Generation of Stable Lipid Raft Microdomains in the Enterocyte Brush Border by Selective Endocytic Removal of Non-Raft Membrane

The small intestinal brush border has an unusually high proportion of glycolipids which promote the formation of lipid raft microdomains, stabilized by various cross-linking lectins. This unique membrane organization acts to provide physical and chemical stability to the membrane that faces multiple deleterious agents present in the gut lumen, such as bile salts, digestive enzymes of the pancreas, and a plethora of pathogens. In the present work, we studied the constitutive endocytosis from the brush border of cultured jejunal explants of the pig, and the results indicate that this process functions to enrich the contents of lipid raft components in the brush border. The lipophilic fluorescent marker FM, taken up into early endosomes in the terminal web region (TWEEs), was absent from detergent resistant membranes (DRMs), implying an association with non-raft membrane. Furthermore, neither major lipid raft-associated brush border enzymes nor glycolipids were detected by immunofluorescence microscopy in subapical punctae resembling TWEEs. Finally, two model raft lipids, BODIPY-lactosylceramide and BODIPY-GM₁, were not endocytosed except when cholera toxin subunit B (CTB) was present. In conclusion, we propose that constitutive, selective endocytic removal of non-raft membrane acts as a sorting mechanism to enrich the brush border contents of lipid raft components, such as glycolipids and the major digestive enzymes. This sorting may be energetically driven by changes in membrane curvature when molecules move from a microvillar surface to an endocytic invagination.     

Lipid rafts in epithelial brush borders: atypical membrane microdomains with specialized functions

Epithelial cells that fulfil high-throughput digestive/absorptive functions, such as small intestinal enterocytes and kidney proximal tubule cells, are endowed with a dense apical brush border. It has long been recognized that the microvillar surface of the brush border is organized in cholesterol/sphingolipid-enriched membrane microdomains commonly known as lipid rafts. More recent studies indicate that microvillar rafts, in particular those of enterocytes, have some unusual properties in comparison with rafts present on the surface of other cell types. Thus, microvillar rafts are stable rather than transient/dynamic, and their core components include glycolipids and the divalent lectin galectin-4, which together can be isolated as "superrafts", i.e., membrane microdomains resisting solubilization with Triton X-100 at physiological temperature. These glycolipid/lectin-based rafts serve as platforms for recruitment of GPI-linked and transmembrane digestive enzymes, most likely as an economizing effort to secure and prolong their digestive capability at the microvillar surface. However, in addition to microvilli, the brush border surface also consists of membrane invaginations between adjacent microvilli, which are the only part of the apical surface sterically accessible for membrane fusion/budding events. Many of these invaginations appear as pleiomorphic, deep apical tubules that extend up to 0.5-1 microm into the underlying terminal web region. Their sensitivity to methyl-beta-cyclodextrin suggests them to contain cholesterol-dependent lipid rafts of a different type from the glycolipid-based rafts at the microvillar surface. The brush border is thus an example of a complex membrane system that harbours at least two different types of lipid raft microdomains, each suited to fulfil specialized functions. This conclusion is in line with an emerging, more varied view of lipid rafts being pluripotent microdomains capable of adapting in size, shape, and content to specific cellular functions.     

Intelectin: a novel lipid raft-associated protein in the enterocyte brush border

Intelectin is a mammalian Ca2+-dependent, D-galactosyl-specific lectin expressed in Paneth and goblet cells of the small intestine and proposed to serve a protective role in the innate immune response to parasite infection. In addition, it is structurally identical to the intestinal lactoferrin receptor known to reside in the enterocyte brush border. To clarify this apparent discrepancy with regard to localization, the aim of this work was to study the cellular and subcellular distribution of small intestinal intelectin by immunofluorescence and immunogold electron microscopy. Secretory granules of lysozyme-positive Paneth cells in the bottom of the crypts as well as goblet cells along the crypt-villus axis were intensively labeled with intelectin antibodies, but quantitatively, the major site of intelectin deposition was the enterocyte brush border. This membrane is organized in stable glycolipid-based lipid raft microdomains, and like the divalent lectin galectin-4, intelectin was enriched in microvillar "superrafts", i.e., membranes that resist solubilization with Triton X-100 at 37 degrees C. This strategic localization suggests that the trimeric intelectin, like galectin-4, serves as an organizer and stabilizer of the brush border membrane, preventing loss of digestive enzymes to the gut lumen and protecting the glycolipid microdomains from pathogens.     

Lipid rafts exist as stable cholesterol-independent microdomains in the brush border membrane of enterocytes

Glycosphingolipid/cholesterol-rich membranes ("rafts")can be isolated from many types of cells, but their existence as stable microdomains in the cell membrane has been elusive. Addressing this problem, we studied the distribution of galectin-4, a raft marker, and lactase, a protein excluded from rafts, on microvillar vesicles from the enterocyte brush border membrane. Magnetic beads coated with either anti-galectin-4 or anti-lactase antibodies were used for immunoisolation of vesicles followed by double immunogold labeling of the two proteins. A morphometric analysis revealed subpopulations of raft-rich and raft-poor vesicles by the following criteria: 1) the lactase/galectin-4 labeling ratio/vesicle captured by the anti-lactase beads was significantly higher (p < or = 0.01) than that of vesicles captured by anti-galectin-4 beads, 2) subpopulations of vesicles labeled by only one of the two antibodies were preferentially captured by beads coated with the respective antibody (p < or = 0.01), 3) the average diameter of "galectin-4 positive only" vesicles was smaller than that of vesicles labeled for lactase. Surprisingly, pretreatment with methyl-beta-cyclodextrin, which removed >70% of microvillar cholesterol, did not affect the microdomain localization of galectin-4. We conclude that stable, cholesterol-independent raft microdomains exist in the enterocyte brush border.     

Lipid raft organization and function in the small intestinal brush border

The enterocyte brush border of the small intestine is a highly specialized membrane designed to function both as a high capacity digestive/absorptive surface of dietary nutrients and a permeability barrier towards lumenal pathogens. It is characterized by an unusually high content of glycolipids (∼30% of the total microvillar membrane lipid), enabling the formation of liquid ordered microdomains, better known as lipid rafts. The glycolipid rafts are stabilized by galectin-4, a 36 kDa divalent lectin that cross-links galactosyl (and other carbohydrate) residues present on membrane lipids and several brush border proteins, including some of the major hydrolases. These supramolecular complexes are further stabilized by intelectin, a 35 kDa trimeric lectin that also functions as an intestinal lactoferrin receptor. As a result, brush border hydrolases, otherwise sensitive to pancreatic proteinases, are protected from untimely release into the gut lumen. Finally, anti-glycosyl antibodies, synthesized by plasma cells locally in the gut, are deposited on the brush border glycolipid rafts, protecting the epithelium from lumenal pathogens that exploit lipid rafts as portals for entry to the organism.     

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

Abstract

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.     

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.     

Microvillar membrane microdomains exist at physiological temperature. Role of galectin-4 as lipid raft stabilizer revealed by "superrafts"

Lipid rafts (glycosphingolipid/cholesterol-enriched membrane microdomains) have been isolated as low temperature, detergent-resistant membranes from many cell types, but despite their presumed importance as lateral sorting and signaling platforms, fundamental questions persist concerning raft function and even existence in vivo. The nonionic detergent Brij 98 was used to isolate lipid rafts from microvillar membrane vesicles of intestinal brush borders at physiological temperature to compare with rafts, obtained by "conventional" extraction using Triton X-100 at low temperature. Microvillar rafts prepared by the two protocols were morphologically different but had essentially similar profiles of protein- and lipid components, showing that raft microdomains do exist at 37 degrees C and are not "low temperature artifacts." We also employed a novel method of sequential detergent extraction at increasing temperature to define a fraction of highly detergent-resistant "superrafts." These were enriched in galectin-4, a beta-galactoside-recognizing lectin residing on the extracellular side of the membrane. Superrafts also harbored the glycosylphosphatidylinositol-linked alkaline phosphatase and the transmembrane aminopeptidase N, whereas the peripheral lipid raft protein annexin 2 was essentially absent. In conclusion, in the microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other membrane microdomain systems.     

Lipid raft organization and function in brush borders of epithelial cells

Polarized epithelial cells of multicellular organisms confront the environment with a highly specialized apical cell membrane that differs in composition and function from that facing the internal milieu. In the case of absorptive cells, such as the small intestinal enterocyte and the kidney proximal tubule cell, the apical cell membrane is formed as a brush border, composed of regular, dense arrays of microvilli. Hydrolytic ectoenzymes make up the bulk of the microvillar membrane proteins, endowing the brush border with a huge digestive capacity. Several of the major enzymes are localized in lipid rafts, which, for the enterocyte in particular, are organized in a unique fashion. Glycolipids, rather than cholesterol, together with the divalent lectin galectin-4, define these rafts, which are stable and probably quite large. The architecture of these rafts supports a digestive/absorptive strategy for nutrient assimilation, but also serves as a portal for a large number of pathogens. Caveolae are well-known vehicles for internalization of lipid rafts, but in the enterocyte brush border, binding of cholera toxin is followed by uptake via a clathrin-dependent mechanism. Recently, 'anti-glycosyl' antibodies were shown to be deposited in the enterocyte brush border. When the antibodies were removed from the membrane, other carbohydrate-binding proteins, including cholera toxin, increased their binding to the brush border. Thus, anti-glycosyl antibodies may serve as guardians of glycolipid-based rafts, protecting them from lumenal pathogens and in this way be part of an ongoing 'cross-talk' between indigenous bacteria and the host.     

Cholera toxin entry into pig enterocytes occurs via a lipid raft- and clathrin-dependent mechanism

The small intestinal brush border is composed of lipid raft microdomains, but little is known about their role in the mechanism whereby cholera toxin gains entry into the enterocyte. The present work characterized the binding of cholera toxin B subunit (CTB) to the brush border and its internalization. CTB binding and endocytosis were performed in organ-cultured pig mucosal explants and studied by fluorescence microscopy, immunogold electron microscopy, and biochemical fractionation. By fluorescence microscopy CTB, bound to the microvillar membrane at 4 degrees C, was rapidly internalized after the temperature was raised to 37 degrees C. By immunogold electron microscopy CTB was seen within 5 min at 37 degrees C to induce the formation of numerous clathrin-coated pits and vesicles between adjacent microvilli and to appear in an endosomal subapical compartment. A marked shortening of the microvilli accompanied the toxin internalization whereas no formation of caveolae was observed. CTB was strongly associated with the buoyant, detergent-insoluble fraction of microvillar membranes. Neither CTB's raft association nor uptake via clathrin-coated pits was affected by methyl-beta-cyclodextrin, indicating that membrane cholesterol is not required for toxin binding and entry. The ganglioside GM(1) is known as the receptor for CTB, but surprisingly the toxin also bound to sucrase-isomaltase and coclustered with this glycosidase in apical membrane pits. CTB binds to lipid rafts of the brush border and is internalized by a cholesterol-independent but clathrin-dependent endocytosis. In addition to GM(1), sucrase-isomaltase may act as a receptor for CTB.     

Anti-glycosyl antibodies in lipid rafts of the enterocyte brush border: a possible host defense against pathogens

The pig small intestinal brush border is a glycoprotein- and glycolipid-rich membrane that functions as a digestive/absorptive surface for dietary nutrients as well as a permeability barrier for pathogens. The present work was performed to identify carbohydrate-binding (lectinlike) proteins associated with the brush border. Chromatography on lactose-agarose was used to isolate such proteins, and their localization was studied biochemically and by immunofluorescence microscopy and immunogold electron microscopy. IgG and IgM were the two major proteins isolated, indicating that naturally occurring anti-glycosyl antibodies are among the major lectinlike proteins in the gut. IgG and IgM as well as IgA were localized to the enterocyte brush border, and a brief lactose wash partially released all three immunoglobulins from the membrane, indicating that anti-glycosyl antibodies constitute a major part of the immunoglobulins at the lumenal surface of the gut. The antibodies were associated with lipid rafts at the brush border, and they frequently (52%) coclustered with the raft marker galectin 4. A lactose wash increased the susceptibility of the brush border toward lectin peanut agglutin and cholera toxin B, suggesting that anti-glycosyl antibodies compete with other carbohydrate-binding proteins at the lumenal surface of the gut. Thus anti-glycosyl antibodies constitute a major group of proteins associated with the enterocyte brush border membrane. We propose they function by protecting the lipid raft microdomains of the brush border against pathogens.     

Apolipoprotein A-1 (apoA-1) deposition in, and release from, the enterocyte brush border: a possible role in transintestinal cholesterol efflux (TICE)?

Transintestinal cholesterol efflux (TICE) has been proposed to represent a non-hepatobiliary route of cholesterol secretion directly "from blood to gut" and to play a physiologically significant role in excretion of neutral sterols, but so far little is known about the proteins involved in the process. We have previously observed that apolipoprotein A-1 (apoA-1) synthesized by enterocytes of the small intestine is mainly secreted apically into the gut lumen during fasting where its assembly into chylomicrons and basolateral discharge is at a minimal level. In the present work we showed, both by immunomicroscopy and subcellular fractionation, that a fraction of the apically secreted apoA-1 in porcine small intestine was not released from the cell surface but instead deposited in the brush border. Cholesterol was detected in immunoisolated microvillar apoA-1, and it was partially associated with detergent resistant membranes (DRMs), indicative of localization in lipid raft microdomains. The apolipoprotein was not readily released from microvillar vesicles by high salt or by incubation with phosphatidylcholine-specific phospholipase C or trypsin, indicating a relatively firm attachment to the membrane bilayer. However, whole bile or taurocholate efficiently released apoA-1 at low concentrations that did not solubilize the transmembrane microvillar protein aminopeptidase N. Based on these findings and the well known role played by apoA-1 in extrahepatic cellular cholesterol removal and reverse cholesterol transport (RCT), we propose that brush border-deposited apoA-1 in the small intestine acts in TICE by mediating cholesterol efflux into the gut lumen.     

Environmental cues and symbiont microbe‐associated molecular patterns function in concert to drive the daily remodelling of the crypt‐cell brush border of the Euprymna scolopes light organ

Recent research has shown that the microbiota affects the biology of associated host epithelial tissues, including their circadian rhythms, although few data are available on how such influences shape the microarchitecture of the brush border. The squid‐vibrio system exhibits two modifications of the brush border that supports the symbionts: effacement and repolarization. Together these occur on a daily rhythm in adult animals, at the dawn expulsion of symbionts into the environment, and symbiont colonization of the juvenile host induces an increase in microvillar density. Here we sought to define how these processes are related and the roles of both symbiont colonization and environmental cues. Ultrastructural analyses showed that the juvenile‐organ brush borders also efface concomitantly with daily dawn‐cued expulsion of symbionts. Manipulation of the environmental light cue and juvenile symbiotic state demonstrated that this behaviour requires the light cue, but not colonization. In contrast, symbionts were required for the observed increase in microvillar density that accompanies post dawn brush‐border repolarization; this increase was induced solely by host exposure to phosphorylated lipid A of symbiont cells. These data demonstrate that a partnering of environmental and symbiont cues shapes the brush border and that microbe‐associated molecular patterns play a role in the regulation of brush‐border microarchitecture.     

Myosin-1A targets to microvilli using multiple membrane binding motifs in the tail homology 1 (TH1) domain

One of the most abundant components of the enterocyte brush border is the actin-based monomeric motor, myosin-1a (Myo1a). Within brush border microvilli, Myo1a carries out a number of critical functions at the interface between membrane and actin cytoskeleton. Proper physiological function of Myo1a depends on its ability to bind to microvillar membrane, an interaction mediated by a C-terminal tail homology 1 (TH1) domain. However, little is known about the mechanistic details of the Myo1a-TH1/membrane interaction. Structure-function analysis of Myo1a-TH1 targeting in epithelial cells revealed that an N-terminal motif conserved among class I myosins and a C-terminal motif unique to Myo1a-TH1 are both required for steady state microvillar enrichment. Purified Myo1a bound to liposomes composed of phosphatidylserine and phosphoinositol 4,5-bisphosphate, with moderate affinity in a charge-dependent manner. Additionally, peptides of the N- and C-terminal regions required for targeting were able to compete with Myo1a for binding to highly charged liposomes in vitro. Single molecule total internal reflection fluorescence microscopy showed that these motifs are also necessary for slowing the membrane detachment rate in cells. Finally, Myo1a-TH1 co-localized with both lactadherin-C2 (a phosphatidylserine-binding protein) and PLCδ1-PH (a phosphoinositol 4,5-bisphosphate-binding protein) in microvilli, but only lactaderin-C2 expression reduced brush border targeting of Myo1a-TH1. Together, our results suggest that Myo1a targeting to microvilli is driven by membrane binding potential that is distributed throughout TH1 rather than localized to a single motif. These data highlight the diversity of mechanisms that enable different class I myosins to target membranes in distinct biological contexts.     

Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells

A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and brush border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveal no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure.     

Binding and cross-linking properties of galectins

The galectins are a family of animal lectins that possess similar carbohydrate binding specificities and conserved consensus sequences. The biological properties of mammalian galectins include the regulation of inflammation, cell adhesion, cell proliferation and cell death. Evidence suggests that the biological activities of the galectins are related to their multivalent binding properties since most galectins possess two carbohydrate recognition domains and are therefore bivalent. For example, galectin-1, which is dimeric, binds and cross-links specific glycoprotein counter-receptors on the surface of human T-cells leading to apoptosis [J. Immunol. 163 (1999) 3801]. Different galectin-1 counter-receptors associated with specific phosphatase or kinase activities formed separate clusters on the surface of the cells as a result of the lectin binding to the carbohydrate chains of the respective glycoproteins. Importantly, monovalent galectin-1 is inactive in this system. This indicates that the separation and organization of signaling molecules that result from galectin-1 binding is involved in the apoptotic signal. The separation of specific glycoprotein receptors induced by galectin-1 binding was modeled on the basis of molecular and structural studies of the binding of lectins to multivalent carbohydrates resulting in the formation of specific two- and three-dimensional cross-linked lattices [Biochemistry 36 (1997) 15073]. In this article, the binding and cross-linking properties of galectin-1 and other lectins are reviewed as a model for the biological signal transduction properties of the galectin family of animal lectins.     

Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells.

A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveals no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure.     

Lattices,rafts, and scaffolds: domain regulation of receptor signaling at the plasma membrane

The plasma membrane is organized into various subdomains of clustered macromolecules. Such domains include adhesive structures (cellular synapses, substrate adhesions, and cell–cell junctions) and membrane invaginations (clathrin-coated pits and caveolae), as well as less well-defined domains such as lipid rafts and lectin-glycoprotein lattices. Domains are organized by specialized scaffold proteins including the intramembranous caveolins, which stabilize lipid raft domains, and the galectins, a family of animal lectins that cross-link glycoproteins forming molecular lattices. We review evidence that these heterogeneous microdomains interact to regulate substratum adhesion and cytokine receptor dynamics at the cell surface.     

The enterocyte microvillus is a vesicle-generating organelle

For decades, enterocyte brush border microvilli have been viewed as passive cytoskeletal scaffolds that serve to increase apical membrane surface area. However, recent studies revealed that in the in vitro context of isolated brush borders, myosin-1a (myo1a) powers the sliding of microvillar membrane along core actin bundles. This activity also leads to the shedding of small vesicles from microvillar tips, suggesting that microvilli may function as vesicle-generating organelles in vivo. In this study, we present data in support of this hypothesis, showing that enterocyte microvilli release unilamellar vesicles into the intestinal lumen; these vesicles retain the right side out orientation of microvillar membrane, contain catalytically active brush border enzymes, and are specifically enriched in intestinal alkaline phosphatase. Moreover, myo1a knockout mice demonstrate striking perturbations in vesicle production, clearly implicating this motor in the in vivo regulation of this novel activity. In combination, these data show that microvilli function as vesicle-generating organelles, which enable enterocytes to deploy catalytic activities into the intestinal lumen.     

Domains of increased thickness in microvillar membranes of the small intestinal enterocyte

Abstract

The apical surface of the enterocyte is sculpted into a dense array of cylindrical microvillar protrusions by supporting actin filaments. Membrane microdomains (rafts) enriched in cholesterol and glycosphingolipids comprise roughly 50% of the microvillar membrane and play a vital role in orchestrating absorptive/digestive action of dietary nutrients at this important cellular interface. Increased membrane thickness is believed to be a morphological characteristic of rafts. Thus, we investigated whether the high contents of lipid rafts in the microvillar membrane is reflected in local variations in membrane thickness. We measured membrane thickness directly from electron micrographs of sections of fixed mucosal tissue. Indeed, mapping of the microvillar membrane revealed a biphasic distribution of membrane thickness. As a point of reference the thickness distribution of the basolateral membrane was clearly monophasic. The encountered domains of increased thickness (DITs) occupied 48% of the microvillar membrane and from the data we estimated the area of a single DIT to have a lower limit of 600 nm². In other experiments we mapped the organization of biochemically defined lipid rafts by immunogold labeling of alkaline phosphatase, a well documented raft marker. Strikingly, the alkaline phosphatase localized to distinct regions of the membrane in a pattern similar to the observed distribution of DITs. Although we were unable to measure membrane thickness directly on the immunogold labeled specimens, and thereby establish an unequivocal connection between DITs and rafts, we conclude that the brush border membrane of the enterocyte contains microdomains distinguishable both by membrane morphology and protein composition.