Kindlin-2 controls bidirectional signaling of integrins

Abstract

Control of integrin activation is required for cell adhesion and ligand-induced signaling. Here we report that loss of the focal adhesion protein Kindlin-2 in mice results in peri-implantation lethality caused by severe detachment of the endoderm and epiblast from the basement membrane. We found that Kindlin-2-deficient cells were unable to activate their integrins and that Kindlin-2 is required for talin-induced integrin activation. Furthermore, we demonstrate that Kindlin-2 is required for integrin outside-in signaling to enable firm adhesion and spreading. Our findings provide evidence that Kindlin-2 is a novel and essential element of bidirectional integrin signaling.

Figure 1.

Peri-implantation lethality and impaired integrin activation in ESCs in the absence of Kindlin-2 expression. (A) Hematoxilin and eosin staining of an E6.5 control and presumptive Kindlin-2^−/− implantation chamber. TUNEL staining (green) and FN staining (red) show apoptosis and disturbed matrix deposition in Kindlin-2^−/− embryos. Nuclei are counterstained with DAPI (blue). (B) Bright-field pictures of wild-type and Kindlin-2^−/− ESCs lines seeded on feeder cells. (C) Western Blot for Kindlin-2 and talin in wild-type and two independent Kindlin-2^−/− ESCs lines. (D) Adhesion assay of wild-type and Kindlin-2^−/− ESCs on different ECM substrates. (E) Integrin surface expression of wild-type (blue), Kindlin-2^−/− (light and dark green), and β1-integrin^−/− (red) ESC lines. A background control is shown in gray. (F) Binding of FNIII7-10 to wild-type and Kindlin-2^−/− ESCs in the presence and absence of MnCl₂. (G) 9EG7 binding on wild-type (blue) and Kindlin-2^−/− (green) ESC lines. A background control is shown in red. (d) Decidua; (epc) ectoplacental cone; (ee) extraembryonic ectoderm; (e) ectoderm; (m) mesoderm.

Figure 2.

Kindlin-2 interacts with integrin tails and is required for integrin activation. (A) GST pull-down assay from ESC lysates with wild-type and mutant GST-tagged β1- and β3-integrin cytoplasmic tails. (B) Pull-down assays with the recombinant GST-tagged wild-type and mutant (QW614/615AA) PTB domain of Kindlin-2 and the recombinant His-tagged β1-integrin cytoplasmatic tail. (C) αIIbβ3-integrin activation in CHO cells transfected with EGFP, EGFP-Kindlin-2, EGFP-Talin head, and EGFP-Kindlin-2 together with the Talin head.

Figure 3.

Kindlin-2 EBs show endoderm and epiblast detachment and diminished integrin activation. (A) Micrographs of methylene blue-stained sections of 4- and 6-d-old wild-type and Kindlin-2^−/− EBs. (B) Cryosections of wild-type and Kindlin-2^−/− EBs were stained with an antibody specific for LN-α1 chain (green) and fluorescently labeled phalloidin to visualize F-actin (red). Nuclei are counterstained with DAPI (blue). (C) TUNEL staining (green), LN-α1 chain (red), and nuclei (blue) of 6-d-old wild-type and Kindlin-2^−/− EBs. (D) Six-day-old wild-type and Kindlin-2^−/− EBs stained with LN-α1 chain (green) and 9EG7 (red) antibodies. Nuclei are counterstained with DAPI (blue). (epi) Epiblast; (end) endoderm.

Figure 4.

Impaired cell spreading of Kindlin-2^−/− endoderm cells. (A) Cryosections of 6-d-old wild-type and Kindlin-2^−/− EBs stained with LN-α1 chain antibody (green) and fluorescently labeled phalloidin to visualize F-actin (red). Nuclei are counterstained with DAPI (blue). (B) Immunofluorescence staining for talin (red) and ILK (green) of wild-type and Kindlin-2^−/− endoderm cells seeded of FN in the presence or absence of MnCl₂. (C) Immunofluorescence staining for paxillin (red) and ILK (green) of wild-type and Kindlin-2^−/− endoderm cells seeded of FN in the presence of MnCl₂. (D) Western blot analysis of ILK in wild-type and Kindlin-2^−/− endoderm cells. (E) Coimmunoprecipitation of ILK and Kindlin-2 from ILK^fl/fl and ILK^−/− fibroblasts.