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First published online 6 January 2004
doi: 10.1242/jcs.00893

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Mechanism of recruiting Sec6/8 (exocyst) complex to the apical junctional complex during polarization of epithelial cells

Charles Yeaman^*,, Kent K. Grindstaff and W. James Nelson

Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, CA 94305-5345, USA

View larger version (81K): [in a new window]   Fig. 3. Effect of sodium bicarbonate concentration on Sec6/8 complex distribution. Confluent MDCK cultures on polycarbonate filters were grown in DMEM containing either 1 g/l (`lo bicarb') or 3.7 g/l (`hi bicarb') sodium bicarbonate for 48 hours. (Top) Cultures were fixed with 4% paraformaldehyde before extraction with buffer containing 1% Triton X-100. Anti-Sec6 monoclonal antibody (9H5) was visualized with FITC-labeled goat anti-mouse antibody. Confocal images were obtained as described in Fig. 1 legend. Scale bar: 5 µm. (Bottom) Triplicate filters of cells grown in hi or lo bicarbonate were extracted successively in Triton X-100 and SDS, as described in Materials and Methods. Sec8 in Triton-soluble (`s') and Triton-insoluble (`p') fractions was quantified by SDS-PAGE and western blotting. Protein levels were quantified using a Molecular Dynamics Phosphorimager. In 1 g/l bicarbonate, Sec8 is enriched at the apical junction (Fig. 1) and is only partially (30%) soluble in Triton X-100. In 3.7 g/l bicarbonate, Sec8 is diffusely distributed along the lateral and basal membranes and is almost entirely (90%) soluble in Triton X-100.

View larger version (40K): [in a new window]   Fig. 6. Sec8 associates with a fraction of ZO-2. (A) MDCK cells were extracted in 1% Triton X-100 either 0 hours (contact-naive) or 48 hours (polarized) after inducing calcium-dependent cell-cell adhesion. Extracts were subjected to immunoprecipitation with specific antibodies to Sec8, Exo70, ZO-1, ZO-2 or occludin. The presence of Sec8 in precipitated immune complexes was assessed by SDS-PAGE followed by immunoblotting with Sec8 antibodies. (B) MDCK cells were homogenized 48 hours after induction of calcium-dependent cell-cell adhesion and junction-enriched membrane fractions were isolated by isopycnic density gradient centrifugation as described in Fig. 2. Membranes were extracted in 1% Triton X-100 and subjected to immunoprecipitation with antibodies specific for Sec8, occludin, claudin-1 or claudin-2. The presence of each of these proteins and of ZO-2 in precipitated immune complexes was assessed by SDS-PAGE followed by immunoblotting with specific antibodies. (C) Polarized MDCK cultures on polycarbonate filters were treated with 2 µM latrunculin B for 1 hour, then fixed with 2% paraformaldehyde before extraction with buffer containing 1% Triton X-100. Anti-Sec6 monoclonal antibody (9H5) was visualized with FITC-labeled goat anti-mouse antibody and anti-ZO-2 polyclonal antibody was visualized with Texas Red-labeled donkey anti-rabbit antibody.

View larger version (78K): [in a new window]   Fig. 1. Sec6/8 complex becomes restricted to apical junctional complex during development of cell polarity. Contact-naive MDCK cells were seeded at confluent density on collagen-coated filters, allowed to attach in low calcium medium for 3 hours, and then switched to high calcium medium for 0, 1, 3, 6, 12, or 24 hours. At each time point, cultures were fixed with 4% paraformaldehyde and extracted with buffer containing 1% Triton X-100. (A,B) Sec6 distribution was compared to that of either E-cadherin (A) or ZO-1 (B). Anti-Sec6 monoclonal antibody (9H5) was visualized with FITC-labeled goat anti-mouse antibody. Rabbit polyclonal antibodies to E-cadherin and ZO-1 were visualized with Texas Red-labeled donkey anti-rabbit antibody. Confocal images in the upper panels were acquired along the x-y axis (en face view) of the cell monolayer. The x-z views, in the lower panels, were constructed by averaging sections over a line at each z position in 0.2 µm steps. Scale bar: 10 µm. (C) Relative pixel intensities of Sec6, E-cadherin and ZO-1 fluorescence at each optical section (1=apical, 19=basal) were averaged from five independently scanned fields.

View larger version (98K): [in a new window]   Fig. 2. Sec6 co-localizes with components of the nectin complex in early cell-cell contacts and polarized MDCK cells. (Top panels) Low-density cultures of MDCK cells were allowed to form calcium-dependent cell-cell contacts for 1 hour, and then fixed with 2% paraformaldehyde before extraction with 1% Triton X-100. Sec6 distribution was compared to that of afadin or nectin-2. (Bottom panel) Confluent MDCK cultures on polycarbonate filters were fixed and extracted 24 hours after induction of calcium-dependent cell-cell adhesion. Sec6 distribution was compared to that of afadin. Anti-Sec6 monoclonal antibody (9H5) was visualized with FITC-labeled goat anti-mouse antibody (in the afadin panels) or with Texas Red-labeled donkey anti-mouse antibody (in the nectin panel). Rabbit polyclonal antibody to afadin was visualized with Texas Red-labeled donkey anti-rabbit antibody, and rat monoclonal antibody to nectin-2 was visualized with FITC-labeled goat anti-rat antibody. Confocal images were obtained as described in Fig. 1 legend. Scale bar: 10 µm.

View larger version (35K): [in a new window]   Fig. 4. Fractionation of MDCK cells in iodixanol gradients. MDCK cells were homogenized either 6 hours or 48 hours after induction of calcium-dependent cell-cell adhesion. Post-nuclear supernatants were mixed with 10%, 20% and 30% (w/v) iodixanol, layered step-wise in centrifuge tubes and centrifuged at 350,000 g for 3 hours. The presence of Sec8, ZO-1, ZO-2, afadin, occludin, claudin-1, claudin-2, E-cadherin, nectin-1 and nectin-2 in gradient fractions was assayed by SDS-PAGE followed by immunoblotting with specific antibodies. Protein levels were quantified using a Molecular Dynamics Phosphorimager. Densities of each fraction were calculated after measuring refractive indices with a refractometer, and are plotted as dotted lines on each graph with values (in g/ml) indicated on the y-axis.

View larger version (41K): [in a new window]   Fig. 5. Fractionation of junctional proteins associated with Sec6/8 complex. (A) Detergent extracts of polarized MDCK cells were fractionated by Superose 6 FPLC as described in Materials and Methods. Fractions 6-28 were divided into equal aliquots, separated by SDS-PAGE, and transferred to Immobilon P membranes. Membranes were probed with antibodies to Sec8, E-cadherin, afadin, ZO-1, ZO-2, ponsin or nectin-2. Protein levels were quantified using a Molecular Dynamics Phosphorimager. The elution profiles of Sec6 and E-cadherin are shown in B. The elution peaks of globular protein standards with known relative molecular masses were also determined: thyroglobulin, Mr=669,000 (fraction 16); apoferritin, Mr=443,000 (fraction 19); catalase, Mr=232,000 (fraction 22); bovine serum albumin, Mr=66,000 (fraction 24). (B) Coimmunoprecipitation of Sec8 with E-cadherin adhesion complex. MDCK cells were extracted either 3.5 hours or 3 days after induction of calcium-dependent cell-cell adhesion and extracts were fractionated by Superose 6 FPLC. Each fraction (10-19) was subjected to immunoprecipitation with anti-E-cadherin E2 antiserum. Immunoprecipitated material was eluted in SDS-PAGE sample buffer and the presence of Sec8 in each fraction was assayed by SDS-PAGE followed by immunoblotting. Protein levels were quantified using a Molecular Dynamics Phosphorimager.

View larger version (49K): [in a new window]   Fig. 7. Sec6/8 complex is associated with E-cadherin and nectin-2. Polarized MDCK cells cultured on polycarbonate filters were surface labeled with either Sulfo-NHS-SS-Biotin (A) or Sulfo-NHS-LC-LC-Biotin (B,C) and extracted with 1% Triton X-100 either directly (no x-link or -DSP) or following (x-link or +DSP) chemical cross-linking with the membrane-permeable cross-linker DSP. Non-biotinylated controls (no biotin) were subjected to cross-linking prior to extraction. (A) Extracts were incubated with avidin-agarose, and the presence of Sec8 in avidin precipitates was assayed by SDS-PAGE followed by immunoblotting with anti-Sec8 antibody. (B,C) Extracts were subjected to immunoprecipitation with anti-Sec8 antibodies, and the presence of biotinylated proteins, Sec8, nectin-2, nectin-1 and E-cadherin in precipitated immune complexes was assessed by SDS-PAGE followed by immunoblotting with HRP-avidin or specific antibodies. `+EGTA' cultures were incubated in LCM + 2 mM EGTA for 6 hours prior to biotinylation.

View larger version (91K): [in a new window]   Fig. 8. E-cadherin and nectin-2 cooperate to recruit Sec6/8 complex to intercellular contacts in fibroblasts. LE cells, or LE cells that had been transiently transfected with pcDNA3.1-IgK-2HA-nectin-2, were cultured without (no Dex) or with (plus Dex) 10-6 M dexamethasone for 18 hours to induce E-cadherin expression. Cells were fixed with 4% paraformaldehyde and then extracted with buffer containing 1% Triton X-100. Anti-Sec8 monoclonal antibody (2E9) was visualized either with FITC- or Texas Red-labeled secondary antibodies. Anti-E-cadherin polyclonal antibody (UVO) was visualized with Texas Red-labeled secondary antibody. Anti-nectin-2 rat monoclonal antibody was visualized with FITC-labeled secondary antibody. Arrows indicate homotypic cell-cell contacts formed between adjacent cells expressing either nectin-2 or E-cadherin only. Arrowheads indicate cell-cell contacts between E-cadherin-expressing cells in which nectin-2 was also expressed. Scale bar: 30 µm.