Human blood group A-like determinants as marker of the intracellular pools of glycoproteins in secretory and absorbing of A+ rabbit jejunum

Human blood group A antigenicity of glycoproteins is retained on epon-embedded jejunum sections after glutaraldehyde fixation and osmium treatment. The intracellular location of molecules bearing these determinants was visualized in the four types of epithelial cells of A+ rabbit jejunum sections with immuno-colloidal gold labeling. The brush border membrane and in particular the glycocalyx of absorbing cells as well as the secretory granules of goblet and Paneth cells were heavily labeled. In enteroendocrine cells, the membrane of secretory granules and not their content was lightly labeled. The differential labeling of secretory or membrane bound glycoproteins is accompanied by different labels of the Golgi complex as expected if labeling of the Golgi saccules was due to the presence of glycoproteins in transit. In all cases the label is primarily concentrated in only half the cisternae on the trans side of the Golgi stacks. In absorbing cells, structures have been revealed in the terminal web that could be related to the brush border membrane and consequently implicated in its biogenesis. The fibrillar material of the glycocalyx appears as highly labeled tangled structures which apparently proceed from densely stained "carrier" vesicles arising from the Golgi apparatus. Vesicles fusing at the lower part of microvilli could result of integration of this material into the lightly labeled vesicles strictly found in the terminal web. These last vesicles could also contain newly synthesized brush border hydrolases.     

Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD

The LKB1 gene encodes a serine/threonine kinase that is mutated in the Peutz-Jeghers cancer syndrome. LKB1 is homologous to the Par-4 polarity genes in C. elegans and D. melanogaster. We have previously reported the identification and characterization of an LKB1-specific adaptor protein, STRAD, which activates LKB1 and translocates it from nucleus to cytoplasm. We have now constructed intestinal epithelial cell lines in which inducible STRAD activates LKB1. Upon LKB1 activation, single cells rapidly remodel their actin cytoskeleton to form an apical brush border. The junctional proteins ZO-1 and p120 redistribute in a dotted circle peripheral to the brush border, in the absence of cell-cell contacts. Apical and basolateral markers sort to their respective membrane domains. We conclude that LKB1 can induce complete polarity in intestinal epithelial cells. In contrast to current thinking on polarization of simple epithelia, these cells can fully polarize in the absence of junctional cell-cell contacts.     

Maintenance of epithelial surface membrane lipid polarity: A role for differing phospholipid translocation rates

Summary Large differences in lipid composition of apical and basolateral membranes from epithelial cells exist. To determine the responsible mechanism(s), rat renal cortical brush border and basolateral membrane phospholipids were labeled using³²P and either [³H]-glycerol or [2-³H] acetate for incorporation and degradation studies, respectively. Brush border and basolateral membrane fractions were isolated simultaneously from the same cortical homogenate. Different phospholipid classes were degraded at variable rates with phosphatidylcholine having the fastest decay rate. Decay rates for individual phospholipid classes were, however, similar in both brush border and basolateral membrane fractions. In phospholipid incorporation studies again, large variations existed between individual phospholipid classes with phosphatidylcholine and phosphatidylinositol showing the most rapid rates of incorporation. Sphingomyelin and phosphatidylserine showed extremely slow incorporation rates and did not enter into the isotopic decay phase for 48 hr. In contrast to degradation studies, however, the same phospholipid class labeled the two surface membrane domains at highly variable rates. The difference in these rates, with the exception of phosphatidylinositol, were identical to the differences in phospholipid compositions between the two membranes. For example, phosphatidylcholine was incorporated into the basolateral membrane 2.5 × faster than into the brush border membrane and its relative composition was 2.5 × greater in the basolateral membrane. The opposite was true for sphingomyelin. These results indicate incorporation and not degradation rates of individual phospholipids play a major role in regulating the differing phospholipid composition of brush border and basolateral membranes.     

Cytoskeletal markers allowing discrimination between brush cells and other epithelial cells of the gut including enteroendocrine cells

Brush cells are specialised epithelial cells scattered throughout the simple epithelia of the respiratory and alimentary tracts. These cells have been suggested to serve a still unknown receptive function and use nitric oxide as a gaseous messenger molecule. At the light microscope level, brush cells can be identified by antibodies against the actin filament crosslinking proteins villin and fimbrin that not only stain the apical tuft of microvilli and their rootlets, but also label projections emanating from the basolateral surface of these cells. Since brush cells contain numerous intermediate filaments and microtubules and display a complicated basolateral cell morphology, we tested in this study whether antibodies against cytokeratin, tubulin and components of the membrane cytoskeleton might provide further markers for these cells at the light microscope level. Here we show that brush cells (identified by villin antibodies) can be discriminated from the neighbouring simple epithelium of the stomach, pancreatic duct and duodenum by particularly strong immunoreactivity with antibodies specific for cytokeratin 18. Tubulin antibodies reacted strongly with the upper half of brush cells in a pattern not observed in the other epithelial cells of these tissues, including enteroendocrine cells of the duodenum. Ankyrin, a protein that links the spectrin-based membrane cytoskeleton to integral proteins of the plasma membrane was revealed as a third cytoskeleton-associated protein, prominently expressed in brush cells where ankyrin is restricted to the basolateral membrane domain. The apparently high concentration of cytokeratin 18, tubulin and ankyrin in brush cells suggests that these cytoskeletal proteins might play a role in the mechanical stability and polarised organisation of these putative receptor cells.Dedicated to Prof. Dr. Drs. h.c. Andreas Oksche on the occasion of his 70th birthday     

Evidence for the transit of aminopeptidase N through the basolateral membrane before it reaches the brush border of enterocytes

Summary In vivo pulse-chase labeling of rabbit jejunum loops was used in conjunction with subcellular fractionation and quantitative immunoprecipitation to determine whether or not the newly synthesized aminopeptidase N transits through the basolateral membrane before it reaches the apical brush border, its final localization. The kinetics of the arrival of the newly synthesized enzyme in the Golgi complex, basolateral and brush border membrane fractions strongly suggest that on leaving the Golgi aminopeptidase N is transiently integrated into the basolateral domain before reaching the brush border.     

Apical sorting by galectin-3-dependent glycoprotein clustering

Epithelial cells are characterized by their polarized organization based on an apical membrane that is separated from the basolateral membrane domain by tight junctions. Maintenance of this morphology is guaranteed by highly specific sorting machinery that separates lipids and proteins into different carrier populations for the apical or basolateral cell surface. Lipid-raft-independent apical carrier vesicles harbour the beta-galactoside-binding lectin galectin-3, which interacts directly with apical cargo in a glycan-dependent manner. These glycoproteins are mistargeted to the basolateral membrane in galectin-3-depleted cells, dedicating a central role to this lectin in raft-independent sorting as apical receptor. Here, we demonstrate that high-molecular-weight clusters are exclusively formed in the presence of galectin-3. Their stability is sensitive to increased carbohydrate concentrations, and cluster formation as well as apical sorting are perturbed in glycosylation-deficient Madin-Darby canine kidney (MDCK) II cells. Together, our data suggest that glycoprotein cross-linking by galectin-3 is required for apical sorting of non-raft-associated cargo.     

Conformational change of rabbit aminopeptidase N into enterocyte plasma membrane domains analyzed by flow cytometry fluorescence energy transfer

Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by cross-linking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.     

Immunolocalization of transferrin and transferrin receptor in mouse small intestinal absorptive cells

The mechanisms by which the duodenal mucosa absorbs iron are unknown. Insorption into absorptive cells of luminal iron bound to transferrin via receptor-mediated endocytosis has been hypothesized, but transferrin and transferrin receptor are absent in apical microvillous brush borders of small bowel biopsies taken from fasted patients and normal volunteers. We hypothesized that a normal iron-containing diet might induce the transient appearance of transferrin and transferrin receptor in apical brush borders of small intestinal absorptive cells in a normal mouse that was provided iron-containing chow until the moment of sacrifice. Light and electron microscopic immunolocalization of transferrin and transferrin receptor in proximal small intestinal absorptive cells was limited to basolateral membranes and coated pits of cells predominantly in the crypts and basal regions of the villi. Transferrin and transferrin receptor were not detected in apical microvillous brush border membranes of these enterocytes. In parallel immunolocalization protocols designed to show the ability to immunodetect other antigens at these locations, maltase and proteoglycan were demonstrated in apical microvillous brush border membranes and in basolateral membranes, respectively, in absorptive cells of small intestinal villous tip, base, and crypt regions. Furthermore, transferrin and transferrin receptor were immunolocalized in hepatocyte sinusoidal microvillus membranes. We conclude that food does not induce the appearance of immunodetectable transferrin and transferrin receptor in the apical microvilli of small intestinal absorptive cells and, therefore, that these iron transport proteins are not involved in the apical microvillous membrane transport of luminal dietary iron.     

Flow cytometry, a very useful technique for the characterization of intestinal membrane vesicles

The plasma membrane of enterocytes comprises two structurally and functionally distinct domains. These are the apical brush border, containing digestive hydrolases and glycocalyx, and the basolateral domain, characterized by other specific markers. Using a fast and easy subcellular fractionation, we purified four membrane vesicle fractions from rabbit small intestinal mucosa: brush border, basolateral, rough endoplasmic reticulum and Golgi + smooth endoplasmic reticulum. Using flow cytometry, the fluorescence polarization of diphenylhexatriene was determined in brush border and in basolateral + Golgi + smooth endoplasmic reticulum membrane fractions in order to investigate changes in the membrane fluidity of both fractions and to compare the results obtained with those of spectroscopic techniques. Moreover, it was possible with flow cytometry to detect and quantify basolateral and brush border markers by using polyclonal and monoclonal antibodies. The advantages of flow cytometry in the detection of brush border membrane markers found in small amounts in the basolateral domain are discussed. Finally, flow cytometry holds great promise for the analysis and sorting of subcellular fractions.     

Truncated brush border myosin I affects membrane traffic in polarized epithelial cells

We investigate, in this study, the potential involvement of an acto-myosin-driven mechanism in endocytosis of polarized cells. We observed that depolymerization of actin filaments using latrunculin A decreases the rate of transferrin recycling to the basolateral plasma membrane of Caco-2 cells, and increases its delivery to the apical plasma membrane. To analyze whether a myosin was involved in endocytosis, we produced, in this polarized cell line, truncated, non-functional, brush border, myosin I proteins (BBMI) that we have previously demonstrated to have a dominant negative effect on endocytosis of unpolarized cells. These non-functional proteins affect the rate of transferrin recycling and the rate of transepithelial transport of dipeptidyl-peptidase IV from the basolateral plasma membrane to the apical plasma membrane. They modify the distribution of internalized endocytic tracers in apical multivesicular endosomes that are accessible to fluid phase tracers internalized from apical and basolateral plasma membrane domains. Altogether, these observations suggest that an acto-myosin-driven mechanism is involved in the trafficking of basolaterally internalized molecules to the apical plasma membrane.     

Myosin II isoforms promote internalization of spatially distinct clathrin-independent endocytosis cargoes through modulation of cortical tension downstream of ROCK2

Although the actomyosin cytoskeleton has been implicated in clathrin-mediated endocytosis, a clear requirement for actomyosin in clathrin-independent endocytosis (CIE) has not been demonstrated. We discovered that the Rho-associated kinase ROCK2 is required for CIE of MHCI and CD59 through promotion of myosin II activity. Myosin IIA promoted internalization of MHCI and myosin IIB drove CD59 uptake in both HeLa and polarized Caco2 intestinal epithelial cells. In Caco2 cells, myosin IIA localized to the basal cortex and apical brush border and mediated MHCI internalization from the basolateral domain, while myosin IIB localized at the basal cortex and apical cell–cell junctions and promoted CD59 uptake from the apical membrane. Atomic force microscopy demonstrated that myosin IIB mediated apical epithelial tension in Caco2 cells. Thus, specific cargoes are internalized by ROCK2-mediated activation of myosin II isoforms to mediate spatial regulation of CIE, possibly by modulation of local cortical tension.     

Selective roles for cholesterol and actin in compartmentalization of different proteins in the Golgi and plasma membrane of polarized cells

To determine the roles of cholesterol and the actin cytoskeleton in apical and basolateral protein organization and sorting, we have performed comprehensive confocal fluorescence recovery after photobleaching analyses of apical and basolateral and raft- and non-raft-associated proteins, both at the plasma membrane and in the Golgi apparatus of polarized MDCK cells. We show that at both the apical and basolateral plasma membrane domains, raft-associated proteins diffuse faster than non-raft-associated proteins and that, different from the latter, they become restricted upon depletion of cholesterol. Furthermore, only transmembrane apical proteins are restricted by the actin network. This indicates that cholesterol-dependent domains exist both at the apical and basolateral membranes of polarized cells and that the actin cytoskeleton has a predominant role in the organization of transmembrane proteins independent of their association with rafts at the apical membrane. In the Golgi apparatus apical proteins appear to be segregated from the basolateral ones in a compartment that is sensitive both to cholesterol depletion and actin rearrangements. Furthermore, consistent with the role of actin rearrangements in apical protein sorting, we found that apical proteins exhibit a differential sensitivity to actin depolymerization in the Golgi of polarized and nonpolarized cells.     

Intracellular redirection of plasma membrane trafficking after loss of epithelial cell polarity

In polarized Madin-Darby canine kidney epithelial cells, components of the plasma membrane fusion machinery, the t-SNAREs syntaxin 2, 3, and 4 and SNAP-23, are differentially localized at the apical and/or basolateral plasma membrane domains. Here we identify syntaxin 11 as a novel apical and basolateral plasma membrane t-SNARE. Surprisingly, all of these t-SNAREs redistribute to intracellular locations when Madin-Darby canine kidney cells lose their cellular polarity. Apical SNAREs relocalize to the previously characterized vacuolar apical compartment, whereas basolateral SNAREs redistribute to a novel organelle that appears to be the basolateral equivalent of the vacuolar apical compartment. Both intracellular plasma membrane compartments have an associated prominent actin cytoskeleton and receive membrane traffic from cognate apical or basolateral pathways, respectively. These findings demonstrate a fundamental shift in plasma membrane traffic toward intracellular compartments while protein sorting is preserved when epithelial cells lose their cell polarity.     

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).     

The uptake of phosphatidylcholine by small intestinal brush border membrane is protein-mediated

Brush border membrane vesicles prepared from rabbit small intestine are essentially free of basolateral membranes and nuclear, mitochondrial, microsomal and cytosolic contaminants. The resulting brush border membrane is unstable due to intrinsic lipases and proteinases. The PC transfer between small unilamellar lipid vesicles or mixed lipid micelles as the donor and the brush border membrane vesicles as the acceptor is protein-mediated. After proteolytic treatment of brush border membrane with papain or proteinase K the PC transfer activity is lost and the kinetics of PC uptake are similar to those measured with erythrocytes under comparable conditions. Evidence is presented to show that the PC transfer activity resides in the apical membrane of the enterocyte and not in the basolateral part of the plasma membrane. Furthermore, the activity is localized on the external surface of the brush border membrane exposed to the aqueous medium with its active centre probably not in direct contact with the lipid bilayer of the membrane. Proteins released from brush border membrane by proteolytic treatment catalyze PC exchange between different populations of small unilamellar vesicles. Furthermore, these protein(s) bind(s) PC forming a PC-protein complex.     

Isolation, purification and characteristics of brush border and basolateral membranes from cattle intestinal epithelium cells

The surface membrane of cattle intestine epithelium cells is separated into vesicated membrane fractions of the brush border and of basolateral membranes. The brush border membrane fraction is deposited with centrifugation (15,000 g) and is localized in the layers of 45, 56.5 and 48% in the density gradient of sucrose (105,000 g). Basolateral membranes, obtained at 70,000 g, in the density gradient of sucrose are in the layers of 30 and 31.5%. The brush border membranes are 8.5 times purified, basolateral membranes--9.1 times with their insignificant contamination with subcellular elements. The both fractions are deprived of mitochondria impurities.     

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.     

Expression and polarized apical secretion in Madin-Darby canine kidney cells of a recombinant soluble form of neutral endopeptidase lacking the cytosolic and transmembrane domains.

Neutral endopeptidase-24.11 (NEP; EC 3.4.24.11) is an abundant metalloendopeptidase of the brush border membrane of kidney proximal tubules. We have recently shown that NEP is delivered directly to the apical domain of the plasma membrane when expressed in polarized Madin-Darby canine kidney (MDCK) cells in culture (Jalal, F., Lemay, G., Zollinger, M., Berteloot, A., Boileau, G., and Crine, P. (1991) J. Biol. Chem. 266, 19826-19832). Here, a soluble form of NEP consisting of the signal peptide of pro-opiomelanocortin fused in-frame with the ectodomain of NEP has been expressed in MDCK cells. Enzymatic assays performed on apical and basolateral culture media of MDCK cells grown on semi-permeable supports indicated that the recombinant enzyme was predominantly released at the apical surface. In contrast, when the chimeric protein was expressed in NIH 3T3 cells or when pro-opiomelanocortin was expressed in MDCK cells, non-polarized secretion was observed into both the apical and basolateral compartments of the culture chamber. Our results suggest that the ectodomain of NEP is sufficient for directing the targeting of this protein to the apical membrane of polarized MDCK epithelial cells.     

Cell membrane fluidity in the intact kidney proximal tubule measured by orientation-independent fluorescence anisotropy imaging.

Membrane fluidity was measured in the isolated perfused proximal tubule from rabbit kidney. The apical and basolateral plasma membranes of tubule cells were stained separately with the fluidity-sensitive fluorophore trimethylammonium-diphenyl-hexatriene (TMA-DPH) by luminal or bath perfusion. Fluorescence anisotropy (r) of TMA-DPH was mapped with spatial resolution using an epifluorescence microscope (excitation 380 nm, emission greater than 410 nm) equipped with rotatable polarizers and a quantitative imaging system. To measure r without the confounding effects of fluorophore orientation, images were recorded with emission polarizer parallel and perpendicular to a continuum of orientations of the excitation polarizer. The theoretical basis of this approach was developed and its limitations were evaluated by mathematical modeling. The tubule inner surface (brush border) was brightly stained when the lumen was perfused with 1 microM TMA-DPH for 5 min; apical membrane r was 0.281 +/- 0.006 (23 degrees C). Staining of the tubule basolateral membrane by addition of TMA-DPH to the bath gave a significantly lower r of 0.242 +/- 0.010 (P less than 0.005); there was no staining of the brush border membrane. To interpret anisotropy images quantitatively, effects of tubule geometry, TMA-DPH lifetime, fluorescence anisotropy decay, and objective-depolarization were evaluated. Steady-state and time-resolved r and lifetimes in the intact tubule, measured by a nanosecond pulsed microscopy method, were compared with results in isolated apical and basolateral membrane vesicles from rabbit proximal tubule measured by cuvette fluorometry; r was 0.281 (apical membrane) and 0.276 (basolateral membrane) (23 degrees C). These results establish a methodology to quantitate membrane fluidity in the intact proximal tubule, and demonstrate a significantly higher fluidity in the basolateral membrane than in the apical membrane.