Analysis of the structure of intramembrane particles of the mammalian urinary bladder.

Abstract

The luminal and discoid vacuole membranes of the superficial cell layer of the transitional epithelium of the mammalian urinary bladder have been studied by thin-sectioning and freeze-fracture-etch (FFE) electron microscope methods. For the FFE studies membranes were deposited on a cationized glass surface, covered by a thin copper disc, and fractured under liquid N2. Specimens were etched at -100°C and replicated at -190°C. A model of the lattice membrane derived from thin sections was used to predict the heights of the fracture faces above the glass surface . A hexagonal pattern of globular intramembrane particles spaced 160 A apart was seen in the external fracture (EF) face plaques as previously described and regarded as the dominant structure. However, very extensive areas of another pattern, seen before in only limited areas, have been found in the EF faces . The pattern consists of a smooth hexagonal lattice with the same space constant as the globular one but a different structure . By image analysis it consists of overlapping domains bordered by shared but incomplete metal rims . Each domain has a central spot of metal encircled by a shadow . The surface of the smooth lattice is partly complementary to the corresponding protoplasmic fracture (PF) face which shows a similar hexagonal lattice with the same space constant . The height of the smooth EF lattice above the glass substrate is the same as the plane of the center of the lipid bilayer predicted by the model . The mean heights of the particles of the globular EF lattice are greater than the total thickness of the membrane as predicted by the model and confirmed by measurements . The globular EF lattice is not complementary and it is concluded that the globular particles do not exist in the native membrane but arise artifactually during the preparatory procedures .Porter and Bonneville (36) first noted the unusual features of the luminal membrane of the mammalian urinary bladder epithelial cell, and it subsequently has been the subject of numerous investigations by groups associated with Porter (11,12,33,37,47) and 29,42,56,57), by ourselves (30,38,53,54), and others (28) . It is now established that 70-75% of the surface area of the luminal membrane and the membranes of the related discoid vacuoles in the luminal epithelial cell display external plaques 0.2-0.5 ,um in diameter comprised of particles in hexagonal array with p6 plane group symmetry spaced -160 A center-to-center . In negatively membranes are more flexible and are often sharply folded, particularly in the discoid vacuoles . For this reason the interplaque membrane is frequently called the "hinge" membrane to distinguish it from the "plaque" or "lattice" membrane .In freeze-fracture-etch (FFE) preparations the 120 A surface particles have a counterpart in an array of globular particles in the external fracture (EF) faces (8, 42). The EF particles usually appear globular and have been said by Staehelin et al . (47) to rise 40 A above the EF face and to fit into corresponding p...

Structural basis of urothelial permeability barrier function as revealed by Cryo-EM studies of the 16 nm uroplakin particle

“…4C): the bottom cytoplasmic zone (C), the transmembrane zone (TM), the trunk zone (TK), and the top joint zone (J). We determined the total height of the urothelial particle to be ~12 nm, which is consistent with previously reported values of 12-13 nm based on thin-section electron microscopy (Staehelin et al, 1972;Hicks et al, 1974;Robertson and Vergara, 1980), 13.2 nm based on cryo-EM (Oostergetel et al, 2001), and 12.5 nm based on atomic force microscopy (Min et al, 2002) (unpublished data). Our recent atomic force microscopy data indicated that the extracellular portion of the urothelial particle is 6.5 nm in height (Min et al, 2002) (Fig.…”

“…4C). This value also compares favorably with reported values of 5 nm (Brisson and Wade, 1983;Walz et al, 1995) or 5.7 nm based on 3D reconstruction of negatively stained images (Taylor and Robertson, 1984); or 6 nm (Hicks et al, 1974) or 6.5 nm (Robertson and Vergara, 1980) based on thin-section electron microscopy. Finally, our atomic force microscopy data indicate that there is a cytoplasmic protrusion of 0.5 nm (Min et al, 2002).…”

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

“…As we and others have shown earlier, each 16-nm particle of the AUM can be resolved into 12 stain-excluding domains, that are arranged in an inner ring of six and outer ring of six (Hicks andKetterer, 1969, 1970;Vergara et al, 1969;Robertson and Vergara, 1980;Brisson and Wade, 1983;Walz et al, 1995). Taylor and Robertson (1984) calculated that the volume of each inner domain is 1.6 times larger than that of an outer domain.…”

Ultracytochemical Demonstration of Functional Groups in the Lumenal Plasma Membrane of Rabbit Transitional Epithelium

“…However, the chemical analysis of the purified lumenal plasma membrane has shown rather high lipid contents in various animals (4,5,16,41). Moreover, the intramembranous particles have been observed recently in the experimental membrane produced with pure lipid mixtures of lecithin and cardiolipin (43,44), and the nature of these particles in the lumenal plasma membrane of the transitional epithelium should carefully be determined (25) . By the ruthenium red staining, the surface coat was stained.…”

“…n plaque regions, methods such as negative staining (14,40,44,45) or freeze-etching (25,29,30) have been applied thus far and no investigation demonstrating macromolecules directly in the lumenal plasma membrane has been reported except for a light microscopic work of Hicks (12). In general, the plasma membrane consists of lipids, proteins and carbohydrates, and the fluid-mosaic model proposed by Singer and Nicolson (31) is generally accepted as a membrane model at present; lipids comprise the bilayer (outer and inner layers) with hydrophilic ends facing outside and hydrophobic tails inside (7,32).…”