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Nat Commun
2016 Mar 08;7:10909. doi: 10.1038/ncomms10909.
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Cadherin-11 localizes to focal adhesions and promotes cell-substrate adhesion.
Langhe RP
,
Gudzenko T
,
Bachmann M
,
Becker SF
,
Gonnermann C
,
Winter C
,
Abbruzzese G
,
Kratzer MC
,
Franz CM
,
Kashef J
.
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Cadherin receptors have a well-established role in cell-cell adhesion, cell polarization and differentiation. However, some cadherins also promote cell and tissue movement during embryonic development and tumour progression. In particular, cadherin-11 is upregulated during tumour and inflammatory cell invasion, but the mechanisms underlying cadherin-11 stimulated cell migration are still incompletely understood. Here, we show that cadherin-11 localizes to focal adhesions and promotes adhesion to fibronectin in Xenopus neural crest, a highly migratory embryonic cell population. Transfected cadherin-11 also localizes to focal adhesions in different mammalian cell lines, while endogenous cadherin-11 shows focal adhesion localization in primary human fibroblasts. In focal adhesions, cadherin-11 co-localizes with β1-integrin and paxillin and physically interacts with the fibronectin-binding proteoglycan syndecan-4. Adhesion to fibronectin mediated by cadherin-11/syndecan-4 complexes requires both the extracellular domain of syndecan-4, and the transmembrane and cytoplasmic domains of cadherin-11. These results reveal an unexpected role of a classical cadherin in cell-matrix adhesion during cell migration.
Figure 1. Xcad-11 is localized in focal adhesions.Xenopus NCC injected with Xcad-11-EGFP, explanted on fibronectin-coated glass dishes and immunostained for (a) β-catenin, (b) paxillin and (c) β1-integrin. (a) A confocal image focused on the apical side of NCC shows co-localization of Xcad-11 with β-catenin at cell–cell contacts. (b,c) TIRF images demonstrating co-localization of Xcad-11 with paxillin and β1-integrin in focal adhesions at the cell substrate. (d) HeLa cells transfected with Xcad-11-EGFP, immunostained for paxillin and imaged by TIRF microscopy display partial localization of Xcad-11 with paxillin at the cell substrate. Scale bars, 20 μm (a); 10 μm (b–d).
Figure 2. Xcad-11 promotes focal adhesion formation and cell–substrate adhesion.(a) NCC explants injected with mbGFP and immunostained for phosphopaxillin. Depletion of Xcad-11 by MO injection leads to loss of phosphopaxillin staining. (b) NCC injected with mbGFP and H2B-cherry or together with Xcad-11 MO. Fluorescence images collected at the same substrate position before (right column) and after (left column) the flipping assay. (c) Statistics for flipping assay (mean±s.d.), n=total number of cells before and after flipping assay. Results of at least three independent experiments were averaged and statistical significance was analysed by Student's t-test. (d) Adhesion forces (mean±s.d.) measured by AFM-based SCFS. At least 10 different cells were measured per condition and statistical significance was assessed according to the Mann–Whitney test. (e) Xcad-11 constructs for reconstitution: extracellular (EC), transmembrane (TM); (p120/ β-cat) p120-binding or β-cat-binding site. (f) Statistics for reconstitution (mean±s.d.), n=total number of cells after flipping assay. Scale bars, 20 μm.
Figure 3. Syn-4 interacts with Xcad-11 and stimulates focal adhesion formation.(a) Co-immunoprecipitation of Syn-4 with different Xcad-11 constructs. (First row) Precipitation of Syn-4 with FLAG antibody from transfected HEK293 cells. Western blot for Xcad-11 (myc) showed successful co-precipitation of Xcad-11, Xcad-11 δe and Xcad-11 δeδc. In the case of Xcad-11 δeδTμ and pCS2+, no co-precipitation with Syn-4 was detected. (Second row) Input for the different Xcad-11 constructs was detected by western blotting. (Third row) Precipitation of different Xcad-11 constructs with myc antibody from transfected HEK293 cells. Western blot for Syn-4 (FLAG) showed successful co-precipitation of Xcad-11, Xcad-11 δe and Xcad-11 δeδc. In the case of Xcad-11 δeδTμ and pCS2+, no co-precipitation with Syn-4 was detected. (Fourth row) Input for Syn-4 was detected by western blotting. Transfection of Xcad-11 alone served as negative control (first panel). (b) Co-immunoprecipitation of Syn-4 with different Xcad-11 constructs analysed for β-catenin (βcat) binding. (First row) Precipitation of different Xcad-11 constructs with myc antibody from transfected HEK293 cells. Western blot for βcat showed successful co-precipitation of Xcad-11, Xcad-11 δe and Xcad-11 δeδTμ . In the case of Xcad-11 δeδc and pCS2+, no co-precipitation with βcat was detected. (Second row) Precipitation of Syn-4 with FLAG antibody showed successful co-precipitation of βcat when Xcad-11 or Xcad-11 δe were co-transfected. (Third row) Input for βcat was detected by western blotting. Transfection of Xcad-11 alone served as negative control (first panel). (c) NCC explants co-injected with mbGFP and Syn-4 MO and immunostained for phosphopaxillin. Depletion of Syn-4 leads to loss of phosphopaxillin staining, which is rescued by co-injection of Syn-4. Scale bars, 20 μm.
Figure 4. Synergistic function of Xcad-11 and Syn-4 in mediating cell-substrate adhesion.(a,c,e) NCC explants co-injected with mbGFP, H2Bcherry and indicated constructs before (right column) and after (left column) the flipping assay. (b,d,f) Statistics for flipping assays corresponding to a,c and e, respectively (mean±s.d.), n=total number of cells after flipping assay. Results of at least three independent experiments were averaged and statistical significance was analysed by Student's t-test. (a,b) Depletion of both Xcad-11 and Syn-4 leads to a dramatic loss of cell–substrate adhesion. (c,d) Neither co-injection of Xcad-11 nor Syn-4 rescues depletion of Syn-4 or Xcad-11, respectively. (e,f) Co-injection of chimera construct Syn4Xcad11 (Syn-4 EC domain and Xcad-11 TM as well as cytoplasmic domain) rescues cell–substrate adhesion in all NC morphant cells. Scale bars, 20 μm.
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