Figure 7. The interaction of Merlin with both β-catenin and Par3 is required for the establishment of functional junctions.
(A) IVTT-produced Merlin or Merlin18-595 was mixed with either GST-β-catenin (top) or Myc-Par3 (middle). Complexes were isolated using either GST beads (top panel) or an anti-Myc antibody (middle panel) and immunoblotted with anti-GST, -Myc, or -Nf2 antibodies. Merlin18-595 can readily bind Myc-Par3 but not GST-β-catenin in vitro. 10% of the input is shown in the bottom panel.
(B) PAM212 keratinocytes were transfected with Myc-Par3, Myc-Par3511-1266 or Myc-Par31-373 and cultured in calcium-containing media for the indicated times. Immunoprecipitation of Par3 followed by immunoblotting with Myc-, AJ- or TJ-specific antibodies revealed that full-length Myc-Par3 and Myc-Par3511-1266 can associate with Merlin and with AJ and TJ proteins. In contrast, Myc-Par31-373 does not associate with Merlin or with AJ and TJ proteins. Note that Myc-Par3511-1266 and Myc-Par31-373 are produced from a Par3 splice variant that does not contain the aPKC-binding site. NRS, normal rabbit serum.
(C) TER was measured across calcium-stimulated PAM212 keratinocyte monolayers that stably express Merlin, Merlin18-595, or Myc-Par31-373. Note that both Merlin18-595 and Myc-Par31-373 dominantly interfered with the establishment of TER. Values = mean +/− SD.
(D–G) Primary wild-type (D and F) or K14-Cre;Nf2lox/lox (E and G) keratinocytes were stimulated with calcium-containing media for 8 hours, and either processed for indirect immunofluorescence (Fix 1, D and E) or subject to a more stringent in situ extraction (Fix 2, F and G). Immunostaining revealed normal localization of Par3 (green) at cell-cell boundaries in K14-Cre;Nf2lox/lox cells that were processed via mild fixation/extraction (Fix 1; D and E). However, Par3 staining in these cells is completely eliminated by more stringent fixation/extraction (Fix 2). In contrast, Par3 localization to cell-cell boundaries is maintained in wild-type keratinocytes in Fix 2. See also Figure S5.