Microvillar Core Research Articles

Chapter 8 The Brush Border of Intestinal Epithelium: A Model System for Analysis of Cell-Surface Architecture and Motility

Publisher Summary This chapter examines a model system for analysis of cell-surface architecture and motility of the brush border of intestinal epithelium. The apical surface of absorptive epithelial cells, such as those lining the lumen of the intestine or proximal tubule of the kidney, consists of a tightly packed array of microvilli variously referred to as the striated, microvillus, or brush border. The brush-border membrane contains a variety of membrane proteins, most of which are enzymes involved in the processing and transport of the nutrients absorbed by the small intestine. Brush borders are isolated from dissociated intestinal epithelial cells by homogenization and differential centrifugation. For cytological experiments, one chicken is used, and for biochemical studies, 5–10 animals are used. Microvilli are freed from the brush borders by homogenization in a Dounce homogenizer, fitted with a tight pestle. Homogenization is continued until the brush borders begin to fragment. As is the case for brush-border isolation, it is important to monitor the progress of homogenization by light microscopy. It is suggested that the contraction of the terminal web was caused by sarcomere-like myosin interactions with the basal ends of the microvillar core filaments and the actin filaments extending from the zonula adherens.

A low molecular weight tracer molecule for immunocytochemistry. Identification of cytoplasmic actin.

Anti actin Fab-fragments were tagged to a small electron dense tracer molecule; ferrocene monocarboxylic acid (230 daltons). The conjugate stains actin filaments, which were found mainly in the core of microvilli.

F-actin binding and bundling properties of fimbrin, a major cytoskeletal protein of microvillus core filaments.

Fimbrin, previously recognized as a major structural protein of the microfilament core bundles of intestinal epithelial cell microvilli, has been purified to homogeneity and characterized. It is a nearly globular monomeric protein of apparent molecular weight 68,000 and has a single calcium binding site (Kd = 9 microM), for which magnesium ions compete. Fimbrin binds to F-actin and this interaction is characterized in detail. Under our optimal binding of conditions, fimbrin induces tightly packed F-actin bundles, similar to the bundles induced by villin, another microvillus structural protein. The formation of mixed fimbrin-villin-actin bundles provides a further step toward the full in vitro reconstitution of microvillus core filaments from its purified individual components. The reconstituted fimbrin-villin-actin bundles do not display the side arms characteristic of isolated microvillus cores. These results are discussed in terms of our current understanding of the organization of the microvillus core filaments and indicate that this structure contains two bundling proteins, villin and fimbrin. The results complement previous studies and now provide a minimal biochemical characterization of all four major actin-associated structural proteins so far identified in core filaments. Three of these (villin, fimbrin, and calmodulin) are calcium-binding proteins, emphasizing the concept of calcium control over submembranous microfilament organization.

Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells.

The bundle of filaments within microvilli of intestinal epithelial cells contains five major proteins including actin, calmodulin, and subunits of 105-, 95-, and 70-kdaltons. It has been previously shown (Howe, C. L., M. S. Mooseker, and T. A. Graves. 1980. Brush-border calmodulin: a major component of the isolated microvillus core. J. Cell Biol. 85: 916-923) that the addition of Ca++ (> 10(-6) M) to microvillus cores causes a rapid, drastic, but at least partially reversible disruption of this actin filament bundle. High-speed centrifugation of microvillus cores treated with Ca++ indicates that several core proteins are solubilized, including 30-50% of the actin and calmodulin, along with much of the 95- and 70-kdalton subunits. Gel filtration of such Ca++ extracts in the presence and absence of Ca++ indicates that microvillar actin "solated" by Ca++ is in an oligomeric state probably complexed with the 95-kdalton subunit. Removal of Ca++ results in the reassembly of F-actin, probably still complexed with 95-kdalton subunit, as determined by gel filtration, cosedimentation, viscometry, and electron microscopy. The 95-kdalton subunit (95K) was purified from Ca++ extracts by DEAE-Sephadex chromatography and its interaction with actin characterized by viscometry, cosedimentation, and EM in the presence and absence of Ca++. In the presence, but not absence, of Ca++, 95K inhibits actin assembly (50% inhibition at 1:50-60 95K to actin) and also reduces the viscosity of F-actin solutions. Similarly, sedimentation of actin is inhibited by 95K, but a small, presumably oligomeric actin- 95K complex formed in the presence of Ca++ is pelletable after long-term centrifugation. In the absence of Ca++, 95K cosediments with F-actin. EM of 95K-actin mixtures reveals that 95K "breaks" actin into small, filamentous fragments in the presence of Ca++. Reassembly of filaments occurs once Ca++ is removed. In the absence of Ca++, 95K has no effect on filament structure and, at relatively high ratios (1:2-6) of 95K to actin, this core protein will aggregate actin filaments into bundles.

Calmodulin-binding proteins of the microfilaments present in isolated brush borders and microvilli of intestinal epithelial cells.

Isolated microfilament cores of intestinal microvilli are known to contain actin and four major associated proteins among which is calmodulin. Immunofluorescence microscopy reveals that calmodulin is present in the microvilli prior to biochemical fractionation of intestinal cells and thus is not bound artifactually during the isolation procedure. Identification of the major microvillus calmodulin-binding protein was achieved by the use of an [125I]calmodulin gel overlay technique. Proteins of microvilli or brush borders were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After removal of sodium dodecyl sulfate, direct binding of radiolabeled calmodulin to the separated polypeptides was assayed by autoradiography. Three calmodulin-binding polypeptides are detected in brush borders. Two polypeptides (apparent Mr = 280,000 and 140,000) show Ca2+ -dependent binding, whereas the third polypeptide (Mr = 110,000) can bind calmodulin in the presence or absence of Ca2+. Microvillus core filaments contain only the latter species. Microvillus cores treated with 25 mM Mg2+ retain calmodulin and the 110,000 polypeptide, whereas the other two associated proteins are greatly reduced, consistent with the hypothesis that the 110,000 protein is the major calmodulin-binding protein of the core filament structure. We discuss the currently documentable structure of the core filaments and evaluate the general usefulness of the calmodulin gel overlay technique.

Villin is a major protein of the microvillus cystoskeleton which binds both G and F actin in a calcium-dependent manner

The cores of the microvilli present on intestinal epithelial cells are currently the only microfilament arrangement which can be isolated ultrastructurally intact and in sufficient quantities for biochemical analysis. We have isolated and characterized villin, a major protein of the microvillus core. Using villin's ability to bind very tightly to immobilized monomeric actin in a calcium-dependent manner, we have developed a method for its rapid purification by affinity chromatography on G actin, which itself was bound to immobilized pancreatic deoxyribonuclease I (DNAase I). The villin-G actin complex on DNAase I is resistant to high ionic strength, and villin, but not actin, is released when the calcium concentration is less than 10 6 M. Purified villin behaves as a globular monomeric protein of molecular weight 95,000, and is free of carbohydrate. Villin also interacts with F actin. In the absence of calcium, villin cross-links F actin having the properties of an F actin bundling or gelation factor. In the presence of calcium (>10 −7 M), villin apparently restricts the polymerization of actin to short filaments which cannot be readily sedimented. The properties of villin are not compatible with its previously suggested role as the cross-filament between the microvillus microfilament core and the plasma membrane, but rather indicate a function as a calcium-dependent F actin-bundling protein. The role of villin is discussed in terms of the other protein components of the microvillus core and in relation to recently described calcium-dependent gelation factors.

Brush-border calmodulin. A major component of the isolated microvillus core.

Calmodulin is present in brush borders isolated from intestinal epithelial cells and is one of the major components of the microvillar filament bundle. Calmodulin was purified from either demembranated brush borders or microvilli by a simple boiling procedure. The boiled supernate derived from the microvillus cores contained one major polypeptide of 20,000 daltons. The supernate from the brush-border preparation contained the 20,000-dalton subunit and a second protein of 30,000 daltons. The 20,000-dalton subunit has been identified as calmodulin by several criteria: (a) heat resistance, (b) comigration with brain calmodulin on alkaline urea gels and SDS gels, both cases in which the 20,000-dalton protein, like calmodulin, exhibits a shift in electrophoretic mobility in the presence of Ca++, and (c) 4--5-fold activation of 3',5'-cyclic nucleotide phosphodiesterase in the presence but not the absence of Ca++. With a cosedimentation assay it was determined that brush-border calmodulin does not bind directly to actin. In the presence of Ca++ (greater than 5 x 10(-7) M) there was a partial release of calmodulin from the microvillus core, along with a substantial conversion of microvillus actin into a nonpelletable from. The dissociation of calmodulin was reversed by removal of Ca++. If microvillus cores were pretreated with phalloidin, the Ca++-induced solubilization of actin was prevented, but the partial dissociation of calmodulin still occurred. The molar ratio of calmodulin:actin is 1:10 in the demembranated brush border and 1:2-3 in the microvillus core. No calmodulin was detected in the detergent-solubilized brush-border membrane fraction.

Carbonic anhydrase, ultrastructural localization in the mouse gastric mucosa and improvements in the technique.

The ultrastructural localization of carbonic anhydrase activity in mouse gastric mucosal cells as revealed by the cobalt bicarbonate histochemical method of Hansson has been made. In addition the effects of fixatives used for ultrastructural studies have been evaluated for reduction of carbonic anhydrase activity; exogenous erythrocyte carbonic anhydrase has been localized in tissues; acetazolamide and potassium cyanate inhibition of activity demonstrated; and an improved method for the osmication of reacted tissues for electron microscopy has been developed. The results indicate that the glutaraldehyde, formaldehyde, picric acid fixative, which retains about 5% of the original carbonic anhydrase activity, is distinctly better for histochemical studies than formaldehyde fixation, which retains about 32% activity. Acetazolamide at 10(-5) M consistently inhibits histochemical reaction, as does 20 mM KCNO, in the incubation medium. Exogenous carbonic anhydrase is readily visualized by the histochemical technique. Electron microscopy of gastric mucosa reacted for carbonic anhydrase activity indicates the focal deposition of the cobalt sulfide reaction product in the cores of microvilli lining the intracellular canaliculi, in the basal and lateral cell folds of parietal cells, and in the microvilli as well as the cytoplasm between mucous granules in the surface mucous cells. In addition, some reaction product was found in the mitochondrial cristae and in some nuclei and intercellular spaces.

Redistribution of actin and fascin in sea urchin eggs after fertilization

Following fertilization, the sea urchin egg cortex undergoes a structural change involving the assembly and organization of actin filaments into microvilli. Antifascin localizes this actin cross-linking protein in the microvilli of the fertilized egg cortex but no organized staining is present in the unfertilized cortex. Determination of the actin content of eggs using the DNAase I inhibition assay indicates that actin is about 1.4% of the total protein. Approximately 90% of this actin is soluble in low calcium isotonic extracts of unfertilized eggs while only 60-65% can be recovered in identical extracts of fertilized eggs. Similar measurements for fascin using a radioimmunoassay indicate this molecule represents about 0.3% of the total egg protein, essentially all of which is recovered in low calcium isotonic extracts of unfertilized eggs. After fertilization only 65-70% of this actin cross-linking protein is in the soluble phase. These results demonstrate a markedly different solubility for actin and fascin after fertilization, when the indirect immunofluorescence staining localizes fascin in the microvilli, and are consistent with the idea that fascin organizes newly polymerized actin filaments into the microvillar cores. A consideration of the amounts of actin and fascin incorporated into the cortex after fertilization and the number of microvilli on the egg surface indicates that the measured values are sufficient to account for the observed microvillar elongation.

Villin: The major microfilament-associated protein of the intestinal microvillus

The major protein associated with actin in the microfilament core of intestinal microvilli has been purified. This protein, for which we propose the name villin, has a polypeptide molecular weight of approximately 95,000. Two arguments suggest that villin may be the microvillus crossfilament protein that links the microfilament core laterally down its length to the cytoplasmic side of the plasma membrane. First, electron microscopy shows that crossfilaments stay attached to isolated membrane-free microvillus cores. Calculation of the expected abundance of the crossfilament protein shows that only villin is present in sufficient quantity to account for these structures. Second, decoration of microvillus cores by antibodies to either actin or villin, followed by ferritin-labeled second antibody in a sandwich procedure, results in specific labeling of the cores in both cases. The antivillin decoration, however, gives rise to a greater increase in diameter, in agreement with a model in which villin projects from the F-actin microfilament core. Villin is distinct from alpha-actinin, a protein suggested to be involved in membrane anchorage of microfilaments in nonmuscle cells. The two proteins differ in molecular weight. Specific antibodies against villin and alpha-actinin show no immunological crossreactivity. Immunofluorescence microscopy reveals that villin is located in the microvilli of the brush border whereas alpha-actinin is absent from the microvilli but is found in the terminal web. In addition, villin is not found in microfilament bundles of tissue culture cells, which are rich in alpha-actinin. Thus, villin and alpha-actinin appear to be immunologically and functionally different proteins.

Amphibian intestinal brush border membranes—I. Isolation from Rana catesbeiana tadpole

1. 1. Intestinal brush border membrane vesicles have been isolated form Rana catesbeiana tadpole. 2. 2. Electron microscopy of brush border membrane vesicles demonstrates a fairly homogenous preparation of vesicles, some of them still containing electron dense material. 3. 3. The dense vesicles probably comprise both microvillus core and membrane. 4. 4. Negative staining of vesicles reveals the presence of knob-like structures (particles) covering the outer surface of the membrane. 5. 5. The membranous fraction is characterized by a high specific activity of alkaline phosphatase, trehalase, glucoamylase, maltase and γ-glutamyltranspeptidase.

Evidence of microfilament‐associated mitochondrial movement

The mitochondria in the lower Malpighian tubule of the insect Rhodnius prolixus can be stimulated by feeding in vivo and by 5-hydroxytryptamine in vitro, to move from a position below the cell cortex to one inside the apical microvilli. During and following their movement into the microvilli, the mitochondria are intimately associated with the microfilaments of the cell cortex and microvillar core bundle. Bridges approximately 14 nm in length and 4 nm in diameter are observed connecting the microvillar microfilaments to the outer mitochondrial membrane and microvillar plasma membrane. Depolymerization of all visible microtubules with colchicine does not inhibit 5-HT-stimulated mitochondrial movement. On the other hand, treatment with cytochalasin B does block mitochondrial movement, suggesting that microfilaments play a role in the mitochondrial motility. We have labeled the microvillar microfilaments, which are 6 nm in diameter, with heavy meromyosin, which supports the contention that they contain actin. A model of the mechanism of mitochondrial movement is presented in which mitochondria slide into position in the microvilli along actin-containing microfilaments in a manner analogous to the sliding actin-myosin model of skeletal muscle.

Purification of microvilli and an analysis of the protein components of the microfilament core bundle

A fast and convenient method for the purification of microvilli from chicken intestinal brush borders is described. The microvilli appear morphologically very similar to those found on intact brush borders. Removal of the microvillus membrane from the microvilli by Triton X-100 treatment reveals compact bundles of microfilaments with small regularly spaced projections along their length. SDS-polyacrylamide gel analysis of the protein components of the brush border, the microvilli and the microvillus core bundles shows that little or no tropomyosin, myosin or filamin is found in the microvillus, whereas polypeptide chains with mobilities characteristic for these proteins are present in the whole brush border. The majority of the microvillus core protein is actin, and the other major protein present has a polypeptide molecular weight of 95 000. Total actin from both brush borders and microvilli, characterized by isoelectric focussing analysis, contained about 40% β actin and 60% γ actin. The presence of both the β and γ cytoplasmic actins in the highly ordered parallel arrays of microfilaments of the microvilli is discussed in light of hypotheses for different functional roles of these two actin species.

Biochemical differentiation of the plasma membrane of the intestinal epithelial cell.

The brush border and basolateral regions of the surface of the intestinal epithelial cell (enterocyte) are exposed to environments which differ even more than those which bathe the different functional regions of the surface of the hepatocyte or the renal tubular cell, and therefore the biochemical differentiation of the enterocyte plasma membrane is also more striking. In addition, it has been easier to study, since the highly adapted brush-border membrane of the enterocyte is one of the easiest of plasma membranes to isolate pure and in quantity (see Isselbacher, 1974). From the study of brush-border preparations a picture has emerged of a membrane with a variety of terminal digestive hydrolases (especially aminopeptidase, disaccharidases and alkaline phosphatase) attached to its luminal surface (Louvard et al., 1975). These are large proteins (Maestracci et al., 1975), which are heavily glycosylated (Kelly & Alpers, 1973; Weiser, 1973; Quaroni et al., 1974) and are moored to the membrane by relatively small hydrophobic polypeptide segments, probably at their N-terminal ends (Maroux & Louvard, 1976). Some of the end-products of digestion that these enzymes generate are then passed across the membrane by transport systems which are closely coupled to the hydrolases and are Na+-independent (Ramaswamy et al., 1976). Some enter the cell via one of several electrogenically driven Na+/solute symport systems (Crane, 1965,1974; Schultz & Curran, 1970; Berger et a[., 1972; Murer & Hopfer, 1974; Hopfer et al., 1973, 1976; Sigrist-Nelson et al., 1976; Alvarado, 1976) and others are taken into the cell by specific transport systems that are independent of Na+ (e.g. fructose; Sigrist-Nelson & Hopfer, 1974). At the cytoplasmic surface of the brush-border membrane, and attached to it by aactinin bridges, is an actin array which forms the microvillus core that also interacts with myosin-like proteins in the terminal web (Mooseker & Tilney, 1975). This raises the exciting, but still largely unexplored, possibility that the brush border is motile (Thuneberg & Rostegaard, 1969; Sandstrom, 1971; Mooseker, 1974) and that this somehow facilitates its digestive and absorptive function, perhaps by preventing the formation of unstirred layers of fluids at either surface of the brush-border membrane. The remainder of the enterocyte plasma membrane (the basolateral region) is separated from the brush border by the terminal bar. Thus, in contrast with the brush border, it interacts with the protective and controlled internal environment of the body and might be expected to be much more similar to the plasma membranes of other cells. It is this region of the plasma membrane which plays the key role in such processes as generating * Present address: Department of Biochemistry, University of Bristol, Medical School, Bristol BS8 1TD. U.K.

Intestinal Epithelial Cell Surface Membrane Glycoprotein Synthesis: I. AN INDICATOR OF CELLULAR DIFFERENTIATION

Abstract Cell surface glycoproteins, which have been implicated in the control and expression of cell turnover, differentiation, and malignant transformation in tissue culture studies, were examined for a possible similar role in the intestinal epithelial cell. This cell is characterized by a rapid cell turnover and by a gradient of differentiation from crypt to villus. A method was developed which isolated epithelial cells from different levels of crypt and villus areas and when radioactive labeled d-glucosamine, l-fucose, or d-galactosamine were given intraperitoneally into a rat, a sharp gradient of radioactive incorporation paralleled the specific activity gradients of sucrase and alkaline phosphatase. Isolated epithelial cells incubated with d-[1-14C]glucosamine also showed that the upper villus cells had a much higher incorporation of d-[1-14C]glucosamine than did crypt cells. Subcellular fractionation of the intestinal cells labeled in vivo demonstrated that the highest specific activity was in the purified microvillus plasma membranes and the lowest specific activity was in the microsomal and cytosol fractions. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of microvillus membranes showed peaks of d-[1-14C]glucosamine incorporation which coincided with peaks of sucrase and alkaline phosphatase activities and most of the bands which stained for protein also stained for glycoprotein. Microvillus core proteins separated on sodium dodecyl sulfate polyacrylamide did not show peaks of d-[1-14C]glucosamine incorporation and did not stain for glycoprotein. These results suggest that the more differentiated upper villus cells incorporate more labeled sugar precursors into membrane glycoproteins than the less differentiated lower villus or crypt cells and that these membrane glycoproteins are associated with membrane-bound enzymes.

Sodium-dependent binding of D-glucose to a filamentous fraction of Tris-disrupted brush borders from hamster jejunum

Tris-disrupted brush borders prepared from hamster jejunum were fractionated by Ficoll density gradient centrifugation. Preferential D-glucose binding to a filamentous, microvillus core fraction was dependent upon the Na + concentration in the incubation medium. No binding occurred when K +, Li +, NH 4 +, or choline was substituted for Na +. It has been postulated that microvillus core filaments are intimately involved in the mechanism of active sugar as well as amino acid transport by the small intestine.

Electron microscopical observations on the brush border of proximal tubule cells of mammalian kidney.

The ultrastructure of the plasma membrane and the core of microvilli of proximal tubule cells has been investigated by electron microscopy using sectioned and negatively stained material. By the technique of negative staining, a particulated coat is disclosed on the outside of the plasma membrane of microvilli of brush borders isolated from rat, rabbit and ox. This coat is composed of 30 to 60 A particles and is 150 to 300 A thick and appears to be a distinguishing feature for the luminal plasma membrane (brush border) of proximal tubule cells. The plasma membrane of the basal part of tubule cells is found to be smooth. By thin sectioning, an axial bundle of 50 to 70 A diameter filaments regularly arranged in an “1+6 configuration”, one axially located filament being surrounded by a ring of six, is disclosed. The distance from the ring of filaments to the inner surface of the plasma membrane is 250–300 A, the diameter of the ring 300 A and the center-to-center distance between filaments 120 A. Negative staining also discloses 60 A filaments in microvilli of isolated brush borders. Broken off, single microvilli (fingerstalls) are observed with thin filaments projecting from their broken ends. Filaments up to 1 μ in length are seen. At high magnification, the filaments appear beaded and show strong resemblance with actin filaments isolated from skeletal muscle. Based on present evidence, it is postulated that microvilli constituting renal brush borders possess contractile properties, which may play a role in the absorption process operating at the luminal part of the cells.

The fine structure of the hepatopancreas of the woodlouse, Oniscus ascellus.

The present ultrastructural study of the hepatopancreas of Oniscus ascellus would seem to indicate that the entire gland functions as a true midgut and is involved in absorption and secretion. Two differentiated cell types, the S and B, are present throughout the gland except for a small area of the posterior or regenerative zone. Ultrastructural features that have been associated with absorption include the presence of a surface enteric coat, regularity of microvillar core filaments, an organelle free region under the microvilli and the uniform distribution of mitochondria below this area. These features are present in both the S and B cell types. Features that are typical of secretory cells include the presence of extensive arrays of parallel rough endoplasmic reticulum, the very active dictyosomes with dilated cisternae and large secretory bodies. These features are present in the B cell. Although occasional arrays of parallel endoplasmic reticulum were noted in the S cell, active dictyosomes with dilated cisternae were not evident. From this ultrastructural study it would appear that the B cell functions in absorption and secretion, while the S cell functions primarily in absorption. A cytochemical study is now in progress.