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R Jahn

Publications and source records attributed to R Jahn.

At least 37 records · Page 2Linked to original sources
PubMed2000

Quantal release of serotonin.

We have studied the origin of quantal variability for small synaptic vesicles (SSVs) and large dense-cored vesicles (LDCVs). As a model, we used serotonergic Retzius neurons of leech that allow for combined amperometrical and morphological analyses of quantal transmitter release. We find that the transmitter amount released by a SSV varies proportionally to the volume of the vesicle, suggesting that serotonin is stored at a constant intravesicular concentration and is completely discharged during exocytosis. Transmitter discharge from LDCVs shows a higher degree of variability than is expected from their size distribution, and bulk release from LDCVs is slower than release from SSVs. On average, differences in the transmitter amount released from SSVs and LDCVs are proportional to the size differences of the organelles, suggesting that transmitter is stored at similar concentrations in SSVs and LDCVs.

Action Potentials↗
PubMed2000

Use of G-protein fusions to monitor integral membrane protein-protein interactions in yeast.

The control of protein-protein interactions is a fundamental aspect of cell regulation. Here we describe a new approach to detect the interaction of two proteins in vivo. By this method, one binding partner is an integral membrane protein whereas the other is soluble but fused to a G-protein gamma-subunit. If the binding partners interact, G-protein signaling is disrupted. We demonstrate interaction between known binding partners, syntaxin 1a with neuronal Sec1 (nSec1), and the fibroblast-derived growth factor receptor 3 (FGFR3) with SNT-1. In addition, we describe a genetic screen to identify nSec1 mutants that are expressed normally, but are no longer able to bind to syntaxin 1a. This provides a convenient method to study interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Intraneural nerve metastasis with multiple mononeuropathies.

Although cancer is a frequent condition, neoplastic involvement of the peripheral nervous system is rare. The mechanisms are heterogeneous and include lesions within the cerebrospinal fluid (CSF) space, local invasion (e.g. brachial plexus), compression, rarely direct infiltration, perineurial spread and even rarer intranerval metastasis. A 47-year-old woman had been treated for a carcinoid 10 years earlier and had received axillar irradiation. At presentation she suffered from weakness of the biceps brachii and was experiencing pain radiating from the axilla into the forearm and thumb. MR scans of the brachial plexus were negative and her symptoms were primarily considered to stem from a postradiation brachial plexopathy, Because of increasing pain, the brachial plexus was explored and a metastasis in the left musculocutaneous nerve was resected. Several months later, numbness and pain appeared in the ulnar nerve and another intrafascicular metastasis in the ulnar nerve was discovered. Resection with preservation of remaining fascicles was performed. This rare case report demonstrates that multiple mononeuropathies, resembling multiplex neuropathy, may be caused by intranerval metastasis.

Brachial Plexus Neuropathies↗
PubMed2000

Live cell imaging by multifocal multiphoton microscopy.

Multifocal multiphoton microscopy (MMM) permits parallel multiphoton excitation by scanning an array of high numerical aperture foci across a plane in the sample. MMM is particularly suitable for live cell investigations since it combines advantages of standard multiphoton microscopy such as optical sectioning and suppression of out-of-focus phototoxicity with high recording speeds. Here we describe several applications of MMM to live cell imaging using the neuroendocrine cell line PC12 and bovine chromaffin cells. Stainings were performed with the acidophilic dye acridine orange and the lipophilic dyes FM1-43 and Fast DiA as well as by transfection of the cells with GFP. In both bovine chromaffin and PC12 cells structural elements of nuclear chromatin and the 3-D distribution of acidic organelles inside the cells were visualized. In PC12 cells differentiated by nerve growth factor examples of neurites were monitored. Stainings of membranes were used to reconstruct the morphology of cells and neurites in three dimensions by volume-rendering and by isosurface plots. 3-D reconstructions were composed from stacks of about 50 images each with a diameter of 30-100 microm that were acquired within a few seconds. We conclude that MMM proves to be a technically simple and very effective method for fast 3-D live cell imaging at high resolution.

Acridine Orange↗
PubMed2000

The R-SNARE endobrevin/VAMP-8 mediates homotypic fusion of early endosomes and late endosomes.

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of the trans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

Animals↗
PubMed1999

Inhibition of SNARE complex assembly differentially affects kinetic components of exocytosis.

In chromaffin cells, an increase in intracellular Ca2+ leads to an exocytotic burst followed by sustained secretion. The burst can be further resolved into two kinetically distinct components, which suggests the presence of two separate pools of vesicles. To investigate how these components relate to SNARE complex formation, we introduced an antibody that blocks SNARE assembly but not disassembly. In the presence of the antibody, the sustained component was largely blocked, the burst was slightly reduced, and one of its kinetic components was eliminated. We conclude that SNARE complexes form before Ca(2+)-triggered membrane fusion and exist in a dynamic equilibrium between a loose and a tight state, both of which support exocytosis. Interaction of the antibody with preformed SNARE complexes favors the loose state.

Adrenal Glands↗
PubMed1999

Subcellular localization of tetanus neurotoxin-insensitive vesicle-associated membrane protein (VAMP)/VAMP7 in neuronal cells: evidence for a novel membrane compartment.

The clostridial neurotoxin-insensitive soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors, tetanus neurotoxin-insensitive (TI)-vesicle-associated membrane protein (VAMP)/VAMP7, SNAP23, and syntaxin 3 have recently been implicated in transport of exocytotic vesicles to the apical plasma membrane of epithelial cells. This pathway had been shown previously to be insensitive to tetanus neurotoxin and botulinum neurotoxin F. TI-VAMP/VAMP7 is also a good candidate to be implicated in an exocytotic pathway involved in neurite outgrowth because tetanus neurotoxin does not inhibit this process in conditions in which it abolishes neurotransmitter release. We have now found that TI-VAMP/VAMP7 has a widespread distribution in the adult rat brain in which its localization strikingly differs from that of nerve terminal markers. TI-VAMP/VAMP7 does not enrich in synaptic vesicles nor in large dense-core granules but is associated with light membranes. In hippocampal neurons developing in vitro, TI-VAMP/VAMP7 localizes to vesicles in the axonal and dendritic outgrowths and concentrates into the leading edge of the growth cone, a region devoid of synaptobrevin 2, before synaptogenesis. After the onset of synaptogenesis, TI-VAMP/VAMP7 is found predominantly in the somatodendritic domain. In PC12 cells, TI-VAMP/VAMP7 does not colocalize with synaptobrevin 2, chromogranin B, or several markers of endocytic compartments. At the electron microscopic level, TI-VAMP/VAMP7 is mainly associated with tubules and vesicles. Altogether, these results suggest that TI-VAMP/VAMP7 defines a novel membrane compartment in neurite outgrowths and in the somatodendritic domain.

Animals↗
PubMed1999

The subcellular localizations of atypical synaptotagmins III and VI. Synaptotagmin III is enriched in synapses and synaptic plasma membranes but not in synaptic vesicles.

Multiple synaptotagmins are expressed in brain, but only synaptotagmins I and II have known functions in fast, synchronous Ca2+-triggered neurotransmitter release. Synaptotagmin III was proposed to regulate other aspects of synaptic vesicle exocytosis, particularly its slow component. Such a function predicts that synaptotagmin III should be an obligatory synaptic vesicle protein, as would also be anticipated from its high homology to synaptotagmins I and II. To test this hypothesis, we studied the distribution, developmental expression, and localization of synaptotagmin III and its closest homolog, synaptotagmin VI. We find that synaptotagmins III and VI are present in all brain regions in heterogeneous distributions and that their levels increase during development in parallel with synaptogenesis. Furthermore, we show by immunocytochemistry that synaptotagmin III is concentrated in synapses, as expected. Surprisingly, however, we observed that synaptotagmin III is highly enriched in synaptic plasma membranes but not in synaptic vesicles. Synaptotagmin VI was also found to be relatively excluded from synaptic vesicles. Our data suggest that synaptotagmins III and VI perform roles in neurons that are not linked to synaptic vesicle exocytosis but to other Ca2+-related nerve terminal events, indicating that the functions of synaptotagmins are more diverse than originally thought.

Animals↗
PubMed1999

Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties.

Assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have recently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes with components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1. When the Q-SNAREs syntaxin 2, 3, and 4, and the R-SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes contained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPase N-ethylmaleimide-sensitive fusion protein and its cofactor alpha-SNAP. Circular dichroism spectroscopy showed that major conformational changes occur during assembly, which are associated with induction of structure from unstructured monomers. Furthermore, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations. We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggesting that specificity of membrane fusion in intracellular membrane traffic is not due to intrinsic specificity of SNARE pairing.

Amino Acid Sequence↗
PubMed1999

The synaptophysin-synaptobrevin complex: a hallmark of synaptic vesicle maturation.

Exocytosis of synaptic vesicles requires the formation of a fusion complex consisting of the synaptic vesicle protein synaptobrevin (vesicle-associated membrane protein, or VAMP) and the plasma membrane proteins syntaxin and soluble synaptosomal-associated protein of 25 kDa (or SNAP 25). In search of mechanisms that regulate the assembly of the fusion complex, it was found that synaptobrevin also binds to the vesicle protein synaptophysin and that synaptophysin-bound synaptobrevin cannot enter the fusion complex. Using a combination of immunoprecipitation, cross-linking, and in vitro interaction experiments, we report here that the synaptophysin-synaptobrevin complex is upregulated during neuronal development. In embryonic rat brain, the complex is not detectable, although synaptophysin and synaptobrevin are expressed and are localized to the same nerve terminals and to the same pool of vesicles. In contrast, the ability of synaptobrevin to participate in the fusion complex is detectable as early as embryonic day 14. The binding of synaptoporin, a closely related homolog of synaptophysin, to synaptobrevin changes in a similar manner during development. Recombinant synaptobrevin binds to synaptophysin derived from adult brain extracts but not to that derived from embryonic brain extracts. Furthermore, the soluble cytosol fraction of adult, but not of embryonic, synaptosomes contains a protein that induces synaptophysin-synaptobrevin complex formation in embryonic vesicle fractions. We conclude that complex formation is regulated during development and is mediated by a posttranslational modification of synaptophysin. Furthermore, we propose that the synaptophysin-synaptobrevin complex is not essential for exocytosis but rather provides a reserve pool of synaptobrevin for exocytosis that can be readily recruited during periods of high synaptic activity.

Aging↗
PubMed1999

A stable interaction between syntaxin 1a and synaptobrevin 2 mediated by their transmembrane domains.

The proteins synaptobrevin (VAMP), SNAP-25 and syntaxin 1 are essential for neuronal exocytosis. They assemble into a stable ternary complex which is thought to initiate membrane fusion. In vitro, the transmembrane domains of syntaxin and synaptobrevin are not required for association. Here we report a novel interaction between synaptobrevin and syntaxin that requires the presence of the transmembrane domains. When co-reconstituted into liposomes, the proteins form a stable binary complex that cannot be disassembled by NSF and that is resistant to denaturation by SDS. Cleavage of synaptobrevin with tetanus toxin does not affect the interaction. Furthermore, the complex is formed when a truncated version of syntaxin is used that contains only 12 additional amino acid residues outside the membrane anchor. We conclude that the interaction is mediated by the transmembrane domains.

Animals↗
PubMed1999

NSF N-terminal domain crystal structure: models of NSF function.

N-ethylmaleimide-sensitive factor (NSF) is a hexameric ATPase essential for eukaryotic vesicle fusion. Along with SNAP proteins, it disassembles cis-SNARE complexes upon ATP hydrolysis, preparing SNAREs for trans complex formation. We have determined the crystal structure of the N-terminal domain of NSF (N) to 1.9 A resolution. N contains two subdomains which form a groove that is a likely SNAP interaction site. Unexpectedly, both N subdomains are structurally similar to domains in EF-Tu. Based on this similarity, we propose a model for a large conformational change in NSF that drives SNARE complex disassembly.

Adenosine Triphosphate↗
PubMed1999

Fusion of endosomes involved in synaptic vesicle recycling.

Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid but not by the slow Ca(2+) chelator EGTA. Endosome fusion was restored by the addition of Ca(2+) with an optimum at a free Ca(2+) concentration of 0.3 x 10(-6) M. Other divalent cations did not substitute for Ca(2+). A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca(2+). We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca(2+) from the endosomal interior.

Animals↗
PubMed1999

Membrane fusion and exocytosis.

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

Exocytosis↗
PubMed1999

Reconstitution of regulated exocytosis in cell-free systems: a critical appraisal.

Regulated exocytosis involves the tightly controlled fusion of a transport vesicle with the plasma membrane. It includes processes as diverse as the release of neurotransmitters from presynaptic nerve endings and the sperm-triggered deposition of a barrier preventing polyspermy in oocytes. Cell-free model systems have been developed for studying the biochemical events underlying exocytosis. They range from semi-intact permeabilized cells to the reconstitution of membrane fusion from isolated secretory vesicles and their target plasma membranes. Interest in such cell-free systems has recently been reinvigorated by new evidence suggesting that membrane fusion is mediated by a basic mechanism common to all intracellular fusion events. In this chapter, we review some of the literature in the light of these new developments and attempt to provide a critical discussion of the strengths and limitations of the various cell-free systems.

Animals↗
PubMed1998

Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs.

SNARE [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four alpha-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.

Amino Acid Sequence↗
PubMed1998

Synaptic transmission: two players team up for a new tune.

An unexpected link has been found between two molecules that were thought to perform seemingly unrelated functions. The AMPA-subclass glutamate receptor GluR2 interacts with the membrane-fusion protein NSF in a manner that appears important for receptor-mediated intracellular signalling.

Carrier Proteins↗