Apical EGF receptors regulate epithelial barrier to gastric acid: endogenous TGF-alpha is an essential facilitator

In previous studies, we found that apical and basolateral EGF receptors (EGFR) on primary canine gastric monolayers decreased paracellular permeability, evident by increased transepithelial electrical resistance (TER) and decreased flux of [(3)H]mannitol (MF). After studying monolayers in Ussing chambers, we now report that treatment with apical, but not basolateral, EGF enhanced tolerance to apical H(+), evident by a slower decay in TER and an attenuated rise in MF. Enhanced tolerance to apical acid was evident within 10 min of treatment with apical EGF. Immunoneutralization of endogenous transforming growth factor (TGF)-alpha accelerated the drop in TER and the rise in MF in response to apical acidification; apical EGF reversed these effects. Study of monolayers cultured in Transwell inserts showed that immunoblockade of basolateral, but not apical, EGFR also impaired the resistance to apical acidification and enhanced MF. We conclude that apical EGFR regulates the barrier to apical acidification via effects on paracellular resistance. Although exogenous basolateral EGF has a less apparent effect on the barrier to acid, endogenous ligand active at basolateral EGFR plays an important role in maintaining the barrier to apical acid. Our data implicate a role for an apical EGFR ligand, which may be EGF or another member of the EGF family.     

THE APICAL MERISTEM OF SPLACHNIDIUM RUGOSUM (PHAEOPHYTA)1

The meristem of Splachnidium rugosum consists of a central apical cell surrounded by a region of actively dividing cells, many of which bear hairs. Conceptacle initials are scattered throughout the surface layer of the meristematic region. Conceptacle initials and apical hairs differentiate adjacent to the apical cell. The apical cell and the conceptacle initials are distinctive, pear-shaped cells possessing similar cytological features that are consistent with significant metabolic activity. They have a nucleus surrounded by dictyosomes, a stellate chloroplast, mitochondria, and numerous vesicles and physodes. When the apical cell is damaged as a result of experimental manipulation, growth ceases. It is inferred that the apical cell controls cell division in the meristematic region and also the differentiation of conceptacle initials and apical hairs. The apical meristems of Splachnidium and species of the Fucales have several important features in common, including the growth-regulatory role of the apical cell and the process of conceptacle initiation. The taxa may possibly have a common evolutionary origin. The problematic and unresolved taxonomic status of Splachnidium is discussed.     

Cell division patterns in the apices of subterranean axis and aerial shoot of Psilotum nudum (Psilotaceae): morphological and phylogenetic implications for the subterranean axis

The cell division pattern in the apical meristem of Psilotum nudum was examined using epi-illumination microscopy and a paraffin method. In the subterranean axis, about half of the derivative cells of the apical cell produce tetrahedral daughter apical cells by the first three or more oblique divisions. Roughly half of these apical cells give rise to the apical meristems of axes, whereas the other half do not. Various relative activities of the mother and daughter apical cells give rise to disordered branching patterns. In the ill-organized apical meristem as well as the leafless and capless structure, the Psilotum subterranean axis differs from the basic organs of vascular plants such as stem and root and seems to be an independent organ. The cell division pattern characteristic of the subterranean axis persists in the young unbranched aerial shoots, although fewer daughter apical cells are produced. Dichotomous branching of the aerial shoots, as in a variety of organs of pteridophytes, involves loss of the mother apical cell followed by appearance of two daughter apical cells.     

Study on membrane recycling in the rat visceral yolk-sac endoderm using concanavalin-A conjugates

The internalization and intracellular movements of apical-cell-membrane material were investigated in the endodermal cells of cultured visceral yolk-sacs of rats (whole-embryo culture; explanted at 10.5 days of gestation and cultured for 24 h) using horseradish peroxidase- and ferritin-labelled concanavalin A (Con-A HRP, Con-A Fer). When visceral yolk-sac endoderm was exposed to Con-A HRP or Con-A Fer for 5 min at 4 degrees C, the apical cell membranes containing a well-developed fuzzy coat were heavily labelled, whereas apical vacuoles, lysosomes and apical canaliculi were not. Incubation of Con-A-labelled endoderm for 5-60 min at 20 degrees and 37 degrees C in Con-A-free serum resulted in a temperature-dependent internalization of membrane-bound lectin into coated vesicles, apical vacuoles and lysosomes, and the apical cell membranes were cleared of the heavy labelling. With increasing incubation time, the number of labelled vacuolar structures and the intensity of their labelling decreased gradually, whereas the number of labelled apical canaliculi increased. Thus, after 30 and 60 min at 37 degrees C, most of the apical canaliculi contained high concentrations of the markers. It was possible to observe labelled apical canaliculi that were in continuity with labelled apical vacuoles and lysosomes as well as with the apical cell membrane. These findings in rat endodermal cells indicate that constituents of the apical cell membrane are internalized in apical vacuoles and lysosomes, and are then brought back to the apical cell membrane by the apical canaliculi, which concentrate and store this membrane material.     

Apical cells of brown algae with particular reference to Sphacelariales, Dictyotales and Fucales

Brown algae show a significant diversity in thallus forms, giving a great number of model systems for the study of many important morphogenetic mechanisms. Thallus growth in brown algae is diffuse, intercalary or apical. The latter takes place by means of one or more apical cells. Among the brown algal groups, Sphacelariales, Dictyotales and Fucales give the best examples of apical growth, and have been repeatedly used for the study of the morphogenetic role of apical cells. In Sphacelariales the apical cells appear strongly polarized, the polarity expressed also on the organization of the microtubule cytoskeleton. These cells show a type of growth that can be compared with tip growth of root hairs, moss protonemata, pollen tubes and fungal hyphae, and is called ‘tip-like growth’. The thallus of Dictyotales grows by the activity of one or more apical cells showing variable degree of polarity. These cells do not exhibit any type of apical growth. In Fucales the vegetative thallus develops by means of an active apical meristem, which includes a large apical cell. This cell does not show polar organization or apical growth. However, in germinating zygotes of Fucales a polar axis is established and during the first stages of development they show a typical tip growth. In the present paper, the available information on the structure and division pattern of apical cells is presented. Their morphogenetic role is discussed, in relation to polarity, cytoskeleton organization, and apical dominance.     

Study on membrane recycling in the rat visceral yolk-sac endoderm using concanavalin-A conjugates

Summary The internalization and intracellular movements of apical-cell-membrane material were investigated in the endodermal cells of cultured visceral yolk-sacs of rats (whole-embryo culture; explanted at 10.5 days of gestation and cultured for 24h) using horseradish peroxidase- and ferritin-labelled concanavalin A (Con-A HRP, Con-A Fer). When visceral yolk-sac endoderm was exposed to Con-A HRP or Con-A Fer for 5 min at 4°C, the apical cell membranes containing a well-developed fuzzy coat were heavily labelled, whereas apical vacuoles, lysosomes and apical canaliculi were not. Incubation of Con-A-labelled endoderm for 5 60 min at 20° and 37°C in Con-A-free serum resulted in a temperature-dependent internalization of membranebound lectin into coated vesicles, apical vacuoles and lysosomes, and the apical cell membranes were cleared of the heavy labelling. With increasing incubation time, the number of labelled vacuolar structures and the intensity of their labelling decreased gradually, whereas the number of labelled apical canaliculi increased. Thus, after 30 and 60 min at 37°C, most of the apical canaliculi contained high concentrations of the markers. It was possible to observe labelled apical canaliculi that were in continuity with labelled apical vacuoles and lysosomes as well as with the apical cell membrane. These findings in rat endodermal cells indicate that constitutents of the apical cell membrane are internalized in apical vacuoles and lysosomes, and are then brought back to the apical cell membrane by the apical canaliculi, which concentrate and store this membrane material.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Rho signaling pathway and apical constriction in the early lens placode

Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase activity. Here we investigate apical constriction during chick lens placode invagination. Inhibition of actin polymerization and myosin II activity by cytochalasin D or blebbistatin prevents lens invagination. To further verify if lens placode invaginate through apical constriction, we analyzed the role of Rho-ROCK pathway. Rho GTPases expression at the apical portion of the lens placode occurs with the same dynamics as that of the cytoskeleton. Overexpression of the pan-Rho inhibitor C3 exotoxin abolished invagination and had a strong effect on apical myosin II enrichment and a mild effect on apical actin localization. In contrast, pharmacological inhibition of ROCK activity interfered significantly with apical enrichment of both actin and myosin. These results suggest that apical constriction in lens invagination involves ROCK but apical concentration of actin and myosin are regulated through different pathways upstream of ROCK. genesis 49:368-379, 2011.     

Intrauterine oxytocin system

At term, uterine epithelial cells express oxytocin (OT) as well as the OT receptor (OTR). Like other epithelial cells, uterine epithelial cells are polarized and sort secretory and membrane components to the apical or the basolateral cell surface. We have studied the subcellular localization of OT-like immunoreactivity (OT-IR) and OTR-IR in rat uterine epithelium by immuno-gold labelling of ultrathin frozen sections. Our observations indicate that OT and OTR are both distributed preferentially to the apical surface of rat uterine epithelial cells. OT-IR showed a 6-fold apical versus basolateral preference and was localized in apical secretory vesicles, suggesting that uterine OT is released by apical exocytosis. OTR-IR was localized to the apical surface with a 9-fold apical versus basolateral preference and was found specifically in association with apical microvilli. The present findings represent the first example of a G protein-coupled receptor that is preferentially localized on the microvillar compartment and support the concept of an autocrine uterine OT system at the apical side of the uterine epithelium.     

Maintaining apical dominance in the fern gametophyte

A kinetic model is developed for cell differentiation in the fern gametophyte to test hypotheses on the role of spatially patterned plasmodesmata networks in development. Of particular interest is the establishment and maintenance of apical cell type in a single cell, with concurrent suppression of this character in all other cells (apical dominance). Steps towards understanding apical cell localization in geometrically simple gametophytes may shed light on the establishment and maintenance of apical meristems in higher plants. The model, based on the plasmodesmata maps of Tilney and colleagues and involving kinetics for a requisite minimum of two morphogens. successfully produces the apical/non-apical cell differentiation patterns of normal development, and redifferentiation due to cell isolation, in six stages from 0-30 d of development. Our results indicate that increasing apical cell plasmodesmata number, as development progresses, is not required for effective transport across apical cell walls in maintaining apical dominance.     

ARNO through its coiled-coil domain regulates endocytosis at the apical surface of polarized epithelial cells

ARNO is a guanine-nucleotide exchange protein for the ARF family of GTPases. Here we show that in polarized epithelial cells, ARNO is localized exclusively to the apical plasma membrane, where it regulates endocytosis. Expression of ARNO stimulates apical endocytosis of the polymeric immunoglobulin receptor, and coexpression of ARF6 with ARNO leads to a synergistic stimulation of apical endocytosis. Expression of a dominant negative ARF6 mutant, ARF6-T27N, antagonizes this stimulatory effect. Deletion of the N-terminal coiled-coil (CC) domain of ARNO causes the mutant ARNO to localize to both the apical and basolateral plasma membranes. Expression of the CC domain alone abolishes ARNO-induced apical endocytosis as well as co-localization of IgA-receptor complexes with ARNO and clathrin. These results suggest that the CC domain contributes to the specificity of apical localization of ARNO through association with components of the apical plasma membrane. We conclude that ARNO acts together with ARF6 to regulate apical endocytosis.     

Comparative morphometrics of the primate apical tuft

The relationship between the structure and function of the primate apical tuft is poorly understood. This study addresses several hypotheses about apical tuft morphology using a large modern primate comparative sample. Two indices of tuft size are employed: expansion and robusticity. First, comparisons of relative apical tuft size were drawn among extant nonhuman primate groups in terms of locomotion and phylogenetic category. Both of these factors appear to play a role in apical tuft size among nonhuman primates. Suspensory primates and all platyrrhines had the smallest apical tufts, while terrestrial quadrupeds and all strepsirrhines (regardless of locomotor category) had the largest tufts. Similarly, hypotheses regarding the apical tufts of hominins, especially the large tufts of Neandertals were addressed using a comparison of modern warm- and cold-adapted humans. The results showed that cold-adapted populations possessed smaller apical tufts than did warm-adapted groups. Therefore, the cold-adaptation hypothesis for Neandertal distal phalangeal morphology is not supported. Also, early modern and Early Upper Paleolithic humans had apical tufts that were significantly less expanded and less robust than those of Neandertals. The hypothesis that a large apical tuft serves as support for an expanded digital pulp is supported by radiographic analysis of modern humans in that a significant correlation was discovered between the width of the apical tuft and the width of the pulp. The implications of these findings for hypotheses about the association of apical tuft size and tool making in the hominin fossil record are discussed.     

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.     

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.     

Src triggers circular ruffling and macropinocytosis at the apical surface of polarized MDCK cells

We addressed the role of Src on cortical actin dynamics and polarized endocytosis in MDCK cells harboring a thermosensitive v-src mutant. Shifting monolayers established at 40 degrees C (non-permissive temperature) to 34 degrees C (permissive temperature) rapidly reactivated v-Src kinase, but tight junctions and cell polarity resisted for >6 h. At this interval, activated v-src was recruited on apical vesicles, induced cortactin-associated apical circular ruffles productive of macropinosomes, thereby accelerating apical pinocytosis by approximately fivefold. Ruffling and macropinosome formation were selectively abrogated by inhibitors of actin polymerization, phosphoinositide 3-kinase, phospholipase C, and phospholipase D, which all returned apical pinocytosis to the level observed at 40 degrees C, underscoring the distinct control of apical micropinocytosis and macropinocytosis. Src promoted microtubule-dependent fusion of macropinosomes to the apical recycling endosome (ARE), causing its strong vacuolation. However, preservation of tubulation and apical polarity indicated that its function was not affected. The ARE was labeled for v-src, Rab11, and rabankyrin-5 but not early endosome antigen 1, thus distinguishing two separate Rab5-dependent apical pathways. The mechanisms of Src-induced apical ruffling and macropinocytosis could shed light on the triggered apical enteroinvasive pathogens entry and on the apical differentiation of osteoclasts.     

Ultrastructure of Stylodinium Iittorale (Dinophyceae) with special reference to the stalk and apical stalk complex

The ultrastructure of Stylodinium littorale Horiguchi et Chihara, a marine, sand-dwelling coccoid dinoflagel-late, was investigated with special emphasis on its stalk and the apical stalk complex. The dinoflagellate alternates between non-motile and motile cells in its life cycle. The non-motile cell possesses a long and distinct stalk. The stalk, consisting of a main cylindrical part and a holdfast, is firmly attached to a thecal plate (the apical pore plate). A part of its proximal portion is hollow and V-shaped in section. The V-shaped hollow space is underlain by a projection from the apical pore plate. An apical stalk complex is present in the motile cells and consists of a large apical pore plate and mucilaginous material. The apical pore plate is depressed into the cell, but has a narrow central tubular projection. The mucilaginous stalk-building material is stored between this plate and the outer plate membrane. The tubular projection of the apical pore plate corresponds to the apical pore of other dinoflagellates and its lumen is filled with electron-dense material. The structure of the apical stalk complex is compared with the homologous structure in Bysmatrum arenicola, the only other example of an apical stalk complex that has been investigated. A general ultrastructural survey revealed that S. littorale possesses a typical dinoflagellate cellular structure.     

Apical and basolateral EGF receptors regulate gastric mucosal paracellular permeability

Previous studies found that monolayers formed from canine oxyntic epithelial cells in primary culture displayed remarkable resistance to apical acidification and both mitogenic and migratory responses to epidermal growth factor (EGF) treatment. In our present studies, we found that EGF increased transepithelial resistance (TER) but not short-circuit current in these monolayers. Parallel effects of EGF on decreasing mannitol flux and increasing TER implicate direct regulation of paracellular permeability. EGF acting at either apical and basolateral receptors rapidly increased TER, but the apical response was sustained whereas the basolateral response was transient. (125)I-labeled EGF binding revealed specific apical binding, but receptor numbers were 25-fold lower than on the basolateral surface. Both apical and basolateral EGF activated tyrosine phosphorylation of EGF receptors (EGFR), beta-catenin, and cellular substrate as evident on confocal microscopy. Although apical EGF activated a lesser degree of receptor autophosphorylation than basolateral EGF, phosphorylation of beta-catenin was equally prominent with apical and basolateral receptor activation. Together, these findings indicate that functional apical and basolateral EGFR exist on primary canine gastric epithelial cells and that these receptors regulate paracellular permeability. The sustained effect of apical EGFR activation and prominent phosphorylation of beta-catenin suggest that apical EGFR may play a key role in this regulation.     

Qualitative inheritance of water-stress induced apical sterility in wheat (Triticum aestivum)

Grain number per unit area is an effective component of grain yield in bread wheat. Water-stress induced apical sterility (tip sterility) reduces the number of grains and, consequently, the grain yield in semi-arid regions with a shortage of available water during the pre-anthesis period. Crosses between apical sterile and apical fertile varieties and selection lines were made and F1, BC1, and F2 populations were subjected to moderate water-stress to study the inheritance of this character. The F2 and BC1 plants were qualitatively categorised into two phenotypes and tested for monohybrid and dihybrid segregation hypotheses. All the spikes of F1 plants obtained from crosses between apical fertile and apical sterile varieties were fully fertile indicating apical fertility is dominant to apical sterility. The F2 segregation Results from crosses between apical fertile lines and Y82187 suggested two complementary dominant genes segregating independently were involved in tolerance to water-stress induced apical sterility. In other words, two dominant genes determine apical fertility in these crosses and if one of these loci is homozygous, recessive waterstress will induce apical sterility. One F2 population segregated for both apical sterility and vernalisation response. Semi-winter plants had more sterile spikelets and the result of chi-square test confirmed monhybrid segregation for vernalisation response.     

Invaginated apical vacuoles in the cells of the proximal convoluted tubule in the rat kidney

Summary Following perfusion fixation of the rat kidney with glutaraldehyde the proximal tubule cells display small apical vacuoles, large apical vacuoles, and apical vacuoles in which a part of the limiting membrane is invaginated into the vacuole. These invaginated apical vacuoles occur more frequently in proximal convoluted tubules than in proximal straight tubules. One tubular cell may contain apical vacuoles of different sizes and stages of invagination, ranging from larger vacuoles with a wide lumen and a small area of invaginated membrane to smaller elements with no apparent lumen and a large area of invaginated membrane. Invaginated apical vacuoles lie either singly in the cytoplasm or close to the membranes of other apical vacuoles, but never in contact with the cell membrane or the membranes of lysosomes, endoplasmic reticulum, Golgi apparatus, mitochondria and peroxisomes.These findings suggest that the invaginated apical vacuoles are not fixation artifacts, but rather develop in living state in cells of the proximal tubule from spherical endocytotic elements.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

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).