselius Foundation, and the Finnish Cancer Organization. J.A. has been supported by the Turku Doctoral Program of Biomedical Sciences and an EMBO Short-Term Fellowship. G.J. is supported by an EMBO Long-Term Fellowship.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Validation of the pFAK-Y397 antibody.
(a,b) Validation of the pFAK-Y397 antibody in Fak−/− and Fak+/+ MEF cell lysates (a) and in TIFFs in the presence of FAK-Y397 phosphorylation inhibitors (1 μM PF271 or PF228) (b) (mean ± SEM, n = 3 independent experiments). Statistics source data can be found in Supplementary Table 2. (c) Quantification of pFAK-Y397 levels following FAK-14 inhibitor (10 μM) treatment. Representative confocal images and ROIs of focal adhesions are shown (mean fluorescence intensity ± SEM, n = 22 cells pooled from two independent experiments). (d) Individual fluorescence density plots of MDA-MB-231 cells plated on crossbow-shaped fibronectin-coated micropatterns and stained for active β1-integrin and pFAK-Y397. Shown are x-axis views. The analysis of these cells was used to generate the 3D probabilistic density plots shown in Fig. 1a. (e) Confocal images of pFAK-Y397 and total or active β1-integrin staining in GFP-Rab21 overexpressing TIFFs plated on fibronectin (45 min). Representative ROIs from endosomes and box plot of the distance between adjacent puncta of active β1-integrin and pFAK or Rab21 in GFP-Rab21-positive endosomes or of pFAK and pFAK outside the endosomes (in pixels) (box plots show the 25th–75th percentiles delineated by the upper and lower limits of the box; the median is shown by the horizontal line inside the box. Whiskers indicate maxima and minima). n = the number of active β1-integrin-pFAK, active β1-integrin-Rab21 and pFAK-pFAK doublets (indicated in the figure) analysed from multiple cells (numbers indicated in the figure) from three independent experiments are indicated. Mann–Whitney test P values are provided.
Supplementary Figure 2 ECM-induced downstream signalling is not due to growth factor receptor activation.
(a) Quantification of kinase activity in NCI-H460 cells plated on collagen. Representative blots are shown (mean ± SEM, n = 3 independent experiments). (b) Quantification of human phospho-receptor tyrosine kinase array in NCI-H460 cells kept in suspension or plated on collagen (45 min). Positive control is an antibody against phospho-tyrosine. Representative blots of cell lysates used for the array are shown (mean phosphoprotein detection ± SEM from two independent experiments). Student’s two-tailed unpaired t-test P values are provided and statistics source data can be found in Supplementary Table 2. Unprocessed original scans of blots are shown in Supplementary Fig. 9.
Supplementary Figure 3 Dynamin inhibition downregulates integrin receptor endocytosis.
(a,b) Integrin receptor endocytosis in NCI-H460 cells plated on collagen for 45 min in the presence of active β1-integrin (9EG7) antibody (45 min) ± dynasore. (a) Representative confocal images and quantification of the proportion of endocytosed integrin receptors based on antibody staining (n(DMSO) = 52 cells, n(dynasore) = 54 cells, pooled from three independent experiments, mean ± SEM). (b) Flow cytometry-based analysis of integrin endocytosis. Receptor internalisation, at the indicated time points, was calculated as an inverse ratio of total 9EG7-labelled integrin remaining on the cell surface compared to time point 0 (mean ± SEM, n = 3 independent experiments). (c) Transferrin endocytosis in NCI-H460 cells plated on collagen for 45 min in the presence of 555-labelled-transferrin (15 min) ± dynasore. Representative confocal images and quantification of the total levels of endocytosed transferrin are shown (n(DMSO) = 35 cells, n(dynasore) = 32 cells, pooled from three independent experiments, mean ± SEM). Student’s two-tailed unpaired t-test P values are provided. Statistics source data can be found in Supplementary Table 2.
Supplementary Figure 4 Inhibition of integrin endocytosis attenuates integrin signalling.
(a,b) Representative blots of kinase activity in TIFFs plated on collagen (a quantified in Fig. 3c) and fibronectin (b quantified in Fig. 3d) ± dynasore. (c,d) Analysis of kinase activity in MDA-MB-231 cells plated on collagen (c) and fibronectin (d) ± dynasore. Representative blots (c,d) and quantification (c) are shown (mean ± SEM, n = 3 independent experiments). (e) Analysis of kinase activity in NCI-H460 cells plated on collagen ± 10 μM Dyngo4a. Representative blots and quantifications are shown (mean ± SEM, n = 3 independent experiments). (f) Analysis of kinase activity in GFP or GFP-Dyn2K44A overexpressing NCI-H460 cells plated on collagen. Representative blot and quantification of normalised band integrated densities are shown (three independent experiments). (g) Flow cytometry quantification of total cell surface β1-integrin levels in NCI-H460 cells held in suspension or plated on collagen (45 min) ± dynasore (mean fluorescence ± SEM, n = 3 independent experiments). (h) Subcellular fractionation of NCI-H460 cells plated on collagen (45 min) ± dynasore. Representative blots of the plasma membrane (PM), cytoplasm (Cyto) and endosomal fractions (Endo) are shown (three independent experiments). (i) Representative immunoblot analysing the recruitment of recombinant FAK to purified endosomes derived from either control- or β1-integrin-silenced Fak−/− MEFs (different siRNA to one used in Fig. 4c; three independent experiments). Please note that silencing of β1-integrin induces cellular levels of β3-integrin. This might contribute to the residual FAK recruitment to the β1-integrin silenced endosomal fraction. Student’s two-tailed unpaired t-test P values are provided. Statistics source data can be found in Supplementary Table 2. Unprocessed original scans of blots are shown in Supplementary Fig. 9.
Supplementary Figure 5 Dynamin inhibition affects cell spreading.
MDA-MB-231 cells (a) or NCI-H460 cells (b) plated on collagen for the indicated times ± dynasore (three independent experiments). (c) Quantification of FAK activation (pFAK-Y397 levels) in NCI-H460 cells incubated with collagen (Col I) or BSA-coated beads in suspension (45 min) ± dynasore (mean ± SEM, n = 3 independent experiments). Student’s two-tailed unpaired t-test P values are provided and statistics source data can be found in Supplementary Table 2. Unprocessed original scans of blots are shown in Supplementary Fig. 9.
Supplementary Figure 6 Rab5, Rab21 and EEA1 are important for integrin-mediated FAK activation.
(a,b) Representative blots and quantification of pFAK levels in GFP and GFP-Rab21 (a) or GFP-Rab5-CA (b) overexpressing MDA-MB-231 cells plated on collagen (n(a) = 3, n(b) = 6 independent experiments, mean ± SEM). (c) Active β1-integrin (12G10) and pFAK-Y397 localization in GFP-EEA1 expressing TIFFs plated on fibronectin (45 min). (d) Representative blot of pFAK-Y397 levels in control or EEA1 smart pool silenced NCI-H460 cells plated on collagen. Student’s two-tailed unpaired t-test P values are provided and statistics source data can be found in Supplementary Table 2. Unprocessed original scans of blots are shown in Supplementary Fig. 9.
Supplementary Figure 7 APPL1 is not important for anoikis suppression.
(a,b) Quantification of caspase-3 positive apoptotic TIFFs ± Dyngo4a (a), and following APPL1 silencing (b) (mean fluorescence ± SEM, n = 3 independent experiments). Student’s t-test P values are provided and statistics source data can be found in Supplementary Table 2.
Supplementary Figure 8 Anchorage-independent growth and metastases of MDA-MB-231 cells are sensitive to FAK and dynamin inhibition and Rab21 silencing.
(a) MDA-MB-231 cells were plated on agar and incubated with the indicated drugs. Anchorage-independent growth was assessed by counting the number of cells per cluster with intact nuclei (mean ± SEM, n = 6 fields of view assessed from three independent experiments). (b) Representative images showing extravasation of siCtrl- and siRab21-treated MDA-MB-231 cells in mouse lungs. Student’s t-test P values are provided.
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Alanko, J., Mai, A., Jacquemet, G. et al. Integrin endosomal signalling suppresses anoikis. Nat Cell Biol 17, 1412–1421 (2015). https://doi.org/10.1038/ncb3250
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