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).

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.

Characterization of MDCK Tet-Off cell lines expressing T7-tagged L-ASIP WT or ΔPB. (A) Schematic structures of the constructs. The aPKC-binding sequence is found in L-ASIP WT but not in L-ASIP ΔPB. (B) Each cell line was cultured with (+) or without (-) 1.0 μg/ml of tetracycline (TC), and 5×10⁴ cells/lane were subjected to western blot analysis with the T7-tag-specific antibody (Omni probe) or anti-aPKCξ 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 ΔPB (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 ΔPB (not shown). Bars, 100 μm; 10 μm (inset).

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-Clear™ filters at a density of 1.5×10⁵ 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 5×10⁴ cells/lane were subjected to western blot analysis with the T7-tag-specific antibody (Omni probe) and anti-aPKCζ antibody.

Insolubilization of occludin after Ca²⁺ switch in MDCK cell lines expressing L-ASIP WT (A,C) or ΔPB (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 ΔPB (D; filled squares), results in the rapid accumulation of occludin in NP-40-insoluble fractions after Ca²⁺ switch.