Extensive Microvilli Research Articles

Circadian rhythms in the Drosophila eye may regulate adaptation of vision to light intensity.

The sensitivity of the eye at night would lead to complete saturation of the eye during the day. Therefore, the sensitivity of the eye must be down-regulated during the day to maintain visual acuity. In the Drosophila eye, the opening of TRP and TRPL channels leads to an influx of Ca++ that triggers down-regulation of further responses to light, including the movement of the TRPL channel and Gα proteins out of signaling complexes found in actin-mediated microvillar extensions of the photoreceptor cells (the rhabdomere). The eye also exhibits a light entrained-circadian rhythm, and we have recently observed that one component of this rhythm (BDBT) becomes undetectable by antibodies after exposure to light even though immunoblot analyses still detect it in the eye. BDBT is necessary for normal circadian rhythms, and in several circadian and visual mutants this eye-specific oscillation of detection is lost. Many phototransduction signaling proteins (e.g., Rhodopsin, TRP channels and Gα) also become undetectable shortly after light exposure, most likely due to a light-induced compaction of the rhabdomeric microvilli. The circadian protein BDBT might be involved in light-induced changes in the rhabdomere, and if so this could indicate that circadian clocks contribute to the daily adaptations of the eye to light. Likewise, circadian oscillations of clock proteins are observed in photoreceptors of the mammalian eye and produce a circadian oscillation in the ERG. Disruption of circadian rhythms in the eyes of mammals causes neurodegeneration in the eye, demonstrating the importance of the rhythms for normal eye function.

MYOFIBROBLASTS HINDER RECOVERY OF HASHIMOTO THYROIDITIS IN THE ULTRASTRUCTURAL LEVEL.

Hashimoto thyroiditis (HT) is an autoimmune disorder associated with hypothyroidism. Lymphocyte infiltration leading to thyroid follicular cell destruction is counteracted by increased collagen production, deposition and scarring. However, only recently a specific subpopulation of modified fibroblasts with contractile properties, namely "myofibroblasts" (MFBs) have been linked to HT. Our ultrastructural study aims to delineate the presence and contribution of MFBs to the fibrotic milieu of HT. Tissue biopsies were obtained from 5 HT-diagnosed patients and specimens were examined using a Transmission Electron Microscope (TEM). Histopathological examination indicated extensive microvilli atrophy and atypical vacuolations of the thyroid follicular cells in the HT samples. In addition to interstitial extravasated lymphocytes, capillaries were encircled by MFBs (mean distance from lumen 1.248± 0.43µm) with the characteristic electron-dense α-smooth muscle actin (α-SMA), confirmable in higher magnifications. Myofibroblastic projections were found to have significantly higher representation near the capillary lumen compared to the impaired endothelial lining (P < 0.01). Our TEM findings suggest that the intrusion of endothelia by myofibroblastic projections can be a significant factor towards the malfunction of follicular cells in HT patients and offer a paradigmal understanding of the ultrastructural interactions that may underlie the HT pathology.

T-cell microvilli constitute immunological synaptosomes that are specialized for intercellular communications

Abstract Lymphocytes contain abundant flexible projections termed microvilli, the inside of which contain many parallel bundles of actin filaments that extend the cell membrane in the form of a finger. Previous reports demonstrated microvilli on T cells have been proposed to survey surfaces of antigen-presenting cells (APC) or facilitate adhesion under flow. However, in addition to their key role in antigen sensing on APCs, whether they serve essential functions during T cell activation remains largely unclear. In this study, we observed that single T cell contacts with APCs occur through microvillar extensions, which appear to serve as locations for sequestration of immunologically important molecules, including TCR complexes, costimulatory and adhesion molecules, and various cytokines. Strikingly, we found that microvilli were separated from the T cell body by the combined action of two independent mechanisms (trogocytosis and membrane budding) and are deposited at the surface of cognate APCs, thereby potentially acting as the most efficient and effective means of delivering T cell messages to cognate APCs. Consistent with this potential role, these T cell microvilli-derived particles (TMPs) were independently capable of activating cognate dendritic cells (DCs). Therefore, our findings suggest that T cell microvilli might serve as ‘immunological synaptosomes’ with TMPs as a novel class of membrane vesicles serving as conveyors of T cell messages or traits to cognate APCs.

Deletion of Dock10 in B Cells Results in Normal Development but a Mild Deficiency upon In Vivo and In Vitro Stimulations.

We sought to identify genes necessary to induce cytoskeletal change in B cells. Using gene expression microarray, we compared B cells stimulated with interleukin-4 (IL-4) and anti-CD40 antibodies that induce B cell spreading, cell motility, tight aggregates, and extensive microvilli with B cells stimulated with lipopolysaccharide that lack these cytoskeletal changes. We identified 84 genes with 10-fold or greater expression in anti-CD40 + IL-4 stimulated B cells, one of these encoded the guanine nucleotide exchange factor (GEF) dedicator of cytokinesis 10 (Dock10). IL-4 selectively induced Dock10 expression in B cells. Using lacZ expression to monitor Dock10 promoter activity, we found that Dock10 was expressed at all stages during B cell development. However, specific deletion of Dock10 in B cells was associated with a mild phenotype with normal B cell development and normal B cell spreading, polarization, motility, chemotaxis, aggregation, and Ig class switching. Dock10-deficient B cells showed lower proliferation in response to anti-CD40 and IL-4 stimulation. Moreover, the IgG response to soluble antigen in vivo was lower when Dock10 was specifically deleted in B cells. Together, we found that most B cell responses were intact in the absence of Dock10. However, specific deletion of Dock10 in B cells was associated with a mild reduction in B cell activation in vitro and in vivo.

Die Belegzelle des Magens: diametrale Bedeutung für die gastroösophageale Refluxkrankheit (GERD)

BACKGROUND: The gastric parietal cell (also called oxyntic cell) is a remarkable physiological entity in view of its regulation and participation in digestion, but also as an exemplary model for its exaggerated biophysical and cell biological processes. Moreover, the ability to pharmacologically control parietal cell activity has proven enormous value in treating a number of pathological conditions. METHODS: This review summarizes what is known about some of the most important physiological activities of the gastric parietal cell. RESULTS: Clues from differences in phenotype between resting and actively secreting cells provide a morphological basis for the massive biological process of intracellular membrane recycling, and explain the regulation by recruitment of the primary proton pump, H,K-ATPase, to the secretory surface. The large changes in membrane surface area require the sub-structural support of the filamentous actin cytoskeleton, which together with a dynamic membrane-cytoskeleton linker protein, known as ezrin, offer the scaffold for large membrane elaborations as well as required plasticity of the resulting microvillar extensions. Studies on basic protein/glycoprotein structure of H,K-ATPase have offered autoprotective mechanisms for the proton pump, and suggest the possibility that similar features in other gastric apical surface proteins may provide a molecular basis to why the stomach does not digest itself. An adjacent anatomical area, the esophagus, does not have the power to resist autodigestion resulting from backflux of gastric (and duodenal) juices; this is the pathological condition called gastroesophageal reflux disease, GERD. Interestingly, GERD related metaplastic changes in the distal esophageal epithelium ultimately result in one of two epithelial phenotypes: one called cardio-oxyntic mucosa, rarely leading to more serious consequences; and the other intestinal mucosa, characterized by goblet cells and having increased probability for progression to dysplasia and adenocarcinoma. CONCLUSIONS: Factors that promote lower esophageal metaplasia and dysplasia have interesting similarities with atrophic gastritis and glandular metaplasia of the gastric mucosa, i.e., intestinalization indicative of adenocarcinoma. These comparisons are discussed for the parietal cell in an unusual environment.

Microvillous Inclusion Disease: Ultrastructural Variability

Microvillous inclusion disease (MVID) is a congenital, usually neonatal, autosomal recessive condition manifested by severe, prolonged secretory diarrhea. Intestinal biopsies reveal extensive microvilli abnormalities, typical inclusions and vesicles mainly of the apical-luminal enterocytes and colonocytes. Although diagnosis can be suspected by special stains of the mucosa (PAS, CD10), the definitive diagnosis, recommended in view of potential intestinal transplantation, requires electron microscopy. In view of the marked variability of ultrastructural changes, extensive illustration is considered valuable for diagnosis. While the pathogenesis is still unknown, a number of images illustrate the suspected “arrested-trafficking” hypothesis of microvillous abnormalities. Others micrographs support the “engulfing” mechanism of inclusion formation. The electron micrographs should help ultrastructural diagnosis in this heterogeneous disease and can confirm diagnosis even in the absence of the typical inclusions.

Quantitative analysis of SR-BI-dependent HDL retroendocytosis in hepatocytes and fibroblasts

Previous studies have suggested that HDL retroendocytosis may play a role in scavenger receptor class B type I (SR-BI)-dependent selective lipid uptake in a cell-specific manner. To investigate this possibility, we developed methods to quantitatively measure HDL uptake and resecretion in fibroblast (COS-7) and hepatocyte (HepG2) cells expressing exogenous SR-BI. Approximately 17% and 24% of HDL associated in an SR-BI-dependent manner with COS-7 and HepG2 cells, respectively, accumulates intracellularly after a 10 min incubation. To determine whether this intracellular HDL undergoes retroendocytosis, we developed a pulse-chase assay whereby internalized biotinylated (125)I-HDL(3) secreted from cells is quantitatively precipitated from cell supernatants using immobilized streptavidin. Our results show a rapid secretion of a portion of intracellular HDL from both cell types (representing 4-7% of the total cell-associated HDL) that is almost complete within 30 min (half-life approximately 10 min). In COS-7 cells, the calculated rate of HDL secretion ( approximately 0.5 ng HDL/mg/min) was >30-fold slower than the rate of SR-BI-dependent selective cholesteryl ester (CE) uptake ( approximately 17 ng HDL/mg/min), whereas the rate of release of HDL from the cell surface ( approximately 19 ng HDL/mg/min) was similar to the rate of selective CE uptake. Notably, the rate of SR-BI-dependent HDL resecretion in COS-7 and HepG2 cells was similar. BLT1, a compound that inhibits selective CE uptake, does not alter the amount of SR-BI-mediated HDL retroendocytosis in COS-7 cells. From these data, we conclude that HDL retroendocytosis in COS-7 and HepG2 cells is similar and that the vast majority of SR-BI-dependent selective uptake occurs at the cell surface in both cell types.

Hepatic SR-BI-mediated cholesteryl ester selective uptake occurs with unaltered efficiency in the absence of cellular energy

Scavenger receptor class B type I (SR-BI) plays a critical role in the delivery of HDL cholesterol and cholesteryl esters (CEs) to liver and steroidogenic tissues by a selective process that does not result in significant degradation of HDL protein. Recently, SR-BI-mediated endocytosis and recycling of HDL have been demonstrated. However, it remains unclear whether efficient SR-BI-mediated selective uptake occurs strictly at the plasma membrane or at additional sites along its endocytic itinerary. To examine the requirement for SR-BI endocytosis in HDL selective uptake, we determined the effects of energy depletion on the levels of cell-associated HDL protein and CE in primary mouse hepatocytes. Compared with CHO cells, we observed a much larger energy-dependent effect on CE uptake in primary mouse hepatocytes. Although varying the levels of caveolin-1 and carboxyl ester lipase altered the efficiency of selective uptake, neither was able to account for the energy-dependent component of HDL-CE uptake. Finally, we demonstrate that the hepatocyte-specific, energy-dependent effects on HDL-apolipoprotein A-I and -CE uptake are independent of SR-BI and are not required to achieve efficient SR-BI-mediated selective uptake of CE. Together, these data support the conclusion that neither the intracellular trafficking of HDL nor any energy-dependent cellular process affects the ability of the cell to maximally acquire CE through SR-BI-mediated selective uptake from HDL.

Phytomonas serpens: cysteine peptidase inhibitors interfere with growth, ultrastructure and host adhesion

In this study, we report the ultrastructural and growth alterations caused by cysteine peptidase inhibitors on the plant trypanosomatid Phytomonas serpens. We showed that the cysteine peptidase inhibitors at 10 μM were able to arrest cellular growth as well as promote alterations in the cell morphology, including the parasites becoming short and round. Additionally, iodoacetamide induced ultrastructural alterations, such as disintegration of cytoplasmic organelles, swelling of the nucleus and kinetoplast–mitochondrion complex, which culminated in parasite death. Leupeptin and antipain induced the appearance of microvillar extensions and blebs on the cytoplasmic membrane, resembling a shedding process. A 40 kDa cysteine peptidase was detected in hydrophobic and hydrophilic phases of P. serpens cells after Triton X-114 extraction. Additionally, we have shown through immunoblotting that anti-cruzipain polyclonal antibodies recognised two major polypeptides in P. serpens, including a 40 kDa component. Flow cytometry analysis confirmed that this cruzipain-like protein has a location on the cell surface. Ultrastructural immunocytochemical analysis demonstrated the presence of the cruzipain-like protein on the surface and in small membrane fragments released from leupeptin-treated parasites. Furthermore, the involvement of cysteine peptidases of P. serpens in the interaction with explanted salivary glands of the phytophagous insect Oncopeltus fasciatus was also investigated. When P. serpens cells were pre-treated with either cysteine peptidase inhibitors or anti-cruzipain antibody, a significant reduction of the interaction process was observed. Collectively, these results suggest that cysteine peptidases participate in several biological processes in P. serpens including cell growth and interaction with the invertebrate vector.

Taste buds on the lips and mouth of some blenniid and gobiid fishes: comparative distribution and morphology

Analysis of taste buds (TB) on the lips and oropharyngeal cavity in several species of gobies (Gobiidae) and blennies (Blenniidae) from the Red Sea, Mediterranean Sea and Seychelles, revealed three types of these organs: types I and II, which protrude above the surrounding epithelium on lobules of various forms, and type III, which terminate on the level of the epithelium. These TB are composed of either light or dark sensory cells with apical microvillar extensions, and of basal cells situated at the TB base. Synaptic junctions occur between the TB cells and the sub‐epithelial sensory nerves. Numerical distribution and morphology of TB on the lips and in the oral cavity of the species studied revealed patterns that are specific on both species and family levels. In most of the gobies the lips, jaws and oral breathing valves are usually covered by numerous lobules, each of which bears papillae with two to seven type I and type II TB, reaching a total of up to 7500 buds on the lips and in the oropharyngeal cavity in these fishes. The number of TB increases with growth (age) of the fish, and the combined and total sensory area of TB in an adult fish can reach up to 80 000 μm2. In contrast, in blennies the anterior region of the oral cavity is seldom lobulated, with far fewer TB; the majority of TB are found in the more posterior region. It is postulated that these differences in TB density and location between gobies and blennies are connected to differences in foraging strategies and diet, and may represent ecomorphological adaptations.

A Sphingomonas bacterium interacting with epithelial cells

Bacteria of the genus Sphingomonas are environmental organisms that have recently been implicated in a variety of community-acquired and nosocomial infections. During studies on bacteria–cell interactions, we incurred a microorganism contaminating our HeLa cell culture, possibly from water utilized for reagent preparation; this bacterium appeared to tightly adhere to cell monolayers and to survive, with only limited growth rate, which did not seem to alter cells as far as shape, growth rate or survival were concerned. The contaminating organism was isolated and partially characterized by morphological, genetic, and biochemical assays. Mechanisms of cell interaction and entry into epithelial cells were investigated by electron microscopy, immunofluorescence, and biochemical inhibitors. Morphological and biochemical features indicated that the microorganism belonged to the genus Sphingomonas. Electron microscopy showed that contact between the Sphingomonas bacterium and epithelial cells leads to a dramatic alteration of the cell surface, with formation of numerous microvillar extensions plus membrane ruffling. Confocal microscopy and the use of inhibitors showed that actin microfilaments were involved during attachment and entry into HeLa cells. Macropinosome formation and an inhibitory effect by amiloride indicate that internalization occurs in part via a macropinocytosis mechanism. Moreover, cholesterol distribution at the site of bacterial binding suggests that Sphingomonas bacteria could use the lipid rafts as initial binding sites.

In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonic stem cells.

To determine whether primate embryonic stem (ES) cell-derived pigment epithelial cells (ESPEs) have the properties and functions of retinal pigment epithelial (RPE) cells in vitro and in vivo. Cynomolgus monkey ES cells were induced to differentiate into pigment epithelial cells by coculturing them with PA6 stromal cells in a differentiating medium. The expanded, single-layer ESPEs were examined by light and electron microscopy. The expression of standard RPE markers by the ESPEs was determined by RT-PCR, Western blot, and immunocytochemical analyses. The ESPEs were transplanted into the subretinal space of 4-week-old Royal College of Surgeons (RCS) rats, and the eyes were analyzed immunohistochemically at 8 weeks after grafting. The effect of the ESPE graft on the visual function of RCS rats was estimated by optokinetic reflex. The expanded ESPEs were hexagonal and contained significant amounts of pigment. The ESPEs expressed typical RPE markers: ZO-1, RPE65, CRALBP, and Mertk. They had extensive microvilli and were able to phagocytose latex beads. When transplanted into the subretinal space of RCS rats, the grafted ESPEs enhanced the survival of the host photoreceptors. The effects of the transplanted ESPEs were confirmed by histologic analyses and behavioral tests. The ESPEs had morphologic and physiological properties of normal RPE cells, and these findings suggest that these cells may provide an unlimited source of primate cells to be used for the study of pathogenesis, drug development, and cell-replacement therapy in eyes with retinal degenerative diseases due to primary RPE dysfunction.

Scavenger receptor BI (SR-BI) clustered on microvillar extensions suggests that this plasma membrane domain is a way station for cholesterol trafficking between cells and high-density lipoprotein.

Receptor-mediated trafficking of cholesterol between lipoproteins and cells is a fundamental biological process at the organismal and cellular levels. In contrast to the well-studied pathway of LDL receptor-mediated endocytosis, little is known about the trafficking of high-density lipoprotein (HDL) cholesterol by the HDL receptor, scavenger receptor BI (SR-BI). SR-BI mediates HDL cholesteryl ester uptake in a process in which HDL lipids are selectively transferred to the cell membrane without the uptake and degradation of the HDL particle. We report here the cell surface locale where the trafficking of HDL cholesterol occurs. Fluorescence confocal microscopy showed SR-BI in patches and small extensions of the cell surface that were distinct from sites of caveolin-1 expression. Electron microscopy showed SR-BI in patches or clusters primarily on microvillar extensions of the plasma membrane. The organization of SR-BI in this manner suggests that this microvillar domain is a way station for cholesterol trafficking between HDL and cells. The types of phospholipids in this domain are unknown, but SR-BI is not strongly associated with classical membrane rafts rich in detergent-resistant saturated phospholipids. We speculate that SR-BI is in a more fluid membrane domain that will favor rapid cholesterol flux between the membrane and HDL.

Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic.

We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day) 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (approximately 34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.

Evolution of cerebral vesicles and their sensory organs in an ascidian larva

AbstractSorrentino M., Manni L., Lane N. J. and Burighel P. 2000. Evolution of cerebral vesicles and their sensory organs in an ascidian larva. —Acta Zoologica (Stockholm) 81: 243–258The ascidian larval nervous system consists of the brain (comprising the visceral ganglion and the sensory vesicle), and, continuous with it, a caudal nerve cord. In most species two organs, a statocyst and an ocellus with ciliary photoreceptors, are contained in the sensory vesicle. A third presumptive sensory organ was sometimes found in an ‘auxiliary’ ganglionic vesicle. The development and morphology of the sensory and auxiliary ganglionic vesicles in Botryllus schlosseri and their associated organs was studied. The sensory vesicle contains a unique organ, the photolith, responding to both gravity and light. It consists of a unicellular statocyst, in the form of an expanded pigment cup receiving six photoreceptor cell extensions. Presumptive mechano‐receptor cells (S1 cells), send ciliary and microvillar protrusions to contact the pigment cup. A second group of distinctive cells (S2), slightly dorsal to the S1 cells, have characteristic microvillar extensions, resembling photoreceptor. We concur with the idea that the photolith is new and derived from a primitive statocyst and the S2 cells are the remnant of a primitive ocellus. In the ganglionic vesicle some cells contain modified cilia and microvillar extensions, which resemble the photoreceptor endings of the photolith. Our results are discussed in the light of two possible scenarios regarding the evolution of the nervous system of protochordates.

Invasive ability of an Escherichia coli strain isolated from the ileal mucosa of a patient with Crohn's disease.

Crohn's disease (CD) is an inflammatory bowel disease in which Escherichia coli strains have been suspected of being involved. We demonstrated previously that ileal lesions of CD are colonized by E. coli strains able to adhere to intestinal Caco-2 cells but devoid of the virulence genes so far described in the pathogenic E. coli strains involved in gastrointestinal infections. In the present study we compared the invasive ability of one of these strains isolated from an ileal biopsy of a patient with CD, strain LF82, with that of reference enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteraggregative (EAggEC), enterohemorrhagic (EHEC), and diffusely adhering (DAEC) E. coli strains. Gentamicin protection assays showed that E. coli LF82 was able to efficiently invade HEp-2 cells. Its invasive level was not significantly different from that of EIEC and EPEC strains (P > 0.5) but significantly higher than that of ETEC (P < 0.03), EHEC (P < 0. 005), EAggEC (P < 0.004) and DAEC (P < 0.02) strains. Strain LF82 also demonstrated efficient ability to invade intestinal epithelial cultured Caco-2, Intestine-407, and HCT-8 cells. Electron microscopy examination of infected HEp-2 cells revealed the presence of numerous intracellular bacteria located in vacuoles or free in the host cell cytoplasm. In addition, the interaction of strain LF82 with epithelial cells was associated with the elongation of microvillar extensions that extruded from the host cell membranes and engulfed the bacteria. This internalization mechanism strongly resembles Salmonella- or Shigella-induced macropinocytosis. The use of cytochalasin D and colchicine showed that the uptake of strain LF82 by HEp-2 cells was mediated by both an actin microfilament-dependent mechanism and microtubule involvement. In addition, strain LF82 survived for at least 24 h in HEp-2 and Intestine-407 cells and efficiently replicated intracellularly in HEp-2 cells. PCR and hybridization experiments did not reveal the presence of any of the genetic determinants encoding EIEC, EPEC, or ETEC proteins involved in bacterial invasion. Thus, these findings show that LF82, which colonized the ileal mucosa of a patient with CD, is a true invasive E. coli strain and suggest the existence of a new potentially pathogenic group of E. coli, which we propose be designated adherent-invasive E. coli.

Schistosoma mansoni: the ultrastructure of larval morphogenesis in Biomphalaria glabrata and of associated host-parasite interactions.

An electron microscopic study has been carried out to describe the transformation of the miracidium of S. mansoni into the mother sporocysts in the susceptible B. glabrata and the associated host-parasite interactions. The miracidium enters the snail host without morphological alterations. Within 3 hr after entering, all the ciliary epidermal plates of the miracidium are discarded. A new tegument is quickly formed by 5 hr postinfection by the expansion of epidermal ridges. The rapid formation of the new tegument reflects the participation of membrane-bound vesicles in the ridge cytons. The membranes of these vesicles become the new tegument membranes with the discharge of their electron-dense contents into the snail tissues. The vesicular contents discharged into the tissues apparently prevent snail amoebocytes (phagocytes) from attachment to the parasite tegument and thus prevent their interference with the further development of the postmiracidium. If a postmiracidium fails to mobilize membrane-bound vesicles in the formation of tegument, the parasite becomes surrounded by closely attached concentric layers of fibroblasts formed by amoebocytes and histiocytes within 24 hr. The membrane-bound vesicles are present in small numbers in the ridge cytons of the miracidium and become numerous in the postmiracidium stage and with many migrate to the ridges through connecting bridges within 24 hr. By 3 days postinfection when extensive microvilli have formed on the tegument the vesicles have disappeared and are replaced by mitochondria, ribosomes and complex carbohydrate particles. Many fibroblasts in the snail connective tissues have phagocytic capacities and are regarded as snail tissue histiocytes or fixed amoebocytes that eventually may become hypertrophic and detached.

Expression of the Amyloid Precursor Protein of Alzheimer's Disease on the Surface of Transfected HeLa Cells

The principal component of the amyloid which accumulates in Alzheimer's Disease brain is a 4-kDa βA4 fragment of the amyloid precursor protein (APP). Although APP has the structural features of an integral transmembrane receptor, there has been limited evidence for expression of APP at the plasma membrane. The function of APP and related molecules is unknown. Using rabbit antisera to purified human brain APP, surface labeling of APP is demonstrable in HeLa cells transfected with the APP695 isoform. Indirect immunofluorescence indicates the presence of APP at the surface of unfixed or aldehyde-fixed cells; preembedding immunoelectron microscopy using 5- or 1-nm gold particles and silver enhancement confirms plasma membrane labeling as well as labeling within intracellular membrane vesicles. Immunolabeling of unfixed cells at 4°C followed by incubation at 37°C shows APP within endocytic vesicles. Transfected HeLa cells with prominent surface APP were larger with more extensive microvilli than nonimmunoreactive HeLa cells. This is consistent with the postulated role of APP as a mediator of cell surface adhesion and membranematrix stabilization.

Epithelial Lining of the Sheep Ampulla Oviduct Undergoes Pregnancy-Associated Morphological Changes in Secretory Status and Cell Height1

The biosynthetic activity of the sheep ampulla oviduct changes during the first few days of pregnancy. An estrogen (E2)-dependent glycoprotein (M(r) 90,000-92,000) is expressed, synthesized, and released when fertilization and embryo development occur in the oviduct. The objective of the present study was to determine whether the secretory epithelial lining of the ampulla, the cellular source of the E2-dependent protein, undergoes morphological alterations during early pregnancy is a contemporaneous manner. Ampulla oviducts obtained from ovariectomized (ovx), estrous (Day 0), and pregnant (Days 1.5, 2, 3, 4, 6, and 16) ewes were analyzed with light and electron microscopy. The mean cell height of epithelial cells from estrus and from Days 1.5, 2, and 3 of pregnancy was significantly (p < 0.05) higher than in ovx animals. A significant reduction in cell height was observed at Day 4 of pregnancy, and this value remained unchanged at Day 6 and Day 16. Ciliated and nonciliated cells were present in the ampulla at all stages examined, with ciliated cells constituting the major cell type. Consistent with previous results, the nonciliated cells obtained from ovx ewes were synthetically inactive and at estrus were characterized by well-developed secretory organelles, including abundant amounts of secretory granules containing the E2-dependent glycoprotein. Extensive microvilli and cytoplasmic blebs protruding into the oviduct lumen distinguished the apical surface of secretory epithelial cells until Day 3. At Day 1.5 the secretory cells contained dilated cisternae of rough endoplasmic reticulum and enlarged Golgi, which were progressively diminished with increasing stages of pregnancy. Exocytosis, fusion of secretory granules, and a reduction in their electron density were seen until Days 3-4. By Day 4 and Day 6, fewer granules were observed in the ampulla cytoplasm and few, if any, were present by Day 16. Increases in the amount of heterochromatin were seen in nuclei at Day 6 and Day 16. Extrusion of epithelial cells into the oviduct-lumen was evident at Day 16 of pregnancy. These data show that 1) the secretory epithelium of the sheep ampulla oviduct undergoes morphological alterations in protein-synthesizing organelles and apical specializations that vary with the stage of pregnancy, 2) the secretory product contained in lamellar granules is released by the process of exocytosis until Days 3-4, and 3) cell death appears to occur at Day 16 by shedding of epithelial cells into the oviduct lumen.

Elevated cAMP levels induce multilayering of MDCK cells without disrupting cell surface polarity.

The effect of hormones on the morphology and cell surface polarity of the epithelial cell line MDCK was examined. When MDCK cells were seeded in high densities in media containing FCS a regular monolayer was formed. However, in serum-free medium supplemented with insulin, transferrin, prostaglandin E1, hydrocortisone and triiodothyronine, the development of a multilayer with intercellular lumina was observed. In hormone-depletion studies we identified PGE1 as the inducer of these multilayers. Since dibutyryl cyclic AMP and the phosphodiesterase inhibitor isobutyl methylxanthine could substitute for PGE1, we conclude that an elevated intracellular cAMP level resulted in formation of the multilayer. Further analysis by electron microscopy and immunocytochemistry revealed a polarized organization of the multilayered cells. Junctional complexes, enclosing microvilli-rich membrane domains, were found at the apices of adjacent cells facing the medium and those surrounding the intercellular lumina. Surprisingly, cells participating in the formation of both the free surface and the surface of the intercellular lumen, exhibited two distinct membranes with microvilli, each separated by junctional complexes. Immunolocalization of membrane marker proteins demonstrated that an apical 114 kDa membrane protein was localized to the free cell surfaces, the same membrane domains where extensive microvilli were also observed. The distribution of a basolateral 58 kDa membrane protein was restricted to sites of cell contact. These results provided evidence that nontransformed epithelial MDCK cells form multilayers in response to elevated cAMP levels; however, they retain the potential of developing cell surface polarity.