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L Edelmann

Publications and source records attributed to L Edelmann.

At least 37 records · Page 2Linked to original sources
PubMed1996

Axonal transport and distribution of synaptobrevin I and II in the rat peripheral nervous system.

Synaptobrevin, a membrane protein of synaptic vesicles that plays a key role in exocytosis, occurs in two closely related isoforms, synaptobrevin I and II. We have analyzed the axonal transport of both isoforms in sciatic nerve and spinal roots. When fast axonal transport was interrupted by crushing, the proteins accumulated continuously proximal to the crush. Accumulation also was observed distal to the crush, but to a lesser extent (47 and 63% of the proximal accumulation for synaptobrevin I and II, respectively). Immunoelectron microscopy revealed that, proximal to the crush, synaptobrevin I and II were associated with small clear vesicles reminiscent of typical synaptic vesicles. Distal to the crush, membranes positive for synaptobrevin I or II were more heterogeneous, including larger membrane profiles that may represent endosomes. In spinal cord, synaptobrevin I and II were colocalized in many terminals. However, labeling for synaptobrevin I was more intense whereas labeling for synaptobrevin II was stronger in dorsal than in ventral horn terminals. Motor endplates contained only synaptobrevin I. In the sciatic nerve, synaptobrevin I was present predominantly in large, myelinated axons, whereas synoptobrevin II was virtually absent but abundant in small- and medium-sized axons. Lumbar sympathectomy, ventral rhizotomy, and double-labeling studies confirmed that synaptobrevin I is present predominantly in motor neurons whereas synaptobrevin II is present in adrenergic and sensory neurons. We conclude that synaptobrevin I and II are transported bidirectionally by fast axonal transport and are expressed heterogeneously in different neurons in the peripheral nervous system of the adult rat, suggesting that these isoforms have special functional roles in different sets of neurons.

Animals↗
PubMed1996

Freeze-dried human leukocytes stabilized with uranyl acetate during low temperature embedding or with OsO4 vapor after embedding.

Two new simple stabilization procedures for freeze-dried biological material are introduced which are compatible with low temperature embedding (LTE) in Lowicryl. The first method uses a Lowicryl K11M/HM20 mixture supplemented with 0.3% uranyl acetate for LTE. For the second method polymerized Lowicryl blocks containing the freeze-dried material are exposed to OsO4 vapor which penetrates into the Lowicryl block and stabilizes the embedded specimen. The quality of structural preservation is demonstrated with human leukocytes.

Acrylic Resins↗
PubMed1995

Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine.

The synaptic vesicle protein synaptobrevin (VAMP) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion. It interacts with the synaptic membrane proteins syntaxin I and synaptosome-associated protein (SNAP)-25 to form a complex which precedes exocytosis [Söllner et al. (1993b) Cell, 75, 409-418]. Here we demonstrate that the majority of synaptobrevin is bound to the vesicle protein synaptophysin in detergent extracts. No syntaxin I was found in this complex when synaptophysin-specific antibodies were used for immunoprecipitation. Conversely, no synaptophysin was associated with the synaptobrevin-syntaxin I complex when syntaxin-specific antibodies were used for immunoprecipitation. Thus, the synaptobrevin pool bound to synaptophysin is not available for binding to syntaxin I and SNAP-25, and vice versa. Synaptobrevin-synaptophysin binding was also demonstrated by chemical cross-linking in isolated nerve terminals. Furthermore, recombinant synaptobrevin II efficiently bound synaptophysin and its isoform synaptoporin, but not the more distantly related synaptic vesicle protein p29. Recombinant synaptobrevin I bound with similar efficiency, whereas the non-neuronal isoform cellubrevin displayed a lower affinity towards synaptophysin. Treatment with high NaCl concentrations resulted in a dissociation of the synaptobrevin-synaptophysin complex. In addition, the interaction of synaptobrevin with synaptophysin was irreversibly abolished by low amounts of SDS, while the interaction with syntaxin I was enhanced. We conclude that synaptophysin selectively interacts with synaptobrevin in a complex which excludes the t-SNAP receptors syntaxin I and SNAP-25, suggesting a role for synaptophysin in the control of exocytosis.

Amino Acid Sequence↗
PubMed1995

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling.

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t-SNAREs participate in SV recycling in what may be functionally distinct forms.

Animals↗
PubMed1995

Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells.

There is strong evidence to indicate that proteins of the synaptobrevin family play a key role in exocytosis. Synaptobrevin 1 and 2 are expressed at high concentration in brain where they are localized on synaptic vesicles. Cellubrevin, a very similar protein, has a widespread tissue distribution and in fibroblasts is localized on endosome-derived, transferin receptor-positive vesicles. Since brain cellubrevin is not detectable in synaptic vesicles, we investigated whether cellubrevin and the synaptobrevins are differentially targeted when co-expressed in the same cell. We report that in the nervous system cellubrevin is expressed at significant levels only by glia and vascular cells. However, cellubrevin is coexpressed with the two synaptobrevins in PC12 cells, a neuroendocrine cell line which contains synaptic vesicle-like microvesicles. In PC12 cells, cellubrevin has a distribution very similar to that of synaptobrevin 1 and 2. The three proteins are targeted to neurites which exclude the transferrin receptor and are enriched in synaptic-like microvesicles and dense-core granules. They are recovered in the synaptic-like microvesicle peak of glycerol velocity gradients, have a similar distribution in isopycnic fractionation and are coprecipitated by anti-synaptobrevin 2 immunobeads. Finally, cellubrevin, like the synaptobrevins, interact with the neuronal t-SNAREs syntaxin 1 and SNAP-25. These results suggest that cellubrevin and the synaptobrevins have similar function and do not play a specialized role in constitutive and regulated exocytosis, respectively.

Amino Acid Sequence↗
PubMed1994

The axonal gamma-aminobutyric acid transporter GAT-1 is sorted to the apical membranes of polarized epithelial cells.

Recent studies suggest that epithelial cells and neurons employ similar mechanisms to target proteins to the distinct subdomains of their polarized cell surface membranes. We have examined the sorting behavior of the neuronal gamma-aminobutyric acid (GABA) transporter GAT-1 expressed by transfection in the polarized epithelial Madin-Darby canine kidney (MDCK) cell line. We find that the GABA transporters endogenously expressed by polarized hippocampal neurons in culture are restricted to axonal plasma membranes. In transfected MDCK cells, the GABA transporter is found to be localized primarily to the apical cell surface when examined by immunocytochemistry, cell surface biotinylation, and transport assay. MDCK cells exposed to hyperosmotic stress express a close relative of GAT-1, the betaine transporter (BGT-1). We find that BGT-1 expressed by transfection in MDCK cells accumulates predominantly at the basolateral cell surface. These observations suggest that the sorting information required for axonal targeting may be similar to that which mediates apical localization in epithelia. Furthermore, it would appear that despite their high degree of homology, the BGT-1 and GAT-1 transporters manifest sorting signals which specify their targeting to distinct cell surface domains.

Animals↗
PubMed1994

A new versatile system for freeze-substitution, freeze-drying and low temperature embedding of biological specimens.

A universal system for freeze-substitution (FS), freeze-drying (FD) and low temperature embedding (LTE) has been developed, suited to perform standardized procedures of cryoprocessing biological and medical specimens as well as systematic studies of dehydration and embedding at various low and high temperatures. In a 35 1 Dewar vessel with 110 mm neck diameter an aluminum tube is mounted to the bottom of the liquid nitrogen (LN2x) reservoir and extends to the lower part of the cylindrical neck. At its top an aluminum plate serves as a contact surface for either the FS chamber or the FD chamber. Fs and subsequent LTE are carried out in an environment of dry cold nitrogen gas provided by evaporating nitrogen from the dewar. Different capsules and moulds may be used for cryodehydration and LTE. FD of bulk specimens or cryosections takes place in an absolutely clean vacuum provided by a cryosorption pump integrated in the FD apparatus. Most of the H2O molecules from the frozen specimen are trapped by large cold surfaces inside the drying chamber. Due to the low LN2 consumption during FS or FD (3-4 1 LN2/day) both procedures may be carried out for 8-10 days without refilling the dewar. A few representative results show that well frozen biological material is stabilized by prolonged FS or FD at temperatures of about -80 degrees C without user of chemical fixatives like OsO4 in the substitution medium during FS or by OsO4 vapor fixation after FD.

Animals↗
PubMed1994

Optimal freeze-drying of cryosections and bulk specimens for X-ray microanalysis.

Electron microscopic investigations of rapidly frozen specimens of striated muscle, either frozen-hydrated or obtained after different dehydration procedures, have shown that the subcellular distribution of the main cellular cation K+ or its surrogates Rb+, Cs+, or Tl+ does not follow the water distribution but follows certain proteins. Conflicting results obtained by X-ray microanalysis of freeze-dried cryosections are explained by showing that freeze-drying of bulk specimens and cryosections must be carried out for rather long periods at low temperature in order to avoid severe shrinkage and ion redistribution artefacts. Proposals for future freeze-drying studies are derived from the concept that cellular water is organized differently from normal free water and that proteins of living cells are able to selectively adsorb alkali-metal ions.

Animals↗
PubMed1993

Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein.

Tetanus toxin inhibits neurotransmitter release by selectively blocking fusion of synaptic vesicles. Recently tetanus toxin was shown to proteolytically degrade synaptobrevin II (also named VAMP-2), a synaptic vesicle-specific protein, in vitro and in nerve terminals. As targets of tetanus toxin, synaptobrevins probably function in the exocytotic fusion of synaptic vesicles. Here we describe a new synaptobrevin homologue, cellubrevin, that is present in all cells and tissues tested and demonstrate that it is a membrane trafficking protein of a constitutively recycling pathway. Like synaptobrevin II, cellubrevin is proteolysed by tetanus toxin light chain in vitro and after transfection. Our results suggest that constitutive and regulated vesicular pathways use homologous proteins for membrane trafficking, probably for membrane fusion at the plasma membrane, indicating a greater mechanistic and evolutionary similarity between these pathways than previously thought.

Amino Acid Sequence↗
PubMed1992

Tetanus toxin action: inhibition of neurotransmitter release linked to synaptobrevin proteolysis.

Tetanus toxin is a potent neurotoxin that inhibits the release of neurotransmitters from presynaptic nerve endings. The mature toxin is composed of a heavy and a light chain that are linked via a disulfide bridge. After entry of tetanus toxin into the cytoplasm, the released light chain causes block of neurotransmitter release. Recent evidence suggests that the L-chain may act as a metalloendoprotease. Here we demonstrate that blockade of neurotransmission by tetanus toxin in isolated nerve terminals is associated with a selective proteolysis of synaptobrevin, an integral membrane protein of synaptic vesicles. No other proteins appear to be affected by tetanus toxin. In addition, recombinant light chain selectively cleaves synaptobrevin when incubated with purified synaptic vesicles. Our data suggest that cleavage of synaptobrevin is the molecular mechanism of tetanus toxin action.

Animals↗
PubMed1992

p53 expression in neoplasms of the uterine corpus.

It has been recognized that mutations in tumor suppressor genes may have an important oncogenic role. Although abnormalities of the p53 tumor suppressor gene have been reported in tumors from various organ systems, p53 expression has not been studied in neoplasms of the uterine corpus. Using a monoclonal antibody to the p53 product, frozen sections of 56 uterine tumors (40 endometrioid endometrial adenocarcinomas, 7 serous endometrial carcinomas, 4 mixed Müllerian tumors, 2 endometrial stromal sarcomas, 1 leiomyosarcoma, 2 leiomyomas) and 2 normal endometria were stained using the immunoperoxidase technique. Staining was evaluated by light microscopic examination; in carcinomas with strong/diffuse reactivity, evaluation was by digitized image analysis. p53 staining of adenocarcinomas was compared statistically to the histologic type, grade, surgical stage, and clinical follow-up. Specific staining was present in the nucleus of malignant tumor cells only. Benign cells did not stain. Strong/diffuse staining was seen in 14 adenocarcinomas and in 2 mixed Müllerian tumors. Weak/focal staining was observed in 14 adenocarcinomas. Serous carcinomas showed strong positivity more frequently than endometrioid endometrial carcinomas. Staining patterns correlated with histologic grade and stage. Image analysis of immunostained p53 correlated with type of adenocarcinoma, but not with grade or stage in the cases measured. p53 was expressed strongly in tumors of five of eight patients who died of adenocarcinoma but in none of five patients with no evidence of disease and a minimum follow-up of 24 months. In addition, 3 of 12 patients with persistent or recurrent disease showed tumors that strongly expressed p53. It is concluded that abnormal expression of p53 occurs frequently in malignant uterine tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients.

The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.

Animals↗
PubMed1991

Freeze-substitution and the preservation of diffusible ions.

Freeze-substitution of biological material in pure acetone followed by low-temperature embedding in the Lowicryls K11M and HM23 yields stable preparations well suited for sectioning and subsequent morphological and microanalytical studies. Transmission electron microscopy of dry-cut sections shows that diffusible cellular thallium ions (T1+) of T1(+)-loaded muscle are localized at similar protein sites in freeze-substituted as in frozen-hydrated preparations. A comparison of X-ray microanalytical data obtained from freeze-dried cryosections and sections of freeze-substituted normal (potassium-containing) muscle shows that K+ ion retention in the freeze-substituted sample is highly dependent on the freeze-substitution procedure used so far, in the best case, about 67% of the cellular K+ is retained after freeze-substitution in pure acetone and low-temperature embedding. It is concluded that the macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze-substitution and low-temperature embedding.

Acetone↗
PubMed1991

Adsorption staining of freeze-substituted and low temperature embedded frog skeletal muscle with cesium: a new method for the investigation of protein-ion interactions.

A new adsorption staining method for transmission electron microscopy is described by means of which cellular adsorption sites of alkali-metal ions can be visualized in freeze-substituted and low temperature embedded biological material. The main features of this staining method are: 1) the use of Cs(+)-ions which are known to accumulate in living cells like K(+)-ions and 2) the removal of the staining solution from thin sections of the embedded material by centrifugal force. It is shown that sections of freeze-substituted and Lowicryl embedded frog skeletal muscle which has not been treated with chemical fixatives can be stained with electron-dense Cs(+)-ions: protein sites of preferential ion adsorption are visualized. These sites are similar to those accumulating monovalent ions in living cells as had been shown previously with frozen-hydrated preparations. An observed pH-dependency of the adsorption staining is consistent with the view that the ion adsorption sites are beta- and gamma-carboxyl groups of cellular proteins. The results obtained so far indicate that the new method can be used to investigate weak interactions between cellular proteins and different ions by electron microscopic methods.

Adsorption↗
PubMed1989

The contracting muscle: a challenge for freeze-substitution and low temperature embedding.

Frog sartorius and semitendinosus muscles are quick-frozen either in the resting state or during contraction by means of a LN2 cooled falling copper block. The frozen specimens are freeze-substituted (acetone + OsO4 + uranyl acetate) in a REICHERT JUNG CS auto and either embedded in Spurr's resin and polymerised at a high temperature (60 degrees C) or embedded and polymerised in the Lowicryls K4M, K11M or HM23 at low temperatures (below -30 degrees C). Excellent morphological results are obtained when freeze-substitution, embedding and polymerisation are all carried out below -50 degrees C. Muscles in which a major portion of cellular K+ ions has been replaced by electron dense Cs+ or Tl+ ions are also cryofixed at rest or during contraction, freeze-substituted in pure acetone for 1 week at -80 degrees C and polymerised in K11M at -60 degrees C. A characteristic uneven distribution of the electron dense ions--known from earlier published control experiments--can be observed in sections of resting muscles. Electrically stimulated muscles show ion redistribution. It is concluded that freeze-substitution and low temperature embedding of quick-frozen contracting muscle may be used to investigate changes of ultrastructure, redistribution of cellular water and intracellular movements of mobile ions during muscle contraction.

Animals↗
PubMed1989

The physical state of potassium in frog skeletal muscle studied by ion-sensitive microelectrodes and by electron microscopy: interpretation of seemingly incompatible results.

According to the commonly accepted membrane pump theory most of cellular K+ ions are freely dissolved in free cellular water; the alternative association-induction hypothesis postulates that the bulk of cellular K+ is adsorbed (weakly bound) to cellular proteins which are maintained in a specific labile state in the cytoplasm of a living cell. K+ activities measured with ion-sensitive microelectrodes in the cytoplasm of frog skeletal muscle seem to confirm the claim that most of cellular K+ ions are free in cellular water. On the other hand, it is evident from electron microscopic ion binding studies that in frog skeletal muscle most of cellular K+ ions are adsorbed to cellular proteins. The conflicting results can be explained with the assumption that a damage of the cytoplasm caused by the impaling microelectrode leads to a liberation of adsorbed ions. The possibility that microelectrodes damage the muscle cytoplasm is tested by using the light microscope. It is found that microelectrodes produce visible traumas which increase with time. Electron microscopic ion binding studies with damaged muscle support the view that monovalent cations are liberated in the disturbed area of a muscle fiber. It is concluded that a K(+)-sensitive microelectrode is not suited to determine the concentration of free K+ ions in intact frog skeletal muscle.

Animals↗
PubMed1988

The cell water problem posed by electron microscopic studies of ion binding in muscle.

The question whether cell K+ is free or bound in the striated frog muscle has been investigated during the last 10 years by different cryomethods and electron microscopy. The results support the view that most of cellular ions are osmotically inactive and that therefore the observed cell water activity must be explained by a model which assumes a specific cell water structure. According to the association-induction hypothesis, cell water is influenced by macromolecules and has low solubilities for Na+ and other solutes which therefore are partly excluded from cellular water. Autoradiography of frozen hydrated Na+ loaded muscles and microanalytical studies with freeze-dried cryosections of ouabain treated muscle support the view that cell water has the proposed Na+ exclusion property. It is concluded that problems such as cell volume regulation and muscle contraction cannot be understood completely without taking into account cellular ion binding and a specific cell water structure; in addition, mainly due to these cell properties it seems to be impossible to avoid volume changes of cells and subcellular compartments during conventional chemical fixation and dehydration of biological specimens.

Animals↗