Characterization of brush borders purified in iso-osmotic medium and microvillar membranes subfractionated from mouse small intestine.
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ABSTRACT:
SUBMITTER: Fujita M
PROVIDER: S-EPMC1163084 | biostudies-other | 1981 Jun
REPOSITORIES: biostudies-other
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Project description:Transporting epithelial cells generate arrays of microvilli, known as a brush border, to enhance functional capacity. To understand brush border formation, we used live cell imaging to visualize apical remodeling early in this process. Strikingly, we found that individual microvilli exhibit persistent active motility, translocating across the cell surface at ∼0.2 μm/min. Perturbation with inhibitors and photokinetic experiments revealed that microvillar motility is driven by actin assembly at the barbed ends of core bundles, which in turn is linked to robust treadmilling of these structures. Actin regulatory factors IRTKS and EPS8 localize to the barbed ends of motile microvilli, where they control the kinetics and nature of movement. As the apical surface of differentiating epithelial cells is crowded with nascent microvilli, persistent motility promotes collisions between protrusions and ultimately clustering and consolidation into higher-order arrays. Thus, microvillar motility represents a previously unrecognized driving force for apical surface remodeling and maturation during epithelial differentiation.
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Project description:BackgroundBrush border microvilli are approximately 1-microm long finger-like projections emanating from the apical surfaces of certain, specialized absorptive epithelial cells. A highly symmetric hexagonal array of thousands of these uniformly sized structures form the brush border, which in addition to aiding in nutrient absorption also defends the large surface area against pathogens. Here, we present a molecular model of the protein cytoskeleton responsible for this dramatic cellular morphology.Methodology/principal findingsThe model is constructed from published crystallographic and microscopic structures reported by several groups over the last 30+ years. Our efforts resulted in a single, unique, self-consistent arrangement of actin, fimbrin, villin, brush border myosin (Myo1A), calmodulin, and brush border spectrin. The central actin core bundle that supports the microvillus is nearly saturated with fimbrin and villin cross-linkers and has a density similar to that found in protein crystals. The proposed model accounts for all major proteinaceous components, reproduces the experimentally determined stoichiometry, and is consistent with the size and morphology of the biological brush border membrane.Conclusions/significanceThe model presented here will serve as a structural framework to explain many of the dynamic cellular processes occurring over several time scales, such as protein diffusion, association, and turnover, lipid raft sorting, membrane deformation, cytoskeletal-membrane interactions, and even effacement of the brush border by invading pathogens. In addition, this model provides a structural basis for evaluating the equilibrium processes that result in the uniform size and structure of the highly dynamic microvilli.
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Project description:Transporting epithelial cells of the gut and kidney interact with their luminal environment through a densely packed collection of apical microvilli known as a brush border (BB). Proper brush border assembly depends on the intermicrovillar adhesion complex (IMAC), a protocadherin-based adhesion complex found at the distal tips of microvilli that mediates adhesion between neighboring protrusions to promote their organized packing. Loss of the IMAC adhesion molecule Cadherin-related family member 5 (CDHR5) results in significant brush border defects, though the functional properties of this protocadherin have not been thoroughly explored. Here, we show that the cytoplasmic tail of CDHR5 contributes to its correct apical targeting and functional properties in an isoform-specific manner. Library screening identified the Ezrin-associated scaffolds EBP50 and E3KARP as cytoplasmic binding partners for CDHR5. Consistent with this, loss of EBP50 disrupted proper brush border assembly with cells exhibiting markedly reduced apical IMAC levels. Together, our results shed light on the apical targeting determinants of CDHR5 and further define the interactome of the IMAC involved in brush border assembly.
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Project description:Rabbit intestinal microvillus membranes possess high-affinity receptors for iron whose activity reflects homeostatic changes in mucosal iron transport. To isolate and characterize these membrane components, purified microvilli were radiolabelled with 59Fe(II) and solubilized in Triton X-100. 59Fe in 105000g supernatants co-eluted with a major broad protein peak (Mr approx. 100000) on gel-permeation chromatography and was rendered diffusible by Pronase digestion but not mild periodate degradation. Fluorescence studies with castor-bean lectin conjugates showed specific binding of this affinity probe exclusively to brush-border membranes in the intestinal epithelium. Affinity chromatography of solubilized membrane proteins showed binding to columns of immobilized lectin. Elution with D(+)-galactose released glycoprotein-bound 59Fe purified up to sevenfold over initial membrane extracts. The lectin bound up to 82% of protein-bound 59Fe. In contrast polyspecific antisera raised against rabbit microvilli in guinea-pigs precipitated less than 10% of solubilized radioactivity. Significantly more protein-bound 59Fe in detergent extracts of microvilli purified from bled animals interacted specifically with the lectin, suggesting that membrane glycoprotein receptors are involved in the homeostatic control of intestinal iron transport.
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Project description:Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen 3H-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given 3H-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given 3H-cholesterol in mice, we found that lumen-to-BBM 3H-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux to dispose of endogenous cholesterol efficiently for therapeutic purposes.
| S-EPMC4811413 | biostudies-literature
Project description:Ca2+ uptake in brush-border vesicles isolated from rat duodena was studied by a rapid-filtration technique. Ca2+ uptake showed saturation kinetics, was dependent on the pH and ionic strength of the medium and was independent of metabolic energy. Uptake activity was readily inhibited by Ruthenium Red, La3+, tetracaine, EGTA, choline chloride and Na+ or K+. The effect of variations in medium osmolarity on Ca2+ uptake and the ionophore A23187-induced efflux of the cation from preloaded vesicles indicated that the Ca2+-uptake process involved binding to membrane components, as well as transport into an osmotically active space. Scatchard-plot analyses of the binding data suggested at least two classes of Ca2+-binding sites. The high-affinity sites, Ka = (2.7 +/- 1.1) x 10(4) M-1 (mean +/- S.D.) bound 3.2 +/- 0.8 nmol of Ca2+/mg of protein, whereas the low-affinity sites (Ka = 60 +/- 6 M-1) bound 110 +/- 17 nmol of Ca2+/mg of protein. In the presence of 100 mM-NaCl, 1.7 and 53 nmol of Ca2+/mg of protein were bound to the high- and low-affinity sites respectively. Decreased Ca2+-uptake activity was observed in vesicles isolated from vitamin D-deficient as compared with vitamin D-replete animals and intraperitoneal administration of 1,25-dihydroxycholecalciferol to vitamin D-deficient rats 16 h before membrane isolation stimulated the initial rate of Ca2+ uptake significantly. The data indicated that Ca2+ entry and/or binding was passive and may involve a carrier-mediated Ca2+-uptake component that is associated with the brush-border membrane. Altering the electrochemical potential difference across the membrane by using anions of various permeability and selected ionophores appeared to increase primarily binding to the membrane rather than transport into the intravesicular space. Since there is considerable binding of Ca2+ to the vesicle interior, a comprehensive analysis of the transport properties of the brush-border membrane remains difficult at present.
| S-EPMC1163010 | biostudies-other
Project description:The epithelial brush border (BB) Na(+)/H(+) exchanger NHE3 is associated with the actin cytoskeleton by binding both directly and indirectly to ezrin; indirect binding is via attachment to NHERF family proteins. NHE3 mobility in polarized epithelial cell BBs is restricted by the actin cytoskeleton and NHERF binding such that only approximately 30% of NHE3 in the apical domain of an OK cell line stably expressing NHERF2 is mobile, as judged by FRAP analysis. Given that levels of NHE3 are partially regulated by changes in trafficking, we investigated whether the cytoskeleton association of NHE3 was dynamic and changed as part of acute regulation to allow NHE3 trafficking. The agonist studied was lysophosphatidic acid (LPA), an inflammatory mediator, which acutely stimulates NHE3 activity by increasing the amount of NHE3 on the BBs by stimulated exocytosis. LPA acutely stimulated NHE3 activity in OK cells stably expressing NHERF2. Two conditions that totally prevented LPA stimulation of NHE3 activity only partially prevented stimulation of NHE3 mobility: the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the NHE3F1 double mutant which has minimal direct binding of NHE3 to ezrin. These results show that LPA stimulation of NHE3 mobility occurs in two parts: (1) PI3K-dependent exocytic trafficking to the BB and (2) an increase in surface mobility of NHE3 in BBs under basal conditions. Moreover, the LPA stimulatory effect on NHE3 mobility required NHERF2. Although NHE3 and NHERF2 co-precipitated under basal conditions, they failed to co-precipitate 30 minutes after addition of LPA, whereas the physical association was re-established by 50-60 minutes. This dynamic interaction between NHERF2 and NHE3 was confirmed by acceptor photobleaching Förster Resonance energy Transfer (FRET). The restricted mobility of NHE3 in BBs under basal conditions as a result of cytoskeleton association is therefore dynamic and is reversed as part of acute LPA stimulation of NHE3. We suggest that this acute but transient increase in NHE3 mobility induced by LPA occurs via two processes: addition of NHE3 to the BB by exocytosis, a process which precedes binding of NHE3 to the actin cytoskeleton via NHERF2-ezrin, and by release of NHERF2 from the NHE3 already localized in the apical membrane, enabling NHE3 to distribute throughout the microvilli. These fractions of NHE3 make up a newly identified pool of NHE3 called the 'transit pool'. Moreover, our results show that there are two aspects of LPA signaling involved in stimulation of NHE3 activity: PI3K-dependent stimulated NHE3 exocytosis and the newly described, PI3K-independent dissociation of microvillar NHE3 from NHERF2.
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Project description:The hydrolysis of NAD by rat intestine was studied to determine the subcellular site of this hydrolysis and to identify the niacin-containing products that are formed. Using [nicotinamide-14C]NAD as substrate, and high pressure liquid chromatography for identification and quantification of products, the present study demonstrates two independent reactions for the hydrolysis of NAD; one that forms nicotinamide through hydrolysis of the ribosyl-pyridinium bond and one that forms nicotinamide mononucleotide through the hydrolysis of the pyrophosphate bond. The nicotinamide mononucleotide is subsequently dephosphorylated to nicotinamide riboside. Enzymes which release nicotinamide mononucleotide and nicotinamide riboside are associated with the brush border membrane as determined by analysis of fractionated intestinal homogenates. The enzyme activity which releases nicotinamide from NAD is associated with the brush border membrane fraction and also with a second cellular particulate fraction. Between pH5 and pH6 NAD is hydrolysed principally to nicotinamide. At pH 7.0 rates of nicotinamide and nicotinamide mononucleotide formation are the same. Above pH 7.0 the formation of nicotinamide mononucleotide is preferred.
| S-EPMC1158334 | biostudies-other
Project description:1. Brush borders were isolated from rabbit kidney-cortex homogenates by rate-zonal centrifugation through a sucrose density gradient in a B-XIV zonal rotor, followed by differential centrifugation. 2. The method of preparation gave brush borders of high purity with a reasonable yield. The morphological appearance supported the evidence from enzymic and chemical investigations, that the brush borders were only slightly contaminated with endoplasmic reticulum, mitochondria, lysosomes and nuclei. 3. The molar ratio of cholesterol to phospholipid lay within the range found in other plasma membranes, but the carbohydrate content was double that found in liver plasma membranes. 4. Alkaline phosphatase, maltase, trehalase and aminopeptidase were major enzymic constituents of the brush borders, and had an approximately equal yield and enrichment, but none of these enzymes fulfilled the criteria for marker enzymes. 5. Mg(2+)-dependent and Na(+),K(+)-dependent adenosine triphosphatases, although found in brush borders, had low yields and low enrichments.
| S-EPMC1174020 | biostudies-other
Project description:Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.
| S-EPMC1164262 | biostudies-other