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Syntaxin 1A is delivered to the apical and basolateral domains of epithelial cells: the role of munc-18 proteins

Joanna Rowe^1,*, Federico Calegari^1,, Elena Taverna¹, Renato Longhi² and Patrizia Rosa^1,

¹ CNR – Cellular and Molecular Pharmacology Center, Department of Medical Pharmacology, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy
² CNR Institute of Biocatalysis and Molecular Recognition, Milan, Italy
^* Present address: Roslin Institute, Roslin, UK
Present address: Max Planck Institute, Dresden, Germany

View larger version (26K): [in a new window]   Fig. 1. Syn1A and munc-18-1 are not immunodetected in epithelial cell lines. Total tissue and cell extracts were prepared from human cerebellum (lane 1), human colon carcinoma derived Caco-2 cells (lane 2), human meningioma (lane 3), canine brain cortex (lane 4), MDCK cells (lane 5) and canine liver (lane 6). Equal amounts of protein were separated on 10% SDS-polyacrylamide gels, transferred onto nitrocellulose and probed with an anti-syn1A monoclonal antibody (Syn1; A), a polyclonal antibody raised against a synthetic peptide of the rat munc-18-1 (Munc-18-1; B) sequence and an anti-munc-18-2 antiserum (Munc-18-2; C). Endogenous syn 1 and munc18-1 could be detected in the human and canine neuronal tissues, whereas the more widespread munc-18-2 protein was immunodetected in all tissues examined with the exception of human meningioma. The positions of molecular mass markers are shown.

View larger version (38K): [in a new window]   Fig. 2. Levels of expression of syntaxins and munc-18 proteins in transfected epithelial cells. Total rat brain extract (lane 4, 15 µg of protein), and total cell extracts (40 µg of protein) prepared from MDCK cells non-transfected (lanes 1 and 6) or transfected with either syn3 (Syn3, lane 2), syn1A (Syn1A, lane 3), syn1A and munc-18-1 (Syn1A, M-18-1, lane 5), or syn1A and munc-18-2 (Syn1A, M-18-2, lane 7), were analyzed by western blotting using antibodies against syn3 (Syn3, lanes 1, 2), syn1 (Syn1, lanes 3,4-7), munc-18-1 (M-18-1, lanes 4 and 5) or munc-18-2 (M-18-2, lanes 6 and 7). After cDNA transfections, syn1A (lane 3) or syn1A and munc-18-1 (lane 5) are immunodetected in MDCK cells. Note the increased levels of syn3 after transfection (compare lanes 1 and 2). Similarly, after cotransfection of munc-18-2 with syn1A (lane 7) the levels of munc-18-2 are also largely increased compared to untransfected MDCK cells (compare lanes 6 and 7).

View larger version (95K): [in a new window]   Fig. 3. In transfected epithelial cells, syn1A, but not syn3, is retained intracellularly in the absence of munc18-1. Non-polarized MDCK (A,A',B) and Caco-2 cells (C,D,E,E') were fixed 24 hours after transfection with syn1A (A,A',C,D,E,E'), or syn3 (B). The cells were immunolabeled using an anti-syn1 monoclonal antibody (B,C) or polyclonal antibodies against syn3 (D). In A,A' and E,E' the cells were double immunolabeled using the monoclonal antibodies against syn1 and a polyclonal antibodies against giantin (A' GIA) and galactosyltransferase (GAL, E'). The cells were analyzed by confocal immunofluorescence microscopy. In A,A' and E,E', asterisks indicate syn 1A transfected cells showing a reticular-like distribution of the t-SNARE (in A) and Golgi complex dispersion (A'). Bars, 20 µm (A,A',D,E,E'); 10 µm (B,C).

View larger version (62K): [in a new window]   Fig. 4. Syn1A transport to the cell surface is rescued by coexpression of munc-18 proteins. Non-polarized MDCK cells were fixed 5 (A-A'') or 48 hours (B-C') after the coexpression of syn1A and munc-18-1 (A-B') or munc-18-2 (C,C'). Cells were double-immunolabeled using the anti-syn1 (SYN1) antibody together with the anti-munc-18-1 (M-18-1), or anti-giantin (GIA) polyclonal antibodies. In C,C', asterisks label cells with syn1A at the plasma membrane and no disturbance of the Golgi complex. Bars, 10 µm.

View larger version (111K): [in a new window]   Fig. 5. Localization of syn1A at the apical and basolateral domains of polarized MDCK cells. MDCK cells cotransfected with syn1A and munc-18-1 were grown on permeable filters for 4 days. Before fixation the apical surface of cell monolayer was labeled using concanavalin A (CONA, A',B') as described in Materials and Methods. Cells expressing syn1A were detected using the anti-syn1 antibody followed by secondary antibody conjugated to rhodamine (SYN1, A,B). Consecutive horizontal optical sections through the cell monolayers were recorded by means of confocal fluorescence microscopy at 0.5 µm intervals. Sections are shown at the level of the apical surface (AP, A,A') and the nucleous (BL, B,B'). Note the presence of syn1A at both the apical and basolateral sides of the expressing cells. Bars, 10 µm.

View larger version (96K): [in a new window]   Fig. 6. Distribution of syn1A and syn3 in polarized Caco-2 cells. Caco-2 cells cotransfected with syn1A and munc-18-1 (A-C' or untransfected (D,D') were grown on permeable filters for 7 days. Transfected cells were double-immunolabeled for syn1A (SYN1, A-C), and untransfected cells were stained for syn3 (SYN3, D). In all cases the apical cell membrane was labeled using concanavalin A (CONA, A'-D'). Consecutive horizontal optical sections through the cell monolayers were recorded as described in Fig. 5. In C-D', confocal sections in the vertical plane are shown with the apical side of the cell monolayer at the top. Note the presence of syn1A at both the apical (AP) and basolateral (BL) membranes, whereas endogenous syn3 appears exclusively at the apical side. Bars, 10 µm.

View larger version (69K): [in a new window]   Fig. 7. Coexpression of munc-18-2 with syn1A does not alter the localization of the t-SNARE in polarized MDCK cells. Cells were cotransfected with syn1A and munc-18-1 (SYN1(M-18-1), A,A', C,C') or munc-18-2 (SYN1 (M-18-2), B,B'). The polarized monolayers were grown as described in Fig. 5. The cells were immunolabeled for syn1A (A,B) or the -subunit of the Na+/K+ ATPase (C) whereas the apical membrane was stained with concanavalin A (CONA, A',B',C'). Confocal sections in the vertical plane are shown with the apical surface (AP) of the cells at the top.

View larger version (57K): [in a new window]   Fig. 8. Detergent insolubility. MDCK cells cotransfected with syn1A and munc-18-1 (A-B') were grown on permeable filters for 5 days and then homogenized at 4°C in buffer containing either 1% (w/v) Triton X-100 or 20 mM CHAPS. Total homogenates were subjected to centrifugation at 15,000 g for 5 minutes (L.S. = low speed) or 100,000 g for 1 hour (H.S. = high speed). The supernatants (S) and corresponding pellets (P) were analyzed by SDS-PAGE. After electrophoresis proteins in the gels were either stained with Coomassie Blue (A) or transferred to filters (B) and probed with anti-munc-18-1 (M-18-1), anti-syn1A (Syn1) and caveolin 1 (Cav-1) antibodies.

View larger version (69K): [in a new window]   Fig. 9. Discontinuous sucrose flotation gradients. MDCK cells transfected with either syn1A and munc-18-1 (a-e) or syn 1A alone (f) and untransfected Caco-2 cells (g) were lysed at 4°C in 1% (w/v) Triton X-100 and then loaded at the bottom of a discontinuous sucrose gradients. After centrifugation proteins in the collected fractions (1-8) and the pellets (P) were separated by SDS-PAGE, blotted onto a membrane and analyzed, using specific antibodies, for the distribution of transferrin receptor (TfR), munc-18-1 (M-18-1), actin, caveolin-1 (Cav), syn1A (Syn1) and syn3 (Syn3). Note that in the presence of munc-18-1 (+M-18-1), a portion of syn1A floats in a low density fraction, which also contains caveolin-1 and syn3.