PubMed Health⌕ Search

Biomedical subjects

W Volknandt

Publications and source records attributed to W Volknandt.

At least 19 recordsLinked to original sources
PubMed1999

Recombinant major vault protein is targeted to neuritic tips of PC12 cells.

The major vault protein (MVP) is the predominant constituent of ubiquitous, evolutionarily conserved large cytoplasmic ribonucleoprotein particles of unknown function. Vaults are multimeric protein complexes with several copies of an untranslated RNA. Double labeling employing laser-assisted confocal microscopy and indirect immunofluorescence demonstrates partial colocalization of vaults with cytoskeletal elements in Chinese hamster ovary (CHO) and nerve growth factor (NGF)-treated neuronlike PC12 cells. Transfection of CHO and PC12 cells with a cDNA encoding the rat major vault protein containing a vesicular stomatitis virus glycoprotein epitope tag demonstrates that the recombinant protein is sorted into vault particles and targeted like endogenous MVPs. In neuritic extensions of differentiated PC12 cells, there is an almost complete overlap of the distribution of microtubules and vaults. A pronounced colocalization of vaults with filamentous actin can be seen in the tips of neurites. Moreover, in NGF-treated PC12 cells the location of vaults partially coincides with vesicular markers. Within the terminal tips of neurites vaults are located near secretory organelles. Our observations suggest that the vault particles are transported along cytoskeletal-based cellular tracks.

Actins↗
PubMed1999

A plethora of presynaptic proteins associated with ATP-storing organelles in cultured astrocytes.

Cultured astrocytes can release a variety of messenger substances via receptor-mediated mechanisms, implicating their potential for regulated exocytosis and the participation of proteins of the SNARE complex. Here we demonstrate the astrocytic expression and organellar association of a large variety of synaptic proteins (synaptobrevin II, synaptotagmin I, synaptophysin, rab3a, synapsin I, SNAP-25, and syntaxin I) and also of the ubiquitous cellubrevin. As revealed by immunoblotting the expression of synaptic proteins was highest within the first few days after plating. Synaptophysin and SNAP-25 showed the most significant decline with prolonged culture time. Rab3a and synaptobrevin II were retained at a high level and synaptotagmin I, synapsin I, and syntaxin I at a lower level until 20 DIV. The immunoreaction for cellubrevin was low at the beginning and increased with prolonged culture time. As revealed by light microscopical immunocytochemistry the proteins are expressed by GFAP-positive astrocytes and associated with organelles of varying size. Immunoelectron microscopical analysis allocates synaptobrevin II and synaptophysin to the membranes of vesicular organelles. Double labeling experiments for pairs of synaptic proteins reveal that individual synaptic proteins can be entirely colocalized or partly reside on different organelles. Subcellular fractionation of astrocyte cultures by sucrose density gradient centrifugation after 2, 6, 13, and 20 DIV showed that the proteins sediment with ATP containing organelles of a broad density range. Our data suggest that messenger substances may be released from cultured astrocytes via receptor-mediated, Ca2+-dependent exocytosis.

Adenosine Triphosphate↗
PubMed1999

Peripheral synapses at identifiable mechanosensory neurons in the spider Cupiennius salei: synapsin-like immunoreactivity.

Indirect immunocytochemical tests were used at the light- and electron-microscopic levels to investigate peripheral chemical synapses in identified sensory neurons of two types of cuticular mechanosensors in the spider Cupiennius salei Keys.: (1) in the lyriform slit-sense organ VS-3 (comprising 7-8 cuticular slits, each innervated by 2 bipolar sensory neurons) and (2) in tactile hair sensilla (each supplied with 3 bipolar sensory cells). All these neurons are mechanosensitive. Application of a monoclonal antibody against Drosophila synapsin revealed clear punctate immunofluorescence in whole-mount preparations of both mechanoreceptor types. The size and overall distribution of immunoreactive puncta suggested that these were labeled presynaptic sites. Immunofluorescent puncta were 0.5-6.8 micrometer long and located 0.5-6.6 micrometer apart from each other. They were concentrated at the initial axon segments of the sensory neurons, while the somata and the dendritic regions showed fewer puncta. Western blot analysis with the same synapsin antibody against samples of spider sensory hypodermis and against samples from the central nervous system revealed a characteristic doublet band at 72 kDa and 75 kDa, corresponding to the apparent molecular mass of synapsin in Drosophila and in mammals. Conventional transmissionelectron-microscopic staining demonstrated that numerous chemical synapses (with at least 2 vesicle types) were present at these mechanosensory neurons and their surrounding glial sheath. The distribution of these synapses corresponded to our immunofluorescence results. Ultrastructural examination of anti-synapsin-stained neurons confirmed that reaction product was associated with synaptic vesicles. We assume that the peripheral synaptic contacts originate from efferents that could exert a complex modulatory influence on mechanosensory activity.

Animals↗
PubMed1999

Axonal transport of ribonucleoprotein particles (vaults).

RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the beta-subunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure-in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon of synaptic vesicles and vault protein. Our data show that ribonucleoprotein-immunoreactive particles can be actively transported within axons in situ from the soma to the nerve terminal and back. The results suggest that the transport of vaults is driven by fast axonal transport motors like the SV2-containing vesicles and mitochondria. Vaults exhibit an anterograde and a retrograde transport component, similar to that observed for the vesicular organelles carrying SV2 and for mitochondria. Although the function of vaults is still unknown studies of the axonal transport of this organelle may reveal insights into the mechanisms of cellular transport of ribonucleoprotein particles in general.

Animals↗
PubMed1998

Formation and sequence analysis of secretoneurin, a neuropeptide derived from secretogranin II, in mammalian, bird, reptile, amphibian and fish brains.

Secretoneurin is a recently-characterized neuropeptide derived from secretogranin II, a protein belonging to the class of chromogranins. We investigated the phylogeny of this peptide by immunoblotting and gel-filtration high performance liquid chromatography followed by radioimmunoassay of brain extracts of various species including chicken, lizard, frog and fish. In addition the amino acid sequence of secretoneurin from pig, hamster, rabbit, guinea-pig and chicken was established by reverse transcriptase polymerase chain reaction. Secretoneurin is strongly conserved during evolution, it is not only expressed in various mammalian species but found also in the brain of birds, reptiles, amphibians and fish. In all these species a significant or near complete processing of secretogranin II to secretoneurin was observed. These data provide significant evidence for the neuropeptide nature of the novel functional peptide.

Amino Acid Sequence↗
PubMed1998

Major vault protein of electric ray is a phosphoprotein.

The major vault protein is the predominant member of a large cytosolic ribonucleoprotein particle, named vaults. Vaults are abundant in nerve terminals of the electric organ of Torpedo marmorata. Negative staining of isolated vaults reveals particle dimensions of 45x65 nm in size. Comparison of the major vault protein (MVP100) from the two electric ray species Torpedo marmorata and Discopyge ommata reveals few microheterogeneities in amino acid sequence. Potential phosphorylation sites for various protein kinases are highly conserved. Phosphorylation studies demonstrate that the major vault protein of Torpedo is a substrate of various protein kinases. MVP100 is phosphorylated by protein tyrosine kinase in vivo and protein kinase C and casein kinase II in vitro. Inhibitors and activators of protein kinases specifically modulate the phosphorylation of MVP100.

Amino Acid Sequence↗
PubMed1997

Analysis of a cDNA encoding the major vault protein from the electric ray Discopyge ommata.

The major vault protein is the predominant constituent of vaults ubiquitous large cytosolic ribonucleoprotein particles. A cDNA clone encoding the 100-kDa major vault protein (MVP100) was isolated from an electric lobe library of Discopyge ommata. The complete nucleotide sequence was determined. Northern blot analysis revealed a 2.8-kb transcript with a high expression in neural tissue. Southern blot analysis indicates that the electric ray MVP100 is a single copy-gene with at least two introns. The primary structure of major vault proteins characterized in slime mold, ray, rat and human is evolutionary highly conserved.

Amino Acid Sequence↗
PubMed1997

Immunocytochemical localization of synaptic proteins at vesicular organelles in PC12 cells.

The distribution of the three synaptic vesicle proteins SV2, synaptophysin and synaptotagmin, and of SNAP-25, a component of the docking and fusion complex, was investigated in PC12 cells by immunocytochemistry. Colloidal gold particle-bound secondary antibodies and a preembedding protocol were applied. Granules were labeled for SV2 and synaptotagmin but not for synaptophysin. Electron-lucent vesicles were labeled most intensively for synaptophysin but also for SV2 and to a lesser extent for synaptotagmin. The t-SNARE SNAP-25 was found at the plasma membrane but also at the surface of granules. Labeling of Golgi vesicles was observed for all antigens investigated. Also components of the endosomal pathway such as multivesicular bodies and multilamellar bodies were occasionally marked. The results suggest that the three membrane-integral synaptic vesicle proteins can have a differential distribution between electron-lucent vesicles (of which PC12 cells may possess more than one type) and granules. The membrane compartment of granules appears not to be an immediate precursor of that of electron-lucent vesicles.

Animals↗
PubMed1996

The major vault protein (MVP100) is contained in cholinergic nerve terminals of electric ray electric organ.

A protein of Mr 100,000 (MVP100) is highly enriched in the electromotor system of electric rays. Biochemical analysis indicates that MVP100 is contained in the cholinergic nerve terminals of Torpedo electric organ as part of a large cytosolic complex. On sucrose density gradient centrifugation MVP100 comigrates with synaptic vesicles or synaptosomes. It can be partially separated from synaptic vesicles by gel filtration or glycerol velocity gradient centrifugation. Within the complex MVP100 behaves like a hydrophobic protein and is protected against proteolytic attack. MVP100 can be immunodetected by an antibody against phosphotyrosine, and it becomes phosphorylated on incubation with [gamma-32P]ATP. By screening an electric ray electric lobe cDNA library the primary structure of MVP100 was analyzed. MVP100 is highly homologous to the major vault proteins of slime mold and rat and to the human lung resistance-related protein. Compared with non-neural tissues the expression of MVP100 is highest in brain and enriched in the electric lobe that contains the somata of the electromotor neurons. Immunoelectron microscopic analysis reveals a close association of MVP100 and synaptic vesicles in the nerve terminals of the electric organ.

Amino Acid Sequence↗
PubMed1996

Two synpatic vesicle proteins of 25 kDa: a comparison of the molecular properties and tissue distribution of svp25 and o-rab3.

Two synaptic vesicle proteins of the electric ray Torpedo--svp25 and o-rab3--are compared with respect to their biochemical properties and tissue distribution. On SDS-PAGE both proteins migrate to the same position of about 25 kDa. As revealed by application of monospecific antibodies and subcellular fractionation both proteins comigrate and cofractionate with the synaptic vesicle compartment. o-Rab3 and svp25 can be separated by lectin chromatography; svp25 is highly glycosylated and binds to concanavalin A sepharose. Upon deglycosylation using glycopeptidase F and O-glycosidase its apparent molecular mass is reduced to about 14 kDa. Partial amino acid sequences obtained by direct microsequencing of purified and deglycosylated svp25 revealed that svp25 is a novel protein that has not yet been characterized in molecular terms. Whereas svp25 was detected in all brain areas investigated, the expression of o-rab3 was found to be restricted to specific regions. An immunoblot analysis demonstrates an exclusive association of both proteins with neural tissues. Our results suggest that cholinergic synaptic vesicles from electric ray electric organ contain at least two membrane-associated proteins of an apparent molecular mass of 25 kDa, the membrane associated o-rab3 and the membrane integral protein svp25. The two proteins can be separated by lectin chromatography for assessment of their biochemical properties.

Animals↗
PubMed1996

Colocalization on the same synaptic vesicles of syntaxin and SNAP-25 with synaptic vesicle proteins: a re-evaluation of functional models required?

Synaptic vesicle docking and calcium dependent exocytosis are thought to require the specific interaction of proteins of the synaptic vesicle membrane (such as VAMP/synaptobrevin and synaptotagmin) and their plasma membrane-located counterparts (such as syntaxin and SNAP-25). When isolating synaptic vesicles by glycerol velocity gradient centrifugation we found cosedimentation of the presumptive presynaptic plasma membrane proteins syntaxin and SNAP-25 with synaptic vesicle membrane proteins. In order to further identify the antibody binding organelles we performed an immunoelectron microscopical analysis of synaptosomal profiles. Syntaxin and SNAP-25 were not only associated with the plasma membrane but to a large extent also with synaptic vesicle profiles. In order to answer the question whether the syntaxin and SNAP-25 containing vesicular compartment would also carry classical synaptic vesicle membrane markers we performed double labeling experiments using poly- and monoclonal antibodies. We found colocalization on the same vesicle not only of SNAP-25 and syntaxin but also of SNAP-25 with the synaptic vesicle membrane proteins SV2 and synaptotagmin and of syntaxin with the vesicular membrane protein synaptophysin. Our results demonstrate that syntaxin and SNAP-25 are colocalized with classical vesicle membrane proteins on the same vesicle and suggest that the functional models for the interaction of presynaptic proteins need to be re-evaluated.

Animals↗
PubMed1995

Calcium-dependent endogenous proteolysis of the vesicle proteins synaptobrevin and synaptotagmin.

The synaptic vesicle integral protein synaptobrevin/VAMP is a target of the clostridial metalloproteases tetanus toxin and botulinum toxins. We provide evidence that synaptobrevin can also be cleaved by an endogenous protease. As revealed by Western blotting proteolysis is calcium-dependent, results in the formation of an 8 kD peptide that becomes apparent within 10 min. Proteolysis can be inhibited by the chelating agents EGTA and EDTA, whereas other protease inhibitors failed to prevent degradation. In addition, a proteolytic degradation of the synaptic vesicle specific protein synaptotagmin could be observed. Other proteins including the synaptic vesicle proteins synapsin I and synaptophysin remained unaltered. Partial calcium-dependent degradation of select synaptic vesicle proteins may play a role in the life cycle of the organelle.

Animals↗
PubMed1995

Inhibition by clostridial neurotoxins of calcium-independent [3H]noradrenaline outflow from freeze-thawed synaptosomes: comparison with synaptobrevin hydrolysis.

Clostridial neurotoxins are known to inhibit regulated, i.e. calcium-dependent exocytosis. In the present study we have investigated their potential role in also inhibiting calcium-independent exocytosis. Synaptosomes from rat forebrain were preloaded with [3H]noradrenaline and permeabilized reversibly by freezing in Ca(2+)-free potassium glutamate containing dimethyl sulfoxide and the toxins to be assayed. Subsequently, outflow of radioactivity was measured in isotonic calcium-free potassium glutamate. The synaptic vesicle protein synaptobrevin-2/VAMP-2 and its toxin-dependent degradation were analysed by Western blotting. The light chain of tetanus toxin reduced the synaptosomal outflow of radioactivity, whereas the activity of the heavy chain was at the detection limit. The respective activities of the dichain toxins from Clostridium tetani and C. botulinum A, B and E were enhanced by pretreatment with dithiothreitol. Reduced single-chain tetanus toxin was less potent than reduced dichain tetanus toxin. Pretreatment with ethylene diamine tetraacetic acid as an inhibitor of Zn(2+)-proteases abolished the actions of the tetanus toxin light chain and of the reduced dichain toxins. Hydrolysis of synaptobrevin-2/VAMP-2 was obtained with tetanus toxin light chain, reduced dichain tetanus toxin and C. botulinum B toxin. Its hydrolysis by single-chain tetanus toxin was less pronounced, and it was absent with botulinum toxins A and E. It is concluded that clostridial neurotoxins can not only inhibit calcium-dependent release but also affect calcium-independent outflow from synaptosomes. Since this is accompanied by selective intrasynaptosomal proteolysis of synaptobrevin, calcium-independent outflow may at least in part involve the vesicular release apparatus.

Animals↗
PubMed1995

The synaptic vesicle and its targets.

Synaptic vesicles play the central role in synaptic transmission. They are regarded as key organelles involved in synaptic functions such as uptake, storage and stimulus-dependent release of neurotransmitter. In the last few years our knowledge concerning the molecular components involved in the functioning of synaptic vesicles has grown impressively. Combined biochemical and molecular genetic approaches characterize many constituents of synaptic vesicles in molecular detail and contribute to an elaborate understanding of the organelle responsible for fast neuronal signalling. By studying synaptic vesicles from the electric organ of electric rays and from the mammalian cerebral cortex several proteins have been characterized as functional carriers of vesicle function, including proteins involved in the molecular cascade of exocytosis. The synaptic vesicle specific proteins, their presumptive function and targets of synaptic vesicle proteins will be discussed. This paper focuses on the small synaptic vesicles responsible for fast neuronal transmission. Comparing synaptic vesicles from the peripheral and central nervous systems strengthens the view of a high conservation in the overall composition of synaptic vesicles with a unique set of proteins attributed to this cellular compartment. Synaptic vesicle proteins belong to gene families encoding multiple isoforms present in subpopulations of neurons. The overall architecture of synaptic vesicle proteins is highly conserved during evolution and homologues of these proteins govern the constitutive secretion in yeast. Neurotoxins from different sources helped to identify target proteins of synaptic vesicles and to elucidate the molecular machinery of docking and fusion. Synaptic vesicle proteins and their markers are useful tools for the understanding of the complex life cycle of synaptic vesicles.

Animals↗
PubMed1994

Two rat homologs of clathrin-associated adaptor proteins.

A cDNA clone predicted to encode a 46,757-Da protein was isolated from a library derived from the electric lobe of the ray Discopyge ommata. Two rat homologs, p47A and p47B, were subsequently isolated. These three proteins share approx. 80% amino acid (aa) identity to each other and have 27-30% aa identity to rat AP50 and mouse AP47, the medium-chain subunits of adaptor complexes associated with clathrin-coated vesicles. These complexes are involved in receptor-mediated pathways of intracellular transport. Rat p47A mRNA is expressed in all tissues examined, including brain, heart, kidney, liver, lung, muscle and spinal cord. Rat p47B mRNA is detected exclusively in brain and spinal cord, and may participate in nervous system-specific functions such as biogenesis or recycling of synaptic vesicles.

Amino Acid Sequence↗
PubMed1994

SV2 and o-rab3 remain associated with recycling synaptic vesicles.

o-rab3 is an electric ray homologue of low molecular weight of GTP-binding proteins thought to be involved in targeting of secretory vesicles to sites of exocytosis. The stimulation-dependent association of o-rab3 with synaptic vesicles was compared with that of the membrane-integral synaptic vesicle protein 2 (SV2). On application of immunoelectron microscopy and the colloidal gold technique, antibodies against either protein labeled the synaptic vesicle membrane compartment. Synaptic vesicles recycled under conditions of low frequency stimulation (0.1 Hz) retained their complement of both SV2 and o-rab3. Isolation of synaptic vesicles by density-gradient centrifugation and subsequent column chromatography yielded no indication of a stimulation-dependent release of o-rab3 from synaptic vesicles. In contrast, multivesicular bodies and vacuoles occasionally observed in the nerve terminals contained SV2 but little if any o-rab3. It is concluded that o-rab3 remains associated with the synaptic vesicle membrane compartment during stimulation-induced cycles of repeated exo- and endocytosis. o-rab3 may be lost once the vesicle enters the prelysosomal pathway.

Animals↗
PubMed1994

Putative synaptic vesicle nucleotide transporter identified as glyceraldehyde-3-phosphate dehydrogenase.

Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain a 34-kDa protein that binds azido-ATP, azido-AMP, and N-ethylmaleimide. The protein was found to share similarities with the mitochondrial ADP/ATP carrier and assumed to represent the synaptic vesicle nucleotide transporter. Synaptic vesicles were purified by sucrose density gradient centrifugation and subsequent chromatography on Sephacryl S-1000 from both Torpedo electric organ and bovine brain cerebral cortex. They contained ATP-binding proteins of 35 kDa and 34 kDa, respectively. ATP binding was inhibited by AMP. Both proteins were highly enriched after column chromatography of vesicle proteins on AMP-Sepharose. Antibodies were obtained against both proteins. Antibodies against the bovine brain synaptic vesicle protein of 34 kDa bound specifically to the 35-kDa protein of Torpedo vesicles. An N-terminal sequence obtained against the 34-kDa protein of bovine brain synaptic vesicles identified it as glyceraldehyde-3-phosphate dehydrogenase. The previously observed molecular characteristics of the putative vesicular nucleotide transporter in Torpedo fit those of glyceraldehyde-3-phosphate dehydrogenase. We, therefore, suggest that the protein previously identified as putative nucleotide transporter is, in fact, glyceraldehyde-3-phosphate dehydrogenase.

Amino Acid Sequence↗