Overexpression of the kinase-deficient mutant of aPKCλ (aPKCkn) in confluent MDCK monolayers disrupts the junctional localization of ASIP and ZO-1 only when the cells are subjected to calcium switch. (a) Confluent MDCK cells seeded on cover slips were infected with adenovirus vector encoding aPKCλkn. 20 h after viral infection, the cells were subjected to calcium switch (+CS) or left untreated (−CS) (see Materials and Methods). After another 20 h, the cells were doubly labeled with anti–aPKCλ and anti–ASIP as indicated at the top (left and middle). Each photograph represents the projected view of optical sections (0.4 μm) obtained from the apical to basal membrane using a laser scanning confocal microscope (unless otherwise noted, the following data were obtained in the same way). Note that fluorescent signals for endogenous aPKCλ are not observed in the photographs because the anti–aPKCλ antibody used (λ2) is not sensitive enough to clearly detect the endogenous protein in MDCK II cells and because the exposure times were adjusted to cover the highly heterogenous level of aPKCλkn expression. (Right) Independent specimens stained by the anti–ZO-1 antibody. Bar, 25 μm. (b) Correlation between the expression level of aPKCλkn and the severity of ZO-1 mislocalization. Based on the fluorescent intensity of aPKCλ, cells were categorized as showing undetectable (−/+), low (+), medium (++), or high (+++) expression, and the ZO-1 staining pattern of the corresponding cell was estimated as complete (white), partially disappeared (hatched), or completely disappeared (black). Original statistical data are presented in Table . (c) LacZ- or aPKCλwt-expressing cells subjected to calcium switch were doubly stained as in a, with anti–aPKCλ and anti–ZO-1 antibodies. Bar, 25 μm.

aPKCkn specifically disturbs the junctional localization of ZO-1 by its dominant negative effect on endogenous aPKC activity. (a) MDCK II cells coinfected with adenovirus vectors encoding aPKCλwt and aPKCλkn were subjected to calcium switch as described in Fig. 1 and, 20 h later, stained with anti–ZO-1 antibody. Total multiplicity of infection was normalized to 120 by mixing with a LacZ-encoding virus vector. The mixed ratios of the each vector are as follows: aPKCλkn:aPKCλwt:LacZ = 2:0:2 (top), 2:1:1 (middle), and 2:2:0 (bottom). Note that increasing amounts of coexpressed aPKCλwt increasingly rescue the phenotype caused by aPKCλkn. (b) MDCK II cells overexpressing aPKCζwt, ζkn, or δkn were similarly prepared as described in Fig. 1 and, 20 h later, doubly stained with anti–PKC antibodies corresponding to each PKC subtype (left) and anti–ZO-1 (right). Bar, 25 μm.

The overexpression of aPKCkn also disrupts the junctional localization of ASIP and ZO-1 in MDCK II cells under normal growth conditions. MDCK II cells infected with the adenovirus vectors indicated were reseeded sparsely (1.7 × 10⁴ cells/cm²) on cover slips and, 40 h later, subjected to immunofluorescent analysis. (Top) The antibodies used are indicated. Bar, 25 μm.

Distribution of various junctional components and F-actin in aPKCλkn-expressing cells. Confluent monolayers of aPKCλkn-expressing MDCK II cells were subjected to calcium switch. 6 h later, ZO-1 distribution was compared with that of other TJ components (a) or adherens junction components (b) by double staining as indicated. For E-cadherin and rhodamine-phalloidin staining, the data for LacZ-expressing cells are also presented for reference. Arrows indicate the positions of aberrant small ring junctional structures, while arrowheads indicate the regions where discrepant staining is observed between ZO-1 and the compared TJ component. Bar, 25 μm. (c and d) Western blot analysis of several junctional proteins in adenovirally infected MDCK II cells. Cells were subjected to calcium switch, lysed 6 h later, and processed for Western blot analysis using the antibodies indicated (left). (c) Typical results of Western blot analysis are presented. The infected viruses are indicated (top). The total amount of protein in each lane was finely adjusted based on the quantitative results of Coomassie brilliant blue staining of the blot membrane (data not shown). Quantified results based on three independent experiments examining the amounts of the indicated proteins in MDCK cells expressing LacZ (white bar), aPKCλwt (hatched bar), or aPKCλkn (black bar) are presented in d. Chemiluminescent signals from the immunostained bands for each junctional protein were quantified using a luminescence Image Analyzer.

Overexpression of aPKCλkn inhibits the development of TER of MDCK II cells after calcium switch. (a) Confluent MDCK II cells grown on a filter were infected with adenovirus vectors, and TER was measured. (Top) The ectopically expressed proteins are as follows: none (X), LacZ (•), aPKCλkn (□), aPKCλwt (▵), and nPKCδkn (○). 2 d after cell seeding (1.5 × 10⁵ cells/cm²) when the TER values reached a plateau, the cells were processed for adenovirus infection (time 0, left) for 2 h in LC medium. Immediately after virus infection, the medium was changed to NC growth medium to allow the cells to complete junctional structure formation before the expression of ectopic proteins. After TER measurement for 45 h, the cells were subjected to calcium switch by changing the medium to LC for 2 h, and then back to NC. The values given represent mean values (1 ± SD) of three parallel cultures. The background resistance obtained from empty filters was deducted. Note that only cells expressing aPKCλkn show a retardation in TER development after calcium switch. (Bottom) MDCK II cells were infected with adenovirus vectors encoding LacZ (•), aPKCλkn (□), or aPKCλwt (▵) as in a. In this case, the cells were kept in fresh LC medium to induce ectopic protein expression in the absence of cell–cell adhesion. 20 h after virus infection, the medium was changed to NC medium, and the development of TER was monitored for 48 h. Note that aPKCλkn-expressing cells show substantial suppression of TER development even 48 h after calcium switch. (b) Overexpression of aPKCλkn increases the paracellular diffusion of FITC-dextran in a molecular mass-dependent manner. Paracellular diffusion of FITC-dextran 40K (left) and 500K (right) across adenovirus-infected MDCK II cells was evaluated. FITC-dextran was added to the medium on the apical side of cells subjected to calcium switch 2 d before. After 3 h incubation at 37°C, the fluorescence intensity of the medium in the basolateral side was measured with a fluorometer. Values given represent the mean values (±SD) of three parallel cultures.

Overexpression of aPKCλkn disrupts apico-basal cell surface polarity of MDCK II cells. (a) Two-dimensional diffusion of ectopically labeled fluorescent lipid from the apical to the basolateral domain. The apical surface of filter-grown MDCK II cell monolayers infected with the indicated adenovirus vectors and subjected to calcium switch were labeled for 10 min on ice with BODIPY-sphingomyelin. The cells were then immediately mounted (0 min) or left for an additional 60 min on ice after extensive washing. The distribution of the fluorescent lipid was then analyzed by taking z-sectional views using a confocal microscope. The upper and lower arrowheads indicate the positions of the apical and basal membranes, respectively. Bar, 25 μm. (b and c) Effect of aPKCλkn on the asymmetric distribution of epithelial polarity markers. Adenovirally infected MDCK II cells were doubly immunostained with anti–aPKCλ and anti–Na⁺, K⁺-ATPase (b) or anti–gp135 antibodies (c) 20 h after calcium switch. Representative xz-sectional views obtained by confocal microscopic analysis are presented. Note that aPKCλkn-expressing cells (arrowheads) exhibit disturbed localization of NKA as well as gp135, proteins that show polarized distributions in LacZ-expressing control cells. Bars, 25 μm.

Ternary complex formation of PAR-6, aPKCλ, and ASIP/PAR-3. (a–c) COS cells were transfected with expression vectors as indicated (top). The cell lysates (Sup) were processed for immunoprecipitation (IP) with anti–Flag (a and c) or anti–T7 (b) monoclonal antibodies, and the coimmunoprecipitated proteins were analyzed using with anti–aPKCλ (ι), anti–T7, anti–Flag, or anti–ASIP (C2-3AP) antibodies. (a) The Flag-PAR-6 immunoprecipitates contained T7-ASIPwt, but not T7-ASIP Δ30 lacking the sequence critical for the interaction with aPKC. (b) ASIPwt was not contained in PAR-6 ΔaPKCBD immunoprecipitates but in PAR-6wt or ΔCRIB/PDZ immunoprecipitates. (c) Coimmunoprecipitation of T7-ASIP with Flag-PAR-6 was enhanced by the coexpression aPKCλwt, but not aPKCλ ΔN47, which lacks the sequences in the PAR-6–binding region. (d) Schematic diagram of the PAR-6–aPKC-ASIP/PAR-3 ternary complex. PAR-6 interacts with ASIP through aPKCλ as a linker.

Identification of endogenous PAR-6 and its association with aPKCλ and ASIP in fully polarized epithelial cells. (a) Identification of endogenous PAR-6 protein in MDCK II cells. Semi-confluent MDCK cells were subjected to immunoprecipitation with 1 μg of affinity-purified anti–PAR-6 rabbit polyclonal antibodies (GW2AP, GC2AP) or control normal rabbit IgG. The resultant immunoprecipitates were subjected to Western blot analysis using the antibodies indicated in parentheses (right). GW2AP and GC2AP specifically immunoprecipitate a 43-kD protein comigrating with PAR-6 expressed in COS cells, which was recognized by N12AP as well as GW2AP. (b) Ternary complex formation of PAR-6, aPKCλ, and ASIP/PAR-3 in vivo. Anti–PAR-6 (GW2AP) immunoprecipitate (IP), prepared as in a, was analyzed using anti–aPKCλ monoclonal or anti–ASIP polyclonal antibody. PAR-6 immunoprecipitates specifically contain endogenous aPKCλ, full-length ASIP, and its splicing variant (bottom band). (c and d) Summary of the yeast two-hybrid assays to analyze the PAR-6–aPKCλ interaction. The interaction was examined by growth on culture plates lacking histidine. The NH₂-terminal region including CR1 and 2 of PAR-6 is sufficient for the interaction with aPKCλ (c), while NH₂-terminal residues 22–113 of aPKCλ are sufficient for the interaction with PAR-6 (d).

Colocalization of PAR-6, aPKCλ, and ASIP/PAR-3 with ZO-1 at the apical end of cell–cell contact region of epithelial cells. (a and b) Immunofluorescence staining of MDCK cells with anti–PAR-6 polyclonal antibodies, GW2AP (a) and GC2AP (b). IgG means an equal amount of normal rabbit IgG used as a negative control. In b, the photograph was taken at higher sensitivity than in a to visualize weak cell–cell staining by GC2AP. (c) Confocal z-sectional view of MDCK cells doubly stained with anti–PAR-6 (GW2AP), anti–aPKCλ (λ1), or anti–ASIP antibodies (green), together with anti–ZO-1 antibody (red). The arrowhead indicates the position of the basal membrane. All three proteins colocalize with ZO-1 to the apical end of lateral membrane. (d) Immunostaining of a frozen section of mouse intestinal epithelium with anti–PAR-6 (GW2AP), anti–aPKCλ, or anti–ASIP antibodies (green). Only merged views with anti–ZO-1 staining (red) are shown. (e) Overexpression of aPKCλkn affects the junctional localization of PAR-6 as well as ZO-1. Bars, 25 μm (a, b, d, and e) and 10 μm (c).