Heterogeneity of microvascular pericytes for smooth muscle type alpha-actin.

Microvascular pericytes are believed to be involved in various functions such as regulation of capillary blood flow and endothelial proliferation. Since pericytes represent a morphologically heterogeneous cell population ranging from circular smooth musclelike to elongated fibroblast-like morphology it is possible that regulation of blood flow (via contractility) and control of endothelial proliferation (as well as other metabolic functions) may be accomplished by different subsets of pericytes. In the present study we provide evidence for heterogeneity of pericytes at the molecular level by using two novel technical approaches. These are (a) immunostaining of whole mounts of the microvascular beds of the rat mesentery and bovine retina and (b) immunoblotting studies of microdissected retinal microvessels. We show that pericytes of true capillaries (midcapillaries) apparently lack the smooth muscle isoform of alpha-actin whereas transitional pericytes of pre- and postcapillary microvascular segments do express this isoform. Thus, regulation of capillary blood flow may be accomplished by the smooth muscle-related pre- and postcapillary pericytes whereas the nonmuscle pericytes of true capillaries may play a role in other functions.

Stress fibers in the splenic sinus endothelium in situ: molecular structure, relationship to the extracellular matrix, and contractility.

In the present study, we investigated structural and functional aspects of stress fibers in a cell type in situ, i.e., the sinus endothelium of the human spleen. In this cell type, stress fibers extend underneath the basal plasma membrane and are arranged parallel to the cellular long axis. Ultrastructurally, the stress fibers were found to be composed of thin actin-like filaments (5-8 nm) and thick myosin-like filaments (10-15 nm X 300 nm). Actin filaments displayed changes in polarity (determined by S-1-myosin subfragment decoration), which may allow a sliding filament mechanism. At their plasmalemmal attachment sites, actin filaments exhibited uniform polarity with the S-1-arrowhead complexes pointing away from the plasma membrane. Fluorescence microscopy showed that the stress fibers have a high affinity for phalloidin and antibodies to actin, myosin, tropomyosin, and alpha-actinin. Vinculin was confined to the cytoplasmic aspect of the plasmalemmal termination sites of stress fibers, while laminin, fibronectin, and collagens were located at the extracellular aspect of these stress fiber-membrane associations. Western blot analysis revealed polypeptide bands that contained actin, myosin, and alpha-actinin to be major components of isolated cells. Exposure of permeabilized cells to MgATP results in prominent changes in cellular shape caused by stress fiber contraction. It is concluded that the stress fibers in situ anchored to cell-to-extracellular matrix contacts can create tension that might allow the endothelium to resist the fluid shear forces of blood flow.

Identification of actin-, alpha-actinin-, and vinculin-containing plaques at the lateral membrane of epithelial cells.

In this paper, a new type of spot desmosome-like junction (type II plaque) is described that is scattered along the entire lateral plasma membrane of rat and human intestinal epithelium. Ultrastructurally type II plaques differed from the classical type of epithelial spot desmosome ("macula adherens", further denoted as type I desmosome) by weak electron density of the membrane-associated plaque material, association of the plaques with microfilaments rather than intermediate filaments, and poorly visible material across the intercellular space. Thus, type II plaques resemble cross-sections of the zonula adherens. Immunofluorescence-microscopic studies were done using antibodies to a main protein associated with the plaques of type I desmosomes (desmoplakin I) and to the three major proteins located at the plaques of the zonula adherens (actin, alpha-actinin, and vinculin). Two types of plaques were visualized along the lateral surface of intestinal and prostatic epithelium: (a) the type I desmosomes, which were labeled with anti-desmoplakin but did not bind antibodies to actin, alpha-actinin, and vinculin, and (b) a further set of similarly sized plaques, which bound antibodies to actin, alpha-actinin, and vinculin but were not stained with anti-desmoplakin. Three-dimensional computer reconstruction of serial sections double-labeled with anti-desmoplakin and anti-alpha-actinin further confirmed that both types of plaques are spatially completely separated from each other along the lateral plasma membrane. The computer graphs further revealed that the actin-, alpha-actinin-, and vinculin-containing plaques have the tendency to form clusters, a feature also typical of type II plaques. It is suggested that the type II plaques represent spot desmosome-like intercellular junctions, which, like the zonula adherens, appear to be linked to the actin filament system. As the type II plaques cover a considerable part of the lateral cell surface, they might play a particular role in controlling cellular shape and intercellular adhesion.

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study.

In the present study we have used immunogold labeling of ultrathin sections of the intact chicken and human intestinal epithelium to obtain further insight into the molecular structure of the brush-border cytoskeleton. Actin, villin, and fimbrin were found within the entire microvillus filament bundle, from the tip to the basal end of the rootlets, but were virtually absent from the space between the rootlets. This suggests that the bulk of actin in the brush border is kept in a polymerized and cross-linked state and that horizontally deployed actin filaments are virtually absent. About 70% of the label specific for the 110-kD protein that links the microvillus core bundle to the lipid bilayer was found overlying the microvilli. The remaining label was associated with rootlets and the interrootlet space, where some label was regularly observed in association with vesicles. Since the terminal web did not contain any significant amounts of tubulin and microtubules, the present findings would support a recently proposed hypothesis that the 110-kD protein (which displays properties of an actin-activated, myosin-like ATPase) might also be involved in the transport of vesicles through the terminal web. Label specific for myosin and alpha-actinin was confined to the interrootlet space and was absent from the rootlets. About 10-15% of the myosin label and 70-80% of the alpha-actinin label was observed within the circumferential band of actin filaments at the zonula adherens, where myosin and alpha-actinin displayed a clustered, interrupted pattern that resembles the spacing of these proteins observed in other contractile systems. This circular filament ring did not contain villin, fimbrin, or the 110-kD protein. Finally, actin-specific label was observed in close association with the cytoplasmic aspect of the zonula occludens, suggesting that tight junctions are structurally connected to the microfilament system.

Localization of myosin, actin, and tropomyosin in rat intestinal epithelium: immunohistochemical studies at the light and electron microscope levels.

Myosin, tropomyosin, and actin were localized in the epithelial cells of rat intestine by means of specific antibodies to chicken gizzard smooth muscle myosin, tropomyosin, and actin by immunohistochemical studies at both the light and electron microscope levels (unlabeled antibody enzyme technique). The pattern of antibody staining was the following (a) Anti-actin was associated with the microfilament bundles of the microvilli in their entire length, as well as with the microfilament network in the terminal web. (b) Anti-myosin was concentrated along the rootlets of the microvillar microfilament bundles and within the filamentous feltwork forming the terminal web. (c) Anti-tropomyosin showed a distribution similar to that of anti-myosin. In addition, the three antibodies also labeled the subplasmalemmal web underneath the cell membrane bordering on the basal lamina. Utilizing the above ultrastructural findings, we wish to propose a functional model of microvillar contraction.

Role of microtubules in polarized delivery of apical membrane proteins to the brush border of the intestinal epithelium.

Colchicine- and vinblastine-induced depolymerization of microtubules (MTs) in the intestinal epithelium of rats and mice resulted in significant delivery of three apical membrane proteins (alkaline phosphatase, sucrase-isomaltase, and aminopeptidase N) to the basolateral membrane domain. In addition, typical brush borders (BBs) occurred at the basolateral cell surface, consisting of numerous microvilli that contained the four major components of the cytoskeleton of apical microvilli (actin, villin, fimbrin, and the 110-kD protein). Formation of basolateral microvilli required polymerization of actin and proceeded at glycocalyx-studded plaques that resembled the dense plaques located at the tips of apical microvilli. BBs from the basolateral membrane became internalized into BB-containing vacuoles which served as recipient organelles for newly synthesized apical membrane proteins. The BB vacuoles fused with each other and finally were inserted into the apical BB. Polarized distribution of Na+,K+-ATPase, a basolateral membrane protein, was not affected by drug-induced depolymerization of MTs. These observations indicate that Golgi-derived carrier vesicles (CVs) containing apical membrane proteins are vectorially guided to the apical cell surface by a retrograde transport along MTs. MTs are uniformly oriented towards a narrow space underneath the apical terminal web (termed subterminal space) that contains MT-organizing properties and controls polarized alignment of MTs. In contrast to apical CVs, targeting of basolateral CVs appears to be independent of MTs but demands a barrier at the apical membrane domain that prevents basolateral CVs from apical fusion (transport barrier hypothesis).

Two novel peripheral membrane proteins, pasin 1 and pasin 2, associated with Na+,K(+)-ATPase in various cells and tissues.

Purification of pig kidney Na+,K(+)-ATPase at low concentrations of SDS (0.5%) allowed copurification of several peripheral membrane proteins. Some of these associated proteins were identified as components of the membrane cytoskeleton. Here we describe two novel globular proteins of of Mr 77,000 (pasin 1) and Mr 73,000 (pasin 2) which copurify and coimmunoprecipitate with Na+,K(+)-ATPase and can be stripped off Na+,K(+)-ATPase microsomes by 1 M KCl. Pasin 1 and pasin 2 were detected by immunoblot analysis in various cells and tissues including erythrocytes and platelets. Immunostaining revealed colocalization of pasin 1 and Na+,K(+)-ATPase along the basolateral cell surface of epithelial cells of kidney tubules and parotid striated ducts (titers of pasin 2 antibodies were too weak for immunocytochemistry). In erythrocytes, pasin 1 and pasin 2 are minor components bound to the cytoplasmic surface of the plasma membrane. Pasin 1 showed the same electrophoretic mobility as protein 4.1b. However, both proteins have different isoelectric points (pasin 1, pI 6; protein 4.1, pI 7), different chymotryptic fragments, and are immunologically unrelated. Short pieces of sequence obtained from pasin 1 and pasin 2 were not found in any other known protein sequence. The occurrence of pasin 1 and pasin 2 in diverse cells and tissues and their association with Na+,K(+)-ATPase suggests a general role of these proteins in Na+,K(+)-ATPase function.

Three different actin filament assemblies occur in every hair cell: each contains a specific actin crosslinking protein.

The apex of hair cells of the chicken auditory organ contains three different kinds of assemblies of actin filaments in close spatial proximity. These are (a) paracrystals of actin filaments with identical polarity in stereocilia, (b) a dense gellike meshwork of actin filaments forming the cuticular plate, and (c) a bundle of parallel actin filaments with mixed polarities that constitute the circumferential filament belt attached to the cytoplasmic aspect of the zonula adhaerens (ZA). Each different supramolecular assembly of actin filaments contains a specific actin filament cross-linking protein which is unique to that particular assembly. Thus fimbrin appears to be responsible for paracrystallin packing of actin filaments in stereocillia; an isoform of spectrin resides in the cuticular plate where it forms the whisker-like crossbridges, and alpha actinin is the actin crosslinking protein of the circumferential ZA bundle. Tropomyosin, which stabilizes actin filaments, is present in all the actin filament assemblies except for the stereocilia. Another striking finding was that myosin appears to be absent from the ZA ring and cuticular plate of hair cells although present in the ZA ring of supporting cells. The abundance of myosin in the ZA ring of the surrounding supporting cells means that it may be important in forming a supporting tensile cellular framework in which the hair cells are inserted.

Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea.

The sensory epithelium of the chick cochlea contains only two cell types, hair cells and supporting cells. We developed methods to rapidly dissect out the sensory epithelium and to prepare a detergent-extracted cytoskeleton. High salt treatment of the cytoskeleton leaves a "hair border", containing actin filament bundles of the stereocilia still attached to the cuticular plate. On SDS-PAGE stained with silver the intact epithelium is seen to contain a large number of bands, the most prominent of which are calbindin and actin. Detergent extraction solubilizes most of the proteins including calbindin. On immunoblots antibodies prepared against fimbrin from chicken intestinal epithelial cells cross react with the 57- and 65-kD bands present in the sensory epithelium and the cytoskeleton. It is probable that the 57-kD is a proteolytic fragment of the 65-kD protein. Preparations of stereocilia attached to the overlying tectorial membrane contain the 57- and 65-kD bands. A 400-kD band is present in the cuticular plate. By immunofluorescence, fimbrin is detected in stereocilia but not in the hair borders after salt extraction. The prominent 125 A transverse stripping pattern characteristic of the actin cross-bridges in a bundle is also absent in hair borders suggesting fimbrin as the component that gives rise to the transverse stripes. Because the actin filaments in the stereocilia of hair borders still remain as compact bundles, albeit very disordered, there must be an additional uncharacterized protein besides fimbrin that cross-links the actin filaments together.

The role of hemoglobin denaturation and band 3 clustering in red blood cell aging.

As hemoglobin begins to denature, it forms hemichromes that cross-link the major erythrocyte membrane-spanning protein, band 3, into clusters. These clusters provide the recognition site for antibodies directed against senescent cells. These antibodies bind to the aged red cell and trigger its removal from circulation.

Colocalization of band 3 with ankyrin and spectrin at the basal membrane of intercalated cells in the rat kidney.

An immunoreactive form of the anion channel protein of erythrocytes, band 3, has been identified in the rat kidney. It is found in the intercalated cells of the distal tubule and collecting ducts. Immunostaining specific for band 3 is confined to the basolateral plasma membrane of these cells, where this protein probably mediates the transport of bicarbonate across the tubular wall. Double-immunolabeling studies demonstrate that band 3 is colocalized with immunoreactive forms of ankyrin and spectrin along the basolateral plasma membrane. The polarized distribution of band 3 may be the result of the association of its cytoplasmic domain with ankyrin, which in turn links band 3 to spectrin and the cytoskeleton. These observations help to explain how the collecting ducts of the kidney can direct the transport of bicarbonate ions, thus maintaining the acid-base balance.

Different Ca2+ affinities and functional implications of the two synaptic adhesion molecules cadherin-11 and N-cadherin.

Cadherins of synaptic complexes are considered to be critically involved in long-term potentiation (LTP). Here we compared biophysical properties of cadherin-11 and N-cadherin, which appear to exert opposing effects on LTP, i.e., inhibition and promotion, respectively. Characterization of cadherin-11 binding by atomic force microscopy and laser tweezers revealed a significantly higher Ca(2+) affinity, with half-maximal binding (K(D)) at 0.11-0.26 mM Ca(2+), as compared to N-cadherin (K(D) approximately 0.7 mM Ca(2+)). Adhesive properties of both cadherins were modulated to a similar degree by manipulation of the actin cytoskeleton indicating to unlikely account for opposing roles in LTP induction. However, differences in Ca(2+) affinity could well explain opposing binding properties during activity-dependent transient reduction of extracellular Ca(2+) ([Ca(2+)](e)) in the synaptic cleft: whereas high frequency stimulation with drop of [Ca(2+)](e) to 0.3-0.8 mM Ca(2+) will result in significant weakening of N-cadherin adhesion, cadherin-11-based adhesion will stay mostly stable. Reduction of N-cadherin adhesion may facilitate synaptic remodeling and LTP induction, while cadherin-11 adhesion with its higher stability at low [Ca(2+)](e) may counteract this process explaining why in cadherin-11-deficient mice LTP is increased rather than decreased.

Replication of Marburg virus in human endothelial cells. A possible mechanism for the development of viral hemorrhagic disease.

Marburg and Ebola virus, members of the family Filoviridae, cause a severe hemorrhagic disease in humans and primates. The disease is characterized as a pantropic virus infection often resulting in a fulminating shock associated with hemorrhage, and death. All known histological and pathophysiological parameters of the disease are not sufficient to explain the devastating symptoms. Previous studies suggested a nonspecific destruction of the endothelium as a possible mechanism. Concerning the important regulatory functions of the endothelium (blood pressure, anti-thrombogenicity, homeostasis), we examined Marburg virus replication in primary cultures of human endothelial cells and organ cultures of human umbilical cord veins. We show here that Marburg virus replicates in endothelial cells almost as well as in monkey kidney cells commonly used for virus propagation. Our data support the concept that the destruction of endothelial cells resulting from Marburg virus replication is a possible mechanism responsible for the hemorrhagic disease and the shock syndrome typical of this infection.

The fodrin-ankyrin cytoskeleton of choroid plexus preferentially colocalizes with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2.

A unique feature of the choroid plexus as a single-layer epithelium is its localization of Na+K(+)-ATPase at its apical (lumenal) surface. In contrast, a band 3 (AE1)-related anion exchanger protein has been localized to the basolateral surface of the choroid plexus. Both Na+K(+)-ATPase and AE1 in other tissues have been shown to bind via ankyrin to the spectrin-actin-based membrane cytoskeleton. Since linkage of integral membrane proteins to the membrane cytoskeleton is important for their restriction to specialized domains of the cell surface, we investigated the polarity of the choroid plexus membrane cytoskeleton. We developed isoform-specific antibodies to confirm the identity of choroid plexus band 3-related polypeptide as AE2. We demonstrated that ankyrin, fodrin/spectrin, actin, myosin, and alpha-actinin are predominantly apical in choroid plexus and preferentially colocalize with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2. Colchicine administration did not alter the polarity of apical cytoskeletal and transport proteins or basolateral AE2 in choroid plexus, suggesting that biosynthetic targeting of these proteins is not microtubule dependent. In choroid plexus papilloma, Na+K(+)-ATPase and AE2 were decreased in amount and failed to preserve their polarized distributions.

Dystrophin in the retina.

Dystrophin is a plasma membrane-associated cytoskeletal protein of the spectrin superfamily. The dystrophin cytoskeleton has been first characterized in muscle. Muscular 427 kDa dystrophin binds to subplasmalemmal actin filaments via its amino-terminal domain. The carboxy-terminus of dystrophin binds to a plasma membrane anchor, beta-dystroglycan, which is associated on the external side with the extracellular matrix receptor, alpha-dystroglycan, that binds to the basal lamina proteins laminin-1, laminin-2, and agrin. In the muscle, the dystroglycan complex is associated with the sarcoglycan complex that consists of several glycosylated, integral membrane proteins. The absence or functional deficiency of the dystrophin cytoskeleton is the cause of several types of muscular dystrophies including the lethal Duchenne muscular dystrophy (DMD), one of the most severe and most common genetic disorders of man. The dystrophin complex is believed to stabilize the plasma membrane during cycles of contraction and relaxation. Muscular dystrophin and several types of dystrophin variants are also present in extramuscular tissues, e.g. in distinct regions of the central nervous systems including the retina. Absence of dystrophin from these sites is believed to be responsible for some extramuscular symptoms of DMD, e.g. mental retardation and disturbances in retinal electrophysiology (reduced b-wave in electroretinograms). The reduced b-wave in electroretinograms indicated a disturbance of neurotransmission between photoreceptors and ON-bipolar cells. At least two different dystrophin variants are present in photoreceptor synaptic complexes. One of these dystrophins (Dp260) is virtually exclusively expressed in the retina. In the neuroretina, dystrophin is found in significant amounts in the invaginated photoreceptor synaptic complexes. At this location dystrophin colocalizes with dystroglycan. Agrin, an extracellular ligand of alpha-dystroglycan, is also present at this location whereas the proteins of the sarcoglycan complex appear to be absent in photoreceptor synaptic complexes. Dystrophin and dystroglycan are located distal from the ribbon-containing active synaptic zones where both proteins are restricted to the photoreceptor plasma membrane bordering on the lateral sides of the synaptic invagination. In addition, some neuronal profiles of the postsynaptic complex also contain dystrophin and beta-dystroglycan. These profiles appear to belong at least in part to projections of the photoreceptor terminals into the postsynaptic dendritic complex. In view of the abnormal neurotransmission between photoreceptors and ON-bipolar cells in DMD patients the dystrophin/beta-dystroglycan-containing projections of photoreceptor presynaptic terminals into the postsynaptic dendritic plexus might somehow modify the ON-bipolar pathway. Another retinal site associated with dystrophin/beta-dystropglycan is the plasma membrane of Müller cells where dystrophin/beta-dystroglycan appear to be present at particular high concentrations. At this location the dystrophin/dystroglycan complex may play a role in the attachment of the retina to the vitreous, and, under pathological conditions, in traction-induced retinal detachment.

Freeze-fracture studies of cytoplasmic inclusions occurring in experimental lipidosis as induced by amphiphilic cationic drugs.

The ultrastructure of cytoplasmic inclusions, which characterize experimental lipidosis as induced by several amphiphilic cationic drugs, was studied by means of freeze-fracturing and thin-sectioning. Retinal and adrenal tissues of rats chronically treated with high oral doses of chlorphentermine were used. In thin sections the cytoplasmic inclusions, which were previously shown to represent lysosomes overloaded with polar lipids, exhibit lamellated or lattice-like internal patterns. The present freeze-fracture observations are interpreted as to indicate that the lamellated inclusions contain polar lipids in the lamellar phase, whereas those with lattice-like patterns contain polar lipids in a hexagonal phase.

Cytoskeleton-membrane connections in the human erythrocyte membrane: band 4.1 binds to tetrameric band 3 protein.

Band 4.1 provides, besides ankyrin, the main linkage between the erythrocyte membrane and its cytoskeleton. Its predominant binding sites in the membrane are located on the glycophorins. However, the cytoplasmic domain of band 3 can also bind band 4.1. We have studied which of the different band 3 oligomers observed (monomers, dimers, tetramers) can act as band 4.1 binding sites, by equilibrium sedimentation experiments on mixtures of purified band 3 and dye-labelled band 4.1 in solutions of a nonionic detergent. At low molar ratios of band 4.1 and band 3, the sedimentation equilibrium distributions obtained could all be perfectly fitted assuming that only two dye-labelled particles were present: uncomplexed band 4.1 and a complex formed between one band 4.1 molecule and one band 3 tetramer. The presence of small amounts of complexes containing band 3 monomers or dimers could not be completely ruled out but is unlikely. On the other hand, stabilized band 3 dimers effectively bound band 4.1. At higher molar band 4.1/band 3 ratio, the band 3 tetramer apparently could bind up to at least four band 4.1 molecules. The band 4.1/band 3 tetramer complex was found to be unstable. The results described, together with those reported previously, point at a prominent role of tetrameric band 3 in ligand binding.

Roles of cytoskeletal and junctional plaque proteins in nuclear signaling.

Cytoplasmic junctional plaque proteins play an important role at intercellular junctions. They link transmembrane cell adhesion molecules to components of the cytoskeleton, thereby playing an important role in the control of many cellular processes. Recent studies on the subcellular distribution of some plaque proteins have revealed that a number of these proteins are able to localize in the nucleus. This dual location indicates that in addition to promoting adhesive interactions, plaque proteins may also play a direct role in nuclear processes, and in particular in the transfer of signals from the membrane to the nucleus. Therefore, translocation of plaque proteins into the nucleus in response to extracellular signals could represent a novel and direct mechanism by which signals can be transmitted from the plasma membrane to the nucleus. This could allow cells to respond to changing environmental conditions in a rapid and efficient way. In addition, conditional sequestration of karyophilic proteins at the sites of cell-cell and cell-substratum adhesion may represent a general mechanism for the regulation of nucleocytoplasmic transport.

Uniform apicobasal polarity of microtubules and apical location of gamma-tubulin in polarized intestinal epithelium in situ.

Polarized differentiation of the intestinal epithelium has been previously shown to depend on an intact microtubular system that is essential for vectorial delivery of apical membrane proteins to the apical cell surfaces. Uniform alignment and polarization of microtubules have been suggested to provide the ultrastructural basis for vectorial transport between the Golgi apparatus and the apical cell surface. In the present study we applied the hook decoration technique to analyse the polarity of microtubules in the rat jejunal epithelium. By immunocytochemistry we studied the subcellular location of gamma-tubulin, an essential component of microtubule-organizing centers. Microtubules were found to be mainly aligned parallel to the apicobasal axis of the cells and to extend from the subterminal space underneath the apical terminal web down to the cellular basis. We found that 98% out of 1122 decorated microtubules displayed uniform apicobasal polarity with the growing ends (plus ends) pointing basally and the non-growing ends (minus ends) pointing towards the cellular apex. No differences were observed with respect to microtubular polarity between the apical, perinuclear and infranuclear cellular portions. Immunostaining specific for gamma-tubulin was restricted to the apical subterminal space underneath the rootlets of microvilli. These findings indicate that the apical subterminal space of enterocytes serves as the predominant if not exclusive microtubule-organizing compartment from which uniformly polarized microtubules grow out with their plus ends towards the cellular basis.

Restriction of the human kidney band 3-like anion exchanger to specialized subdomains of the basolateral plasma membrane of intercalated cells.

In this study, the kidney analog of the erythrocyte anion exchanger, band 3, served as the first example of an anion translocating membrane protein in a nucleated cell type to be localized at the ultrastructural level. Kidney band 3 was found to be confined to the basolateral membrane of the intercalated cells in the human collecting duct. The immunogold label displayed a striking non-uniform distribution along the basolateral plasma membrane with a preferential concentration at pleated areas of the membrane surface. The pleated portions are suggested to represent specialized subdomains to which the band 3 analog might be restricted by linkage via ankyrin to the spectrin-based membrane cytoskeleton. The immunolabel did not extend apically to the level of the zonula adherens and zonula occludens indicating that tight junctions might not be important for maintaining the polarized distribution of this integral membrane protein. Association of antibody label with the rough endoplasmic reticulum and other types of cytoplasmic membranes indicate pathways in the biosynthesis and degradation of this anion exchanger.