Intracellular redirection of plasma membrane trafficking after loss of epithelial cell polarity

Abstract

In polarized Madin-Darby canine kidney epithelial cells, components of the plasma membrane fusion machinery, the t-SNAREs syntaxin 2, 3, and 4 and SNAP-23, are differentially localized at the apical and/or basolateral plasma membrane domains. Here we identify syntaxin 11 as a novel apical and basolateral plasma membrane t-SNARE. Surprisingly, all of these t-SNAREs redistribute to intracellular locations when Madin-Darby canine kidney cells lose their cellular polarity. Apical SNAREs relocalize to the previously characterized vacuolar apical compartment, whereas basolateral SNAREs redistribute to a novel organelle that appears to be the basolateral equivalent of the vacuolar apical compartment. Both intracellular plasma membrane compartments have an associated prominent actin cytoskeleton and receive membrane traffic from cognate apical or basolateral pathways, respectively. These findings demonstrate a fundamental shift in plasma membrane traffic toward intracellular compartments while protein sorting is preserved when epithelial cells lose their cell polarity.

Figure 1

Syntaxin 11 is endogenously expressed in epithelial cell lines. Total membrane lysates were prepared from the following cultured cell lines: canine kidney epithelium–derived MDCK cells, MDCK cells stably transfected with human syntaxin 11, human colon carcinoma–derived Caco-2 and HT-29 cells, human liver epithelial HepG2 cells, and human HeLa cells. Equal amounts of protein were separated on a 12% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with an affinity-purified polyclonal antibody against an N-terminal peptide of the human syntaxin 11 sequence. Endogenous syntaxin 11 could be detected in MDCK, Caco-2, and HeLa cells. Identical signals were obtained with Western blots probed with antisera from two additional rabbits. All bands disappeared after competition with the syntaxin 11 peptide (our unpublished results). The electrophoretic mobility of syntaxin 11 in HeLa cells and exogenously expressed syntaxin 11 in MDCK cells is higher than that of endogenous syntaxin 11 in MDCK and Caco-2 cells.

Figure 2

Syntaxin 11 is localized at the plasma membrane in polarized but not in nonpolarized MDCK cells. A mixed population of MDCK cells stably expressing human syntaxin 11 was grown either for 3 d on polycarbonate filters (A–D) or for 2 h on glass coverslips (E). The cells were stained with antibodies against syntaxin 11 (A, C, and E, green), the lateral plasma membrane protein E-cadherin (B and D), or the late endosomal/lysosomal protein LAMP-2 (E, red). B and D also show nuclear DNA staining with propidium iodide. A and B show horizontal confocal optical sections just above the nuclei. C and D show vertical optical sections with the apical plasma membrane at the top. Syntaxin 11 colocalizes with E-cadherin at the lateral plasma membrane. E shows a representative horizontal optical section through the middle of the cells. Syntaxin 11 does not significantly colocalize with LAMP-2. The absence of a syntaxin 11 signal in neighboring nonexpressing cells (A and E) demonstrates the specificity of the antibody. Bars, 5 μm.

Figure 3

All plasma membrane t-SNAREs relocalize from intracellular compartments to their final plasma membrane domains during development of MDCK cells into a polarized monolayer. MDCK cells stably expressing syntaxins 2, 3, 4, and 11 and SNAP-23 were seeded at high density onto polycarbonate filters. At the indicated times, the cells were fixed and stained with affinity-purified antibodies against the respective SNAREs (green) as well as an antibody against the tight junction protein ZO-1 (red). Nuclei were stained with propidium iodide (red). Confocal optical sections through the monolayers are shown with the apical side on top. Once the cells have established contacts, the tight junctions can be seen as red dots at the junction between the apical and basolateral plasma membranes. At the earliest time, large intracellular vacuoles are occasionally detected (arrows), most frequently with syntaxin 3 and SNAP-23. While the distribution of all studied SNAREs is predominantly intracellular at 2 h, it shifts to a predominantly plasma membrane localization during the course of 7 d. Note that starting at d 1, syntaxin 3 is always excluded from the basolateral plasma membrane, whereas syntaxin 4 is always excluded from the apical plasma membrane. Bar, 5 μm.

Figure 4

In nonpolarized MDCK cells, apical t-SNAREs localize to the VAC, whereas basolateral SNAREs are excluded from it. MDCK cells stably expressing syntaxins 2, 3, 4, and 11 and SNAP-23 were seeded onto glass coverslips in regular medium. After 2 h, the cells were switched to LCM and incubated for 16 h. Cells were fixed, permeabilized, and stained for the individual SNAREs (left column) and with an antibody against an endogenous apical plasma membrane protein, gp135 (right column). gp135 localizes to the VAC. Syntaxins 2, 3, and 11 and SNAP-23 colocalize with gp135 in VACs. In contrast, syntaxin 4 is excluded from VACs (arrows indicate VACs for better orientation). Syntaxins 2 and 11 and SNAP-23 are also found in large intracellular compartments that exclude gp135 (arrows) in addition to gp135-positive VACs. Bars, 5 μm.

Figure 5

Endogenous syntaxin 3 localizes to VACs in Caco-2 cells. The human colon carcinoma cell line Caco-2 was grown under low-calcium conditions as described in Figure 4. Endogenously expressed syntaxin 3 (A) and the microvillar protein villin (B) were labeled with the appropriate antibodies. Syntaxin 3 is strongly enriched in VACs that are also positive for villin, indicating that VACs are lined by microvilli (arrows). Bar, 5 μm.

Figure 6

VACs are not related to lysosomes. MDCK cells expressing syntaxin 3 were grown in LCM for 16 h, fixed, permeabilized, and stained for syntaxin 3 (A) and the late endosomal/lysosomal protein LAMP-2 (B). Note that large vacuoles stain for syntaxin 3 but not LAMP-2 (arrows). Bar, 5 μm.

Figure 7

Syntaxin 4 is a marker for a novel intracellular organelle in nonpolarized MDCK cells. MDCK cells expressing syntaxin 4 were grown in LCM (A–F and I–T) or regular medium (G and H) for 16 h, fixed, permeabilized, and stained for syntaxin 4 (left columns). Cells were colabeled for the following antigens: (B) antigen 6.23.3, a 58-kDa basolateral plasma membrane protein; (D) Na/K-ATPase; (F and H) E-cadherin; (J) F-actin (staining with fluorescent phalloidin); (L) fodrin; (N) cytokeratin (antibody against pan-cytokeratin); (P) the Golgi protein Golgin-97; (R) γ-tubulin to stain the MTOCs; and (T) ubiquitin. Note that cells in E and F were grown in LCM and show very low E-cadherin signals, in contrast to cells in G and H, which were grown in regular medium. Micrographs for panels F and H were recorded at identical imaging settings for comparison of signal intensities. Note also that cells in S and T were grown in the presence of the proteasome inhibitor acetyl-leucyl-leucyl-norleucinal (ALLN) to allow the accumulation of aggresomes. Arrows at identical locations between panels are drawn for better orientation. Bars, 5 μm.

Figure 8

Intracellular syntaxin 4–positive organelles can be observed under normal extracellular calcium conditions in cells that are not within a monolayer. MDCK cells expressing syntaxin 4 were grown for 16 h in medium containing a normal calcium concentration. The medium for A–D contained 5% FCS, whereas the medium for E and F was serum-free. Cells were costained for syntaxin 4 (left column) and gp135 (right column). Syntaxin 4 frequently localizes to intracellular organelles in single cells (C) as well as in cells at the periphery of a colony (A, arrow) regardless of the presence of serum. In contrast, syntaxin 4 is mostly restricted to the plasma membrane in cells in the midst of a colony. Prominent intracellular gp135-positive VACs are frequent in cells grown in the absence of serum (E and F, arrows) but rare when serum is included. Bars, 5 μm.

Figure 9

The “apical” and “basolateral” intracellular compartments receive membrane traffic from cognate polarized trafficking pathways. MDCK cells or MDCK cells stably expressing the wild-type pIgR (wt-pIgR), signalless pIgR (SL-pIgR), or a GPI-anchored mutant form of pIgR (GPI-pIgR) were grown in LCM for 16 h. IgA, 50 μg/ml polymeric IgA was added during the 16-h incubation of cells expressing wild-type pIgR. Tf, 1 μg/ml iron-loaded caninetransferrin was added to MDCK cells during the incubation. The cells were fixed, permeabilized, and stained with an antibody against the endogenous apical plasma membrane protein gp135 to label VACs and propidium iodide as a nuclear stain (right column, as indicated). The pIgR was detected with the use of an antibody against the ectodomain (left column, top three panels). IgA, transferrin, syntaxin 4, and gp80/clusterin were detected with the use of specific antibodies. Note that wt-pIgR, SL-pIgR, and GPI-pIgR are transported to the VAC (arrows). Also, IgA added to the medium of wt-pIgR–expressing cells is transported to the VAC (arrows), indicating that wt-pIgR is first transported to the plasma membrane after biosynthesis, where it can bind IgA, internalize it, and transport it into the VAC. In contrast, internalized transferrin does not reach the VAC (arrow). Instead, it is deposited into a syntaxin 4–positive compartment (F). The endogenous soluble protein gp80/clusterin, which is normally secreted apically and basolaterally (∼2:1 ratio) in polarized MDCK cells, is transported to both VACs (G) and syntaxin 4–positive organelles (H) in nonpolarized cells, suggesting that it reaches these compartments directly after biosynthesis. The panels in the left column show the same fields as those in the right column. Arrows are drawn for better orientation. The outline of one cell in G is drawn for clarity. Bars, 5 μm.

Figure 10

Quantitation of surface transport of the secreted protein gp80/clusterin in polarized and nonpolarized MDCK cells. MDCK cells (A) or MDCK cells stably expressing SL-pIgR (B) were grown as either a polarized monolayer on polycarbonate filters in regular medium or as nonpolarized cells on glass coverslips in LCM. Proteins were pulse-labeled with [³⁵S]cysteine. (A) The secretion of radiolabeled gp80 was monitored by collecting apical or basolateral medium (polarized) or total medium (nonpolarized) at different times and quantitation by SDS-PAGE and phosphorimaging and is represented as a percentage of total radiolabeled gp80 (intracellular plus secreted). Apically and basolaterally secreted gp80 was added to yield total secretion of gp80 from polarized cells. Note that after 3 h, gp80 secretion was nearly complete in polarized cells, whereas ∼15% of gp80 remained intracellular in nonpolarized cells. (B) The surface delivery of newly synthesized SL-pIgR was monitored by the addition of a low concentration of V8 protease to the chase medium, which efficiently released the extracytoplasmic domain of pIgR into the medium. Quantitation was as described above. Note that the kinetics of SL-pIgR delivery in polarized and nonpolarized cells is nearly identical, indicating that the majority of SL-pIgR is delivered directly to the plasma membrane. Experiments were done in triplicate, and error bars represent SDs. Error bars were omitted when they were smaller than the symbol.