Search

Purpose: Localized renal cell carcinoma is usually curable by nephrectomy. However, a large fraction of patients already present with metastatic disease, which results in a poor outcome. Currently no clinically relevant screening assay is available to detect early stage renal cell carcinoma. We investigated whether urinary extracellular matrix (ECM) proteins and/or matrix metalloproteinase (MMP) activity may be valuable as a noninvasive indicator of early stage renal cell carcinoma. Materials and Methods: Urine specimens from preoperative patients with renal cell carcinoma and healthy controls were collected. The urinary excretion of the ECM proteins collagen IV, laminin and fibronectin was investigated by immunoblotting. MMP activity was assessed by gelatin zymography and by a fluorescence based microtiter plate activity assay. Results: The full-length forms of all 3 ECM proteins investigated were significantly decreased or absent in renal cell carcinoma urine. Based on criteria established in this study this finding would lead to the correct detection of 95% of patients with renal cell carcinoma (21 of 22) with a false-positive rate of 4.5% (1 of 22 controls). All 11 nonmetastatic cases of the lowest clinical stage (T1N0M0) were correctly identified. The absence of urinary ECM proteins was due to significantly increased urinary MMP activity. Conclusions: Analysis of decreased urinary ECM proteins and analysis of increased MMP activity may have value for the development of a sensitive, high throughput molecular screening assay to detect early stage renal cell carcinoma.

The SNARE membrane fusion machinery controls the fusion of transport vesicles with the apical and basolateral plasma-membrane domains of epithelial cells and is implicated in the specificity of polarized trafficking. To test the hypothesis that differential expression and localization of SNAREs may be a mechanism that contributes to cell-type-specific polarity of different proteins, we studied the expression and distribution of plasma-membrane SNAREs in the retinal pigment epithelium (RPE), an epithelium in which the targeting and steady-state polarity of several plasma membrane proteins differs from most other epithelia. We show here that retinal pigment epithelial cells both in vitro and in vivo differ significantly from MDCK cells and other epithelial cells in their complement of expressed t-SNAREs that are known — or suggested — to be involved in plasma membrane trafficking. Retinal pigment epithelial cells lack expression of the normally apical-specific syntaxin 3. Instead, they express syntaxins 1A and 1B, which are normally restricted to neurons and neuroendocrine cells, on their apical plasma membrane. The polarity of syntaxin 2 is reversed in retinal pigment epithelial cells, and it localizes to a narrow band on the lateral plasma membrane adjacent to the tight junctions. In addition, syntaxin 4 and the v-SNARE endobrevin/VAMP-8 localize to this sub-tight junctional domain, which suggests that this is a region of preferred vesicle exocytosis. Altogether, these data suggest that the unique polarity of many retinal pigment epithelial proteins results from differential expression and distribution of SNAREs at the plasma membrane. We propose that regulation of the expression and subcellular localization of plasma membrane SNAREs may be a general mechanism that contributes to the establishment of distinct sorting phenotypes among epithelial cell types.

The apical- and basolateral-specific distribution of target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) of the syntaxin family appear to be critical for polarity in epithelial cells. To test whether differential SNARE expression and/or subcellular localization may contribute to the known diversity of trafficking phenotypes of epithelial cell types in vivo, we have investigated the distribution of syntaxins 2, 3, and 4 in epithelial cells along the renal tubule. Syntaxins 3 and 4 are restricted to the apical and basolateral domains, respectively, in all cell types, indicating that their mutually exclusive localizations are important for cell polarity. The expression level of syntaxin 3 is highly variable, depending on the cell type, suggesting that it is regulated in concert with the cellular requirement for apical exocytic pathways. While syntaxin 4 localizes all along the basal and lateral plasma membrane domains in vivo, it is restricted to the lateral membrane in Madin-Darby canine kidney (MDCK) cells in two-dimensional monolayer culture. When cultured as cysts in collagen, however, MDCK cells target syntaxin 4 correctly to the basal and lateral membranes. Unexpectedly, the polarity of syntaxin 2 is inverted between different tubule cell types, suggesting a role in establishing plasticity of targeting. The vesicle-associated (v)-SNARE endobrevin is highly expressed in intercalated cells and colocalizes with the H+-ATPase in α- but not β-intercalated cells, suggesting its involvement in H+-ATPase trafficking in the former cell type. These results suggest that epithelial membrane trafficking phenotypes in vivo are highly variable and that different cell types express or localize SNARE proteins differentially as a mechanism to achieve this variability.

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.

The terminal step of cytokinesis in animal cells is the abscission of the midbody, a cytoplasmic bridge that connects the two prospective daughter cells. Here we show that two members of the SNARE membrane fusion machinery, syntaxin 2 and endobrevin/VAMP-8, specifically localize to the midbody during cytokinesis in mammalian cells. Inhibition of their function by overexpression of nonmembrane-anchored mutants causes failure of cytokinesis leading to the formation of binucleated cells. Time-lapse microscopy shows that only midbody abscission but not further upstream events, such as furrowing, are affected. These results indicate that successful completion of cytokinesis requires a SNARE-mediated membrane fusion event and that this requirement is distinct from exocytic events that may be involved in prior ingression of the plasma membrane.