Synaptic vesicles retain their identity through the endocytic cycle

After fusion of synaptic vesicles with presynaptic membrane and secretion of the contents of the vesicles into the synaptic cleft (a process known as exocytosis), the vesicular membrane is retrieved by endocytosis (internalization) for re-use. Several issues regarding endocytosis at central synapses are unresolved, including the location of membrane retrieval (relative to the active zone, where exocytosis occurs), the time course of various endocytic steps, and the recycling path taken by newly endocytosed membranes. The classical model of synaptic-vesicle recycling, proposed by analogy to other cellular endocytic pathways, involves retrieval of the membrane, fusion of the membrane with endosome-like compartments and, finally, budding of new synaptic vesicles from endosomes, although the endosomal station may not be obligatory. Here we test the classical model by using the fluorescent membrane dye FM1-43 with quantitative fluorescence microscopy. We find that the amount of dye per vesicle taken up by endocytosis equals the amount of dye a vesicle releases on exocytosis; therefore, we conclude that the internalized vesicles do not, as the classical picture suggests, communicate with intermediate endosome-like compartments during the recycling process.     

A coupled proton-transfer and twisting-motion fluorescence probe for lipid bilayers

A new and sensitive molecular probe, 2-(2'-hydroxyphenyl)imidazo[1, 2-a]pyridine (HPIP), for monitoring structural changes in lipid bilayers is presented. Migration of HPIP from water into vesicles involves rupture of hydrogen (H) bonds with water and formation of an internal H bond once the probe is inside the vesicle. These structural changes of the dye allow the occurrence of a photoinduced intramolecular proton-transfer reaction and a subsequent twisting/rotational process upon electronic excitation of the probe. The resulting large Stokes-shifted fluorescence band depends on the twisting motion of the zwitterionic phototautomer and is characterized in vesicles of dimyristoyl-phosphatidylcholine and in dipalmitoyl-phosphatidylcholine at the temperature range of interest and in the presence of cholesterol. Because the fluorescence of aqueous HPIP does not interfere in the emission of the probe within the vesicles, HPIP proton-transfer/twisting motion fluorescence directly allows us to monitor and quantify structural changes within bilayers. The static and dynamic fluorescence parameters are sensitive enough to such changes to suggest this photostable dye as a potential molecular probe of the physical properties of lipid bilayers.     

Separation of megakaryocytes from mouse bone marrow by density gradient centrifugation

Freeze-etch preparations of mesothelial cells taken from the peritoneum of mouse reveal the presence of vesicles invaginating the apical and the basal cell surfaces. These vesicles are scarcely seen within the cytoplasm. Long tortuous tubular profiles extend for considerable distance within the cytoplasm and are frequently associated with the vesicles. The possible nature and role of the vesicles and the tubules in transport phenomena across the mesothelial barrier, are discussed in relation to the pore theory advanced by physiologists and the "stomata" concept observed by early German and contemporary anatomists. "Occludens" junctions of the leaky type are seen though their macular or zonular nature is yet to be established.     

Membrane fusion is induced by a distinct peptide sequence of the sea urchin fertilization protein bindin

Fertilization in the sea urchin is mediated by the membrane-associated acrosomal protein bindin, which plays a key role in the adhesion and fusion between sperm and egg. We have investigated the structure/function relationship of an 18-amino acid peptide fragment "B18," which represents the minimal membrane binding motif of the protein and resembles a putative fusion peptide. The peptide was found to mimic the behavior of its parent protein bindin with respect to (a) its high affinity for lipid bilayers, (b) the ability to aggregate and fuse vesicles, (c) the binding of Zn2+ by a histidine-rich motif, (d) the tendency to self-assemble, and (e), as indicated earlier, the adhesion to cell surface polysaccharides. Fluorescence and light scattering assays were used here to monitor peptide-induced lipid mixing, leakage, and aggregation of large unilamellar sphingomyelin/cholesterol vesicles. For these activities, B18 requires the presence of Zn2+ ions, with which it forms oligomeric complexes and assumes a partially alpha-helical conformation, as observed by circular dichroism. We conclude that aggregation and fusion involves a "trans-complex" between peptides on apposing vesicles that are connected by Zn2+ bridges.     

Endocytosis and exocytosis events regulate vesicle traffic in endothelial cells

We used water-soluble styryl pyridinium dyes that fluoresce at the membrane-water interface to study vesicle traffic in endothelial cells. Cultured endothelial cells derived from bovine and human pulmonary microvessels were incubated in styryl probes, washed to remove dye from the plasmalemmal outer face, and observed by digital fluorescence microscopy. Vesicles that derived from plasmalemma by endocytosis were filled with the styryl dye. These vesicles were distributed throughout the cytosol as numerous particles of heterogeneous diameter and brightness. Vesicle formation was activated 2-fold following addition of extracellular albumin whereas a control protein, immunoglobulin G, had no effect. Dye uptake was abrogated by labeling at low temperatures and inhibitors of phosphoinositide-3-kinase (PI 3-kinase). Tyrosine kinase inhibitors (genistein and herbimycin A) prevented the albumin-induced vesicle formation. Cytochalasin B prevented vesicle redistribution indicating involvement of actin filaments in translocation of endosomes away from sites of vesicle formation. Styryl dye was lost from cells by exocytosis as evident by the disappearance of discrete fluorescent particles. N-ethylmaleimide and botulinum toxin types A and B caused cells to accumulate increased number of vesicles suggesting that exocytosis was regulated by NSF-dependent SNARE mechanism. The results suggest that phosphoinositide metabolism regulates endocytosis in endothelial cells and that extracellular albumin activates endocytosis by a mechanism involving tyrosine phosphorylation, whereas exocytosis is a distinct process regulated by the SNARE machinery. The results support the hypothesis that albumin regulates its internalization and release in vascular endothelial cells via activation of specific endocytic and exocytic pathways.     

Biologically active Fas antigen and its cognate ligand are expressed on plasma membrane-derived extracellular vesicles

Exfoliation of plasma membrane components is a directed process that consumes energy and requires active cell metabolism. Proteins involved in regulating the survival and proliferation of eukaryotic cells are released on exfoliated vesicles. We examine here whether the Fas receptor and its cognate ligand (FasL) are present on vesicles shed from high metastatic potential CX-1 cells and low metastatic potential MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation at 2 hours and cumulative levels of extracellular vesicles in serum-free medium conditioned by CX-1 cells are increased by 1.8-fold and 1.6-fold, respectively, relative to that in medium conditioned by MIP-101 cells. Although vesicles shed from both cancer cell lines contain Fas antigen, the amount of Fas per vesicle and the percentage of vesicles containing Fas are increased for vesicles isolated from MIP-101 cells, relative to those from CX-1 cells, as determined by immunogold particle labeling and electron microscopy and by immunofluorescence microscopy and flow cytometry. Results of metabolic labeling with 35S-methionine indicate that Fas biosynthesis is reduced by up to 3.3-fold for CX-1 cells, relative to that of MIP-101 cells, consistent with the finding of decreased Fas on vesicles shed from the plasma membrane of CX-1 cells. Although mRNA for soluble Fas receptor is detectable in both cell lines, depletion of shed vesicles from serum-free medium by ultracentrifugation removes all detectable biological activity. FasL is detected on vesicles exfoliated from HuT 78 cells by immunoelectron microscopy and Western blot analysis. FasL-bearing vesicles induce apoptosis of Fas-expressing cancer cells at the same level as observed by treatment with monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellular vesicles from MIP-101 but not from CX-1 cells protect the CX-1 cell line from FasL-induced and anti-Fas-mediated apoptosis, indicating that Fas present on shed vesicles is biologically active. We conclude that the Fas antigen and its cognate ligand are exfoliated from the cell surface in a bioactive configuration. Exfoliation may provide a mechanism for long-range signal-directed apoptosis while maintaining Fas/FasL on a membrane surface.     

Preparation of inside-out vesicles of pig lymphocyte plasma membrane

Between 30 and 50% of pig lymphocyte plasma membrane vesicles were not bound by concanavalin A (Con A)-Sepharose. Various results suggest that the Con A-unretarded fraction represents "inside-out" membrane vesicles. First, an alternative cell surface ligand, anti-lymphocytic serum, gave a similar fractionation to Con A. Second, lack of binding by Con A was not due to lack of carbohydrate or to masking of carbohydrate by extraneous protein, because the unfractionated membrane and the unretarded fraction had similar carbohydrate and polypeptide compositions. Third although the carbohydrate of the unretarded membrane vesicles was accessible to 125I-labelled Con A and to release by soluble trypsin, it was not accessible to ferritin-Con A or trypsin-Sepharose. Fourth, antisera against the external surface of the Con A-unretarded vesicles strongly agglutinated the unretarded membrane, but caused negligible agglutination of whole lymphocytes. When attached to Sepharose these antisera bound all of the Con A-unretarded fraction, but failed to bind the membrane that adhered to Con A-Sepharose.     

Involvement of bristle coat structures in surface membrane formations and membrane interactions during coenocytotomic cleavage in caps of Acetabularia mediterranea

In the multinucleate cap rays of the green alga Acetabularia mediterranea the cell surface increases dramatically within a short time period during the final stages of coenocytotomic cleavage. In early stages of cyst formation the cytoplast is traversed by numerous large and prolate cleavage vesicles which are characterized by typical columellar or spinous coat structures. The cleavage vesicles are closely associated with the surface of plastids and, to a lesser degree, of mitochondria. This intimate association seems to be mediated by regularly spaced, densely stained intermembranous cross-bridge structures and is maintained throughout cleavage. These cleavage vesicles contain a finely fibrillar material structurally similar to the hyaline layer of mucilage that fills the space between the plasma membrane and cell wall. They line up with invaginations of the plasmalemma and vacuole membranes and, together with smaller vesicles interspersed, constitute preformed "perforation lines" for the final separation of the coenoblast portions. Equidistantly spaced plaques of attachment of such vesicles with surface membrane are described. We hypothesize (a) that the cleavage vesicle membrane is the immediate precursor to the new postcoenocytotomic surface membrane, (b) that the cleavage vesicle coat structures are integrated into the subsurface coat of the plasma membrane, (c) that growth of the laterally attached cleavage vesicles by intussusception of small fuzzy-coated vesicles is confined to their "free ends," (d) that the intermembranous cross-bridge elements are related to bristle coat structures and play a role in the establishment of the cleavage lines, and (e) that the coenocytotomic cleavage process is organized so that adjacent plastids are separated in a way that guarantees the inclusion of several plastids in each cyst.     

Inequalities in health related to women''s marital, parental, and employment status--a comparison between the early 70s and the late 80s, Norway

There is abundant evidence that the pathophysiology leading to neuronal death during post-ischemic brain reperfusion involves radical-mediated damage. Although the ultrastructural alterations accompanying brain ischemia and reperfusion are well characterized, little is known about the ultrastructural alterations that are specific to radical damage. This study examines in differentiated and undifferentiated neuroblastoma B-104 cells the viability (by dye exclusion) and ultrastructural consequences of radical damage initiated by 50 microM cumene hydroperoxide (CumOOH). Differentiation was most notably associated with formation of neurites and an extensive cytoskeletal feltwork. CumOOH-induced cell death was increased after differentiation and was blocked by the iron chelator DETAPAC. The ultrastructural characteristics of radical damage here included: (1) plasmalemmal holes that appear to undergo "patching" by well-organized membrane whorls, (2) accumulation of numerous free ribosomes, (3) markedly increased vesicular trafficking about the Golgi accompanied by Golgi transformation from cisternal organization to clusters of vacuoles with numerous fusing vesicles, (4) development of large multi-layered vacuoles that include damage membranes and organelles and appear to undergo extrusion from the cell, and (5) a general loss of cytoplasmic volume. These ultrastructural alterations developed more rapidly and were consistently more advanced in differentiated cells throughout the 6-h time course. In differentiated cells radical damage also induced the disorganization and subsequent loss of the extensive feltwork of cytoskeletal elements. There was little damage to the membranes of the nuclear envelope and mitochondria. Our observations in this system are strikingly similar to ultrastructural alterations in Golgi and ribosomal organization seen in vulnerable neurons during post-ischemic brain reperfusion and suggest that these alterations during reperfusion reflect the consequence of radical-mediated damage.     

Glycoprotein containing vesicles in the surface epithelial cells of the ascending colon of the rat

Recently, radioautographic studies have shown that cell coat glycoproteins are transported to the cell surface by vesicles both in the amoeba (Flickinger, '75) and in the epithelial cells of the ascending colon of the mouse (Michaels and Leblond, '76). In the current morphological and cytochemical study of the surface epithelial cells of the rat ascending colon, it is shown that filamentous material, resembling the cell coat, is contained in saccules toward the mature face of the Golgi apparatus and vesicles close to the apparatus and near the terminal web. The vesicles are limited by a unit membrane composed of asymmetric osmiophilic leaflets and similar to the plasma membrane. When stained by the periodic acid-chromic acid-silver methenamine technique, silver was precipitated on the cell components containing the filamentous material indicating the presence of glycoproteins. Narrow invaginations from the cell surface that may correspond to vesicles undergoing exocytosis were also positive for glycoproteins. The distribution of the filamentous material that was glycoprotein positive parallels the pathway followed by material that had been found to be labeled with a tritiated glycoprotein precursor (3H-fucose) in the epithelial cells of the ascending colon of the mouse. It is suggested that the system of vesicles in the rat colon cells is acting in a manner similar to the vesicles in the mouse cells to transport cell coat glycoproteins from the Golgi apparatus to the cell surface.     

Membrane-destabilizing properties of C2-ceramide may be responsible for its ability to inhibit platelet aggregation

We have studied the effects of short-chain ceramides on platelet structure and function. N-Acetylsphingosine (C2-ceramide), a cell-permeable short-chain analogue, and N-acetyldihydrosphingosine (C2-dihydroceramide), which lacks the 4-5 double bond, have been investigated. C2-Ceramide (15 microM) inhibited ADP-induced aggregation by 50% at a platelet concentration of 1.25 x 10(8)/mL, while it took twice that concentration to inhibit aggregation by 50% when the platelet concentration was doubled. This indicates that the effect of C2-ceramide on ADP-induced platelet aggregation depends on the ratio of ceramide to total platelet lipid, with a ratio of 0.2 giving significant inhibition. C2-Ceramide at a ceramide: lipid ratio of 0.2 caused platelets to form fenestrations and pseudopodia which were longer and thinner than those caused by agonists such as ADP or thrombin. C2-Dihydroceramide had no effect on ADP-induced aggregation or platelet morphology at any ceramide:lipid ratio. Platelet lysis was induced by C2-ceramide at higher ceramide:lipid ratios (0.5), whereas C2-dihydroceramide did not induce lysis, suggesting that C2-ceramide is able to destabilize membranes. This was tested directly by assessing whether the ceramides induced leakage of 6-carboxyfluorescein from lipid vesicles. C2-Ceramide caused nearly total leakage of dye from the vesicles at a ceramide:lipid ratio of 10. The leakage caused by C2-dihydroceramide at a ceramide:lipid ratio of 10 was equal to that induced by C2-ceramide at a ratio of 0.2 (approximately 3%). The ability of the ceramides to destabilize membranes was also examined by measuring changes in fluorescence anisotropy of the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into lipid vesicles. C2-Ceramide induced a larger decrease in anisotropy than a detergent (Triton X-100) which is known to lyse membranes. C2-Dihydroceramide did not alter membrane fluidity. The ability of C2-ceramide to cause platelet fenestrations, formation of irregular platelet pseudopodia, platelet lysis, lipid vesicle leakage, and increases in the fluidity of lipid vesicles all suggest that C2-ceramide inhibits platelet aggregation because it destabilizes the platelet membrane. C2-Dihydroceramide did not inhibit platelet aggregation and lacked the nonspecific effects on membranes that C2-ceramide possessed, suggesting that C2-dihydroceramide is not an appropriate control for the nonspecific effects of C2-ceramide.     

The role of lipoproteins in the delivery of tumour-targeting photosensitizers

1. Serum lipoproteins play an important role in the in vivo transport of several porphyrinoid derivatives having a moderate or high degree of hydrophobicity. 2. There appears to exist a correlation between the extent of photosensitizer association with low-density lipoproteins (LDL) and the efficiency of tumour targeting by some classes of photosensitizers, such as differently sulphonated porphyrins and phthalocyanines, haematoporphyrin dialkylethers and unsubstituted phthalocyanines and naphthalocyanines. 3. In all cases, LDL-carried photosensitizers are preferentially released to malignant cells; hence, direct cell damage appears to be the major determinant of tumour damage consequent to photodynamic therapy. 4. Present evidence suggests that the LDL-associated photosensitizer is accumulated by tumour cells largely via a receptor-mediated endocytotic process. 5. Thus, the use of delivery systems for orientating a systemically injected photosensitizer towards lipoproteins has been explored; promising results have been obtained by incorporation of the dye into liposomal vesicles, oil emulsions or inclusion complexes, as well as by precomplexation of the dye with LDL. 6. Moreover, a suitable choice of the chemical constituents of the delivery system and the experimental conditions allows one to modulate the photosensitizer distribution among the different lipoproteins. 7. The occurrence of tumour-targeting strategies other than the LDL pathway is briefly discussed.     

Are exocytosis mechanisms neurotransmitter specific?

Neurotransmission is a multistage regulated process in which a variety of active molecules contained in vesicles are liberated in response to specific stimuli from different types of neurone or related cells. This includes the release of fast neurotransmitters such as amino acids and acetylcholine from central and peripheral synapses, but also that of relatively slow-acting polypeptides from central and peripheral neurones or neuroendocrine cells. Considerable progress has been made over recent years in the understanding at a molecular level of the mechanism of regulated exocytosis, a crucial phase in this phenomenon. The currently proposed overall mechanism, which incorporates the "SNARE" hypothesis for vesicle-membrane docking and fusion, is based on data from experimental models ranging from brain synaptosomes to mast cells. Since the kinetics of the models studied and the physiological effects of the neurotransmitters implicated vary so much, it is pertinent to question whether a general mechanism can be proposed from such experimental data. This review examines known differences in putative exocytotic mechanisms for the various systems studied and attempts to relate these to the nature of the active substances released. Differences exist in each step of the exocytosis process and include the channel through which Ca2+ enters to trigger it or the internal Ca2- source, the type of vesicle in which the transmitter is packaged, the way vesicles are translocated to the surface membrane or how they dock and fuse with it. Major differences have been reported in release mechanisms of different types of vesicle, but minor differences also exist within the same vesicle class. Thus small synaptic vesicles and large dense core vesicles are translocated by distinct processes and the Ca2+ channels, Ca2+ sensors and docking proteins involved in other steps are not identical in all neuronal phenotypes. It may be concluded that each of these differences has evolved to accommodate the different physiological requirements of the neuromodulator released.     

The eleven stages of the cell cycle, with emphasis on the changes in chromosomes and nucleoli during interphase and mitosis

Since we had subdivided the cell cycle into 11 stages--four for mitosis and seven for the interphase--and since we had experience in detecting DNA in the electron microscope (EN) by the osmium-amine procedure of Cogliati and Gauthier (Compt. Rend. Acad. Sci., 1973;276:3041-3044), we combined the two approaches for the analysis of DNA-containing structures at all stages of the cell cycle. Thin Epon sections of formaldehyde-fixed mouse duodenum were stained by osmium-amine for electron microscopic examination of the stages in the 12.3-hr long cell cycle of mouse duodenal crypt columnar cells. In addition, semi-thin Lowicryl sections of mouse duodenal crypts and cultured rat kidney cells were stained with the DNA-specific Hoechst 33258 dye and examined in the fluorescence microscope. The DNA detected by osmium-amine is in the form of nucleofilaments, seen at high magnification as long rows of 11 nm-wide rings (consisting of stained DNA encircling unstained histones). At all stages of the cycle as well as in nondividing cells, nucleofilaments are of three types: 'free,' 'attached' to chromatin accumulations, and 'compacted' in all chromatin accumulations, the form of dense spirals within. At stage I of the cycle, besides free and attached nucleofilaments, compacted ones are observed in the three heterochromatin forms (peripheral, nucleolus-associated, clumped). Soon after the S phase begins, chromatin 'aggregates' appear, which are small at stage II, mid-sized at stage III, and large at stage IV. Chromatin 'bulges' also appear at stage III and enlarge at stage IV, while heterochromatins disappear. At stage V, aggregates and bulges accrete into 'chromomeres,' a process responsible for the apparent chromosome condensation observed at prophase. The chromomeres gradually line up in rows and, at stage VIa (prometaphase), approach one another within each row and coalesce to build up the metaphase chromosomes which are fully formed at stage VIb (metaphase). Daughter chromosomes arising at stage VII (anaphase) are eventually packed into a chromosomal mass at each pole of the cell. During stage VIII (telophase), the chromosomal mass is split into large chunks. In the course of the G1 phase, the chunks thin out to give rise to irregular 'bands' at stage IX, the bands are then cleaved into central and peripheral fragments at stage X, and finally the central fragments are replaced by free nucleofilaments and clumps at stage XI, while the peripheral fragments are replaced by peripheral heterochromatin. The "nucleoli" at stages I-III are associated with stained heterochromatin but otherwise appear as unstained lucent areas, except for weakly stained patches composed of histone-free DNA filaments. During stage IV, nucleoli lose patches and associated heterochromatin, while weakly lucent, pale vesicles appear within nucleoli and in the nucleoplasm. By the end of substage VIa, nucleoli generally disappear, while pale vesicles persist around the chromosomes appearing at substage VIb. At stages VIII and IX, the vesicles seem to become strongly lucent and, at stages IX and X, they associate and fuse to yield homogeneous lucent areas, the 'prenucleolar bodies,' which include histone-free DNA patches. During stage XI, groups of these bodies associate to give rise to nucleoli. In conclusion, the cell cycle DNA changes can be classified into 4 broad periods (Fig. 6): 1) Stage I is a 2-hr long interphase "pause," during which the stained DNA shows no signs of either chromosome condensation or decondensation, while the overall nuclear pattern is similar to that in nondividing cell nuclei. Nucleoli are fully developed. 2) From stage II to VIa, the "chromosome condensation" period extends over about 7 hr, during which the events are interpreted as follows. Throughout the S phase (stages II-IV), newly-synthesized segments of nucleofilaments approach one another, adhere and thus build aggregates and later bulges on nuclear matrix sites. (ABSTRACT TRUNCATED)     

Vectorial release of poliovirus from polarized human intestinal epithelial cells

Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.     

Expression of the beta 4 integrin subunit in the mouse heart during embryonic development: retinoic acid advances beta 4 expression

Dye transfer between lens fiber cells and between lens epithelial cells and underlying fiber cells was studied using a wide dynamic range-cooled CCD camera, H2O immersion objectives and image analysis techniques. Each lens was decapsulated by a new technique which leaves the epithelial cells adherent to the lens fiber mass. Lucifer Yellow CH was injected into either single epithelial cells or single fiber cells using the standard whole cell configuration of the patch voltage clamp technique. The results demonstrate extensive dye communication between fiber cells at the lens posterior surface, anterior surface, and equatorial surface. Dye transfer between deep fiber cells was also observed. Dye transfer between approximately 10% of epithelial cells and their underlying fiber cells was apparent when care was taken to yield wide dynamic range images. This was required because the relatively high concentration of dye in the epithelial cell masks the presence of much lower dye concentrations in the underlying fiber cell. A mathematical model which includes dye concentration, time, and spatial spread suggests that those epithelial cells that are coupled to an underlying fiber cell are about as well dye coupled as the epithelial cells themselves. The relatively low dye concentration in a fiber cell is due to its larger volume and diffusion of the dye along the axis of the fiber away from the fiber/epithelial junction.     

The fine structure of the interrenal cells of the duck (Anas platyrhynchos) with evidence for possible exocytotic release of steroids

The duck interrenal cell possesses ultrastructural characteristics common to other steroid-secreting cells. Lipid droplets and mitochondria are abundant and lie principally at the apical end of the cell. Lipid droplets are not membrane-limited. Cisternae of smooth endoplasmic reticulum that are occasionally continuous with the less abundant rough endoplasmic reticulum are a prominent feature of the interrenal cell. Tubular profiles of rough endoplasmic reticulum often lie tangentially to mitochondria and ribosomes are either free, grouped in polyribosomal clusters, or bound to the endoplasmic reticulum. Mitochondria possess tubular cristae in the inner regions of the gland and frequently contain a paracrystalline array of small 10nm (o.d.) tubules and less frequently a hexagonal array of 40 nm trilaminar rings. Other cytoplasmic components include dense bodies, residual bodies, microtubules, microfilaments and specialized single membrane-bound vesicles. Gap junctions, intermediate junctions and interdigitating processes constitute the main intercellular associations. No tight junctions were identified. The single membrane-bound vesicles which are occasionally filled with a low electron-dense, lipid-like material form septate-like "junctions" with the plasma membrane. The septa bridge an intracellular gap of 15-17 nm. The vesicles are usually located near the subendothelial space at the basal and basilateral regions of the cell. Occasionally, vesicles fuse with the plasma membrane. It is suggested that these vesicles represent morphological evidence for the exocytotic release of steroid hormones.     

Use of fluorescent probes to follow membrane traffic in nerve terminals

Optical tracers in conjunction with fluorescence microscopy have become widely used to follow the movement of synaptic vesicles in nerve terminals. The present review discusses the use of these optical methods to understand the regulation of exocytosis and endocytosis of synaptic vesicles. The maintenance of neurotransmission depends on the constant recycling of synaptic vesicles and important insights have been gained by visualization of vesicles with the vital dye FM1-43. A number of questions related to the control of recycling of synaptic vesicles by prolonged stimulation and the role of calcium to control membrane internalization are now being addressed. It is expected that optical monitoring of presynaptic activity coupled to appropriate genetic models will contribute to the understanding of membrane traffic in synaptic terminals.     

Coulometric detection in high-performance liquid chromatographic analysis of cholesteryl ester hydroperoxides

From the secretion of neurotransmitters via synaptic vesicles to the expulsion of cellular waste via contractile vacuoles, exocytosis and its sequel, endocytosis, are being explored with a variety of new optical tools. Fluorescent markers, especially styryl dyes such as FM1-43 (which reversibly labels endosomal membranes), have been used to follow exo- and endocytic events in many cell types. Even though the development of new dyes is still largely empirical, some theoretical principles have emerged to guide future dye chemistry. Moreover, advances in optical imaging technology that augment conventional fluorescence microscopy are appearing. For example, interference reflection microscopy (which requires no flurophore) and total internal reflection microscopy have recently been used to observe single exocytic events at the contact point between a glass coverslip and the plasma membrane.