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Figure 1: Subcellular localization of wild-type and K256E α-actinin-4 in mouse podocytes and effects on cell spreading. (a) Differentiated podocytes were infected with adenoviruses encoding HA-tagged α-actinin-4 K256E and wild type. Cells were fixed and stained with an anti-HA antibody to detect α-actinin-4, and phalloidin to detect F-actin. Uninfected cells were treated in the same manner and serve as control. Wild-type α-actinin-4 is predominantly found with membrane-associated cortical actin and process-like projections with limited expression along stress fibers. Conversely, α-actinin-4 K256E is predominantly associated with stress fibers and is excluded from the cell periphery. (b) A differentiated mouse podocyte expressing GFP-tagged wild-type or K256E α-actinin-4. The wild type expressing cell forms numerous α-actinin-4-containing lamellipodia in response to serum stimulation. While α-actinin-4 K256E readily decorates actin stress fibers, it, nevertheless, forms large aggregates, and the cells fail to produce lamellipodia. (c, d) Differentiated mouse podocytes expressing GFP alone (control), wild-type, or K256E α-actinin-4 were trypsinized, held in suspension for 15 min, replated onto collagen I-coated coverslips, and analyzed by fluorescence microscopy at 3 and 6 h. Expression of α-actinin-4 K256E significantly reduced the number of spreading cells. After 6 h, wild-type α-actinin-4 was localized to cortical actin, whereas K256E α-actinin-4 remained condensed within the cell. (b) versus control and wild-type of corresponding time point. Panels (a), (c), and (d) of the figure are adapted from [33] with permission from the Nature Publishing Group.