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Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation

¹ Department of Molecular Biology, Yokohama City University School of Medicine, Kanazawa-ku, Yokohama 236-0004, Japan
² Department of Pathology, Yokohama City University School of Medicine, Kanazawa-ku, Yokohama 236-0004, Japan
³ Department of Anatomy, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan

View larger version (44K): [in a new window]   Fig. 1. (A) Full length ASIP/PAR-3 (ASIP180k) and its splice variant with a shorter C-termini (ASIP150k). The region recognized by each antibody (C2-3AP, A2-2AP and Yap) is indicated; Yap recognizes the specific C-terminal sequence of ASIP150k. CR, conserved region. (B) Expression of ASIP/PAR-3 in various rat tissues. Total extracts from various rat tissues (lung, glandular stomach, kidney, prostate, ovary, uterus), intestinal epithelial cell scrapings (IEC), and COS1 cells transfected with T7-ASIP150k or T7-ASIP180k as positive controls were subjected to SDS-PAGE (8% polyacrylamide gel) followed by western blot analysis with the affinity-purified anti-ASIP antibodies (C2-3AP, A2-2AP and Yap). C2-3AP recognizes bands of 180 kDa (filled arrowheads; lung, glandular stomach, kidney, prostate, ovary and uterus) and 150/160 kDa (unfilled arrowheads; IEC, kidney and prostate), which are recognized by A2-2AP and Yap, respectively.

View larger version (73K): [in a new window]   Fig. 2. ASIP/PAR-3 distributes at the cell-cell junctions of various rat epithelial cells in a manner different from that of ZO-1. The differential distributions of ASIP/PAR-3 and ZO-1 are compared in rat forestomach (A-C), small intestine (E-G), and renal cortex (I-K). The samples were double stained with affinity-purified anti-ASIP antibody (C2-3AP, green) and anti-ZO-1 (red). In merged views (C,G,K), the yellow and orange signals show the co-localizations of ASIP/PAR-3 and ZO-1. In stratified squamous epithelium, ASIP/PAR-3 localizes at cell-cell junctions from the basal layer to the granular layer, and some cytoplasmic punctate patterns are observed (A). The localization of ZO-1 in the same field as in A is shown in B. The signal intensity of ZO-1 in the cell-cell junctions was much higher in the spinous layer than in the basal layer. ZO-1 is also detected in blood vessels (B, arrowhead), whereas ASIP/PAR-3 is not. As shown in the merged view (C) and indicated schematically in D, the relative signal intensities of ASIP/PAR-3 and ZO-1 differ along with epithelial differentiation. (D) Phase contrast image. Asterisk, lumen; BL, basal layer; GL, granular layer; HL, horny layer; LP, lamina propria; SL, spinous layer. In small intestine, ASIP/PAR-3 is concentrated in the subapical domain of epithelial cell-cell junctions (E) in a manner different from that of ZO-1 (F). As indicated in (B), ZO-1 signals are also detected in blood vessels (F, arrowheads). Unfilled arrowheads in G indicate that the signal intensity of ASIP/PAR-3 (green) in crypts is relatively higher than that of ZO-1 (red), whereas ZO-1 signal intensities are relatively higher in villus (asterisk). (H) Phase contrast image of the same field as E. As shown in I-K, the signal intensity of ASIP/PAR-3 (green) in proximal renal tubules (pt) is the same as in distal tubules (dt); however, that of ZO-1 (red) is much higher in distal renal tubules (dt) than proximal tubules (pt). (L) Schematic structure of proximal and distal renal tubules in a nephron. The number of TJ strands in epithelial cells of the distal renal tubule is much higher than in the proximal tubule. Bars, 40 µm (A), 20 µm (E), 10 µm (I).

View larger version (117K): [in a new window]   Fig. 3. Immunogold electron microscopy of ASIP/PAR-3 (A-D) and ZO-1 (C,D) in epithelial cells of distal (A,C) and proximal (B,D) renal tubules. Ultrathin cryosections of rat renal tubular epithelial cells were labeled with anti-ASIP/PAR-3 pAb (A,B; 5 nm gold particles). In both the distal (A) and proximal (B) tubules, gold particles for ASIP/PAR-3 are concentrated exclusively in the cytoplasm of TJ. Gold particles are constantly observed at the apical edge of TJ, and are also frequently found at the basal edge of TJ. (C,D) Double immunolabeling of renal tubular epithelium for ASIP/PAR-3 and ZO-1. In contrast to ASIP/PAR-3 (10 nm gold particles), ZO-1 (5 nm gold particles) distributes alongside TJ in the distal (C) and proximal (D) tubules. Apical is up and basal is down in these figures (A-D). TJ, tight junction. Bars, 100 nm.

View larger version (72K): [in a new window]   Fig. 4. Characterization of MDCK Tet-Off cell lines expressing T7-tagged L-ASIP WT or PB. (A) Schematic structures of the constructs. The aPKC-binding sequence is found in L-ASIP WT but not in L-ASIP PB. (B) Each cell line was cultured with (+) or without (-) 1.0 µg/ml of tetracycline (TC), and 5x104 cells/lane were subjected to western blot analysis with the T7-tag-specific antibody (Omni probe) or anti-aPKC antibody. The expression of T7-tagged ASIP/PAR-3 in each cell line was strongly induced by the withdrawal of TC, whereas 1.0 µg/ml of TC completely repressed the expression to under the detectable levels. (C-E) Immunofluorescent images show that the overexpressed T7-tagged ASIPs localize mainly at cell-cell contacts of MDCK cells cultured without TC (TC0). Confluent monolayers of MDCK cells expressing T7-L-ASIP WT (C) or T7-L-ASIP PB (D) were labeled by the Omni probe pAb. 1 µg/ml of TC (TC1) completely repressed the expressions of L-ASIP WT (E) and L-ASIP PB (not shown). Bars, 100 µm; 10 µm (inset).

View larger version (44K): [in a new window]   Fig. 5. Induced overexpression of ASIP/PAR-3 accelerates TER development after cell plating. (A-C) There is no significant difference in cell growth between cells cultured with or without 10 ng/ml of doxycycline (DC). (D-F) TER development of each cell line was measured at different times after plating on Transwell-ClearTM filters at a density of 1.5x105 cells/cm2. Cells were maintained with or without 10 ng/ml of DC for at least 3 days before plating. Every point is the mean±s.e.m. of three groups of cells on independent filters. P-values were calculated with a two-sided t-test and statistical significance was considered at P<0.05 (D, asterisks). (G) Expressions of the T7-tagged ASIPs and endogenous aPKC at the different times after plating. Cells were maintained with or without 10 ng/ml of DC and 5x104 cells/lane were subjected to western blot analysis with the T7-tag-specific antibody (Omni probe) and anti-aPKC antibody.

View larger version (54K): [in a new window]   Fig. 6. Insolubilization of occludin after Ca2+ switch in MDCK cell lines expressing L-ASIP WT (A,C) or PB (B,D). MDCK cells cultured with or without 10 ng/ml of DC were harvested at different times after Ca2+ switch, and the NP-40-insoluble fractions were subjected to western blot analysis with anti-ASIP (C2-3AP) or anti-occludin pAb. CBB staining of tubulins on the blotted membranes is shown as a control. The results are representative of three independent experiments. NP-40-insoluble occludin was detected by chemiluminescence ECL, and the resulting signals were quantified directly with LAS-1000 plus system (FUJI Photo Film, Tokyo, Japan) in a fluorescence image mode and normalized to the amount of tubulins in each lane (C,D). Each point is the mean±s.e.m. of three independent experiments. P-values were calculated with a two-sided t-test and statistical significance was considered at P<0.05 (asterisk). Induced overexpression of L-ASIP WT (C; filled circles), but not PB (D; filled squares), results in the rapid accumulation of occludin in NP-40-insoluble fractions after Ca2+ switch.

View larger version (75K): [in a new window]   Fig. 7. Localization of Ser827 phosphorylated ASIP/PAR-3 in mature and immature cell-cell contacts. ASIP/PAR-3 phosphorylated at Ser827 (A,D) and overexpressed T7-tagged ASIP/PAR-3 (B,E) were labeled with Cy3 and FITC, respectively. Four hours after Ca2+ switch, the overexpressed ASIP/PAR-3 (green; B,C) in mature cell-cell contacts overlaps the immunoreactivities of ASIP/PAR-3 phosphorylated at Ser827 (red; A,C, arrows). In contrast, 1 hour after switching, the immunoreactivities for ASIP/PAR-3 phosphorylated at Ser827 predominantly occupy the most apical tip of the immature cell-cell contacts (D,F; filled arrowhead), whereas overexpressed L-ASIP WT is detected also in the region slightly basal to the apical tip (E, F; unfilled arrowhead). The merged view of an X-Z optical section is shown in F with filled and unfilled arrowheads corresponding to the levels of the X-Y optical sections shown in G and H, respectively. The distance between the two X-Y optical sections is 1.75 µm. Merged X-Z views of other representatives are shown in I, J and K. Anti-S827-P antibody strongly stains cell-cell junctions of MDCK cells expressing T7-tagged ASIP/PAR-3 (L), whereas the antigen peptide abolishes the signals (M) in cell-cell junctions where T7-tagged ASIP/PAR-3 is concentrated (N). The distribution of overexpressed T7-tagged ASIP/PAR-3 in the same field as in M is shown in N. Bars, 10 µm.