Adjacent Microvilli Research Articles

Prey capture and phagocytosis in the choanoflagellate Salpingoeca rosetta.

Choanoflagellates are unicellular and colonial aquatic microeukaryotes that capture bacteria using an apical flagellum surrounded by a feeding collar composed of actin-filled microvilli. Flow produced by the apical flagellum drives prey bacteria to the feeding collar for phagocytosis. We report here on the cell biology of prey capture in rosette-shaped colonies and unicellular “thecate” or substrate attached cells from the choanoflagellate S. rosetta. In thecate cells and rosette colonies, phagocytosis initially involves fusion of multiple microvilli, followed by remodeling of the collar membrane to engulf the prey, and transport of engulfed bacteria into the cell. Although both thecate cells and rosette colony cells produce ∼70 nm “collar links” that connect and potentially stabilize adjacent microvilli, only thecate cells were observed to produce a lamellipod-like “collar skirt” that encircles the base of the collar. This study offers insight into the process of prey ingestion by S. rosetta, and provides a context within which to consider potential ecological differences between solitary cells and colonies in choanoflagellates.

Intestinal Brush Border Assembly Driven by Protocadherin-Based Intermicrovillar Adhesion

Transporting epithelial cells build apical microvilli to increase membrane surface area and enhance absorptive capacity. The intestinal brush border provides an elaborate example with tightly packed microvilli that function in nutrient absorption and host defense. Although the brush border is essential for physiological homeostasis, its assembly is poorly understood. We found that brush border assembly is driven by the formation of Ca(2+)-dependent adhesion links between adjacent microvilli. Intermicrovillar links are composed of protocadherin-24 and mucin-like protocadherin, which target to microvillar tips and interact to form a trans-heterophilic complex. The cytoplasmic domains of microvillar protocadherins interact with the scaffolding protein, harmonin, and myosin-7b, which promote localization to microvillar tips. Finally, a mouse model of Usher syndrome lacking harmonin exhibits microvillar protocadherin mislocalization and severe defects in brush border morphology. These data reveal an adhesion-based mechanism for brush border assembly and illuminate the basis of intestinal pathology in patients with Usher syndrome. PAPERFLICK:

Random amplification of polymorphic DNA (RAPD) typing of carnobacteria isolated from hindgut chamber and large intestine of Atlantic cod ( Gadus morhua l.)

Autochthonous and allochthonous bacteria were isolated from hindgut chamber and large intestine of fed and starved Atlantic cod ( Gadus morhua L.). All bacterial strains isolated from hindgut chamber belong to carnobacteria. However, only 10.2% of the bacteria strains from the large intestine belong to carnobacteria. Random amplification of polymorphic DNA (RAPD) analysis using three selective primers, was performed to further identify the carnobacteria strains. Nine of these were isolated from hindgut chamber contents, ten associated with epithelial cells of the hindgut chamber, and six isolated from the large intestines of fed and starved fish. The 25 isolates segregated into eight clusters. The major cluster comprised nine strains isolated from the hindgut chamber of both fed and starved fish showing low similarity with the reference strains. The other strains isolated from the hindgut were located in clusters showing high similarity with Carnobacterium gallinarum or Carnobacterium piscicola. Strains isolated from large intestine appeared more divergent and were located in five different clusters. Autochthonous (indigenous) bacteria were clearly demonstrated in the hindgut chamber as transmission electron microscopy revealed rod-shaped bacteria between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in the hindgut chamber.

Cholera Toxin Entry into Pig Enterocytes Occurs via a Lipid Raft- and Clathrin-Dependent Mechanism

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

Deep-apical tubules: dynamic lipid-raft microdomains in the brush-border region of enterocytes.

The brush border of small intestinal enterocytes is highly enriched in cholesterol- and glycosphingolipid-containing membrane microdomains, commonly termed as lipid 'rafts'. Functionally, transcytosis of IgA and exocytosis of newly made brush-border proteins in enterocytes occur through apical lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized by the non-permeable surface marker Ruthenium Red in the brush-border region of the cells. The surface-connected tubules were labelled by antibodies to caveolin-1 and the glycolipid asialo G(M1), and they were sensitive to cholesterol depletion by methyl-beta-cyclodextrin, indicating the presence of raft microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50-100 nm, and penetrated up to 1 microm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as the resident brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to cholesterol depletion suggests that deep-apical tubules function as a cell-surface membrane reservoir for cholesterol and for rapid adaptive changes in the size of microvilli at the brush border.

Ultrastructural features of the vas deferens from patients undergoing vasectomy and vasectomy reversal.

Despite more than 30 million vasectomies, the ultrastructural features of the epithelium of the vas deferens (VD) of healthy fertile men, as well as the effects of vasectomy at both proximal (testicular) and distal (abdominal) regions of the VD relative to the initial site of incision, have yet to be fully elucidated. In the present study, the VD from 22 fertile men undergoing vasectomy and 7 vasectomized men undergoing vasectomy reversal were examined by light and transmission electron microscopy. In fertile men, aside from cellular organelles involved in endocytosis and merocrine secretion, the epithelial principal cells showed protrusions of their apical cytoplasm between adjacent microvilli, referred to as "apical blebs." The latter contained solely numerous ribosomes/polysomes and few endoplasmic reticulum (ER) cisternae, unlike the presence of lysosomes, lipofuscin granules, mitochondria, and the Golgi apparatus in the apical principal cell cytoplasm, suggesting the segregation of organelles within blebs. Many apical blebs presented a bulbous extremity with a thin stalklike attachment connecting them to the apical principal cell surface, while others appeared to be isolated and well removed from it, suggesting that blebs are capable of detaching and being liberated into the lumen. We hypothesize that apical blebs represent a type of secretion, referred to as "apocrine secretion." In men undergoing vasectomy reversal, the VD proximal (testicular) to the vasectomy site showed a reduction in the size of principal cells and their microvilli and in the number of apical blebs. In contrast, the lumen of the VD distal (abdominal) to the vasectomy site was virtually abolished, with the epithelium reduced to a flattened layer of cells showing a paucity of organelles and no apical blebs, suggesting that these cells become undifferentiated in the absence of seminal fluids. Taken together, these data may explain, in part, the decreased pregnancy rate noted after vasectomy reversal despite a patent anastomosis.

Lipid Rafts Exist as Stable Cholesterol-independent Microdomains in the Brush Border Membrane of Enterocytes

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

Morphological and functional abnormalities in the ileum of rats with spontaneous hypertension: studies on SGLT1 protein.

Na+-dependent D-glucose and D-galactose transport was studied in the ileal brush-border membrane vesicles from both spontaneously hypertensive rats (SHR) and their normotensive genetic control Wistar-Kyoto (WKY) rats. Initial rates and accumulation ratios of the transport of both monosaccharides were significantly lower in the hypertensive rats compared to the WKY rats. In order to determine whether such modifications are related to morphological abnormalities, ileal epithelium of SHR and WKY rats was examined by light and electron microscopy. In addition, immunohistochemical and immunocytochemical localization of Na+-glucose cotransporter (SGLT1) was performed. Light microscopy studies showed hypertrophy in the ileal villi of hypertensive rats, with an increase in the villus width when compared to those from normotensive rats. Immunohistochemical studies of SGLT1 showed the protein localized in the apical membrane of the absorptive epithelial cells, along the entire villus. No changes between SHR and WKY rats were noted in the intensity and distribution of the SGLT1 protein along the villus-crypt axis. Electron microscopy studies showed a patchy loss of microvilli in the ileal enterocytes of SHR, compared to those from WKY rats. Immunocytochemical studies of SGLT1 were carried out by the immunogold method. Colloidal gold particles were localized at the ileal microvilli of normotensive rats. No significant presence of SGLT1 was found in the smooth apical surface of ileum from hypertensive rats, although most adjacent microvilli were marked. Morphological changes were accompanied by modifications in the sugar transport and in the immunolocalization of SGLT1 in the ileal epithelium of SHR.

Ultrastructural and functional changes in the jejunal epithelium of spontaneously hypertensive rats

Ultrastructural studies on the epithelium, sugar transport and immunocytochemistry of Na +-glucose cotransporter (SGLT1) were carried out in the jejunum of Spontaneously Hypertensive Rats (SHR) and their normotensive genetic control, Wistar-Kyoto (WKY) rats. Electron microscopy studies showed a regular brush-border membrane in the jejunal enterocytes of WKY rats, with colloidal gold particles, representing SGLT1, localized at the microvilli of the absorptive epithelial cells. However, a patchy loss of microvilli was detected in the jejunal sections from SHR, with no presence of colloidal gold particles, indicating the absence of the SGLT1 protein. Most adjacent microvilli were normal in size like those found in WKY rats, and SGLT1 labeling was observed. All these changes were accompanied by a reduction in Na +-dependent D-glucose and D-galactose uptakes in the jejunal BBMVs isolated from SHR, when compared to WKY rats. We conclude that ultrastructural changes were paralleled by modifications in the sugar transport and in the localization of SGLT1 in the jejunal epithelium of SHR.

Epithelium-associated bacteria in the gastrointestinal tract of Arctic charr (Salvelinus alpinus L.). An electron microscopical study.

The primary aim was to use transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to define the location of epithelium-associated bacteria in the digestive tract of the salmonid fish, Arctic charr (Salvelinus alpinus). TEM and SEM examination of the gastrointestinal tract demonstrated substantial numbers of ovoid and rod-shaped bacterial cells associated with the microvillous brush borders of enterocytes. Bacteria were found at the tips of microvilli as well as between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in two regions (pyloric caeca and midgut). Electron microscope examination of the gut is an important tool for evaluating the microbial ecology of the fish digestive tract ecosystem. The results of the current study clearly demonstrate that the intestine is involved in bacterial endocytosis.

Morphological and Functional Abnormalities in the Ileum of Rats with Spontaneous Hypertension: Studies on SGLT1 Protein

Background: Na+-dependent D-glucose and D-galactose transport was studied in the ileal brush-border membrane vesicles from both spontaneously hypertensive rats (SHR) and their normotensive genetic control Wistar-Kyoto (WKY) rats. Initial rates and accumulation ratios of the transport of both monosaccharides were significantly lower in the hypertensive rats compared to the WKY rats. Methods: In order to determine whether such modifications are related to morphological abnormalities, ileal epithelium of SHR and WKY rats was examined by light and electron microscopy. In addition, immunohistochemical and immunocytochemical localization of Na+-glucose cotransporter (SGLT1) was performed. Results: Light microscopy studies showed hypertrophy in the ileal villi of hypertensive rats, with an increase in the villus width when compared to those from normotensive rats. Immunohistochemical studies of SGLT1 showed the protein localized in the apical membrane of the absorptive epithelial cells, along the entire villus. No changes between SHR and WKY rats were noted in the intensity and distribution of the SGLT1 protein along the villus-crypt axis. Electron microscopy studies showed a patchy loss of microvilli in the ileal enterocytes of SHR, compared to those from WKY rats. Immunocytochemical studies of SGLT1 were carried out by the immunogold method. Colloidal gold particles were localized at the ileal microvilli of normotensive rats. No significant presence of SGLT1 was found in the smooth apical surface of ileum from hypertensive rats, although most adjacent microvilli were marked. Conclusion: Morphological changes were accompanied by modifications in the sugar transport and in the immunolocalization of SGLT1 in the ileal epithelium of SHR.

Filamentous structure of the external surface of abalone eggs revealed by quick-freeze deep-etch technique.

The external surface of abalone eggs was examined by thin section and quick-freeze, deep-etch electron microscopy. In thin sections, networks of fine filaments were found interconnecting the adjacent microvilli on the surface of unfertilized eggs. Quick-freeze, deep-etch electron microscopy revealed the three-dimensional structure of these networks of filaments on the external surface of the egg. Mainly two networks of filaments were identified; one was composed of thicker (14-19 nm) filaments interconnecting with the neighboring microvilli nearly horizontally, and the other was composed of thinner (8-14 nm) branched filaments closely surrounding the microvilli surface as well as highly interconnecting neighboring microvilli in a polygonal pattern. The overall structure of the filamentous network on the egg surface showed no distinct alteration after fertilization. These networks of filaments observed on the egg surface may play a key role in sperm-egg interaction.

X-ray diffraction study of the live squid retina.

In previous studies of invertebrate rhabdomes by X-ray diffraction, glutaraldehyde fixation of the retina was used because this tissue is very labile and, without fixation, disintegrates within an hour of dissection. However, with conventional X-ray apparatus more than ten hours exposure time was needed to record a diffraction pattern. In this study, X-ray diffraction patterns from unfixed squid retina could be successfully obtained by use of synchrotron radiation and a storage phosphor screen as detector. The diffraction spots were indexed on a two-dimensional hexagonal lattice of 60 nm. X-ray data was analysed by comparing Patterson functions calculated from the diffraction intensities with those based on model building. The hexagonal shape of microvillar cross section was suggested by the systematic weakness of (0, k) reflections beyond k = 4 and the appearance of the six symmetry-related diffuse maxima around (4 nm)-1. The best-fitting model showed a large gap between adjacent microvilli (approximately 12 nm), which has been expected (for ionic current flow through the inter-microvillus space to generate the membrane potential) but not observed with the chemically fixed retina, possibly due to an artifact of fixation. Also, the existence of massive inter-microvillus material, scarcely observed by conventional electron microscopy, has been confirmed.

Submicrovillar tubules in distal segments of squid photoreceptors detected by rapid freezing

Invertebrate phototransduction is believed to involve an inositol trisphosphate (InsP3)-mediated release of calcium from intracellular storage compartments. Although light-induced production of InsP3 has been demonstrated for squid retinas, morphological evidence for the presence of internal calcium stores has been lacking. Because squid retinas are about 1 mm thick and composed of densely packed receptor cells, conventional aldehyde fixatives may not penetrate rapidly enough to preserve subcellular organelles. To reduce the time for fixative penetration, receptor cells were isolated from intact retinas before fixation, but these techniques provided little improvement in the preservation of membrane-bound compartments. Alternatively, the distal ends of the receptors were ultra-rapidly frozen by dropping 1 mm2 pieces of intact retina against a liquid helium-cooled copper block. Electron micrographs of thick sections from rapidly frozen and freeze-substituted retinas showed elongated saccules oriented parallel to the long axis of the receptor cell and located about 40 nm from the microvillar openings. Freeze-fracture and etch views of rapidly frozen cells showed that the saccules are 130 nm diameter tubules and extend for at least several micrometers along the length of the receptor cell. We call these organelles submicrovillar tubules (SMT). The gap between the SMT and the plasma membrane contains a network of filaments that appear to be actin. Freeze-fracture and etch views of the rhabdomeres also indicate that adjacent microvilli are separated by a 6-8-nm-wide extracellular space along most of their length. This space is spanned by extracellular connections linking adjacent microvilli. The position and orientation of the SMT suggest that these organelles may serve the same function as the more voluminous and highly convoluted submicrovillar cisternae found in other invertebrates. The SMT is likely to be the intracellular compartment that stores and releases calcium as part of the InsP3-mediated light response.

Organoid reorganization of human tumors under in vitro conditions

In the present study we describe a new method to cultivate human tumors, which allows organoid differentiation under in vitro conditions. Diverse tumors of different origin and various histopathology which had been heterotransplanted to athymic mice were dissociated into single cells and seeded at high cell density onto a membrane filter consisting of cellulose nitrate at the gas-medium interface. Within a few days, the tumor cells reorganized and differentiated into organoid structures which exhibited the typical histological characteristics of the original tissues. Due to the formation of organoid aggregates, which was also previously seen with normal fetal cells, this type of culture has been described as 'organoid culture'. In the case of adenocarcinomas of the lung and the colon including the rectum, glandular structures with central lumina, adjacent microvilli, and junctional complexes were formed. Numerous specific intercellular contacts such as desmosomes and tight junctions occurred as well as interdigitations of adjacent cell membranes. In a tumor of the rectum, a typical brush border differentiated at the surface of the reorganized tumor-tissue aggregate. Epidermoid carcinomas of the head and neck developed structures resembling the spinous layer of the epidermis, exhibiting numerous desmosomes and intracytoplasmic bundles of tonofilaments radiating into the desmosomes. Most tumors produced a fragmentary monolayered or multilayered basal lamina of similar morphological appearance as under in vivo conditions. These results illustrate the organoid reorganization and differentiation of human tumor cells under the experimentally rather simple conditions of the organoid culture systems and clearly demonstrate that this in vitro system comes close to the in vivo situation as far as certain differentiation phenomena are concerned.

The extracellular matrix of Xenopus laevis eggs: a quick-freeze, deep-etch analysis of its modification at fertilization.

Eggs of the amphibian, Xenopus laevis, were quick-frozen, deep-etched, and rotary-shadowed. The structure of the extracellular matrix surrounding these eggs, including the perivitelline space and the vitelline envelope (VE), was visualized in platinum replicas by electron microscopy. The perivitelline space contains an elaborate filamentous glycocalyx which connects microvillar tips to the plasma membrane, to adjacent microvilli, and to the overlying VE. The VE is comprised of two layers, the innermost of which is a thin network of horizontal fibrils lying on the tips of the microvilli. The outermost is a thicker layer of large, cable-like fibers which twist and turn throughout the envelope. Upon fertilization, three dramatic modifications of the matrix occur. A thin sheet of smooth material, termed the smooth layer, is deposited on the tips of the microvilli and separates the egg from the overlying envelopes. The VE above is transformed from a thick band of cable-like fibers to concentric fibrous sheets, the altered VE. Finally, an ornate band of particles, corresponding to the fertilization layer in previous studies, is deposited at the altered VE/jelly interface. The altered VE and the fertilization layer comprise the fertilization envelope, which effects the structural block to polyspermy.

Evidence for surface entrapment of cholesterol-rich lipoproteins in luteinized ovary.

Previous studies showed that perfused 125I-labeled low and high density lipoproteins (LDL, HDL) have affinity for specialized microvillar regions of luteal cells in hormone-primed, luteinized rat ovaries. In the current report, we re-examined the interaction of cholesterol-rich lipoproteins with these specialized plasma membrane regions using native lipoproteins visualized as discrete particles by standard electron microscopic techniques. In ovaries perfused with the various lipoproteins, spherical particles (varying in size from 12 to 28 nm depending on the particle used) were found over the surfaces of all luteal cells and filling up extensive "channel" space formed by the apposed plasma membranes of adjacent microvilli or cytoplasmic surfaces. Only 30% of these tissue-associated particles were removable after prolonged washing with perfused media or heparin. Few intact particles were found inside the cells, despite the fact that the lipoproteins induced a substantial hormone response by the ovary. To determine the total protein internalized by cells during the course of the experiments, parallel biochemical experiments were carried out with nonreleasable (14C-sucrose-coupled) human LDL. Of the total bound 14C-sucrose LDL, only 8.5% was degraded (trichloroacetic acid-soluble) and presumed internalized by the cells. Thus, while large numbers of cholesterol-rich lipoprotein particles interact with the luteal cell surface in specialized microvillar channels and elicit a progesterone response, relatively few intact lipoprotein particles appear to enter the cells to be degraded. We speculate that in the luteinized ovary, a large majority of the lipoprotein-cholesterol transfer occurs at the surface of the luteal cells, and that the membranes of the microvillar channels are involved in this process.

The paracrystalline structure of an insect rhabdomere (Calliphora erythrocephala)

By use of a modified fixation technique, the receptor cells of the compound eye of the blowfly Calliphora erythrocephala were found to contain a regular, paracrystalline array of alternating rows of hexagonally shaped microvilli. The receptor cells R1 to R6 have a cell-specific number of microvilli per row in a cross section. Every microvillus has a filament cluster connecting the axial skeleton with the microvillar membrane. This cluster is preferentially right-left oriented relative to the longitudinal axis of the microvillar array. Three adjacent microvilli are interconnected by an electron-dense substance. A mirroring technique indicated that this intermicrovillar structure consists of three subunits, although these subunits could not be conclusively demonstrated by classical densitometry or image subtraction techniques. The electron-dense substance can be seen in all cross sections of the proximal and distal parts of the microvilli. They are cylindrical structures separating the microvilli along their entire length. It is suggested that these cylindrical aggregates contain an enzymatic complex separating the rhodopsin-containing microvillar membrane into six compartments.

Prokaryotic-eukaryotic cell junctions: Attachment of spirochetes and flagellated bacteria to primate large intestinal cells

The attachment sites of two types of commensal gram-negative bacteria to surface epithelial cells in human rectum and monkey colon were studied by electron microscopy of thin sections and freeze-fracture replicas. Spirochetes and flagellates are known to coexist in the brush border region of primate colonic epithelial cells, but their mode of attachment has not been described in detail. Both elongated, spiral organisms adhere by one end to columnar cell apical surfaces, oriented parallel to host cell microvilli. The attached end of the flagellate scarcely indents the host cell membrane and does not adhere to adjacent microvilli. The attachment site involves a circumferential cuff of bacterial outer membrane which is separated from host cell membrane by a uniform 6-nm gap filled with fibrillar cell coat material. Peroxidase passed freely through this gap. In freeze-fracture replicas, the zone of adherence is marked by a loose band of heterogeneous particles on the inner leaflet of the outer bacterial membrane. The spirochete, in contrast, pushes a long sleeve of host cell membrane into the terminal web cytoplasm and makes close contact with the invaginated columnar cell membrane and with adjacent microvilli. Freeze-fracture reveals that at the point where the spirochete body passes into the columnar cell glycocalyx, a series of irregularly spaced circumferential particle arrays appears on the inner leaflet of the bacterial cell outer membrane. These arrays extend over the entire embedded portion of the organism, but are replaced at the embedded tip by a distinct, closely packed series of circular ridges and grooves. Columnar cell membranes show no intra or submembrane specialization at the attachment sites of either organism. Autoradiographs reveal that radiolabeled glucosamine and N-acetylmannosamine (but not sulfate or fucose) may be incorporated by the bacteria under organ culture conditions, and that maturation of the columnar cell glycocalyx is a crucial factor in bacterial attachment.

The morphology and fine structure of the larval midgut of a moth ( Manduca sexta) in relation to active ion transport

Light and electron microscopic examination of the midgut of Manduca sexta has shown that the organization of this tissue is more complex than was originally believed. The midgut can be divided into anterior, middle and posterior regions on the basis of the pattern of folding of the epithelial sheet, and variations in the structure of goblet and columnar cells which occur along its length. The columnar cells show gradual structural changes from the anterior to the posterior end of the midgut. For example, the microvilli in the anterior region form a dense, interconnecting network from which vesicles break off. This organization becomes less obvious through the middle region, until by the posterior region each microvillus is unconnected to adjacent microvilli along its entire length and vesicles are no longer produced. Two distinct types of goblet cells are found. In the anterior and middle regions the goblet cells have a large basally located cavity, but in the posterior region the cavity occupies only the apical half of the cell. In both cases the cavity is formed by invagination of the apical membrane, which is studded with small particles implicated in active ion transport. In the anterior and middle regions this membrane is closely associated with mitochondria, but not in the posterior region. The significance of the observed structural differences is discussed in relation to active ion transport.