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doi: 10.1242/10.1242/jcs.00116

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Retinal pigment epithelial cells exhibit unique expression and localization of plasma membrane syntaxins which may contribute to their trafficking phenotype

¹ Department of Cell Biology, Lerner Research Institute, Cleveland, Ohio 44195, USA
² Urological Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA
³ Department of Ophthalmic Research, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA

View larger version (35K): [in a new window]   Fig. 1. Comparison of SNARE expression in RPE-J cells and rat kidney or brain by western blot analysis. Equal protein amounts of total homogenates of differentiated RPE-J cells, rat kidney or rat brain (1/10 of the protein) were separated by SDS-PAGE and analyzed by western blot with specific antibodies as indicated. Note that syntaxin 3 is virtually undetectable in RPE-J cells. By contrast, the expression levels of syntaxins 2 and 4 are roughly comparable between RPE-J and rat kidney. SNAP-23, cellubrevin and endobrevin are expressed in both kidney and RPE-J. RPE-J cells also express the `neuron-specific' t-SNAREs syntaxin 1A and 1B but there is no detectable SNAP-25.

View larger version (30K): [in a new window]   Fig. 2. RT-PCR analysis of syntaxin isoform expression in RPE-J cells. mRNA of syntaxin 3 and of different alternatively spliced isoforms of syntaxin 2 were amplified by RT-PCR. For syntaxin 2 isoform determination, two primer pairs were used that had been previously shown to distinguish between isoforms 2A, 2B, 2C and 2D (Quinones et al., 1999). Primer combination 21 generates the following PCR products: syntaxin 2A (200 bp), 2D (228 bp), no products for syntaxin 2B and 2C. Primer combination 22 generates the following PCR products: syntaxin 2A (700 bp), 2B (600 bp), 2C (170 bp), 2C 170 bp), 2D (728 bp). The respective positions of the different products are indicated by arrows. A shows that transcripts for syntaxin 2A, B and C can be detected in rat kidney. Rat brain expresses all four syntaxin 2 isoforms as previously reported. As shown in B, reaction products for syntaxins 2A and 2B were detected from RPE-J cells but not 2C, 2D. For the detection of syntaxin 3A transcripts, a single primer pair was used. B shows that syntaxin-3A-specific reaction product was detected in the positive control (lane `syn3', rat syntaxin 3A cDNA as template), whereas no syntaxin 3A product could be detected from RPE-J cells. As a negative control, no products were detected in samples that were not reverse transcribed (-RT).

View larger version (133K): [in a new window]   Fig. 3. Localization of syntaxins 2 and 4 in RPE-J cells. Polarized monolayers of RPE-J cells were cultured on Transwell filters, and syntaxin localizations were analyzed by double-label confocal immunofluorescence microscopy. Panels A-C and D-F, respectively, represent the same fields of xy confocal sections. Small panels underneath are xz confocal sections. A shows immunostaining for syntaxin 2, B for the tight junction protein ZO-1 and C the color-merged image. D shows immunostaining for syntaxin 4, E for the basolateral plasma membrane protein EMMPRIN and F the color-merged image. Note that syntaxin 2 and ZO-1 colocalize throughout most of the areas of cell-cell contact. However, the localization of ZO-1 at the tight junctions is more uniform. There are areas of intense syntaxin 2 staining, whereas others are nearly devoid of it. Syntaxin 4 and EMMPRIN both colocalize in a relatively uniform fashion at the areas of cell-cell contact. Bars, 5 µm.

View larger version (130K): [in a new window]   Fig. 4. Expression and subcellular localization of SNAREs in vivo. Rat eye tissue sections were double-labeled with specific antibodies and analyzed by confocal fluorescence microscopy. In all panels, the respective SNARE is in green, staining for the double-label is in red and the nuclear stain is in blue. (A) Syntaxin 3 (green), collagen-IV (red). Note that syntaxin 3 is expressed in the photoreceptor inner segments (IS) but is not detectable in the photoreceptor outer segments (OS) or the RPE cells. RPE cells are identified by the collagen-IV-positive basement membrane (BM). (B) Syntaxin 1A (green), collagen-IV (red). Note the expression of syntaxin 1A at the apical plasma membrane of RPE cells (arrowhead) that is in contact with the photoreceptor outer segments, which themselves are negative. (C) Syntaxin 1B (green), occludin (red). Note the syntaxin 1B signal on the apical plasma membrane of the RPE cells (arrowhead) above the tight junctions identified by occludin-staining. (D) Syntaxin 2 (green), occludin (red). Note that syntaxin 2 localizes to a narrow band just underneath the occludin-positive tight junctions. Syntaxin 2 does not overlap with occludin but is instead always towards the basolateral side of the cells, indicating that it localized to a region of the lateral plasma membrane that is adjacent to the tight junctions. (E) Syntaxin 4 (green), occludin (red). Similar to syntaxin 2, syntaxin 4 localizes to a narrow band underneath the occludin-positive tight junctions (arrowhead). In addition, syntaxin 4 staining is evident at the basal plasma membrane and in intracellular vesicles that are often perinuclear. (F) Endobrevin (green), occludin (red). Note that endobrevin localizes to narrow a band underneath the tight junctions (arrowhead) in addition to localizing in small vesicles that are diffusely distributed throughout the cytoplasm of RPE cells.

View larger version (28K): [in a new window]   Fig. 5. Schematic models of syntaxin distribution and polarized trafficking pathways in MDCK and RPE cells. The subcellular localizations of syntaxins are indicated as reported previously in MDCK cells (Low et al., 1996) and in the present work in RPE cells. In MDCK cells, trafficking from the TGN to the apical plasma membrane (route A) was previously shown to involve syntaxin 3 (Low et al., 1998a), whereas trafficking from the TGN to the basolateral plasma membrane (route B) was shown to involve syntaxin 4 (Lafont et al., 1999). By contrast, basolateral-to-apical transcytosis is independent of syntaxin 3 in MDCK cells (Low et al., 1998a). The absence of syntaxin 3 in RPE cells suggests that route A does not exist in this cell type but that probably an alternative route designated A2 exists. This alternative route may depend on syntaxin 1A and/or 1B, which are expressed in RPE cells and localize to the apical plasma membrane. In RPE cells, syntaxin 2 is exclusively localized to a region below the tight junctions, in contrast to its distribution along both plasma membrane domains in MDCK cells. Syntaxin 4 localizes all along the lateral membrane in MDCK cells, and post-Golgi transport vesicles carrying basolateral cargo can fuse along the entire lateral membrane (Kreitzer et al., submitted). By contrast, in RPE cells syntaxin 4 localizes to the same narrow band underneath the tight junctions as does syntaxin 2. In addition, syntaxin 4 localizes to the basal membrane. This suggests that syntaxin 4 may serve two different trafficking pathways that lead to either location (designated B1 and B2). These syntaxin-4-dependent routes may originate from the TGN and/or endosomes.

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