Supplementary MaterialsSupplemental data for this article can be accessed around the publisher’s website
Supplementary MaterialsSupplemental data for this article can be accessed around the publisher’s website. was shown to suppress proliferation in an Ocln-dependent manner. Blockade of protein kinase C-zeta (PKC-) diminished transepithelial electrical resistance (TER) of HPAFII monolayers that was not corrected by dobutamine treatment while the loss of TER resulting from inhibition of ROCK1 could be partially recovered. Examination of normal and cancerous human pancreatic biopsies showed that this cellular localization of Ocln, c-Yes, YAP, and TEAD were similar to HPAFII for normal cells TC21 and AsPc1 for cancerous cells. Together, these results suggest a link between Hpo and signals emanating from cell-cell contacts involving Ocln that may regulate pancreatic cell proliferation through the coordination of planar cell polarity with apical-basal polarity events. binding partner of YAP (Aragona et?al., 2013), we examined whether Ocln could function as a potential sensor to regulate proliferation signals involved in apical-basal epithelial cell polarity. We tested this hypothesis using 2 human pancreatic cancer cells lines with different potentials to polarize to form polarized monolayers on permeable supports (Kim et?al., 1989). We first characterized the cellular distribution for proteins of interest in HPAFII cells produced at low cell density on plastic where nascent lateral cell-cell contacts had begun to be established. Immunofluorescence exhibited YAP was extensively EC1167 co-localized with c-Yes. This co-localization occurred primarily in the cytoplasm; some YAP, but very little c-Yes was observed in the nucleus (Fig.?1A1). Ocln/c-Yes co-localizations were observed primarily at cell-cell contacts but these appeared to occur to a lesser extent than c-Yes/YAP co-localizations and showed similarities to the Ocln/YAP distribution: mostly cytoplasmic co-localizations (Fig.?1A2). Co-localization of Ocln with YAP was less striking relative to Ocln/c-Yes and c-Yes/YAP interactions (Fig.?1A3). Further, c-Yes/YAP co-localizations appeared to EC1167 be primarily in the cytoplasm while Ocln/YAP co-localizations were observed more frequently at leading edge surfaces of these small cell colonies. TEAD/Ocln co-localizations were predominately in the cytoplasm of HPAFII cells, with limited interactions at the cell surface (Fig.?1A4). Higher magnification analysis showed TEAD/Ocln co-localizations to be incomplete in their overlap (Fig.?1A4), unlike the more complete overlay observed for c-Yes/YAP, c-Yes/Ocln and YAP/Ocln co-localizations (Fig.?1A1CA3). Open in a separate window Physique 1. Distribution and co-localization of c-Yes, Ocln, YAP, and TEAD in HPAFII cells 0.001. (D) Immunoblot analysis of TEAD in the nuclear fractions of HPAF II cells as a consequence of 10 or 20?M dobutamine exposure. Quantification of TEAD expression values are mean of 3 impartial experiments SEM; * 0.05. Dobutamine-induced redistribution of Ocln has functional consequences Dobutamine treatment of HPAFII cells produced at low cell densities resulted in a dose-dependent shift in Ocln immunofluorescence from the cytoplasm to the cell surface (Fig.?2). Concomitant with this shift in Ocln localization, there was an increase in co-localizations involving c-Yes and Ocln that was associated predominantly with EC1167 the membrane fraction of these cells (Fig.?3A, B). These results are interesting in light of previous findings showing that c-Yes is usually associated with Ocln at assembling TJ structures and is dissociated from Ocln at times when TJ structures are disassembling (Chen et?al., 2002b). Thus, suppression of YAP translocation to the nucleus by dobutamine treatment is usually associated with increased levels of c-Yes/Ocln co-localization in polarizing HPAFII cells. Open in a separate window Physique 3. Redistribution of Ocln and c-Yes in HPAFII cells and restriction of tricellulin to tri-cellular contacts following dobutamine treatment. (A) Confocal microscopy showing c-Yes and Ocln distribution in HPAFII cells. Cells were treated with 10 or 20 M dobutamine for 24?h period prior to c-Yes and Ocln cell distribution analysis. Scale bar, 20?m. (B) Immunoprecipitation of Ocln followed by an immunoblot analysis for c-Yes in nuclear, cytoplasmic, and membrane fractions of non-treated and 20?M dobutamine treated HPAFII cells. (C) Confocal images showing distribution of Ocln and tricellulin (Tric) in HPAFII cells treatment with 20?M dobutamine for 24?h. Scale bar, 20?m. (D) Immunoprecipitation of Ocln followed by an immunoblot analysis for Tric in nuclear, cytoplasmic, and membrane fractions of HPAFII cells following 20?M dobutamine exposure. 0.05. (I) Immunoblot for Ocln in HPAFII cells present in isolated fractions before and after treatment with dobutamine. (J) TER measurements in HPAFII confluent monolayers seeded.