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Fig. 3. Apical targeting of the C-terminus of Shrm alters cell morphology. (A) Schematic of the chimeric protein consisting of Endolyn and the C-terminus of Shrm (Endo-ShrmC) and the control chimeric protein consisting of Endolyn and the C-terminus of Shrm truncated to remove ASD2 (Endo-ShrmC
). (B,C) T23 MDCK cells on Transwell filters were transiently transfected with expression vectors for Endo-ShrmC (B) or Endo-ShrmC (C) and stained to detect Shrm (green) and ZO-1 (red). (D) Equal amounts (30 µg) of total cell lysate from induced and un-induced T23-Endo-ShrmC cells were assayed by western blot analysis using anti-Shrm sera. Filters were re-probed to detect ß-catenin to verify equal protein loading. (E,F) Endo-ShrmC cells were grown on Transwell filters in the presence (E) or absence (F) of doxycyclin and stained to detect ZO-1 (green, E' and F'), E-cadherin (red, E'' and F''), or Shrm (blue, E''' and F'''). Boxed regions in (F) and (F') are enlarged in the insets. Arrows denote finger-like structures. (G,H) Lateral (G' and H') and apical (G'' and H'') optical sections from the un-induced (G' and G'') and induced (H' and H'') cells shown in panels E and F, respectively. The lateral sections are from the mid-way point of the apical-basal axis and the sub-apical sections are taken at the level of the AJC as defined by ZO-1 staining. In all panels, scale bar represents 15 µm. Open arrowheads denote position of the X-Z projections shown beneath relevant panels.
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