Electron microscopic studies of coated membranes in two types of gill epithelial cells of lamprey

Summary Coated membranes in two types of gill epithelial cell of adult lamprey, Lampetra japonica, were studied by electron microscopy. The type 3 gill epithelial cells possess well-developed microvilli or microfolds, apical vesicles and abundant mitochondria. The cytoplasmic surface of the microvillous plasma membrane is covered by a coat of regularly spaced particles with a center-to-center distance of about 15 nm. Each particle consists of a bulbous free end, about 10 nm in diameter, and a connecting piece, about 5 nm long. Apical vesicles are covered by a surface coat which consists of fine filamentous material but lack any special coating on their cytoplasmic surface.The type 4 cells (chloride cells) are characterized by apical vesicles, abundant mitochondria and cytoplasmic tubules. These tubules possess a coat on their luminal surface which consists of spirally wound parallel rows of electron-dense materials. The rows are about 16 nm apart and wound at a pitch of about 45°. The cytoplasmic surface of these tubules does not display a special coat. These coated membranes are assumed to be the sites of active ion transport across the plasma membrane. In particular, particles in type 3 cells and linear coat materials in chloride cells may be either loci of transport enzymes or energy generating systems. Apical vesicles lack any coating on their cytoplasmic surface but a fine filamentous coat is present on their luminal surface. They contain intraluminal vesicles and are continuous with apical ends of cytoplasmic tubules.     

Elektronenmikroskopische Untersuchungen von Differenzierungsvorgängen bei Moosen

Summary In meristematic cells of the gemma of Riella helicophylla and in young bud cells from the protonema of Funaria hygrometrica the cell plate is formed by fusion of small vesicles originating from the Golgi apparatus. These spherical vesicles of about 0.1 m diameter have an electron dense centre, probably consisting of pectic substances or their precursors. The endoplasmic reticulum producing multivesicular bodies participate in cell plate formation too. Another cytoplasmic component forming the cell plate are coated vesicles, the origin of which is the Golgi apparatus and perhaps also the endoplasmic reticulum. In view of these observations the question of whether the endoplasmic reticulum or the Golgi apparatus forms the cell plate must be answered in this way: both endoplasmic reticulum and Golgi apparatus supply material for growth of the cell plate. Multivesicular bodies, coated vesicles and other small vesicles of unknown nature participate in the formation of the primary wall.

---

---

Zum Teil finanziert mit Sondermitteln des Landes Niedersachsen an Prof. Dr. M. Bopp.     

Self-organizing actin waves as planar phagocytic cup structures

Actin waves that travel on the planar membrane of a substrate-attached cell underscore the capability of the actin system to assemble into dynamic structures by the recruitment of proteins from the cytoplasm. The waves have no fixed shape, can reverse their direction of propagation and can fuse or divide. Actin waves separate two phases of the plasma membrane that are distinguished by their lipid composition. The area circumscribed by a wave resembles in its phosphoinositide content the interior of a phagocytic cup, leading us to explore the possibility that actin waves are in-plane phagocytic structures generated without the localized stimulus of an attached particle. Consistent with this view, wave-forming cells were found to exhibit a high propensity for taking up particles. Cells fed rod-shaped particles produced elongated phagocytic cups that displayed a zonal pattern that reflected in detail the actin and lipid pattern of free-running actin waves. Neutrophils and macrophages are known to spread on surfaces decorated with immune complexes, a process that has been interpreted as “frustrated” phagocytosis. We suggest that actin waves enable a phagocyte to scan a surface for particles that might be engulfed.Key words: actin waves, Dictyostelium, membrane tension, pattern formation, phagocytosis, PI3-kinase, PI(3,4,5)P3, self-organization     

Intracellular compartmentation of two enzymes of berberine biosynthesis in plant cell cultures

Out of the eight enzymes involved in the biosynthesis of the isoquinoline alkaloid berberine, at least, two enzymes, berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase, are exclusively located in a vesicle with a specific gravity of =1.14 g·cm^–3 as shown by direct enzymatic assay as well as immunoelectrophoresis. Electronmicroscopic examination of the enzyme-containing particulate preparation from Berberis wilsoniae var. subcaulialata cultured cells demonstrated that it is composed mainly of membranous vesicles. The protein composition of this preparation reveals the presence of only about 20 separable proteins, of which two major ones are berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase. Incubation of these vesicles with the substrate (S)-reticuline in the presence and absence of S-adenosyl-l-methionine leads to the formation of a red product which was identified as dehydroscoulerine. If the cytoplasmic enzyme S-adenosyl-l-methionine:(S)-scoulerine-9-O-methyltransferase is added to the vesicle preparation in the presence of (S)-reticuline and S-adenosyl-l-methionine, not dehydroscoulerine but columbamine, the immediate precursor of berberine is formed. Some of the quaternary alkaloids are located inside the vesicles; fusion of these vesicles leads to vacuoles containing the quaternary alkaloids. These vesicles are the first highly specific and unique compartment serving only alkaloid biosynthesis; they are found in members of four different plant families and in cell cultures as well as in differentiated tissue.Abbreviations BBE berberine bridge enzyme - STOX (S)-tetrahydroprotoberberine oxidase Dedicated to Professor Karl Decker, Freiburg, on the occasion of his 60^th birthday     

The noodle defense

Macrophages ingest and kill microbes by phagocytosis and delivery to lysosomes. In this issue, Prashar et al. (2013, J. Cell Biol. http://dx.doi.org/10.1083/jcb.201304095) demonstrate that the elongated morphology of filamentous bacteria does not prevent ingestion by macrophages or the fusion of lysosomes, but creates a chimeric, unclosed phagolysosomal compartment whose leakiness blunts the toxicity of lysosomal enzymes, thereby increasing bacterial survival.The tremendous variety of morphologies among microorganisms may allow them to thwart ingestion by the amoeboid cells that patrol pond water and the tissues of animals. Indeed, filament formation by bacteria can limit uptake by phagocytes (Justice et al., 2008). In this issue, Prashar et al. provide mechanistic support for the concept that a filamentous morphology allows bacteria to resist death by phagocytosis.In the normal sequence of events in phagocytosis, outlined in the late nineteenth century and refined by 130 years of microscopy and cell biology, phagocytic cells such as macrophages engage particles by receptor-mediated binding to ligands on microbe surfaces. Receptor signaling leads to the formation of actin-rich, cup-shaped extensions of the cell surface, which enclose particles into membrane-bound intracellular organelles (Swanson, 2008). These phagosomes then mature by progressive fusion with other membranous compartments in the cell, including secretory granules, endosomes, and lysosomes (Flannagan et al., 2009). The mixing of phagosomal and lysosomal contents kills ingested microbes by delivering them into an acidic, hydrolase-rich environment. Ingestion, maturation, and killing have long been considered sequential, nonoverlapping activities.But to be a useful hunting tool or a robust arm of host defense, phagocytosis must overcome microbes in all their various microscopic forms (Fig. 1). To ask how the framework for killing by phagocytosis is altered when a macrophage engages an elongate object, Prashar et al. (2013) examined phagocytosis of filamentous bacteria, which begins after macrophage exploratory movements locate a free filament end (Möller et al., 2012). Once engaged, the macrophage constructed an elongated tubular phagocytic cup comprised of a sleeve of plasma membrane with an actin-rich cuff, which pulled the filament into the cell as if sucking in a long spaghetti noodle. The cup membrane remained contiguous with the plasma membrane until it reached the distal end of the filament, at which point the cup closed into a discrete phagosome inside the cell. Earlier fluorescence microscopy of phagocytosis by macrophages and by Dictyostelium discoideum amebae had shown that membrane phospholipid and protein profiles change even before a phagocytic cup closes into the cell (Botelho et al., 2000; Dormann et al., 2004; Mercanti et al., 2006; Golebiewska et al., 2011). Prashar et al. (2013) identified similar lateral heterogeneity in membrane organization. Remarkably, fusion with early endosomes, late endosomes, and lysosomes began before filaments were fully internalized into the cell, such that the various stages of phagosome maturation were arranged along the length of the tubular phagocytic cups. This extends the earlier studies by showing that the mechanisms which maintain distinct organelle identities do not require organelles to be physically separate from each other inside the cell.Open in a separate windowFigure 1.Extreme phagocytosis. H.S. Jennings’ account (Jennings, 1976) of observations by Rhumbler in 1898: “Ameba verrucosa coiling up and ingesting a filament of Oscillaria. The animal settles upon the middle of an Oscillaria filament, envelopes it, and lengthens out along it (a). Then one end bends over (b), so that a loop is formed in the filament (c). The amoeba then stretches out on the filament again, bends it over anew, and the process is repeated until the filament forms a close coil within the amoeba (c to g).”The delayed closure of elongated phagocytic cups compromised macrophage antimicrobial activities. The actin cuff around the filament formed a tight ring that could limit egress of large dextrans delivered into the phagocytic cup from lysosomes. Nonetheless, while the phagocytic cup membranes remained contiguous with the plasma membrane, protons and lysosomal enzymes leaked out. Complete cup closure was required for phagosome acidification and the full degradative capacity of macrophage defenses. This suggests that adopting a filamentous morphology allows bacteria to lessen the toxicity of microbicidal compounds delivered into the phagosome. Consistent with this idea, Prashar et al. (2013) determined that the survival and growth of Legionella pneumophila in macrophages correlated with filament length.Studies of phagocytosis are undoubtedly important for understanding host defense against infections, but they also provide a vantage point for asking how cells organize cytoplasm for the complexities of microscopic life. Fc receptor–mediated phagocytosis of particles coated with IgG proceeds by a zipper-like mechanism, in which the patterns of IgG ligands on a particle surface guide the distribution of phagocyte signaling that shapes a phagosome (Swanson and Baer, 1995). This localized signaling is modulated by feedback regulation related to the physical properties of the particle. For example, macrophages presented with long, rod-shaped particles coated with IgG respond differently depending on the orientation of their initial contact with those particles. Particles contacted end-on are readily ingested, but particles contacted along their long face are not (Champion and Mitragotri, 2006). This regulation of Fc receptor signaling by surface topology could explain why macrophages ingest filaments only after locating a filament end. But it is a puzzling observation, not least because a macrophage will engage a planar surface coated with IgG as if to engulf it—a response termed “frustrated phagocytosis” (Wright and Silverstein, 1984). Why should phagocytosis proceed on an impossibly large planar surface but not against the side of a rod-shaped particle? Although still unresolved, these fascinating questions about shape-sensing in phagocytosis have been addressed by recent theoretical and experimental studies (Clarke et al., 2010; Dieckmann et al., 2010; Tollis et al., 2010).Another puzzling relationship between Fc receptor signaling and the physical dimensions of the prey concerns the role of phosphatidylinositol 3-kinase (PI3K) in phagocytosis. PI3K is required for phagocytosis of microspheres larger than 3 µm in diameter, but not for phagocytosis of smaller microspheres (Araki et al., 1996; Cox et al., 1999). This suggests that phagocytosis is regulated by a PI3K-dependent feedback related to particle size. How does that work? It could be that PI3K relieves a feedback inhibition of Fc receptor signaling that begins only after some delay, such that it becomes rate-limiting only for the phagocytosis of larger particles that take more time to ingest. Prashar et al. (2013) excluded that possibility by examining the effects of PI3K inhibition on the phagocytosis of filaments. In the presence of the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, phagocytosis of IgG-opsonized sheep erythrocytes was inhibited but the phagocytosis of filaments was not. This suggests that formation of the narrow aperture for sucking in a noodle occurs below the PI3K-dependent size threshold. Moreover, once a phagocytic response was initiated, it continued for as long as necessary to ingest the filament, showing that PI3K-dependent regulation of phagocytosis is spatial rather than temporal. The 3′ phosphoinositide products of PI3K may be part of a system for gauging the three-dimensional distribution of phagocytic receptor signaling, a mechanism of spatial integration that regulates cellular commitment to phagocytosis (Zhang et al., 2010). In the battles with the exotic geometries of microbial life, it may be necessary for a phagocyte to decide whether a particle is small enough to eat or should instead be held outside and engaged as extracellular prey. Accordingly, the narrow end of a filament presents a stimulus that is below the size threshold for PI3K-dependent regulation, so the macrophage begins slurping it in, with a considerable amount of lysosomal spittle dribbling out.     

Endocytosis in the ureteric duct epithelium of the hagfish (Myxine glutinosa L.)

Summary Solutions containing ferritin or thorotrast particles were microperfused through the ureteric duct of the hagfish. The markers were taken up by the epithelial cells by way of endocytosis and were transported in bulk in apical vesicles. Newly formed apical vesicles containing marker showed bristle coating on the cytoplasmic side of their limiting membrane. This coating appeared to be lost during the movement of vesicles deeper into the cytoplasm.The findings indicate that the epithelial cells in the ureteric duct have capablity for extensive bulk uptake of macromolecules from the luminal fluid. The mechanisms involved in absorption appear to be similar to those in proximal convoluted tubules of mammals.The apical dense tubules observed with some fixation techniques appear to represent collapsed endocytotic vesicles.The authors are indebted to Finn Walwig, Cand. real., Marine Biology Station, University of Oslo, Dröbak, Norway for kindly supplying the hagfishes used in this study. The technical assistance of Miss Signe Fjeldsenden and Miss Britt-Marie Pettersson is gratefully acknowledged.This work has been supported by grants from the Karolinska Institutet Medical School, Stockholm, Sweden (Therese och Johan Anderssons Minne).     

Signaling pathways in phagocytosis

Phagocytosis is an uptake of large particles governed by the actin-based cytoskeleton. Binding of particles to specific cell surface receptors is the first step of phagocytosis. In higher Eucaryota, the receptors able to mediate phagocytosis are expressed almost exclusively in macrophages, neutrophils, and monocytes, conferring immunodefence properties to these cells. Receptor clustering is thought to occur upon particle binding, that in turn generates a phagocytic signal. Several pathways of phagocytic signal transduction have been identified, including the activation of tyrosine kinases and (or) serine/threonine kinase C in pivotal roles. Kinase activation leads to phosphorylation of the receptors and other proteins, recruited at the sites of phagocytosis. Monomeric GTPases of the Rho and ARF families are likely to be engaged downstream of activated receptors. The GTPases, in cooperation with phosphatidylinositol 4-phosphate 5-kinase and phosphatidylinositol 3-kinase lipid modifying enzymes, can modulate locally the assembly of the submembranous actin filament system leading to particle internalization. BioEssays 21:422–431, 1999. © 1999 John Wiley & Sons, Inc.     

The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

Differences of adenylate cyclase localization in guinea-pig peritoneal macrophages under different physiological conditions: An ultracytochemical study

Summary The ultrastructural localization of adenylate cyclase activity has been investigated in unfixed guinea-pig peritoneal macrophages in different physiological states (such as suspension, adhesion and phagocytosis) using a medium containing 5-adenylyl-imidodiphosphate (AMP-PNP) as the substrate.Adenylate cyclase activity was observed in cytoplasmic vacuoles of macrophages in suspension; in the perinuclear space, endoplasmic reticulum, Golgi complex and pseudopods of adherent macrophages; and surrounding phagocytosed polystyrene particles. The activity was inhibited by Alloxan added to the incubation medium and no staining was observed when AMP-PNP was omitted from the medium.The segregation of this enzyme to phagocytic vacuoles and pseudopods may have significant implications in understanding cyclic nucleotide function in adhesion and phagocytosis.     

Ultrastructural study on the release of neurosecretory material from the sinus gland of the land crab,Gecarcinus lateralis

Summary Sites of release of neurosecretory material were examined in a neurohemal organ of decapod crustaceans, the sinus gland of the land crab, Gecarcinus lateralis. Such discharge into the circulation seems to occur primarily at interfaces between the neurosecretory axons and the acellular stromal sheath which is interposed between parenchyma and hemolymph. The evidence obtained from electron micrographs of adult specimens indicates that more or less intact secretory granules are released into the extraaxonal space primarily by the process of exocytosis. Synaptic-type vesicles are clustered in parts of neurosecretory axons facing the stromal sheath. Such vesicles are thought to result from rearrangement of membranes temporarily fused at the release site and to a minor degree from fragmentation of neurosecretory vesicles within the axon. The presence of nonmarginal vesicles and the occasional appearance of free intraaxonal dense material are interpreted as indications of a second, probably less frequent, mode of release of neurosecretory material.Supported by grants AM-3984, NB-00840, and NB-05219 from the U.S.P.H.S., administered by Dr. Berta Scharrer.I am greatly indebted to Mrs. Sarah Wurzelmann for her excellent technical assistance. I thank Dr. Dorothy E. Bliss for contributing the animals used in this study, and Mr. Murray Altmann for technical advice.     

The Ultrastructure of Mouse Lung: The Alveolar Macrophage

Free alveolar macrophages of normal mouse lung have been studied in the electron microscope. The tissue was obtained from several young adult white mice. One other animal was instilled intranasally with diluted India ink 1½ hours prior to the removal of the lung. Thin sections of the osmium-fixed, methacrylate-embedded tissue were examined either in an RCA EMU 2 electron microscope or in a Siemens and Halske Elmiskop I b. A few thick sections obtained from the same embeddings were stained for iron. The normal alveolar macrophages, which are usually in contact with the alveolar epithelium, were found to contain a variety of inclusion bodies, along with the usual cytoplasmic components like mitochondria, endoplasmic reticulum, and Palade granules. Another typical component of the cytoplasm of these cells which appears as small (~6 mµ) very dense granules of composite fine structure is interpreted as ferritin. It is assumed that this ferritin is formed from red blood cells ingested by the alveolar macrophages. The macrophages in the alveoli were found to phagocytize intranasally instilled India ink particles. Such cells, with engulfed India ink particles, were often of more rounded form and the particles were frequently seen lying inside membrane-bound vacuoles or vesicles of the cytoplasm. The membrane of a few vesicles containing India ink particles was seen as the invaginated portion of the cell plasma membrane, and in one instance these same vesicles were seemingly interconnected with a rough surfaced cisterna of the endoplasmic reticulum. The process of phagocytosis is recognized as related to the "normal" process of pinocytosis.     

In vivo effects of lipopolysaccharide on lymphoid and non-lymphoid cells in the mouse spleen

Summary In the present study the effects of lipopolysaccharide (LPS) on the cellular composition and phagocytosis of India ink in the inner parts of the periarteriolar lymphocyte sheaths (PALS) are described.Staining for B-, T-lymphocytes, and reticulin fibers in the spleen of normal and LPS-injected mice shows that the B-dependent follicular area is increased in size after LPS administration. However, the number of T-lymphocytes in the inner PALS is reduced markedly and a relatively high number of B-lymphocytes can be found in this area. The significance of this phenomenon is discussed.In untreated mouse spleen, carbon particles become localized in strongly acid-phosphatase (AP)-positive macrophages of the red pulp, marginal zone and white pulp 24 h after an intravenous injection of India ink. All these macrophages contain numerous carbon particles. After LPS pretreatment, the phagocytosis of carbon particles in the inner PALS is dramatically diminished, although many strongly AP-positive macrophages can be found in this area. The phagocytosis of carbon particles in the other compartments of the spleen did not change. It appears that injection of 2 g LPS or more is sufficient to induce this phenomenon which is most significant when LPS is injected 24 or 48 h before exposure to India ink.Abbreviations LPS lipopolysaccharide - PALS periarteriolar lymphocyte sheath - AP acid phosphatase - IDC interdigitating cells     

Fc receptor-mediated phagocytosis requires CDC42 and Rac1.

At the surface of phagocytes, antibody-opsonized particles are recognized by surface receptors for the Fc portion of immunoglobulins (FcRs) that mediate their capture by an actin-driven process called phagocytosis which is poorly defined. We have analyzed the function of the Rho proteins Rac1 and CDC42 in the high affinity receptor for IgE (FcepsilonRI)-mediated phagocytosis using transfected rat basophil leukemia (RBL-2H3) mast cells expressing dominant inhibitory forms of CDC42 and Rac1. Binding of opsonized particles to untransfected RBL-2H3 cells led to the accumulation of F-actin at the site of contact with the particles and further, to particle internalization. This process was inhibited by Clostridium difficile toxin B, a general inhibitor of Rho GTP-binding proteins. Dominant inhibition of Rac1 or CDC42 function severely inhibited particle internalization but not F-actin accumulation. Inhibition of CDC42 function resulted in the appearance of pedestal-like structures with particles at their tips, while particles bound at the surface of the Rac1 mutant cell line were enclosed within thin membrane protrusions that did not fuse. These phenotypic differences indicate that Rac1 and CDC42 have distinct functions and may act cooperatively in the assembly of the phagocytic cup. Inhibition of phagocytosis in the mutant cell lines was accompanied by the persistence of tyrosine-phosphorylated proteins around bound particles. Phagocytic cup closure and particle internalization were also blocked when phosphotyrosine dephosphorylation was inhibited by treatment of RBL-2H3 cells with phenylarsine oxide, an inhibitor of protein phosphotyrosine phosphatases. Altogether, our data show that Rac1 and CDC42 are required to coordinate actin filament organization and membrane extension to form phagocytic cups and to allow particle internalization during FcR-mediated phagocytosis. Our data also suggest that Rac1 and CDC42 are involved in phosphotyrosine dephosphorylation required for particle internalization.     

Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast

Summary The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparentK _m approx. 150 m) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparentK _d 0.98±0.21nm). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparentK _i values were 0.23±0.012, 0.36±0.035, 0.78±0.1, 0.70±0.12 (mm), and 0.12 and 4.2±1.4 (nm). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brushborder membrane vesicles (apparentK _d 1.05±0.13nM and apparentK _i values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14±0.045, 0.54±0.046, 1.26±0.20, 1.09±0.18mm and 0.14 and 3.7±0.5nm, respectively). Exposure of both membrane vesicles to UV light in the presence of [³H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparentM _r on SDS gel electropherograms of 77,000–45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surface of the human placental syncytiotrophoblast posses broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters.     

Monoclonal antibodies to a fern spermatozoid detect novel components of the mitotic and cytokinetic apparatus in higher plant cells

Summary The aim of this study was to search for uncharacterized components of the plant cytoskeleton using monoclonal antibodies raised against spermatozoids of the fernPteridium (Marc et al. 1988). The cellular distribution of crossreacting immunoreactive material during the division cycle in wheat root tip cells was determined by immunofluorescence microscopy and compared to the fluorescence pattern obtained with antitubulin. Five antibodies are of special interest. Pas1D3 and Pas5F4 detect a diffuse cytoplasmic material, which, during mitosis, follows the distribution of microtubules (MTs) at the nuclear surface and in the preprophase band (PPB), spindle and phragmoplast. The immunoreactive material codistributes specifically with MT arrays of the mitotic apparatus and does not associate with interphase cortical MTs. Pas5D8 is relevant to the PPB and spatial control of cytokinesis. It binds in a thin layer at the cytoplasmic surface throughout the cell cycle, except when its coverage is transiently interrupted by an exclusion zone at the PPB site and later at the same site when the phragmoplast fuses with the parental cell wall.Pas2G6 reacts with a component of basal bodies and the flagellar band in thePteridium spermatozoid and recognizes irregularly shaped cytoplasmic vesicles in wheat cells. During interphase these particles form a cortical network.Pas6D7 binds to dictyosomes and dictyosome vesicles. At anaphase the vesicles accumulate at the equator and subsequently condense into the cell plate.Abbreviations MT microtubule - PPB preprophase band     

Ultrastructure of transitional epithelium of man

Summary Blocks of human normal renal pelvis and ureter obtained at the time of surgery were fixed in glutaraldehyde and osmium with or without ruthenium red, for electron microscopic observations. The transitional epithelium is arranged in three cell layers: basal, intermediate and superficial. All epithelial cells show numerous microvilli and contain the characteristic vesicles of transitional epithelium, bundles of cytoplasmic filaments, microtubules and numerous free ribosomes. The epithelial extracellular compartment is notably large and appears as an intricate, tridimensional network of canaliculi and cisternae which are wider in the intermediate and superficial layers and in which microvilli and cytoplasmic folds of vicinal cells are often attached or interdigitated. At these sites there are desmosomes.The surface of all transitional epithelial cells is covered by a fibrillar mucous coat which is more developed at the plasmalemma of the free border of luminal cells in which microvilli are also seen. Ruthenium red stains selectively the plasmalemma and the mucous coat of the free surface of the epithelium, indicating the presence of an acid polysaccharide. With this technic (Luft, 1965), it is observed, radiating from the plasmalemma, branching filaments which measure 100 Å in diameter forming a zone of varying density which is about 400 m wide and which corresponds, at the light microscopic level, to the luminal border of the transitional epithelial cells in which a sialomucin has been identified. The slender filaments have a beaded appearance. At the free border, superficial cells are attached by functional complexes in which tight junctions seal the epithelial intercellular space, which is opened at the level of the basement membrane where only desmosomes are observed.The ultrastructure of human transitional epithelium of urinary tract resembles the duct cells of the salt gland of certain marine birds (Fawcett, 1962) and the amphibian epidermis (Farquhar and Palade, 1965) in which there are active processes of transport. The mucous surface coat, selectively stained by the ruthenium red, contains a sialomucin (Monis and Dorfman, 1965, 1967).The ultrastructure and histochemistry of the mucous fluffy coat of man transitional epithelium and the observations of Porter and Tamm (1955), on the ultrastructure of preparations of the Tamm and Horsfall mucoprotein (1952) are bases for suggesting that transitional epithelium of urinary tract of man is the site of biosynthesis of certain urinary mucoids. Present investigations are directed to obtain evidence to substantiate this hypothesis.General Abbreviations B basal cell - E exfoliating cell - I intermediate cell - L lumen - S superficial cell - SC surface coat - bm basement membrane - ci cell infolding - d desmosome (macula adhaerens) - f fibroblast - fi cytoplasmic filaments - is intercellular space - jc junctional complex - ly lysosome - lym lymphocyte - mt microtubules - m mitochondria - mv microvilli - n nucleus - r ribosomes - rv round vesicle - zo zonula occludens - za zonula adherens Dr. Monis wishes to thank Dr. E. De Robertis for the use of the electron microscope facilities of the Instituto de Anatomía General y Embriologia, Facultad de Medicina, Universidad de Buenos Aires. — Prof. E. Trabucco and Dr. R. J. Borzone (Cátedra de Clinica Genitourinaria de la Facultad de Medicina, Universidad de Buenos Aires) generously supplied the specimens which were the bases of this study. — Thanks are due to Mrs. A. M. Novara and Mrs. Defilippi-Novoa for efficient technical help and to Miss Rosa Gentile for secretarial assistance. Photomicrography by Mr. M. A. Saenz.Dr. Zambrano is investigator (CNICT).     

The appearance of a type of cortical vesicles subsequent to fertilization of the sea urchin egg,their character and possible function

Summary Three different types of vesicles present before fertilization in the sea urchin egg were examined. The a-type corresponds to rough endoplasmic vesicles; the vesicles of b-type are rather smooth but may have vestigial granules within their membranes; the c-type belongs to the multivesicular bodies. Upon fertilization, vesicles appear in the outer cortical zone (vesicles of d-type). The majority of them arises by budding from the vesicles ofb-type. The budding occurs mainly at the basis of or within ridges of the cell surface; they may also be present in broader microvilli. The distance between the ridges was varied by early transfer of the eggs to calcium-free sea water. An inducing effect of the ridges on location of theb-vesicles and on the formation ofd-vesicles by budding could thus be demonstrated.Thed-vesicles appearing upon fertilization resemble in size and structure Golgi vesicles formed by budding from Golgi bodies present in the interior cortex or below it. Also theb-vesicles have their counterpart in the Golgi bodies. Theb andd-vesicles are therefore regarded as a Golgi system in which theb-vesicles represent dispersed Golgi bodies and thed- vesicles Golgi vesicles. Thed-vesicles may be designated as cortical Golgi vesicles, (c.G.v.).In thec-vesicles i.e. the multivesicular bodies (m.v.b.), a nucleid was observed which may be subdivided. A compartmentation of m.v.b. was observed which may lead to a detachment of vesicles of about the same size as thed-vesicles (c.G.v.) but probably of a different character.The differentiation of the fertilization membrane of the sea urchin egg occurs in two stages: the assembly and the solidification stage. The tentative conclusion is drawn that a secretion from the c.G.v. functions as an agent in the solidification process. The c.G.v. may also act upon the hyaline layer, both in its early formation and its maintenance during the course of embryonic development.The material for the present investigation was collected at Kristineberg Zoological Station and at Stazione Zoologica, Naples. The writer is indebted to the authorities of these stations for generous helop. Financial support was received from the Swedish Natural Sciences Research Council and the Swedish Cancer Society for which I express my gratitude.I also express my gratitude to Dr.Björn Afzelius for surveying the electron microscopic part of the work, to Dr.Jane Baxandall for permission to study her original electron micrographs and to Dr.Elsa Wicklund and Dr. L.Contoli for contribution to the experiments on the effect of calcium-free sea water. I thank Mrs.Astri Runnwström and Mrs.AnneMarie Ede for their able assistance.This research was carried out in association with the Research group of embryology for the study of cellular differentiation of the Department of Zoology, University of Rome (Director Professor P.Pasquini).     

The ultrastructure of mouse lung: the alveolar macrophage

Free alveolar macrophages of normal mouse lung have been studied in the electron microscope. The tissue was obtained from several young adult white mice. One other animal was instilled intranasally with diluted India ink 1(1/2) hours prior to the removal of the lung. Thin sections of the osmium-fixed, methacrylate-embedded tissue were examined either in an RCA EMU 2 electron microscope or in a Siemens and Halske Elmiskop I b. A few thick sections obtained from the same embeddings were stained for iron. The normal alveolar macrophages, which are usually in contact with the alveolar epithelium, were found to contain a variety of inclusion bodies, along with the usual cytoplasmic components like mitochondria, endoplasmic reticulum, and Palade granules. Another typical component of the cytoplasm of these cells which appears as small ( approximately 6 mmicro) very dense granules of composite fine structure is interpreted as ferritin. It is assumed that this ferritin is formed from red blood cells ingested by the alveolar macrophages. The macrophages in the alveoli were found to phagocytize intranasally instilled India ink particles. Such cells, with engulfed India ink particles, were often of more rounded form and the particles were frequently seen lying inside membrane-bound vacuoles or vesicles of the cytoplasm. The membrane of a few vesicles containing India ink particles was seen as the invaginated portion of the cell plasma membrane, and in one instance these same vesicles were seemingly interconnected with a rough surfaced cisterna of the endoplasmic reticulum. The process of phagocytosis is recognized as related to the "normal" process of pinocytosis.     

A Ca-dependent K channel in “luminal” membranes from the renal outer medulla

Summary This paper describes properties of⁸⁶Rb fluxes through K channels in luminal membrane vesicles prepared from rabbit renal outer medulla. By measuring⁸⁶Rb uptake against an opposing chemical gradient of K ions, using membranes loaded with KCl, a transient accumulation of isotope is observed, which is blocked by Ba ions. This is the behavior expected of a conductive Rb flux through a Ba-sensitive K channel. The⁸⁶Rb accumulation is driven by an electrical diffusion potential as shown in experiments using either vesicles loaded with different anions, or an outwardly directed Li gradient with a Li ionophore. The vesicles containing the channel show a cation selectivity with the order Rb > K > Cs > Li > Na > choline. The Ba-sensitive Rb flux is dependent on Ca within the vesicles, with a very high affinity estimated asK _0.5 10 to 100nm. The vesicles appear to be right-side-out. The Ba-sensitive⁸⁶Rb uptake is also inhibited by quinineK _0.5 30 m but is insensitive to tetraethyl ammonium ions and apamin. These isotope flux experiments complement electrophysiological experiments in providing independent evidence for the existence of K channels in the luminal surface of cells of this ascending limb of the loop of Henle. The very high Ca affinity suggests that cytoplasmic Ca could play an important role in regulation of transepithelial salt flux in this region of the nephron.