Asymmetric Unit Membrane Research Articles

ANTIGENIC AND ULTRASTRUCTURAL MARKERS ASSOCIATED WITH UROTHELIAL CYTODIFFERENTIATION IN PRIMARY EXPLANT OUTGROWTHS OF MOUSE BLADDER

The purpose of this study was to establish an in vitro culture model that closely resembles whole mouse urothelial tissue. Primary explant cultures of mouse bladder were established on porous membrane supports and explant outgrowths were analysed for morphology and the presence of antigenic and ultrastructural markers associated with urothelial cytodifferentiation. When examined at the ultrastructural level, the cultured urothelium was polarized and organized as a multilayered epithelium. Differentiation was found to increase from the porous membrane towards the surface and from the explant towards the periphery of the culture. Scanning and transmission electron microscopical analysis of the most superficially-located cells revealed four successive differentiation stages: cells with microvilli, cells with ropy microridges, cells with rounded microridges, and highly-differentiated cells with asymmetric unit membrane (AUM) plaques forming rigid microridges and fusiform vesicles. The more highly-differentiated cells were numerous at the periphery of the culture, but rare close to the explant. Epithelial organization was stabilized by well developed cell junctions. Immunolabeling demonstrated that superficial urothelial cells in culture: (1) develop tight junctions, E-cadherin adherens junctions and abundant desmosomes and (2) express uroplakins and cytokeratin 20 (CK 20). Using a culture model of primary explant outgrowth we have shown that non-differentiated mouse urothelial cells growing on a porous membrane show a high level of de novo differentiation.

Uroplakin and androgen receptor expression in the human fetal genital tract: insights into the development of the vagina.

Although a dual origin of the vagina has been popularized, other theories support a müllerian or wolffian duct origin or various combinations of these structures and the urogenital sinus. Uroplakins are specialized membrane proteins of the urothelial plaque, constituting the asymmetrical unit membrane of the bladder, and represent specific molecular markers of urothelial differentiation. We hypothesize that the epithelium of the dorsal wall of the urogenital sinus is involved in the formation of the sinovaginal bulbs and will express uroplakins. In addition, localization of the androgen receptor and its temporal expression during development may in part explain the varied effects of androgens on the lower female genital tract in congenital adrenal hyperplasia. Lower genitourinary tracts from 4 human female fetuses (9 to 18 weeks) were serially sagittally sectioned. Representative sections were stained with hematoxylin and eosin, rabbit antibodies against panuroplakin and antibodies to the androgen receptor. At 9 weeks of gestation the urogenital sinus showed evidence of evagination and the formation of the sinovaginal bulbs. The urothelium of the entire urogenital sinus expressed uroplakins including the region of the dorsal wall involved in evagination and formation of the sinovaginal bulbs. The müllerian ducts were in direct contact with the area of urogenital sinus evagination but were not in continuity with the sinus. Androgen receptors were expressed in the epithelium and the stroma of the urogenital sinus, sinovaginal bulbs, and müllerian and wolffian ducts. By 14 weeks androgen receptor expression was almost absent in the urothelium of the urogenital sinus, and the epithelium and surrounding stroma of the lower vagina and müllerian ducts. The area of evagination of the urogenital sinus expresses uroplakins, is involved in the formation of the sinovaginal bulbs and further substantiates the urogenital sinus origin of the lower vagina. Since testosterone inhibits formation of the lower vagina, the timing of exposure to systemic testosterone in congenital adrenal hyperplasia will determine the phenotypic appearance of the external genitalia and effect of testosterone on the development of the lower genital tract. If exposure to testosterone occurs after 12 weeks only clitoromegaly occurs. Androgen receptor is absent in the urogenital sinus urothelium, vaginal epithelium and müllerian ducts by 14 weeks, suggesting that these tissues become androgen insensitive and vaginal development will proceed normally after that critical time.

UROPLAKIN AND ANDROGEN RECEPTOR EXPRESSION IN THE HUMAN FETAL GENITAL TRACT: INSIGHTS INTO THE DEVELOPMENT OF THE VAGINA

Purpose: Although a dual origin of the vagina has been popularized, other theories support a mullerian or wolffian duct origin or various combinations of these structures and the urogenital sinus. Uroplakins are specialized membrane proteins of the urothelial plaque, constituting the asymmetrical unit membrane of the bladder, and represent specific molecular markers of urothelial differentiation. We hypothesize that the epithelium of the dorsal wall of the urogenital sinus is involved in the formation of the sinovaginal bulbs and will express uroplakins. In addition, localization of the androgen receptor and its temporal expression during development may in part explain the varied effects of androgens on the lower female genital tract in congenital adrenal hyperplasia.Materials and Methods: Lower genitourinary tracts from 4 human female fetuses (9 to 18 weeks) were serially sagittally sectioned. Representative sections were stained with hematoxylin and eosin, rabbit antibodies against panuroplakin and ant...

Urothelial cell detachment and differentiation in urinary bladder

In developing and in repairing bladder, proliferation of the transitional urothelium is followed by cell detachment--desquamation or apoptosis. Proliferation results in formation of terminally differentiated superficial cells and this process may be followed by checking the cells on the presence of differentiation markers. The formation of an asymmetric unit membrane (AUM) structure (plaque) on the cell surface is in correlation with urothelial differentiation. Thus, the microstructure of the luminal surface of the urinary bladder provides a very convenient differentiation biomarker. The surface of immature cells showed a pattern of microvilli. The progress of differentiation was associated with microvili arranged in rows finally forming the characteristic pattern of ridges in terminally differentiated cells. These results demonstrate that the characteristic surface pattern and the AUM plaque formation in the apical plasma membrane of superficial urothelial cells are associated with specific morphology, and patterns and thus help detect differentiation level of cell.

Urothelial cell detachment and differentiation in urinary bladder.

In developing and in repairing bladder, proliferation of the transitional urothelium is followed by cell detachment - desquamation or apoptosis. Proliferation results in formation of terminally differentiated superficial cells and this process may be followed by checking the cells on the presence of differentiation markers. The formation of an asymmetric unit membrane (AUM) structure (plaque) on the cell surface is in correlation with urothelial differentiation. Thus, the microstructure of the luminal surface of the urinary bladder provides a very convenient differentiation biomarker. The surface of immature cells showed a pattern of microvilli. The progress of differentiation was associated with microvilli arranged in rows finally forming the characteristic pattern of ridges in terminally differentiated cells. These results demonstrate that the characteristic surface pattern and the AUM plaque formation in the apical plasma membrane of superficial urothelial cells are associated with specific morphology, and patterns and thus help detect differentiation level of cell.

The effect of lamina propria on the growth and differentiation of urothelial cells in vitro.

The effect of lamina propria on the growth and differentiation of the mouse urinary bladder urothelial cells in vitro has been studied by light and electron microscopy using morphological and immunohistochemical methods. Three different types of urothelial cultures were maintained on a porous membrane in serum-free medium for 10 days. Our results showed that urothelial cells in culture types I and II were organised as the multilayer epithelium showing terminal differentiation, from the porous membrane toward the surface. On the surface of culture type I and II, superficial urothelial cells were highy differentiated. They formed tight junctions and had plaques of an asymmetric unit membrane, a hallmark of terminal differentiation in bladder superficial cells. These cultured superficial cells were also cytokeratin 20 positive. On the contrary, urothelial cells in culture type III have shown a limited capacity for survival and have grown as poorly differentiated monolayer. These results revealed that reciprocal intercellular signalling between the lamina propria and urothelial cells is essential to achieve terminal differentiation of urothelial cells in vitro.

Actin filaments during terminal differentiation of urothelial cells in the rat urinary bladder

The localisation of actin filaments was studied in rat urothelial cells during differentiation which accompanied regeneration after cell damage induced by cyclophosphamide (CP). By immunofluorescence it was established that actin filaments equally stained along the cell circumference in basal and intermediate cells, while basolateral cell membrane expression was found in terminally differentiated superficial cells. During regeneration, after CP treatment, simple urothelial hyperplasia developed with smaller cuboidal superficial cells, in which actin filaments were equally distributed under the apical and basolateral plasma membranes. As demonstrated by immunoelectron microscopy, the apical surface of these superficial cells was covered with microvilli containing bundles of actin filaments. Within 1 week, the urothelium reverted to its normal three-layer thickness. Superficial cells became larger and flattened and the unthickened apical plasma membrane matured into a thick asymmetric unit membrane. Concomitantly actin filaments disappeared from apical areas of superficial cells while remaining abundant at basolateral areas. Our results indicate that in the urothelium subcellular distribution of actin filaments can be considered as a marker of cell differentiation.

Immunohistochemical Analysis of Uroplakins, Urothelial Specific Proteins, in Ovarian Brenner Tumors, Normal Tissues, and Benign and Neoplastic Lesions of the Female Genital Tract

Uroplakins are the characteristic integral membrane proteins in terminally differentiated, superficial urothelial asymmetric unit membrane. Brenner tumors of the ovary and Walthard cell nests of Fallopian tubes have been considered to represent urothelial differentiation in the female genital tract, but no definitive differentiation marker has been demonstrated supporting such a conclusion. An immunohistochemical analysis was performed to assess the expression of uroplakins in these lesions as well as in various benign and neoplastic lesions and normal tissues of the female genital tract. Focal expression of uroplakins was observed on the luminal surface of ovarian Brenner tumor cells forming microcysts in all 5 cases examined. In contrast, uroplakins were slightly expressed in only 1 of 12 cases of Walthard cell nests, even in the presence of microcyst formation. Uroplakins were not expressed in other benign or malignant lesions or normal tissues of the female genital tract. These results support the hypothesis that the Brenner tumor and possibly Walthard cell nests represent urothelial (transitional cell) differentiation.

Urothelial hinge as a highly specialized membrane: detergent-insolubility, urohingin association, and in vitro formation

Urothelial surface is covered by numerous plaques (consisting of asymmetric unit membranes or AUM) that are interconnected by ordinary looking hinge membranes. We describe an improved method for purifying bovine urothelial plaques using 2% sarkosyl and 25 m M NaOH to remove contaminating membrane and peripheral proteins selectively. Highly purified plaques interconnected by intact hinge areas were obtained, indicating that the hinges are as detergent-insoluble as the plaques. These plaque/hinge preparations contained uroplakins, an as yet uncharacterized 18-kDa plaque-associated protein, plus an 85-kDa glycoprotein that is known to be hinge-associated in situ. Examination of the isolated, in vitro-resealed bovine AUM vesicles by quick-freeze deep-etch showed that each AUM particle consists of a 16-nm, luminally exposed “head” anchored to the lipid bilayer via a 9-nm transmembranous “tail”, and that an AUM plaque can break forming several smaller plaques separated by newly formed particle-free, hinge-like areas. These data lend support to our recently proposed three-dimensional model of mouse urothelial plaques. In addition, our findings suggest that urothelial plaques are dynamic structures that can rearrange giving rise to new plaques with intervening hinges; that the entire urothelial apical surface (both plaque and hinge areas) is highly specialized; and that these two membrane domains may be equally important in fulfilling some of the urothelial functions.

Primary Uroepithelial Cultures: A MODEL SYSTEM TO ANALYZE UMBRELLA CELL BARRIER FUNCTION

Despite almost 25 years of effort, the development of a highly differentiated and functionally equivalent cell culture model of uroepithelial cells has eluded investigators. We have developed a primary cell culture model of rabbit uroepithelium that consists of an underlying cell layer that interacts with a collagen substratum, an intermediate cell layer, and an upper cell layer of large (25-100 micrometer) superficial cells. When examined at the ultrastructural level, the superficial cells formed junctional complexes and had an asymmetric unit membrane, a hallmark of terminal differentiation in bladder umbrella cells. These cultured "umbrella" cells expressed uroplakins and a 27-kDa uroepithelial specific antigen that assembled into detergent-resistant asymmetric unit membrane particles. The cultures had low diffusive permeabilities for water (2.8 x 10(-4) cm/s) and urea (3.0 x 10(-7) cm/s) and high transepithelial resistance (>8000 Omega cm2) was achieved when 1 mM CaCl2 was included in the culture medium. The cell cultures expressed an amiloride-sensitive sodium transport pathway and increases in apical membrane capacitance were observed when the cultures were osmotically stretched. The described primary rabbit cell culture model mimics many of the characteristics of uroepithelium found in vivo and should serve as a useful tool to explore normal uroepithelial function as well as dysfunction as a result of disease.

Immunophenotype of transitional metaplasia of the uterine cervix.

Transitional, glandular, and squamous epithelia show distinctive patterns of expression of cytokeratins 13, 17, 18, and 20. Furthermore, some markers, such as cytokeratin (CK) 20 and the asymmetric unit membrane, are related to terminal urothelial differentiation, their expression limited to superficial and occasional intermediate cells. In this study, seven cases fulfilling the morphologic criteria of transitional metaplasia of the uterine cervix were investigated with these markers to determine whether there was evidence of true urothelial-type differentiation in these lesions. Results were consistent and showed clear differences between the squamous, glandular, and metaplastic transitional areas of the cervix. In particular, the expression of CK13, CK17, and CK18 in the metaplastic areas was identical to the pattern observed in normal urothelium. However, full urothelial differentiation was not observed, as CK20 and asymmetric unit membrane were never expressed in the transitional metaplasia. Based on these findings, the term of immature transitional metaplasia would be a more accurate description of this entity.

Cloning of the human uroplakin 1B cDNA and analysis of its expression in urothelial-tumor cell lines and bladder-carcinoma tissue.

The human uroplakin 1B (UPK1B) gene codes for a structural protein which is a terminal differentiation component of the asymmetric unit membrane on the apical surface of the mammalian bladder. UPK1B is a member of the tetraspan family of proteins, many of which have de-regulated patterns of expression in cancer. Using polymerase-chain-reaction techniques, we have cloned a partial human UPK1B cDNA which codes for the putative full open reading frame for the UPK1B protein. The deduced human UPK1B protein sequence has 92% and 93% amino-acid homology with bovine UPK1b and mink TI1 proteins respectively. Using Northern analysis, we show that the human UPK1B gene is highly expressed in normal human urothelium. However, expression of UPK1B mRNA was undetectable or markedly reduced in 11 out of 16 samples of transitional-cell-bladder-carcinoma tissue and in all 5 bladder-carcinoma cell lines when compared with normal urothelial tissue. The molecular mechanism of down-regulation of RNA expression does not appear to involve gross gene rearrangements or allelic loss.

Uroplakins and cytokeratins in the regenerating rat urothelium after sodium saccharin treatment.

A sodium saccharin (NaSac) diet was used to induce cell damage and regeneration in the urothelium of the male rat urinary bladder. Foci of terminally differentiated superficial cell exfoliation were detected after 5 weeks and their number increased after 10 and 15 weeks of the diet. At the sites of superficial cell loss, regenerative simple hyperplasia developed. Within 5 weeks of NaSac removal, regeneration re-established normal differentiated urothelium. In order to follow urothelial differentiation during regeneration we studied the expression of uroplakins and cytokeratins by means of immunocytochemistry and immunohistochemistry, respectively. Normal urothelium was characterised by terminally differentiated superficial cells which expressed uroplakins in their luminal plasma membrane and cytokeratin 20 (CK20) in the cytoplasm. Basal and intermediate cells were CK20 negative and cytokeratin 17 (CK17) positive. In hyperplastic urothelium all cells synthesised CK17, but not CK20. Differentiation of the superficial layer was reflected in three successive cell types: cells with microvilli, cells with rounded microridges and those with a rigid-looking plasma membrane on the luminal surface. The cells with microvilli did not stain with anti-uroplakin antibody. When the synthesis of uroplakins was detected rounded microridges were formed. With the elevated expression of uroplakins the luminal plasma membrane becomes rigid-looking which is characteristic of asymmetric unit membrane of terminally differentiated cells. During differentiation, synthesis of CK17 ceased in superficial cells while the synthesis of CK20 started. These results indicate that during urothelial regeneration after NaSac treatment, specific superficial cell types develop in which the switch to uroplakin synthesis and transition from CK17 to CK20 synthesis are crucial events for terminal differentiation.

Histology and ultrastructure of the urothelium lining the ureter and the renal pelvis in sows.

The histology and ultrastructure of the urothelium lining the ureter and renal pelvis in sows was examined using light microscopy, and scanning and transmission electron microscopy. The epithelium of both the ureter and the renal pelvis were similar and consisted of the basal, the intermediate and the superficial cells. The luminal surface of the superficial cells was characterized by microplicae. Transmission electron microscopy showed that the luminal membrane of these cells showed trilaminar asymmetric unit membrane. Tight junctions linked the lateral parts of the superficial cells, while the intermediate and the basal cells were connected to each other and the basal cells with the basal membrane through desmosomes and hemidesmosomes, respectively. Clusters of mitochondria were observed throughout the urothelium. Unlike that of the basal cells, the Golgi complex and the endoplasmic reticulum were well developed in the superficial and intermediate cells. Lysosomes and vesicles were observed only in the superficial and the intermediate cells. Fine cytoplasmic filaments, although very few in the basal cells, were observed throughout the epithelium. The results are compared and discussed with the findings reported on the urotherlium of other mammalian species and humans.

Reorganisation of the urothelial luminal plasma membrane in the cyclophosphamide treated rats.

The luminal surface of differentiated urothelium is characterised by the presence of asymmetric unit membrane (AUM) which contains four major integral proteins, uroplakins. Cyclophosphamide (CP) causes extensive denudation of the urothelium that is followed by regeneration. In this study the differentiation of the urothelial luminal plasma membrane was examined in CP treated rats by electron microscopy. Single dose of cyclophosphamide was injected and rats were killed after 1, 3, 7, 14 or 28 days. One day after the treatment only few cells remained in the urothelium. They had a flat surface and did not stain with anti-AUM antibody. During regeneration cells with short or pleomorphic microvilli and latter cells with ropy and leafy microridges appeared. Cells with either type of microvilli were not labelled whereas cells with microridges were labelled with anti-AUM. At day 28 the luminal surface was normal and labelled. These results show a good correlation of morphogenesis and the expression of uroplakins.

Formation of asymmetric unit membrane during urothelial differentiation.

Mammalian urothelium undergoes unique membrane specialization during terminal differentiation making numerous rigid-looking membrane plaques (0.3-0.5 micron diameter) that cover the apical cell surface. The outer leaflet of these membrane plaques is almost twice as thick as the inner leaflet hence the name asymmetric unit membrane (AUM). Ultrastructural studies established that the outer leaflet of AUM is composed of 16 nm particles forming two dimensional crystals, and that each particle forms a 'twisted ribbon' structure. We showed recently that highly purified bovine AUMs contain four major integral membrane proteins: uroplakins Ia (27 kD), Ib (28 kD), II (15 kD) and III (47 kD). Studies of the protease sensitivity of the different subdomains of uroplakins and other considerations suggest that UPIa and UPIb have 4 transmembrane domains, while UPII and UPIII have only one transmembrane domain. Chemical crosslinking studies showed that UPIa and UPIb, which share 39% amino acid sequence, are topologically adjacent to UPII and UPIII, respectively, thus raising the possibility that there exist two biochemically distinct AUM particles, i.e., those containing UPIa/UPII vs. UPIb/UPIII. Bovine urothelial cells grown in the presence of 3T3 feeder cells undergo clonal growth forming stratified colonies capable of synthesizing and processing all known uroplakins. Transgenic mouse studies showed that a 3.6 kb 5'-flanking sequence of mouse uroplakin II gene can drive the expression of bacterial LacZ gene to express in the urothelium. Further studies on the biosynthesis, assembly and targeting of uroplakins will offer unique opportunities for better understanding the structure and function of AUM as well as the biology of mammalian urothelium.

Distinctive ultrastructural pathology of nonulcerative interstitial cystitis: new observations and their potential significance in pathogenesis.

Ultrastructural study of the bladder in interstitial cystitis has, so far, been limited, mainly to the urothelium. The present study was conducted first to study in detail the ultrastructural features of all tissue components of the bladder wall in nonulcerative interstitial cystitis and second to derive clues from the observed changes to pathogenesis of the disease. Endoscopic biopsies of urothelium with attached suburothelium, and muscularis, were obtained from both lesional and nonlesional areas in 5 female patients with unequivocal clinical diagnosis of interstitial cystitis. The specimens were processed for electron microscopic study by standard methods and subjected to comprehensive ultrastructural study of urothelium, suburothelium, detrusor muscle cells, intrinsic blood vessels, and intrinsic nerves. A distinctive combination of peculiar muscle cell profiles, injury of intrinsic vessels and nerves in muscularis and suburothelium, and discohesive urothelium was observed in lesional and less markedly in nonlesional samples of all specimens. Marked edema of various tissue elements and cells appeared to be a common denominator of many observed changes. Edema of muscle cells resulted in characteristic querciphylloid profiles, so designated because of peripheral bosselation of cell sarcoplasm with a lobed perimeter resembling that of an oak leaf. Urothelial changes disrupted the true permeability barrier, consisting of asymmetric unit membrane and triple epithelial junctions of surface (umbrella) cells. Vascular lesions included endothelial cell injury and suggested sluggishness of intrinsic microcirculation. Neural changes included a combination of degenerative and regenerative features, some expressing neural plasticity. The observed ultrastructural changes appear to be sufficiently distinctive to be diagnostic in specimens submitted for pathologic confirmation of nonulcerative interstitial cystitis. The changes do not support a primary pathogenetic role of mast cells or a selectively deficient glycosaminoglycan layer. They do suggest, however, a pathogenesis based on a potentially self-perpetuating process of neurogenic inflammation that can trigger a biologically potent cascade of events, including a leaky urothelium and mast cell activation. As proposed, neurogenic inflammation consolidates various proposals advanced as the pathogenesis of interstitial cystitis and can readily accommodate infectious, immunologic, and autoimmunologic mechanisms as factors that contribute to development or chronicity of the disease.

Ultrastructural study of the noninvolved urothelium of tumor-bearing patients after two years of intravesical interferon therapy.

Seventeen patients with transitional cell carcinoma of the urinary bladder were studied. Twelve patients did not have a recurrence 2 years after a transurethral resection (TUR) followed by interferon (IFN)-alpha 2b treatment. This observation led us to study the ultrastructural morphology of the noninvolved urothelium in 8 of the above 12 patients. All patients had a primary solitary grade I or II tumor. Topical therapy was started after TUR. Each patient received a total of 22 instillations of 50 MU IFN-alpha 2b in 12 months according to the standard procedure. After the first year, a repetitive dose of 50 MU IFN-alpha 2b was administered every 2 months for a period of 1 more year. At the end of therapy, certain ultrastructural modifications were observed indicating a partial restoration of the urothelium: the existence of asymmetric unit membrane, a well-developed Golgi apparatus and an increase of the filaments. The cells were joined to each other with well-developed tight junctions. Tubuloreticular inclusions were also observed. Prevention of recurrence by restoring the morphology of the noninvolved urothelium in response to IFN treatment deserves further examination.

Selective Interactions of UPIa and UPIb, Two Members of the Transmembrane 4 Superfamily, with Distinct Single Transmembrane-domained Proteins in Differentiated Urothelial Cells

The transmembrane 4 (TM4) superfamily contains many important leukocyte differentiation-related surface proteins including CD9, CD37, CD53, and CD81; tumor-associated antigens including CD63/ME491, CO-029, and SAS; and a newly identified metastasis suppressor gene R2. Relatively little is known, however, about the structure and aggregation state of these four transmembrane-domained proteins. The asymmetrical unit membrane (AUM), believed to play a major role in stabilizing the apical surface of mammalian urothelium thus preventing it from rupturing during bladder distention, contains two TM4 members, the uroplakins (UPs) Ia and Ib. In association with two other (single transmembrane-domained) membrane proteins, UPII and UPIII, UPIa and UPIb form 16-nm particles that naturally form two-dimensional crystalline arrays, thus providing unique opportunities for studying membrane structure and function. To better understand how these proteins interact to form the 16-nm particles, we analyzed their nearest neighbor relationship by chemical cross-linking. We show here that UPIa and UPIb, which share 39% of their amino acid sequence, are cross-linked to UPII and UPIII, respectively. We also show that UPIa has a propensity to oligomerize, forming complexes that are stable in SDS, and that UPII can be readily cross-linked to form homodimers. The formation of UPII homodimers is sensitive, however, to octyl glucoside that can solubilize the AUMs. These data suggest that there exist two types of 16-nm AUM particles that contain UPIa/UPII or UPIb/UPIII, and support a model in which the UPIa and UPII occupy the inner and outer domains, respectively, of the UPIa/UPII particle. This model can account for the apparent "redundancy" of the uroplakins, as the structurally related UPIa and UPIb, by interacting with different partners, may play different roles in AUM formation. The model also suggests that AUM plaques with different uroplakin compositions may differ in their assembly, and in their abilities to interact with an underlying cytoskeleton. Our data indicate that two closely related TM4 proteins, UPIa and UPIb, can be present in the same cell, interacting with distinct partners. AUM thus provides an excellent model system for studying the targeting, processing, and assembly of TM4 proteins.

Towards the Molecular Architecture of the Asymmetric Unit Membrane of the Mammalian Urinary Bladder Epithelium: A Closed “Twisted Ribbon” Structure

The asymmetric unit membrane (AUM) forms numerous plaques covering the apical surface of mammalian urinary bladder epithelium. These plaques contain four major integral membrane proteins called uroplakins Ia, Ib, II and III, which form particles arranged in a well-ordered hexagonal lattice with p6 symmetry and a lattice constant of 16.5 nm. Bovine AUM plaques negatively stained with anionic sodium silicotungstate revealed structural detail to 3.1 nm resolution. Correlation averaging resolved each particle into 12 stain-excluding domains arranged in two concentric rings (inner ring radius (rin)=3.7 nm, outer ring radius (rout)=6.6 nm), each with six domains which were rotated by roughly 30° relative to each other.Negative staining with cationic uranyl formate increased the resolution to 2.2 nm and unveiled distinct connections between adjacent AUM particles. These connections may provide a molecular basis for the observed insolubility of the plaques in many detergents. Examination of the luminal face of freeze-dried/unidirectionally metal-shadowed AUM plaques established a left-handed vorticity of the 16 nm protein particles, whereas the cytoplasmic face exhibited no significant surface corrugations. Three-dimensional reconstruction from sodium silicotungstate-stained specimens revealed the AUM particles to be built of six “V-shaped” subunits anchored upright in the membrane. The mass density distribution within uranyl formate-stained AUM particles was similar except that the inner tip of each V was bridged to the outer tip of an adjacent V, so that the 16 nm AUM particle appeared as a continuous, “twisted ribbon” embracing a central cavity. Finally, mass measurements of unstained/freeze-dried plaques by scanning transmission electron microscopy yielded a total mass of 1,120 kDa per membrane-bound AUM particle. By imposing constraints on the possible uroplakin stoichiometries within AUM plaques, these data provide a first glimpse of the molecular architecture of the 16 nm particles constituting the plaques.