1. Ezrin in its open, activated conformation binds and colocalizes with MISP at the cell cortex.
Ezrin in its open, activated conformation binds and colocalizes with MISP at the cell cortex. (A) Flag–MISP and GFP–ezrin were co-expressed in HEK293T cells. Complexes were immunoprecipitated with Flag-M2 antibody (Flag-IP) or anti-GFP antibody (GFP-IP), and analyzed by western blotting using the indicated antibodies. (B) Co-IP of endogenous ezrin from HeLa cells as analyzed by western blotting using anti-MISP or -ezrin antibodies. IgG, control immunoglobulin. (C) Domain structure of full-length ezrin (comprising the N-terminal located FERM domain, the α-tail and the C-ERMAD domain. Indicated residue T567 gets phosphorylated, whereas R579 is important for actin binding and prevents binding between the N- and C-termini. In vitro GST pulldown (GST-PD) of recombinant GST-MISP and His-tagged ezrin constructs. Purified proteins were incubated, subjected to GST pulldown and analyzed by western blotting using the indicated antibodies. (D,E) Co-staining of MISP (magenta) and ezrin (green) antibodies in a Caco-2 BBe cyst (D) or a mitotic HeLa cell (E). DNA is stained with Hoechst (blue). Localization is either shown in a maximum projection of three planes or in a single confocal plane. Insets show higher magnification of the framed region. Scale bars: 5 µm.
Yvonne T. Kschonsak, and Ingrid Hoffmann J Cell Sci 2018;131:jcs214544
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