Electron microscope observations of the melanocyte of the human epidermis

Proteins and colloidal materials, administered orally to suckling rats and mice, were ingested by columnar absorptive cells of the jejunum and ileum, but not of the duodenum. Bovine gamma globulin and ovalbumin were identified in the apical cytoplasm by staining with fluorescent antibody; trypan blue, Evans blue, saccharated iron oxide, and colloidal gold were detected intracellularly by their color, specific staining, and appearance in the electron microscope. Each substance was segregated in membrane-enclosed vacuoles, apparently part of a system of potentially interconnecting vacuoles and tubules in the apical cytoplasm which is continuous in places with the apical cell membrane. We postulate that ingestion of foreign materials was accomplished by pinocytosis, that is, by invagination of the apical cell membrane to form vacuoles containing material from the intestinal lumen. Approximately 18 days after birth columnar absorptive cells lost the ability to ingest proteins and colloids, and no longer contained large vacuoles and numerous tubules. At this age rats and mice lose the ability to absorb antibodies from the intestine in an immunologically intact form, and we conclude that cellular ingestion is part of the mechanism of absorption of intact proteins in suckling animals. Particulate fat apparently is absorbed in both newborn and adult animals by micropinocytosis. Thus adult animals may not have lost the capacity for pinocytosis, but rather have become selective as to what substances provoke it. Cortisone acetate, administered subcutaneously to rats 8 to 10 days old alters the columnar absorptive cells within 72 hours so that they resemble the cells in adult animals and no longer ingest proteins.     

The Ingestion of Proteins and Colloidal Materials by Columnar Absorptive Cells of the Small Intestine in Suckling Rats and Mice

Proteins and colloidal materials, administered orally to suckling rats and mice, were ingested by columnar absorptive cells of the jejunum and ileum, but not of the duodenum. Bovine gamma globulin and ovalbumin were identified in the apical cytoplasm by staining with fluorescent antibody; trypan blue, Evans blue, saccharated iron oxide, and colloidal gold were detected intracellularly by their color, specific staining, and appearance in the electron microscope. Each substance was segregated in membrane-enclosed vacuoles, apparently part of a system of potentially interconnecting vacuoles and tubules in the apical cytoplasm which is continuous in places with the apical cell membrane. We postulate that ingestion of foreign materials was accomplished by pinocytosis, that is, by invagination of the apical cell membrane to form vacuoles containing material from the intestinal lumen. Approximately 18 days after birth columnar absorptive cells lost the ability to ingest proteins and colloids, and no longer contained large vacuoles and numerous tubules. At this age rats and mice lose the ability to absorb antibodies from the intestine in an immunologically intact form, and we conclude that cellular ingestion is part of the mechanism of absorption of intact proteins in suckling animals. Particulate fat apparently is absorbed in both newborn and adult animals by micropinocytosis. Thus adult animals may not have lost the capacity for pinocytosis, but rather have become selective as to what substances provoke it. Cortisone acetate, administered subcutaneously to rats 8 to 10 days old alters the columnar absorptive cells within 72 hours so that they resemble the cells in adult animals and no longer ingest proteins.     

Visualization of endocytosed markers in freeze-fracture studies of toad urinary bladder

Antidiuretic hormone (ADH) stimulation increases the apical membrane water permeability of granular cells in toad urinary bladder. This response correlates closely with the fusion of tubular cytoplasmic vesicles with the membrane and delivery of intramembrane particle (IMP) aggregates from the tubules (aggrephores) to the apical membrane. These aggregates are believed to be associated with the channels responsible for the water permeability increase. Removal of ADH triggers apical membrane endocytosis and disappearance of aggregates from the apical membrane. However, it has been unclear whether aggregate disappearance is due to disassembly of aggregates within the apical membrane or to their endocytic retrieval as intact structures. Using colloidal gold and horseradish peroxidase to follow endocytosis from the apical surface after ADH removal, we have directly observed in cross-fractured bladder cells the intramembrane structure of intracellular vesicles that contain these fluid-phase markers. Under these conditions, intact aggregates can be identified in the membrane of tubular endocytosed vesicles. This directly demonstrates that conditions which lower apical membrane water permeability cause the tubular aggrephores to "shuttle" intact aggregates from the apical membrane back into the cytoplasm. An additional population of vesicles with tracer are found which are spherical and display structural features of the apical membrane, as well as occasional aggregates. These vesicles may be responsible for retrieval of aggregates from the surface apical membrane.     

Function of the cytoskeleton in cells with microridges from the oral epithelium of the carp Cyprinus carpio

Superficial cells of the oral mucosal epithelium in the carp and the cytoskeleton of the epithelial cells are examined by scanning and transmission electron microscopy. Microridges are formed on the surface of the epithelium. Epithelial cells contain two types of vesicles: mucous secretory vesicles and coated vesicles. Most of the mucous vesicles are situated in the center of the cell near the Golgi apparatus. In freeze-fracture replicas, intramembranous particles are abundant in the membranes of the secretory vesicles but rare in the apical plasma membrane. Coated vesicles are situated in the apical and subapical cytoplasm. A great number of thick filaments, considered to be keratin filaments, run randomly throughout the cell to form a meshwork. Thick filaments, which are sparse in the central cytoplasm, are connected to the membranes of the secretory vesicles and other membranous organelles. A layer of closely packed thin filaments, considered to be actin filaments, is found just beneath the apical plasma membrane. Microtubules also occur in the apical cytoplasm and run almost parallel to the cell surface. Both kinds of vesicles are connected to the thin and thick filaments. Their functional significance in the regulation of membrane at the free surface is discussed.     

Membrane protein trafficking through the common apical endosome compartment of polarized Caco-2 cells.

By raising monoclonal antibodies to the apical surface of Caco-2 cells we have identified a membrane protein (p100) that internalizes and recycles constitutively between the apical plasma membrane and endosomes in the apical cytoplasm. By applying tracers bound to the transferrin receptor, which internalizes and recycles back to the basolateral border, we demonstrate that the apical endosomes containing p100 include a subset of multivesticular bodies (MVB), which are also accessible to proteins arriving from the basolateral endosome. Tracers bound to EGF receptors and alpha-2-macroglobulin, which internalize from the basolateral border and are degraded, probably in lysosomes, also pass through the p100-containing MVB. These studies therefore suggest that the apical cytoplasm of Caco-2 cells contains a population of MVB capable of receiving membrane proteins trafficking in from both apical and basolateral borders and then routing them to a variety of cell surface and intracellular destinations. The differential distribution of apical and basolateral tracers within the 50-nm-diameter tubules connected to these p100-positive apical MVB suggests that the destination of proteins trafficking from the MVB back to apical and basolateral surfaces is determined by the tubules to which they gain access.     

Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells

Targeted delivery of proteins to distinct plasma membrane domains is critical to the development and maintenance of polarity in epithelial cells. We used confocal and time-lapse total internal reflection fluorescence microscopy (TIR-FM) to study changes in localization and exocytic sites of post-Golgi transport intermediates (PGTIs) carrying GFP-tagged apical or basolateral membrane proteins during epithelial polarization. In non-polarized Madin Darby Canine Kidney (MDCK) cells, apical and basolateral PGTIs were present throughout the cytoplasm and were observed to fuse with the basal domain of the plasma membrane. During polarization, apical and basolateral PGTIs were restricted to different regions of the cytoplasm and their fusion with the basal membrane was completely abrogated. Quantitative analysis suggested that basolateral, but not apical, PGTIs fused with the lateral membrane in polarized cells, correlating with the restricted localization of Syntaxins 4 and 3 to lateral and apical membrane domains, respectively. Microtubule disruption induced Syntaxin 3 depolarization and fusion of apical PGTIs with the basal membrane, but affected neither the lateral localization of Syntaxin 4 or Sec6, nor promoted fusion of basolateral PGTIs with the basal membrane.     

The distribution of MUC1, an apical membrane glycoprotein, in mammary epithelial cells at the resolution of the electron microscope: implications for the mechanism of milk secretion

The distribution of the glycoprotein, mucin 1 (MUC1), was determined in lactating guinea-pig mammary tissue at the resolution of the electron microscope. MUC1 was detected on the apical plasma membrane of secretory epithelial cells, the surface of secreted milk-fat globules, the limiting membranes of secretory vesicles containing casein micelles and in small vesicles and tubules in the apical cytoplasm. Some of the small MUC1-containing vesicles were associated with the surfaces of secretory vesicles and fat droplets in the cytoplasm. MUC1 was detected in much lower amounts on basal and lateral plasma membranes. By quantitative immunocytochemistry, the ratio of MUC1 on apical membranes and milk-fat globules to that on secretory vesicle membranes was estimated to be 9.2:1 (density of colloidal gold particles/microm membrane length). The ratio of MUC1 on apical membranes compared with basal/lateral membranes was approximately 99:1. The data are consistent with a mechanism for milk-fat secretion in which lipid globules acquire an envelope of membrane from the apical surface and possibly from small vesicles containing MUC1 in the cytoplasm. During established lactation, secretory vesicle membrane does not appear to contribute substantially to the milk-fat globule membrane, or to give rise in toto to the apical plasma membrane.     

Immunolocalization of a mammalian aquaporin 3 homolog in water-transporting epithelial cells in several organs of the clawed toad Xenopus laevis

Nucleotide sequences of cDNA were used to construct antibodies against an aquaporin (AQP) expressed in the clawed toad, Xenopus laevis, viz., Xenopus AQP3, a homolog of mammalian AQP3. Xenopus AQP3 was immunolocalized in the basolateral membrane of the principal cells of the ventral skin, the urinary bladder, the collecting duct and late distal tubule of the kidney, the absorptive epithelial cells of the large intestine, and the ciliated epithelial cells of the oviducts. Therefore, we designated this AQP as basolateral Xenopus AQP3 (AQP-x3BL). The intensity of labeling for AQP-x3BL differed between the ventral and dorsal skin, with the basolateral membrane of the principal cells in the ventral skin showing intense labeling, whereas that in the dorsal skin was lightly labeled. AQP-x3BL was also immunolocalized in the basolateral membrane of secretory cells in the small granular and mucous glands of the skin. As AQP-x5, a homolog of mammalian AQP5, is localized in the apical membrane of these same cells, this provides a pathway for fluid secretion by the glands. Although Hyla AQP-h2 is translocated from the cytoplasm to the apical membrane of the Hyla urinary bladder in response to arginine vasotocin (AVT), AQP-h2 immunoreactivity in Xenopus bladder remains in the cytoplasm and barely moves to the apical membrane, regardless of AVT stimulation. AQP-x3 is localized in the basolateral membrane, even though the AVT-stimulated AQP-h2 does not translocate to the apical membrane. These findings provide new insights into AQP function in aquatic anurans.     

The effect of thioglycolate on intermediate filaments and membrane translocation in rat urothelium during the expansion-contraction cycle

Summary The functional role of cytokeratin intermediate filaments in the translocation of asymmetric membrane plaques between cytoplasm and surface of apical urothelial cells was investigated during contraction and expansion of rat urinary bladders. A stereological investigation of electron micrographs provided estimations of surface area, volume, and number of discoidal vesicles and infoldings per unit volume of urothelial apical cell cytoplasm. Contracted and distended bladders incubated in 0.01 M sodium bicarbonate were compared to identical preparations experimentally incubated in 5 mM thioglycolic acid. The latter reagent disrupts the intermediate filament network by reducing sulfhydryl bridges. Densities of discoidal vesicles in cells contracted after incubation in thioglycolate were similar to density estimations in cells expanded under control conditions. Similarly, densities of vesicles in cells expanded after exposure to thioglycolate were comparable in number to those in normally contracted cells. Thus, membrane translocation to and from the luminal surface was blocked by thioglycolate treatment. The lack of normal membrane transfer at the luminal surface induces apical cells exposed to experimental conditions to undergo extraordinary adjustments in response to external pressures of bladder contraction and distension. During contraction, the apical-intermediate cell interface unfolded while the luminal surface ballooned out into the lumen. In distended bladders, large intercellular spaces formed between apical cells along their lateral margins. The results support a model published earlier implicating the filament network as a critical mediator of membrane translocation.     

Low intracellular pH induces redistribution of fodrin and instabilization of lateral walls in MDCK cells.

We have studied the effect of intracellular pH on the establishment and maintenance of the cellular polarity in MDCK cells by utilizing nigericin which causes lowering of the cytoplasmic pH. At pH below 6.5, MDCK cells lost their polarized morphology and became roundish, with an increased apical area and shortened and unstable lateral walls. The lateral wall marker proteins uvomorulin and Na,K-ATPase remained segregated to the lateral walls in the acidified cells, as shown by immunofluorescence microscopy. Fodrin, on the other hand, was released from its normal basolateral residence and was found in the cytoplasm. Actin, which normally co-localizes with fodrin along the basolateral walls, showed a dotty distribution in the cytoplasm of acidified cells, while stress fibers remained intact. Microtubular network appeared flattened, but the Golgi complex retained its apical position. The pH change-induced alterations were readily reversible, as the normal basal-apical polarity (columnar shape, distinct apical and lateral domains with apposing and stiff lateral membranes) was reformed within 10 minutes after restoring the normal pH gradient across the cell membrane. This coincided with the translocation of fodrin from the cytoplasm to the lateral walls. The results show that lowering of intracellular pH leads to temporary segregation of fodrin from the other components of the membrane skeleton assembly, and that association of fodrin with the lateral walls seems to be a prerequisite for their close apposition and for the maintenance of normal basal-axial polarity.     

Analysis of biochemical markers of MCF-7 cell apoptosis induced by a recombinant analogue of lactaptin

Earlier, a pro-apoptotic peptide lactaptin was isolated from human milk and characterized and its recombinant analogue RL2 was generated. Both peptides were demonstrated to be capable of apoptotic cell death induction in human breast adenocarcinoma MCF-7 cells. In the work, biochemical markers of RL2-induced MCF-7 apoptosis are analyzed. Activation of initiator and effector caspases, as well as apoptotic changes in mitochondrial membrane potential and cytoplasm membrane composition in cells treated with RL2, were analyzed using flow cytometry and Western blot techniques. MCF-7 cell death induced by RL2 was found to be associated with phosphatidylserine exposure on the surface of the plasma membrane. Also, RL2 was demonstrated to induce dissipation of the mitochondrial membrane potential and activation of initiator caspases 8 and 9 and an effector caspase 7.     

Localization of MCC (mutated in colorectal cancer) in various tissues of mice and its involvement in cell differentiation.

Localization of the MCC (mutated in colorectal cancer) gene product, a cell cycle-regulating protein mutated in several colorectal tumors, in various mouse tissues was examined by immunohistochemistry and immunoelectron microscopy. MCC was localized on microvilli and in the apical cytoplasm in renal proximal tubule epithelial cells and pancreatic acinar cells. In hepatocytes, MCC was exclusively detected on microvilli. MCC was highly expressed in the cerebral cortex and the molecular layer of the cerebellar cortex and was partially associated with membrane organelles in neuronal elements. Adrenal chromaffin cells showed little expression of MCC. MCC was localized to the cell margins of ependymal cells, thyroid follicular cells, and anterior pituitary cells. In parotid acinar cells, only the apical surface was immunopositive. MCC was not expressed in skeletal and cardiac muscle. MCC was present at lateral cell borders in the duodenum and colon epithelium. In addition, the apical cytoplasm of colon epithelial cells exhibited intense immunoreactivity. The amount of MCC increased during differentiation of NGF-treated PC12 cells. In conclusion, MCC was expressed in differentiated cells and was associated with the plasma membrane and membrane organelles. In addition to the negative regulation of the cell cycle, MCC may be involved in cell differentiation.     

Effects of stimulation on the ultrastructure and Na, K, Cl composition of the fundus of the rat plantar sweat gland

Initial stimulation of the rat plantar sweat gland with pilocarpine caused a variable degree of distension of the apical membrane of the secretory cell. This appeared to be a process of filtration of secretory cell cytoplasm through the apical terminal web. Further stimulation resulted in luminal dilatation, cytoplasmic depletion, and morbidity of some cells. These morphological changes in the footpad gland, which thus can no longer be considered as eccrine, were accompanied by a fall in potassium and a rise in sodium concentration within the secretory cells. The mode of secretion induced by pharmacological stimulation was fundamentally the same as that in the glands of species responsive to thermal stimulation.     

Ultrastructure of the ovary of Dermatobia hominis (Diptera: Cuterebridae). I. Development during the 3rd larval instar

The ultrastructure and distribution of gonial and somatic cells in the ovary of Dermatobia hominis was studied during the 3rd larval instar. In larvae weighing between 400 and 500 mg, the ovary is partially divided into basal and apical regions by oblong somatic cells that penetrate from the periphery; these cells show ovoid nucleus and cytoplasm full of microtubules. In both regions, gonial cells with regular outlines, large nucleus and low electron-density cytoplasm are scattered among the interstitial somatic cells. These later cells have small nucleus and electrondense cytoplasm. Clear somatic cells with small nucleus and cytoplasm of very low electron-density are restrict to the apical region of the gonad. Degenerating interstitial somatic cells are seen in the basal portion close to the ovary peduncle. During all this larval period the morphological features of the ovary remain almost the same. At the end of the period there is a gradual deposition of glycogen in the cytoplasm of the somatic cells, increase in the number and density of their mitochondria plus nuclear modification as membrane wrinkling and chromatin condensation in masses.     

Fine structure of the larval midgut of the fly Rhynchosciara and its physiological implications

The midgut of Rhynchosciara americana larvae consists of a cylindrical ventriculus from which protrudes two gastric caeca formed by polyhedral cells with microvilli covering their apical faces. The basal plasma membrane of these cells is infolded and displays associated mitochondria which are, nevertheless, more conspicuous in the apical cytoplasm. The anterior ventricular cells possess elaborate mitochondria-associated basal plasma membrane infoldings extending almost to the tips of the cells, and small microvilli disposed in the cell apexes. Distal posterior ventricular cells with long apical microvilli are grouped into major epithelial foldings forming multicellular crypts. In these cells the majority of the mitochondria are dispersed in the apical cytoplasm, minor amounts being associated with moderately-developed basal plasma membrane infoldings. The proximal posterior ventriculus represents a transition region between the anterior ventriculus and the distal posterior ventriculus. The resemblance between the gastric caeca and distal posterior ventricular cells is stressed by the finding that their microvilli preparations display similar alkaline phosphatase-specific activities. The results lend support to the proposal, based mainly on previous data on enzyme excretion rates, that the endo-ectoperitrophic circulation of digestive enzymes is a consequence of fluid fluxes caused by the transport of water into the first two thirds of midgut lumen, and its transference back to the haemolymph in the gastric caeca and in the distal posterior ventriculus.     

Electron microscopic research on the mitochondria-rich bladder cells of the frog

The ultrastructural peculiarities of mitochondria-rich cells of the frog urinary bladder are analysed using three electron microscopic methods: ultrathin sections, scanning electron microscopy, freeze fracture. The mitochondria and tubular and vesicular structures are most abundant in the apical region of cytoplasm. The P-face (PF) of the apical plasma membrane is characterized by the presence of rod-shaped intramembrane particles (IMP), whereas the E-face (EF) possesses complementary pits. Depending on the distribution density of the rod-shaped IMP, three types of cells are described. The apical plasma membrane has an invert distribution of the globular IMP: a great quantity of IMP on the EF and a few particles on the PF. This structure of the apical plasma membrane is supposed to correlate with its very low water permeability. Using filipin as a marker of cholesterol localization, it has been shown that the mitochondria-rich cell apical membrane contains more cholesterol than that of the granular cells. The nature of the rod-shaped IMP and their role in the transmembrane ion transport have been discussed.     

Distribution of microtubules and microfilaments in exocrine (ventral prostatic epithelial cells and pancreatic exocrine cells) and endocrine cells (cells of the adenohypophysis and islets of Langerhans). The relationship between cytoskeletons and epithelial-cell polarity

We investigated the distribution of microtubules and microfilaments in some exocrine and endocrine cells in rats. Microtubules were stained by applying an immunofluorescent technique using antibodies against beta-tubulin, while microfilaments were stained with rhodamine-phalloidin, which binds selectively to polymerized actin filaments. In the cytoplasm of some exocrine cells (pancreatic acinar cells and ventral prostatic epithelial cells), the microtubules were distributed longitudinally from the apical region to the basal region, but no microtubules were found in the nuclear region. In exocrine cells, most of the microfilaments were localized beneath the apical plasma membrane. In some endocrine cells (those of the adenohypophysis and the islets of Langerhans), the microtubules exhibited a radial or reticular distribution in the cytoplasm, and intense fluorescence was observed in the perinuclear region. The immunofluorescence produced by the antibodies against beta-tubulin was more intense in endocrine cells than in exocrine cells. The microfilaments observed in the endocrine cells studied were homogenously distributed beneath the plasma membrane. Dot-like rhodamine-phalloidin staining was often observed in the cytoplasm of both the exocrine and endocrine cells. The present study clearly demonstrated marked differences in the distribution of cytoskeletal elements in exocrine and endocrine cells, and these may reflect differences in the secretory direction of such cells as well as in epithelial-cell polarity.     

EBV up-regulates cytochrome c through VDAC1 regulations and decreases the release of cytoplasmic Ca2+ in the NPC cell line

EBV (Epstein-Barr virus) is considered to be a major factor that causes NPC (nasopharyngeal carcinoma), which is one of the sneakiest cancers frequently occurring in Southeast Asia and Southern China. Apoptosis and pro-apoptotic signals have been studied for decades; however, few have extended the prevailing view of EBV to its impact on NPC in perspective of apoptosis. One of the important proteins named VDAC1 (voltage-dependent anion protein 1) on the mitochondrial outer membrane controls the pro-apoptotic signals in mammalian cells. The impact of EBV infection on VDAC1 and related apoptotic signals remains unclear. In order to study the VDAC1's role in EBV-infected NPC cells, we employ siRNA (small interfering RNA) inhibition to analyse the release of Ca2+ and Cyto c (cytochrome c) signals in the cytoplasm, as they are important pro-apoptotic signals. The results show a decrease of Ca2+ release and up-regulation of Cyto c with EBV infection. After siRNA transfection, the dysregulation of Cyto c is neutralized, which is evidence that the level of Cyto c release in virus-infected NPC cells is the as same as that of non-infected NPC cells. This result indicates that EBV infection changes the cytoplasmic level of Cyto c through regulating VDAC1. In summary, this study reports that EBV changes the release of Ca2+ and Cyto c in the cytoplasm of NPC cells, and that Cyto c changes are mediated by VDAC1 regulation.     

The effect of salinity acclimation on the ultrastructure of the gills of Gammarus oceanicus (Segerstråle, 1947) (Crustacea: Amphipoda)

Summary Acclimation to low salinity induces changes in the ultrastructure of the gill cells of the marine euryhaline amphipod, Gammarus oceanicus. The gills are composed of a single cell type. In 100% artificial sea water, these cells contain moderate numbers of mitochondria which are randomly distributed in the cytoplasm. The plasma membrane is extensively invaginated at the apical, lateral, and basal surfaces. Acclimation to 20% artificial sea water induces a further invagination at the apical cell membrane to form an elaborate apical labyrinth. The extracellular spaces between the folds in the basal cell membrane dilate to 1500 Å or more. Mitochondria are more abundant and in many cells they undergo a change in conformation. The mitochondria are crowded into thin leaflets of cytoplasm between the dilated basal invaginations or into the narrow space between apical and basal cell membranes. Consequently, they lie in close contact with the plasma membrane over much of their surface.Supported in part by grants from the United States Public Health Service, 5 RO1 AM13455-03 and PHS FR-07085-04, and by a grant from the National Research Council of Canada administered by Dr. G. P. Morris.     

Molecular motors are differentially distributed on Golgi membranes from polarized epithelial cells

Microtubules (MT) are required for the efficient transport of membranes from the trans-Golgi and for transcytosis of vesicles from the basolateral membrane to the apical cytoplasm in polarized epithelia. MTs in these cells are primarily oriented with their plus ends basally near the Golgi and their minus-ends in the apical cytoplasm. Here we report that isolated Golgi and Golgi-enriched membranes from intestinal epithelial cells possess the actin based motor myosin-I, the MT minus- end-directed motor cytoplasmic dynein and its in vitro motility activator dynactin (p150/Glued). The Golgi can be separated into stacks, possessing features of the Golgi cisternae, and small membranes enriched in the trans-Golgi network marker TGN 38/41. Whereas myosin-I is present on all membranes in the Golgi fraction, dynein is present only on the small membrane fraction. Dynein, like myosin-I, is associated with membranes as a cytoplasmic peripheral membrane protein. Dynein and myosin-I coassociate with membranes that bind to MTs and cross-link actin filaments and MTs in a nucleotide-dependent manner. We propose that cytoplasmic dynein moves Golgi membranes along MTs to the cell cortex where myosin-I provides local delivery through the actin- rich cytoskeleton to the apical membrane.