Electron tomography of fusiform vesicles and their organization in urothelial cells.

The formation of fusiform vesicles (FVs) is one of the most distinctive features in the urothelium of the urinary bladder. FVs represent compartments for intracellular transport of urothelial plaques, which modulate the surface area of the superficial urothelial (umbrella) cells during the distension-contraction cycle. We have analysed the three-dimensional (3D) structure of FVs and their organization in umbrella cells of mouse urinary bladders. Compared to chemical fixation, high pressure freezing gave a new insight into the ultrastructure of urothelial cells. Electron tomography on serial sections revealed that mature FVs had a shape of flattened discs, with a diameter of up to 1.2 µm. The lumen between the two opposing asymmetrically thickened membranes was very narrow, ranging from 5 nm to 10 nm. Freeze-fracturing and immunolabelling confirmed that FVs contain two opposing urothelial plaques connected by a hinge region that made an omega shaped curvature. In the central cytoplasm, 4-15 FVs were often organized into stacks. In the subapical cytoplasm, FVs were mainly organized as individual vesicles. Distension-contraction cycles did not affect the shape of mature FVs; however, their orientation changed from parallel in distended to perpendicular in contracted bladder with respect to the apical plasma membrane. In the intermediate cells, shorter and more dilated immature FVs were present. The salient outcome from this research is the first comprehensive, high resolution 3D view of the ultrastructure of FVs and how they are organized differently depending on their location in the cytoplasm of umbrella cells. The shape of mature FVs and their organization into tightly packed stacks makes them a perfect storage compartment, which transports large amounts of urothelial plaques while occupying a small volume of umbrella cell cytoplasm.

Urothelial plaque formation in post-Golgi compartments.

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.

Golgi apparatus fragmentation as a mechanism responsible for uniform delivery of uroplakins to the apical plasma membrane of uroepithelial cells.

BACKGROUND INFORMATION: The GA (Golgi apparatus) has an essential role in membrane trafficking, determining the assembly and delivery of UPs (uroplakins) to the APM (apical plasma membrane) of superficial UCs (uroepithelial cells) of urinary bladder. UPs are synchronously and uniformly delivered from the GA to the APM by DFVs (discoidal- or fusiform-shaped vesicles); however, the mechanism of UP delivery is not known. We have used the culture model of UCs with the capacity to undergo terminal differentiation to study the process of uniform delivery of DFVs to the APM and to elucidate the mechanisms involved. RESULTS: By three-dimensional localization using confocal microscopy of immunofluorescence-labelled GA-related markers [GM130 (cis-Golgi matrix protein of 130 kDa), GS15 (Golgi Snare 15 kDa), GS28 and giantin], uroepithelial differentiation-related markers (UPs), MTs (microtubules; α-tubulin) and intermediate filaments [CK7 (cytokeratin 7) and CK20], we found that in non-differentiated, UP-negative UCs the GA is mostly organized as a single ribbon-like structure close to the nucleus, whereas in differentiated, UP-positive UCs the GA is fragmented and spread almost through the entire cell. The FRAP (fluorescence recovery after photobleaching) experiments on the UCs transfected with GalT (trans-Golgi/TGN enzyme ß1,4-galactosyltransferase) fused to fluorescent protein showed that Golgi-resident enzyme cycles freely within ribbon-like GA but not within fragmented GA. By CLEM (correlative light-electron microscopy), we examined the GA fragments in cells expressing UPs. We found that GA fragments are fully functional and similar to the GA fragments that are formed after nocodazole treatment. Furthermore, we demonstrated that the reorganization of GA into a fragmented form is associated with the impairment of the MT organization in the basal, central and subapical cytoplasm and the accumulation of intermediate filaments in the apical cytoplasm that could affect the kinetics of MT star leading to the peripheral fragmentation of the GA in the differentiated UCs. CONCLUSIONS: The fragmentation of the GA and the subsequent spreading of GA to the cell periphery represent one of the key events that promote the uniform delivery of UPs over the entire APM of differentiating UCs and thus are of major importance in the final proper formation and maintenance of the blood-urine barrier.

Formation and maintenance of blood-urine barrier in urothelium.

Blood-urine barrier, which is formed during differentiation of superficial urothelial cells, is the tightest and most impermeable barrier in the body. In the urinary bladder, the barrier must accommodate large changes in the surface area during distensions and contractions of the organ. Tight junctions and unique apical plasma membrane of superficial urothelial cells play a critical role in the barrier maintenance. Alterations in the blood-urine barrier function accompany most of the urinary tract diseases. In this review, we discuss recent discoveries on the role of tight junctions, dynamics of Golgi apparatus and post-Golgi compartments, and intracellular membrane traffic during the biogenesis and maintenance of blood-urine barrier.

Melatonin prevents the development of hyperplastic urothelium induced by repeated doses of cyclophosphamide.

Repeated cyclophosphamide (CP) chemotherapy increases the risk of developing bladder cancer, which could be due to the extremely rapid proliferation of urothelial cells observed in hyperplastic urothelium induced by CP treatment. We investigated the effect of melatonin on the development of urothelial hyperplasia induced by repeated CP treatment. Male ICR mice were injected with CP (150 mg/kg) or melatonin (10 mg/kg) with CP once a week for 3, 4 and 5 weeks. Transmission and scanning electron microscopy, immunohistochemistry and Western blot analysis were used to study the ultrastructure, apoptosis, proliferation and differentiation of urothelial cells. Repeated doses of CP caused the development of hyperplastic urothelium with up to ten cell layers and increased proliferation and apoptotic indices regarding Ki-67 and active caspase-3 immunohistochemistry, respectively. Scanning electron microscopy observations, cytokeratin and asymmetrical unit membrane immunohistochemistry and Western blot analysis showed a lower differentiation state of superficial urothelial cells. Melatonin co-treatment prevented the development of hyperplastic urothelium, statistically significantly decreased proliferation and apoptotic indices after four and five doses of CP and caused higher differentiation state of superficial urothelial cells.

Apoptosis and desquamation of urothelial cells in tissue remodeling during rat postnatal development.

Postnatal rat urothelium was studied from day 0 to day 14, when intense cell loss as part of tissue remodeling was expected. The morphological and biochemical characteristics of urothelial cells in the tissue and released cells were investigated by light and electron microscopy, by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, by annexin V/propidium iodide assay, and by immunofluorescent detection of active caspases and tight-junction protein occludin. Intense apoptosis and massive desquamation were detected between postnatal days 7 and 10. During this period, active caspases and TUNEL-positive cells were found in the urothelium. Disassembled cell-cell junctions were detected between cells. The majority of desquamated cells expressed no apoptotic cell morphology, but were active caspase positive and TUNEL positive. Ann+/PI- apoptotic bodies and desquamated Ann+/PI+ cells were detected in the lumen. These results indicate that apoptosis and desquamation participate in urothelial cell loss in the rat early postnatal period, indispensable for fast urothelial remodeling during development.

Endocytotic activity of bladder superficial urothelial cells is inversely related to their differentiation stage.

The composition of the apical plasma membrane of bladder superficial urothelial cells is dramatically modified during cell differentiation, which is accompanied by the change in the dynamics of endocytosis. We studied the expression of urothelial differentiation-related proteins uroplakins and consequently the apical plasma membrane molecular composition in relation to the membrane-bound and fluid-phase endocytosis in bladder superficial urothelial cells. By using primary urothelial cultures in the environment without mechanical stimuli, we studied the constitutive endocytosis. Four new findings emerge from our study. First, in highly differentiated superficial urothelial cells with strong uroplakin expression, the endocytosis of fluid-phase endocytotic markers was 43% lower and the endocytosis of membrane-bound markers was 86% lower compared to partially differentiated cells with weak uroplakin expression. Second, superficial urothelial cells have 5-15-times lower endocytotic activity than MDCK cells. Third, in superficial urothelial cells the membrane-bound markers are delivered to lysosomes, while fluid-phase markers are seen only in early endocytotic compartments, suggesting their kiss-and-run recycling. Finally, we provide the first evidence that in highly differentiated cells the uroplakin-positive membrane regions are excluded from internalization, suggesting that uroplakins hinder endocytosis from the apical plasma membrane in superficial urothelial cells and thus maintain optimal permeability barrier function.

Apical plasma membrane traffic in superficial cells of bladder urothelium.

Superficial urothelial cells that line the urinary bladder accommodate cyclical changes in organ volume while maintaining a permeability barrier between urine and tissue fluids. The specific apical plasma membrane traffic is necessary for their proper function. The composition of the apical plasma membrane is dramatically modified during differentiation of bladder urothelial cells, most notably by assembly of urothelial plaques containing uroplakins. However, the assembly of uroplakins into plaques, their insertion and removal from the apical surface, and the regulation of these processes are still poorly understood. This review examines the traffic (exocytosis/endocytosis) of the apical plasma membrane during differentiation of urothelial cells and focuses on the physiological and clinical significance of the apical plasma membrane traffic in bladder superficial urothelial cells.

Rapid differentiation of superficial urothelial cells after chitosan-induced desquamation.

Superficial cell desquamation followed by differentiation of newly exposed superficial cells induces regeneration of the urinary bladder epithelium, urothelium. In the present work, chitosan was evaluated as a new inducer of urothelial cell desquamation, in order to study the regeneration of mouse urothelial cells in vivo. Intravesical application of chitosan dispersion caused complete removal of only the superficial layer of cells within 20 min of treatment. Differentiation of the new superficial layer was followed by the appearance and distribution of three urothelial differentiation markers, tight junction protein ZO1, cytokeratin 20 and the maturation of the apical plasma membrane. The arrangement of ZO1 into continuous lines in individual cells of the intermediate layer was already found after 10 min of chitosan application, when desquamation had just started. The appearance of the apical membrane changed from microvillar to typically scalloped within 20 min of regeneration, while complete arrangement of the cytokeratin 20 network took 60 min. These findings provide a new perspective on the rate of the differentiation process in the urothelium and make chitosan a new and a very controllable tool for studies on urothelial regeneration.

Redistribution of cytosolic FGFR1 after induced migration of urothelial cells in culture.

The distribution of cytosolic fibroblast growth factor receptor 1 (FGFR1) was studied in correlation to cell migration in urothelial cell line g/G. Cell motility was analysed with a new method using consecutive series of photographs of cells relocated on CELLocate coverslips and with image analysis software. The results confirmed that FGF1 stimulated cell motility only when cells were grown on collagen I coating. During the transition from sessile to motile cell phenotype a complete redistribution of cytosolic FGFR1 was revealed. In sessile cells, FGFR1 had a filamentous distribution and its location matched cytokeratin 7. In cells of the migrating phenotype, the distribution of FGFR1 was diffuse, mainly located in cytosol. Our data reveal that the location of cytosolic FGFR1 depends on the motile characteristics of the cell. The results also indicate that attachment of cells to collagen I is crucial for the induction of urothelial cell motility with FGF1.

Effect of melatonin on apoptosis, proliferation and differentiation of urothelial cells after cyclophosphamide treatment.

Melatonin was recently shown to have protective effects against cyclophosphamide (CP)-induced hemorrhagic cystitis (HC) by diminishing bladder oxidative stress. HC is accompanied by destruction of the bladder urothelium and followed by apoptosis and rapid regeneration via proliferation and differentiation of urothelial cells, reaching complete restoration of normal urothelium in three weeks. Therefore, the effect of melatonin on apoptosis, proliferation and differentiation of urothelial cells, during destruction and regeneration of the urothelium three-weeks after a single dose CP treatment, was studied. F344 male rats were injected intraperitoneally with saline (control group) or melatonin (Mel group) or a single dose of CP (100 mg/kg; CP group) or melatonin (10 mg/kg) with CP (Mel + CP group). Melatonin co-treatment with CP significantly reduced apoptosis and increased proliferation of urothelial cells at day 1 and thus prevented extensive loss of cells from the urothelium. However, proliferation indices at days 4 and 7 after melatonin and CP co-treatment suddenly dropped and therefore the development of hyperplasia was prevented. Melatonin co-treatment with CP also resulted in earlier differentiation of superficial urothelial cells. Melatonin seems to have protective effect against CP-induced urothelial damage and a favorable impact on regeneration and restoration of normal urothelium, since it reduces the number of apoptotic and proliferating urothelial cells and results in their earlier differentiation.

Mitochondrial localization of nitric oxide synthase in partially differentiated urothelial cells of urinary bladder lesions.

In urothelium, nitric oxide (NO) produced by NO synthase (NOS) plays various roles during disease processes. Because diseases influence the expression of uroplakins, the main urothelial differentiation-related proteins, we compared their expression and localization with that of inducible NOS (iNOS) in bladder outlet obstruction caused by benign prostatic hyperplasia and in noninvasive urothelial neoplasms (papilloma, low-grade, and high-grade papillary carcinoma). In all cases, we found areas with terminal and areas with partial cell differentiation. Terminally differentiated urothelium was uroplakin positive and iNOS negative. Areas of partial differentiation contained superficial cells with altered apical surface morphology and with no or weak uroplakin staining. These areas showed elevated iNOS staining. By immunoelectron microscopy, it was demonstrated for the first time that iNOS was localized in mitochondria of urothelial cells that show partial differentiation. These results suggest that various urinary bladder lesions alter the normal differentiation pathway of urothelial superficial cells, which induces the expression of NOS in mitochondria of partially differentiated cells.

Distribution of junction- and differentiation-related proteins in urothelial cells at the leading edge of primary explant outgrowths.

Leading edge cells, which are located at the forefront of a wound margin, play a significant role in coordinating the wound healing process. In this study, leading edge cells of the urothelial explant outgrowth, resembling leading edge cells during urothelial full-thickness wound healing in vivo, were analyzed for expression and distribution of junction and differentiation-related proteins. Ultrastructural and immunofluorescence studies revealed that urothelial cells at the leading edge expressed ZO-1, claudin-4, occludin, E-cadherin, cytokeratin 7 and cytokeratin 20, while no expression of claudin-8 was noted. ZO-1, claudin-4, occludin and E-cadherin were localized along the cell membranes where neighbouring leading edge cells were in contact. Cytokeratin 7 was detected as filaments and cytokeratin 20 as small dots and sparse filaments. In conclusion, we detected early expression of ZO-1, claudin-4 and occludin at the urothelial leading edge, predicating the later formation of tight junctions as a necessary stage for the differentiation process that subsequently begins. The expression of occludin and cytokeratin 20 in urothelial cells at the leading edge suggests that leading edge cells may develop into fully differentiated superficial cells.

Amphiphile-induced tubular budding of the bilayer membrane.

Amphiphile-induced tubular budding of the erythrocyte membrane was studied using transmission electron microscopy. No chiral patterns of the intramembraneous particles were found, either on the cylindrical buds, or on the tubular nanoexovesicles. In agreement with these observations, the tubular budding may be explained by in-plane ordering of anisotropic membrane inclusions in the buds where the difference between the principal membrane curvatures is very large. In contrast to previously reported theories, no direct external mechanical force is needed to explain tubular budding of the bilayer membrane.

Urothelial injuries and the early wound healing response: tight junctions and urothelial cytodifferentiation.

Using primary explant cultures of mouse bladder, the early response of the urothelium after superficial and full-thickness injuries was investigated. In such an in vitro wound healing model, explant surfaces with a mostly desquamated urothelial superficial layer represented superficial wounds, and the exposed lamina propria at the cut edges of the explants represented full-thickness wounds. The urothelial cell ultrastructure, the expression and subcellular distribution of the tight junctional protein occludin, and differentiation-related proteins CK 20, uroplakins, and actin were followed. Since singular terminally differentiated superficial cells remained on the urothelium after superficial injury (i.e., original superficial cells), we sought to determine their role during the urothelial wound-healing process. Ultrastructural and immunocytochemical studies have revealed that restored tight junctions are the earliest cellular event during the urothelial superficial and full-thickness wound-healing process. Occludin-containing tight junctions are developed before the new superficial cells are terminally differentiated. New insights into the urothelium wound-healing process were provided by demonstrating that the original superficial cells contribute to the urothelium wound healing by developing tight junctions with de novo differentiated superficial cells and by stretching, thus providing a large urothelial surface with asymmetric unit membrane plaques.

Differentiation of epithelial cells in the urinary tract.

Uroplakins, cytokeratins and the apical plasma membrane were studied in the epithelia of mouse urinary tract. In the simple epithelium covering the inner medulla of the renal pelvis, no uroplakins or cytokeratin 20 were detected and cells had microvilli on their apical surface. The epithelium covering the inner band of the outer medulla became pseudostratified, with the upper layer consisting of large cells with stalks connecting them to the basal lamina. Uroplakins and cytokeratin 20 were not expressed in these cells. However, some superficial cells appeared without connections to the basal lamina; these cells expressed uroplakins Ia, Ib, II and III and cytokeratin 20, they contained sparse small uroplakin-positive cytoplasmic vesicles and their apical surface showed both microvilli and ridges. Cytokeratin 20 was seen as dots in the cytoplasm. This epithelium therefore showed partial urothelial differentiation. The epithelium covering the outer band of the outer medulla gradually changed from a two-layered to a three-layered urothelium with typical umbrella cells that contained all four uroplakins. Cytokeratin 20 was organized into a complex network. The epithelium possessed an asymmetric unit membrane at the apical cell surface and fusiform vesicles. Umbrella cells were also observed in the ureter and urinary bladder. In males and females, the urothelium ended in the bladder neck and was continued by a non-keratinized stratified epithelium in the urethra in which no urothelial cell differentiation markers were detected. We thus show here the expression, distribution and organization of specific proteins associated with the various cell types in the urinary tract epithelium.

Differentiation-dependent localisation of tissue-type plasminogen activator in human bladder urothelium.

Human bladder urothelium is able to secrete tissue-type plasminogen activator (tPA). The aim of our study was to analyse localisation of tPA antigen in comparison to differentiation state of cells in samples of histologically normal urothelium and non-invasive tumours of the human urinary bladder. Twenty-five samples of normal urothelium and 31 non-invasive papillary tumours from 36 patients were examined. The presence of tPA antigen was evaluated immunohistochemically. Differentiation of superficial cells was assessed by the presence of urothelial cell differentiation markers, uroplakins (UPs; immunohistochemistry) and cell's apical surface architecture (scanning electron microscopy). All tissue samples stained anti-tPA positive. In normal urothelium, the intensity of anti-tPA staining was the strongest in superficial cells, which were well-differentiated. In tumours, all cell layers stained anti-tPA positive. The intensity of anti-tPA positive reaction in the upper cell layer correlated with the percentage of anti-UP positive superficial cells. Superficial cells showed various differentiation states. The localisation of tPA antigen in human in vivo tissue is not confined to the well-differentiated superficial cells. Our results suggest a positive correlation between tPA secretion and cell differentiation.

What determines differentiation of urothelial umbrella cells?

Cytokeratins, uroplakins and the asymmetric unit membrane are biochemical and morphological markers of urothelial differentiation. The aim of our study was to follow the synthesis, subcellular distribution and supramolecular organization of differentiation markers, cytokeratins and uroplakins, during differentiation of umbrella cells of mouse bladder urothelium. Regenerating urothelium after destruction with cyclophosphamide was used to simulate de-novo differentiation of cells, which was followed from day 1 to day 14 after cyclophosphamide injection. Cytokeratin 7 and uroplakins co-localized in the subapical cytoplasm of superficial cells from the early stage of differentiation on. At early stages of superficial cell differentiation cytokeratin 7 was filamentary organized, and rare uroplakins were found on the membranes of relatively small cytoplasmic vesicles, which were grouped in clusters under the apical membrane. Later, cytokeratin 7 gradually reorganized into a continuous trajectorial network, and uroplakins became organized into plaques of asymmetric unit membrane, which formed fusiform vesicles. After insertion of fusiform vesicles into the apical plasma membrane, the surface acquired microridged appearance of umbrella cells. Cytokeratin 20 appeared as the last differentiation marker of umbrella cells. Cytokeratin 20 was incorporated into the pre-existing trajectorial cytokeratin network. These results indicate that differentiation of urothelial cells starts with the synthesis of differentiation-related proteins i.e., cytokeratins and uroplakins, and later with their specific organization. We consider that the umbrella cell has reached its final stage of differentiation when uroplakins form plaques of asymmetric unit membrane that are inserted into the apical plasma membrane and when cytokeratin 20 becomes included in a trajectorial cytokeratin network in the subapical area of cytoplasm.

Spherocyte shape transformation and release of tubular nanovesicles in human erythrocytes.

We have studied dodecylmaltoside-induced echinocyte-spheroechincyte-spherocyte shape transformation and membrane vesiculation using transmission electron microscopy (TEM) on freeze-fracture replicas. It is indicated that spherical erythrocyte shape at higher dodecylmaltoside concentration is formed due to loss of membrane in the process where small, mostly tubular nanovesicles are released predominantly from the top of echinocyte and spheroechinocyte spicules.

Temporal and spatial dimensions of postnatal growth of the mouse urinary bladder urothelium.

Postnatal growth and renewal of mouse urothelium start on the day of birth. In the present study, temporal and spatial dimensions of urothelial growth were studied during the first two postnatal weeks. Quantitative analysis showed that the rate of urothelial cell proliferation is significantly higher during all 14 postnatal days than in adult mice. Three peaks of proliferative and mitotic activity were revealed: on the day of birth and postnatal day 1, on days 6 and 7, and on day 14. The high proliferation rate around the day of birth and at postnatal days 6 and 7 coincides with cell death in the urothelium. Semiquantitative analysis showed that during all 14 postnatal days, the urothelial proliferative response is mostly confined to the basal cell layer. Urothelial cells divide predominantly in parallel to the plain of the urothelium on all chosen postnatal days. Increased portions of urothelial cells, dividing perpendicularly to the urothelium were observed only on the day of birth and on postnatal day 7. Our results suggest that postnatal growth of mouse urothelium is particularly the result of an increasing number of cells in individual cell layers and not the result of an increasing number of cell layers.