Urothelial plaques are specialized membrane domains in urothelial superficial (umbrella) cells, composed of highly ordered uroplakin particles. We investigated membrane compartments involved in the formation of urothelial plaques in mouse umbrella cells. The Golgi apparatus did not contain uroplakins organized into plaques. In the post-Golgi region, three distinct membrane compartments containing uroplakins were characterized: i) Small rounded vesicles, located close to the Golgi apparatus, were labelled weakly with anti-uroplakin antibodies and they possessed no plaques; we termed them "uroplakin-positive transporting vesicles" (UPTVs). ii) Spherical-to-flattened vesicles, termed "immature fusiform vesicles" (iFVs), were uroplakin-positive in their central regions and contained small urothelial plaques. iii) Flattened "mature fusiform vesicles" (mFVs) contained large plaques, which were densely labelled with anti-uroplakin antibodies. Endoytotic marker horseradish peroxidase was not found in these post-Golgi compartments. We propose a detailed model of de novo urothelial plaque formation in post-Golgi compartments: UPTVs carrying individual 16-nm particles detach from the Golgi apparatus and subsequently fuse into iFV. Concentration of 16-nm particles into plaques and removal of uroplakin-negative membranes takes place in iFVs. With additional fusions and buddings, iFVs mature into mFVs, each carrying two urothelial plaques toward the apical surface of the umbrella cell.
COBISS.SI-ID: 28976345
In superficial umbrella cells of normal urothelium, uroplakins (UPs) are assembled into urothelial plaques, which form fusiform vesicles (FVs) and microridges of the apical cell surface. Altered urothelial differentiation causes changes in the cell surface structure. Here, we investigated ultrastructural localization of UPIa, UPIb, UPII and UPIIIa in normal and cyclophosphamide-induced preneoplastic mouse urothelium. In normal urothelium,terminally differentiated umbrella cells expressed all four UPs, which were localized to the large urothelial plaques covering mature FVs and the apical plasma membrane. The preneoplastic urothelium contained two types of superficial cells with altered differentiation: (1) poorly differentiated cells with microvilli and small, round vesicles that were uroplakin-negative; no urothelial plaques were observed in these cells; (2) partially differentiated cells with ropy ridges contained uroplakin-positive immature fusiform vesicles and the apical plasma membrane. Freeze-fracturing showed small urothelial plaques in these cells. We concluded that in normal urothelium, all four UPs colocalize in urothelial plaques. However, in preneoplastic urothelium, the growth of the uroplakin plaques was hindered in the partially differentiated cells, leading to the formation of immature FVs and ropy ridges instead of mature FVs and microridges. Our study demonstrates that despite a lower level of expression, UPIa, UPIb, UPII and UPIIIa maintain their plaque association in urothelial preneoplastic lesions.
COBISS.SI-ID: 29577177
The aim of our study was to investigate the association of desmosomal proteins with cholesterol-enriched membrane domains, commonly called membrane rafts, and the influence of cholesterol on desmosome assembly in epithelial MDCK cells (clone MDc-2). Biochemical analysis proved an association of desmosomal cadherin desmocollin 2 (Dsc2) in cholesterol-enriched fractions that contain membrane raft markers caveolin-1 and flotillin-1 and the novel raft marker ostreolysin. Cold detergent extraction of biotinylated plasma membranes revealed that ~ 60% of Dsc2 associates with membrane rafts while the remainder is present in non-raft and cholesterol-poor membranes. The results of immunofluorescence microscopy confirmed colocalization of Dsc2 and ostreolysin. Partial depletion of cholesterol with methyl-beta-cyclodextrin disturbs desmosome assembly, as revealed by sequential recordings of live cells. Moreover, cholesterol depletion significantly reduces the strength of cell-cell junctions and partially releases Dsc2 from membrane rafts. Our data indicate that a pool of Dsc2 is associated with membrane rafts; particularly with the ostreolysin type of membrane raft and that intact membrane rafts are necessary for desmosome assembly. Taken together, these data suggest cholesterol as a potential regulator that promotes desmosome assembly.
COBISS.SI-ID: 27616729