Figure 6.
Dynamic localization of MST1/YAP during differentiation. (A–C) Subcellular localization of MST1 and YAP in response to differentiation stimuli. (A) Fractionation experiments were conducted with primary keratinocytes from wild-type or mutant embryos with or without Ca2+ for 24 h. Fractionated lysates were subjected to western blot analysis with the indicated antibodies. N, nuclear; C, cytoplasmic. (B) WW45-deficient keratinocytes were co-transfected with the indicated plasmids. After 24 h of transfection, cells were maintained in Ca2+ medium for a further 24 h before harvesting for fractionation experiments. Western blot analysis was performed with the indicated antibodies. Note that the major cytoplasm translocation of YAP and nuclear translocation of MST1 were detected only in the presence of intact WW45. (C) Primary keratinocytes were cultured with or without Ca2+ for 24 h and then subjected to immunostaining with anti-MST1, anti-YAP and anti-p-YAP. Note the phospho-dependent translocation of YAP and MST1 in Ca2+-induced differentiated control cells but not in mutant cells. (D) Immunoperoxidase staining for MST1, YAP and p-YAP in wild-type (a–f) and mutant (a′–f′) epithelium at E17.5. Note the intense nuclear detection of YAP and cytoplasmic detection of MST1 in most mutant epithelial cells, which is in contrast to the dynamic distributions according to differentiation stage in control epithelial cells. Serine 127 phospho-specific immunoreactivity was cytoplasm-specific in the differentiating zones in the control, whereas the signal was absent from the mutant cells. Counterstaining was performed with haematoxylin. (a–c, a′–c′) Skin; (d–f, d′–f′) small intestine. Scale bar: 10 μm (C) and 100 μm (D).