Sequestration Revisited: Integrating Traditional Electron Microscopy,De Novo Assembly and New Results

Electron microscopy analysis of the autophagic sequestration membrane (SM) in various metazoan cell types after different fixation methods shows that: (1) the growing SM cannot derive from preformed rough surfaced endoplasmic reticulum (RER) membranes by transformation; (2) the empty cleft between the two layers of the SM after aldehyde fixation is an artifact of sample preparation; (3) the SM emerges from and grows de novo in cytoplasmic areas where membranous precursors cannot be identified by traditional electron microscopy; (4) the growing SM consists of two tightly packed membrane layers with a sharp bend at the edge; (5) changes in the environment of the growing SM participate in the determination of the size and shape of the autophagosome.     

Plasma membrane regeneration in the myxomyceteBadhamia utricularis

Summary Plasmodial strands of the myxomyceteBadhamia utricularis were injured and, after fixation and sectioning, examined using light and transmission electron microscopy. Injury results in the formation of a cytoplasmic droplet that rapidly regenerates a plasma membrane by the apparent fusion of vesicles at its surface. During the same time several structurally distinct layers form within the droplet. Some of these internal layers, which persist for several minutes after injury, may also arise from vesicle fusion. These results have differences from those reported for the relatedPhysarum polycephalum.     

Morphogenesis of endoplasmic reticulum in Xenopus oocytes after microinjection of rat liver smooth microsomes

We have determined the kinetics of endoplasmic reticulum (ER) reconstitution following insertion of rat-liver smooth microsomes (SM) into Xenopus oocyte cytoplasm using electron microscopy as well as cytochemistry and thick-section 3-dimensional reconstruction. Oocytes were fixed 0, 10, 20, 40, 80, and 120 min after microinjection with SM and processed for thin- and thick-section electron microscopy. At 0 min postinjection, rat liver SM were observed as small vesicles and were loosely dispersed amongst oocyte organelles. At 10 min, tubules were discerned among many elongate vesicles; and these structures comprised large cytoplasmic regions delimited by mitochondria and yolk platelets. By 20 min, segregation of transplanted organelles yielded yolk-platelet-free regions composed of few vesicles but increasingly numerous, long and anastomosing tubules. By 40 min, a network with numerous tubular branches and fenestrations was observed among the few remaining vesicles. By 80 min, transformation of rat liver SM into a complex network of branching and anastomosing tubules was complete. Three-dimensional reconstruction revealed the network to be composed of interconnecting elements consisting of anastomosing tubules. The reconstituted network of anastomosing tubules in Xenopus oocytes was compared to the network of anastomosing tubules in rat liver hepatocytes and was found to be essentially identical. Network formation occurred in oocytes pretreated with either vinblastine (40 microM) or nocodazole (0.166 microM), and network organization was maintained in oocytes treated with the same drugs after microinjection and reconstitution. We conclude that SM retain sufficient molecular information for rapid self-assembly into structures resembling those in the cells from which they were derived. Both the assembly and maintenance of ER structure in oocyte cytoplasm are microtubule-independent. The formation of such structures following microinjection of SM into living cells provides a unique assay for this type of membrane subfraction.     

A new set of vesicles in Giardia lamblia

In the present study it was demonstrated the existence of a new set of membrane-bounded vesicles in Giardia lamblia. They were found in dividing and non-dividing trophozoites studied by routine transmission electron microscopy, freeze-fracture and Thiéry's technique. Encysting cells were not studied. These vesicles appear different to the previously reported components of the Giardia endomembranous system, such as the endoplasmic reticulum (ER), lysosome-like peripheral vesicles (PV), and the encystation-specific vesicles (ESV) that appear during trophozoite differentiation into cysts. They measure 100-150 nm in diameter, and thus are smaller than the peripheral vesicles, and the encystation-specific vesicles (ESV). They were found in clusters, scattered throughout the cytoplasm, but preferentially located close to the nuclei, axonemes, median bodies, and ER profiles. These internal vesicles are roughly spherical, and their contents present different electron densities and are more electrondense than those of the peripheral vesicles. They appeared to be budding from the outer nuclear membrane envelope. These cytoplasmic vesicles were found only in cells with very good fixation. Only few cells in the same preparation exhibited these vesicles.     

Isolation of plasma membrane fractions from green wheat leaves by free-flow electrophoresis or two-phase partition alone or in combination

Plasma membrane vesicles were isolated from shoots of light-grown wheat seedlings by preparative free-flow electrophoresis, aqueous polymer two-phase partition or both. Plasma membrane vesicles were identified from staining of thin sections prepared for electron microscopy with phosphotungstic acid at low pH. The orientation of the plasma membrane vesicles was determined from latency and trypsin sensitivity of K⁺ Mg²⁺ATPase and of glucan synthase II, and concanavalin A-peroxidase binding and membrane asymmetry visualized by electron microscopy. The K⁺Mg²⁺ATPase and of glucan synthase II activities of plasma membrane fractions isolated by two-phase partition were latent and trypsin resistant. The vesicles bound concanavalin A-peroxidase strongly and exhibited a cytoplasmic side-in morphology. These fractions of cytoplasmic side-in vesicles were less than 10% contaminated by cytoplasmic side-out vesicles. By free-flow electrophoresis, two populations of vesicles which stained with phosphotungstic acid at low pH, designated D and E, were obtained. The vesicle population with the lower electrophoretic mobility, fraction E, contained plasma membrane vesicles with properties similar to those of the plasma membrane vesicles obtained after two-phase partition. The phosphotungstic-reactive vesicles with greater electrophoretic mobility, fraction D, were concanavalin A unreactive with the cytoplasmic membrane leaflet outwards. Less than 50% of the K⁺Mg²⁺-ATPase activity of this fraction was latent and trypsin sensitive. The vesicles of fraction D appeared to be preferentially cytoplasmic side-out. The electrophoretic mobilities of cytoplasmic side-out (non-latent glucan synthase II activity) and cytoplasmic side-in (latent glncan synthase II activity) plasma membrane vesicles isolated from a frozen and thawed wheat plasma membrane fraction, corresponded with the mobilities of fraction D and E, respectively, again showing that the plasma membrane vesicles with the lesser electrophoretic mobility were cytoplasmic side-in. The cytoplasmic side-in and cytoplasmic side-out vesicles therefore showed opposite eletrophoretic mobilities compared with a previous free-flow electrophoretic separation of soybean plasma membranes. The majorities of the plasma membrane vesicles of both fractions D and E entered the upper phase upon two-phase partition with the phase composition used for purification of wheat plasma membranes. Thus, neither electrophoretic mobility nor phase partitioning characteristics can be used as the only criteria for assignment of vesicle orientation.     

Role of bristle-coated membrane in the uptake of ferritin by rat macrophages

The distribution of horse ferritin in rat peritoneal macrophages was studied by electron microscopy. In the first few minutes after exposure of the cells to ferritin either in vivo or in vitro, the protein is found in invaginations of the surface membrane not yet isolated from the external medium. These caveolae are characterized by the presence of a prominent bristle coat on their cytoplasmic aspect and a less distinct fuzzy coat on the surface exposed to the exterior of the cell. At later stages ferritin is found in bristle-coated vesicles and later in larger smooth-surfaced vacuoles. Counts of bristle-coated membrane sites before and after exposure to ferritin suggest that ferritin is selectively bound to preformed patches of bristle-coated surface membrane which subsequently invaginate to form the coated vesicles. The question of possible induction of such sites by the presence of ferritin is raised but cannot be settled by the available data.     

TISSUE PREPARATION AND FINE STRUCTURE OF THE RADICLE APEX FROM COTTON SEEDS

A comparative methodological study was made of the fine structure of apical cortical cells in excised radicles from cotton (Gossypium hirsutum L. var M-8) seeds. Radicles from dry seed had 12% moisture content and were prepared for electron microscopy using several different techniques. These included different methods of chemical fixation or freeze-fracture and etching of unfixed tissue for transmission electron microscopy (TEM) and cryofracturing of fixed and dehydrated radicles for scanning electron microscopy (SEM). Cortical cells had a similar appearance regardless of the method used in tissue preparation. Cell walls had a pronounced waviness which was particularly evident in SEM images of cells lining the elongated intercellular air spaces. The plasma membrane (PM) delimited the cytoplasm of each cell as an intact unit membrane. Single layers of tightly-packed lipid bodies (LB) were apposed to the PM and protein bodies (PB). Distension of cells, membranous organelles and LB was observed in radicles fixed by immersion in aqueous solutions, suggesting that a certain amount of hydration occurred during fixation. This interpretation was supported by the compact appearance of cells and organelles in tissue prepared by freeze-etch or vapor fixation. We conclude that freeze-fracture and etching of unfixed tissue provided the best information for cell morphology and structure of membranes and organelles in dry tissue. Complementary data on the fine details of nuclei and cytoplasmic organelles were best observed with TEM of fixed tissue. These data when viewed collectively indicate the advantage of using several techniques to obtain analogous and complementary information essential for establishing a baseline level of information on the fine structure of cells in dry tissue.     

Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli.

Dimethyl sulfoxide reductase is a trimeric, membrane-bound, iron-sulfur molybdoenzyme induced in Escherichia coli under anaerobic growth conditions. The enzyme catalyzes the reduction of dimethyl sulfoxide, trimethylamine N-oxide, and a variety of S- and N-oxide compounds. The topology of dimethyl sulfoxide reductase subunits was probed by a combination of techniques. Immunoblot analysis of the periplasmic proteins from the osmotic shock and chloroform wash fluids indicated that the subunits were not free in the periplasm. The reductase was susceptible to proteases in everted membrane vesicles, but the enzyme in outer membrane-permeabilized cells became protease sensitive only after detergent solubilization of the E. coli plasma membrane. Lactoperoxidase catalyzed the iodination of each of the three subunits in an everted membrane vesicle preparation. Antibodies to dimethyl sulfoxide reductase and fumarate reductase specifically agglutinated the everted membrane vesicles. No TnphoA fusions could be found in the dmsA or -B genes, indicating that these subunits were not translocated to the periplasm. Immunogold electron microscopy of everted membrane vesicles and thin sections by using antibodies to the DmsABC, DmsA, DmsB subunits resulted in specific labeling of the cytoplasmic surface of the inner membrane. These results show that the DmsA (catalytic subunit) and DmsB (electron transfer subunit) are membrane-extrinsic subunits facing the cytoplasmic side of the plasma membrane.     

Immunoelectron microscopic localization of the F1F0 ATPase (ATP synthase) on the cytoplasmic membrane of alkalophilic Bacillus firmus RAB

Evidence that the F1F0 ATPase (ATP synthase) of alkalophilic Bacillus firmus RAB is localized exclusively on the cytoplasmic membrane was obtained by immunogold electron microscopy using a highly specific polyclonal antibody against the beta subunit of Escherichia coli F1F0 ATPase. The energetic problem faced by cells of B. firmus RAB growing oxidatively at pH 10.5 despite a low protonmotive force across the cytoplasmic membrane cannot, therefore, be circumvented by localization of energy transducing functions on hypothetical internal membranes.     

Studies on scale structure in the common goby Pomatoschistus microps Krøyer

A histological study on scale structure in the common goby, Pomatoschistus microps (Krøyer) has established a similarity to other teleost scales, but yielded two new findings. First, the osseous layer of the scale was formed of successive and parallel calcified bands that may reflect a rhythm in scale growth. Second, extracellular matrix vesicles (100–200 nm) were identified by electron microscopy, at the calcifying area in the anterior growing edge of the scale; distribution of these vesicles corresponded closely to the pattern of scale calcification. A proposal on the mechanism of growth and calcification of scale has been made. The surface topography of the scale has also been examined by scanning electron microscopy. The raised edge of the circulus is formed of minute knobs, which may be used as criterion in fish taxonomy.     

Incorporation of cholesterol in sphingomyelin- phosphatidylcholine vesicles has profound effects on detergent-induced phase transitions

Vesicle <--> micelle transitions are important phenomena during bile formation and intestinal lipid processing. The hepatocyte canalicular membrane outer leaflet contains appreciable amounts of phosphatidylcholine (PC) and sphingomyelin (SM), and both phospholipids are found in the human diet. Dietary SM enrichment inhibits intestinal cholesterol absorption. We therefore studied detergent-induced vesicle --> micelle transitions in SM-PC vesicles. Phase transitions were evaluated by spectrophotometry and cryotransmission electron microscopy (cryo-TEM) after addition of taurocholate (3-7 mM) to SM-PC vesicles (4 mM phospholipid, SM/PC 40%/60%, without or with 1.6 mM cholesterol). After addition of excess (5-7 mM) taurocholate, SM-PC vesicles were more sensitive to micellization than PC vesicles. As shown by sequential cryo-TEM, addition of equimolar (4 mM) taurocholate to SM-PC vesicles induced formation of open vesicles, then (at the absorbance peak) fusion of bilayer fragments into large open structures (around 200 nm diameter) coexisting with some multilamellar or fused vesicles and thread-like micelles and, finally, transformation into an uniform picture with long thread-like micelles. Incorporation of cholesterol in the SM/PC bilayer changed initial vesicular shape from spherical into ellipsoid and profoundly increased detergent resistance. Disk-like micelles and multilamellar vesicles, and then extremely large vesicular structures, were observed by sequential cryo-TEM under these circumstances, with persistently increased absorbance values by spectrophotometry. These findings may be relevant for bile formation and intestinal lipid processing. Inhibition of intestinal cholesterol absorption by dietary SM enrichment may relate to high resistance against bile salt-induced micellization of intestinal lipids in presence of the sphingolipid.     

Cytochemical study of liposome and lipid vesicle phagocytosis

Electron microscopy cytochemistry has been used to study the cytoplasmic location of liposomes and lipid vesicles following specific antibody-dependent phagocytosis. The vesicle compositions were 94–99 mol% ‘fluid’ lipid (egg phosphatidylcholine or dimyristoylphosphatidylcholine at 37°C or ‘solid’ lipid (dipalmitoylphosphatidylcholine at 37°C). In some cases, 4 mol% phosphatidylserine was included in the vesicle membrane so as to vary the surface charge density. These vesicles undergo specific antibody-dependent phagocytosis by RAW264 macrophages when the lipid membranes contain 1–2 mol% dinitrophenyl lipid hapten in the presence of rabbit anti-dinitrophenyl IgG antibody. Internalized lipid vesicles can be visualized with the electron microscope when ferritin is trapped in the internal aqueous compartments prior to internalization. The lipid vesicles were demonstrated to be internal to the macrophage plasma membranes by selectively staining the plasma membranes with Ruthenium red. The cytoplasmic location of vesicles and liposomes was studied by electron microscopic staining for activities of the following enzymes: (1) acid phosphatase; (2) inorganic trimetaphosphatase; (3) adenosine triphosphatase; and (4) glucose-6-phosphatase. The first two enzymatic activities were found in association with ferritin-containing vesicles after antibody-dependent phagocytosis, showing the formation of vesicle-containing phagolysosomes. Adenosine triphosphatase and glucose-6-phosphatase were primary not associated with the vesicles, suggesting a minimal association of vesicles with plasma membrane, Golgi, endoplasmic reticulum and perinuclear cisternae. Phagosome-lysosome fusion did not appear to depend on the type of target lipid vesicle or liposome, on the ‘fluidity’ of the target membrane, or the presence of phosphatidylserine in the target membrane.     

Permeation of bacterial cells, permeation of cytoplasmic and artificial membrane vesicles, and channel formation on lipid bilayers by peptide antibiotic AS-48.

Peptide AS-48 induces ion permeation, which is accompanied by the collapse of the cytoplasmic membrane potential, in sensitive bacteria. Active transport by cytoplasmic membrane vesicles is also impaired by AS-48. At low concentrations, this peptide also causes permeability of liposomes to low-molecular-weight compounds without a requirement for a membrane potential. Higher antibiotic concentrations induce severe disorganization, which is visualized under electron microscopy as aggregation and formation of multilamellar structures. Electrical measurements suggest that AS-48 can form channels in lipid bilayers.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : I. Structure and Formation of Mesosomes

Cells of Chondrococcus columnaris were sectioned and examined in the electron microscope after fixation by two different methods. After fixation with osmium tetroxide alone, the surface layers of the cells consisted of a plasma membrane, a dense layer (mucopeptide layer), and an outer unit membrane. The outer membrane appeared distorted and was widely separated from the rest of the cell. The intracytoplasmic membranes (mesosomes) appeared as convoluted tubules packaged up within the cytoplasm by a unit membrane. The unit membrane surrounding the tubules was continuous with the plasma membrane. When the cells were fixed with glutaraldehyde prior to fixation with osmium tetroxide, the outer membrane was not distorted and separated from the rest of the cell, structural elements (peripheral fibrils) were seen situated between the outer membrane and dense layer, and the mesosomes appeared as highly organized structures produced by the invagination and proliferation of the plasma membrane. The mesosomes were made up of a series of compound membranes bounded by unit membranes. The compound membranes were formed by the union of two unit membranes along their cytoplasmic surfaces.     

Calcium-Dependent Self-Association of Synaptotagmin I

Abstract: Synaptotagmin I, an integral membrane protein of secretory vesicles, appears to have an essential role in calcium-triggered hormone and neurotransmitter release. The large cytoplasmic domain of synaptotagmin I has two C2 domains that are thought to mediate calcium and phospholipid binding. A recombinant protein (p65 1–5) comprised of the cytoplasmic domain was previously shown to aggregate purified chromaffin granules and artificial phospholipid vesicles in a calcium-dependent manner. p65 1–5 may be able to aggregate membrane vesicles by a self-association reaction. This hypothesis led us to investigate the ability of synaptotagmin I protein fragments to multimerize in vitro. We found that p65 1–5, in the absence of membranes, was able to self-associate to form large aggregates in a calcium-dependent manner as shown by light-scattering assays and electron microscopy. In addition, a recombinant protein comprised of only the second half of the cytoplasmic domain, including the second C2 domain, was also able to self-associate and aggregate phospholipid vesicles in a calcium-dependent manner. A recombinant protein comprised of only the first C2 domain was not able to self-associate or aggregate vesicles. These results suggest that synaptotagmin I is able to bind calcium in the absence of membranes and that the second half of the cytoplasmic domain is able to bind calcium and mediate its multimerization in a calcium-dependent manner. The ability of synaptotagmin I protein fragments to multimerize in a calcium-dependent manner in vitro suggests that multimerization may have an important function in vivo.     

Interactions between pH-sensitive liposomes and model membranes

The structure and dynamics of two different pH-sensitive liposome systems were investigated by means of cryo-transmission electron microscopy and different photophysical techniques. Both systems consisted of dioleoylphosphatidylethanolamine (DOPE) and contained either oleic acid (OA) or a novel acid-labile polyethylene glycol-conjugated lipid (DHCho-MPEG5000) as stabiliser. Proton induced leakage, lipid mixing and structural changes were studied in the absence and presence of EPC liposomes, as well as in the presence of liposomes designed to model the endosome membrane. Neither DHCho-MPEG5000- nor OA-stabilised liposomes showed any tendency for fusion with pure EPC liposomes or endosome-like liposomes composed of EPC/DOPE/SM/Cho (40/20/6/34 mol.%). Our investigations showed, however, that incorporation of lipids from the pH-sensitive liposomes into the endosome membrane may lead to increased permeability and formation of non-lamellar structures. Taken together the results suggest that the observed ability of DOPE-containing liposomes to mediate cytoplasmic delivery of hydrophilic molecules cannot be explained by a mechanism based on a direct, and non-leaky, fusion between the liposome and endosome membranes. A mechanism involving destabilisation of the endosome membrane due to incorporation of DOPE, seems more plausible.     

Cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor cluster in cell cultures

Whole-mount stereo electron microscopy has been used to examine the cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor (AChR) clusters in cultures of Xenopus nerve and muscle cells. The cells were grown on Formvar-coated gold electron microscope (EM) finder grids. AChR clusters were identified in live cultures by fluorescence microscopy after labeling with tetramethylrhodamine-conjugated alpha-bungarotoxin. After chemical fixation and critical-point drying, the cytoplasmic specializations of identified cells were examined in whole mount under an electron microscope. In the presynaptic nerve terminal opposite to the AChR cluster, synaptic vesicles were clearly suspended in a lattice of 5-12- nm filaments. Stereo microscopy showed that these filaments directly contacted the vesicles. This lattice was also contiguous with the filament bundle that formed the core of the axon. At the AChR cluster, an increased cytoplasmic density differentiated this area from the rest of the cytoplasm. This density was composed of a meshwork of filaments with a mean diameter of 6 nm and irregularly shaped membrane cisternae 0.1-0.5 micron in width, which resembled the smooth endoplasmic reticulum. These membrane structures were interconnected via the filaments. Organelles that were characteristic of the bulk of the sarcoplasm such as the rough endoplasmic reticulum and the polysomes, were absent from the cytoplasm associated with the AChR cluster. These results indicate that the cytoskeleton may play an important role in the development and/or the maintenance of the neuromuscular synapse, including the release of transmitter in the nerve terminal and the clustering of AChRs in the postsynaptic membrane.     

Interaction of Actinobacillus actinomycetemcomitans outer membrane vesicles with HL60 cells does not require leukotoxin

Outer membrane derived vesicles (MVs) secreted by Actinobacillus actinomycetemcomitans JP2 contain a membranolytic leukotoxin and are toxic to human HL60 cells. To determine how MVs interact with human target cells, HL60 cells were incubated with vesicles, reacted with anti-vesicle antibodies and a FITC-labelled reporter, and visualized by confocal scanning laser microscopy. Target cells rapidly became reactive with anti-vesicle antibodies upon exposure to vesicles. Confocal microscopy showed that labelling occurred primarily in the cytoplasmic membrane and that very little internal fluorescence was observed. The cytoplasmic membrane of HL60 cells was also strongly labelled after exposure to MVs that contained the fluorescent phospholipid, SP-DiOC18. In contrast, incubation of cells with free SP-DiOC18 resulted primarily in the labelling of internal structures of HL60 cells. These results suggest that A. actinomycetemcomitans MVs associate with, or are incorporated into the cytoplasmic membrane of HL60 cells. The leukotoxin is a membranolytic cytotoxin and cells exposed to MVs were lysed by vesicle-associated toxin in a time and dose-dependent manner. However, cells became reactive with anti-vesicle antibodies when MVs were added in the presence of inhibitors of leukotoxin-mediated lysis or when sublytic doses of MVs were analysed. In addition, MVs produced by an isogenic leukotoxin-deficient strain of A. actinomycetemcomitans JP2 were non-toxic but rapidly interacted with HL60 cells. These results suggest that A. actinomycetemcomitans MVs can deliver leukotoxin to HL60 cells but that the association of vesicles with the cytoplasmic membrane occurs independently of the leukotoxin polypeptide.     

Electron microscopic and biophysical studies of liposome membrane structures to characterize similar features of the membranes of Streptomyces hygroscopicus

To characterize the novel non-planar plasma membrane structure of bacteria (wafer structure), liposome membranes from the bacterial lipid mixture and individual lipid fractions were prepared and investigated by freeze-fracture electron microscopy, microcalorimetry and 31P-NMR spectroscopy. The phospholipid content of the membranes is essential for the formation of the non-planar membrane structure and there is no indication that the formation of the structure is connected with temperature-induced lipid phase transition processes. An exaggerated form of the wafer structure (raspberry structure) is also visible and additionally, in both cases, many small spherical vesicles are observed. We suggest that both membrane features of the liposomal and bacterial membranes are induced by these vesicles, forming a hexagonal or cubic organization of vesicles on the cytoplasmic surface of the biological membrane, and in between the multilamellae in the artificial membranes.     

ARTIFACTUAL LOCALIZATION OF FERRITIN IN THE CILIARY EPITHELIUM IN VITRO

The accumulation of ferritin by the ciliary epithelium of the adult albino rabbit has been studied by electron microscopy. The experiments have been carried out under in vitro conditions, such that any uptake observed should be the result of passive diffusion of the tracerparticles rather than the product of active metabolic processes. The cells were fixed in osmium tetroxide and embedded in Araldite. Ferritin was found localized in three areas: in rows of apparent vesicles, free in the cytoplasmic matrix, and in the basement membrane. Some of the conclusions reached are as follows. The appearance of tracer in rows of vesicles is not in itself an adequate demonstration of pinocytosis. The permeability of the plasma membrane is drastically increased by osmium tetroxide fixation, so that tracer particles are free to diffuse across the membrane and wander through the cytoplasm. These results indicate the serious danger of being misled by artifacts when colloidal particles are used as tracers.