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E D Gundelfinger

Publications and source records attributed to E D Gundelfinger.

At least 73 records · Page 4Linked to original sources
PubMed1997

Functional expression of rat synapse-associated proteins SAP97 and SAP102 in Drosophila dlg-1 mutants: effects on tumor suppression and synaptic bouton structure.

The synapse-associated proteins SAP97 and SAP102 are mammalian proteins that are structurally related to the Drosophila tumor suppressor protein DlgA. Previous analyses revealed that DlgA is essential for the integrity of epithelia and neuromuscular synapses. Here we show that synaptic bouton structure is severely affected in mutant larvae carrying the dlg-1(XI-2) allele. We have tested SAP97 and SAP102 for functional homology to DlgA by heterologous expression in Drosophila. Both SAP97 and SAP102 can suppress tumor formation in dlg-1 mutant flies and mimic DlgA at larval neuromuscular junctions. Neuronal expression of SAP97 or SAP102 is required for morphological restoration of synaptic boutons, indicating that presynaptic DlgA function is essential for establishing structurally intact motor nerve terminals at larval neuromuscular junctions.

Adaptor Proteins, Signal Transducing↗
PubMed1997

The neuronal calcium-sensor protein VILIP modulates cyclic AMP accumulation in stably transfected C6 glioma cells: amino-terminal myristoylation determines functional activity.

VILIP (visinin-like protein) is a member of the neuronal subfamily of EF-hand calcium sensor proteins. Members of this family are involved in the calcium-dependent regulation of the desensitization of signal cascades in retinal photoreceptors. To gain insight into the function of VILIP in cell signaling, we have transfected wild-type VILIP and mutant VILIP lacking the myristoylation consensus sequence into C6 glioma cells. Expression of wild-type VILIP did not significantly influence the desensitization of beta-adrenergic receptors, which are coupled to adenylyl cyclase in C6 cells. However, VILIP expression increased the beta-adrenergic receptor-stimulated cyclic AMP (cAMP) level in these cells severalfold. The stimulatory effect was also observed after direct stimulation of the adenylyl cyclase with forskolin, indicating that VILIP acts downstream of receptor and G protein in the beta-adrenergic signaling pathway in C6 cells. In contrast, the nonmyristoylated mutant of VILIP reduced cellular cAMP levels in C6 cells. Myristoylated wild-type VILIP was associated in a calcium-dependent manner with membrane fractions during subcellular fractionation, presumably owing to a calcium-myristoyl switch. In contrast, association of nonmyristoylated mutant VILIP with membranes was strongly reduced. Thus, myristoylation and most likely the calcium-dependent membrane association of VILIP are important prerequisites for the activating effect of wild-type VILIP on cAMP accumulation in C6 cells. These results suggest that VILIP acts as a calcium sensor molecule that modulates cell signaling cascades, possibly by direct or indirect regulation of adenylyl cyclase activity.

Animals↗
PubMed1996

The neuronal EF-hand Ca(2+)-binding protein VILIP: interaction with cell membrane and actin-based cytoskeleton.

VILIP is a member of the visinin/recoverin family of neuronal EF-hand Ca(2+)-binding proteins. Cell fractionation revealed cytoplasmic, membrane- and cytoskeleton-associated pools of VILIP. The association with the cytoskeletal protein fraction is Ca(2+)-dependent and may be mediated by direct interaction with actin. This is concluded from the observations that (i) Ca(2+)-loaded recombinant VILIP binds actin in an overlay assay; (ii) in the presence of Ca(2+), beta-actin co-immunoprecipitates with native VILIP from brain extracts, and (iii) actin and VILIP are co-localized in PC12 cells stably transfected with VILIP cDNA. The interaction of VILIP with the cortical cytoskeleton through actin may facilitate a Ca(2+)-dependent recruitment of VILIP to the cell membrane.

Actins↗
PubMed1996

Distribution of visinin-like protein (VILIP) immunoreactivity in the hippocampus of the Mongolian gerbil (Meriones unguiculatus).

Visinin-like protein (VILIP) is a neuronal EF-hand Ca(2+)-binding protein. In the chick brain, it is widely expressed, e.g. in neurons of the visual pathway and the cerebellum. In the cerebellum, a presynaptic localization of VILIP in glutamatergic parallel- and climbing-fiber terminals has been observed. Here, we describe the distribution of immunoreactivity (IR) detected by antibodies against chick VILIP in the gerbil hippocampus at the light and electron microscopic level. VILIP antibodies stain neurons in the whole hippocampal formation including pyramidal cells in the CA1 and CA3 region of the Ammon's horn and granule cells of the dentate gyrus. In CA3 neurons, VILIP-IR is localized in dendrites and dendritic spines.

Animals↗
PubMed1996

Localization of the neural calcium-binding protein VILIP (visinin-like protein) in neurons of the chick visual system and cerebellum.

The visinin-like protein (VILIP) is a member of a recently discovered family of calcium sensors specifically expressed in neurons. Family members contain four potential calcium-binding domains commonly referred to as "EF-hand motifs". VILIP interacts in a calcium-dependent manner with the actin-based neuronal cytoskeleton and modulates the phosphorylation of G-protein-coupled receptors, i.e., rhodopsin, in vitro. Here, we have used antisera against VILIP to study its distribution in the chick brain. Immunostaining of subsets of neurons is observed throughout the brain. Generally, the distribution of VILIP coincides well with the distribution of VILIP transcripts as detected previously by in situ hybridization. The most intense expression is detected in the visual system and the cerebellum. In the visual system, neurons of the nuclei of the ascending tecto-fugal pathway are stained, as are the pretectal, isthmic, and oculomotor nuclei. VILIP immunoreactivity is found in cell bodies, dendrites, and synaptic structures. Thus, VILIP appears to be an excellent marker for the characterization of neurons of the visual pathway. In the cerebellum, VILIP immunoreactivity is detected in deep cerebellar nuclei and in a subset of granule cells, Golgi type II cells, basket cells, and stellate cells, whereas it is completely absent from Purkinje cells. Intense punctate staining in the molecular layer suggests that VILIP is transported from deep cerebellar nuclei and from granule cells to the glutamatergic climbing-fiber and parallel-fiber synapses, respectively, both of which terminate on Purkinje-cell dendrites. The localization of VILIP in these presynaptic terminals has been confirmed at the electron-microscopic level.

Animals↗
PubMed1996

Protein components of a rat brain synaptic junctional protein preparation.

Antisera against a rat brain synaptic protein preparation, the postsynaptic density (PSD) fraction, were used to isolate cDNA clones by expression screening of a rat brain cDNA library. About one fifth of more than 200 analyzed cDNAs encoding potential synapse-associated proteins were previously unknown. Identifiable proteins include, among others, components of the pre- and postsynaptic cytoskeleton, synaptic vesicle proteins and several protein kinases and kinase substrates. This demonstrates that both pre- and postsynaptic elements purify with the PSD fraction.

Animals↗
PubMed1996

SAP102, a novel postsynaptic protein that interacts with NMDA receptor complexes in vivo.

Synapse-associated proteins (SAPs) are constituents of the pre- and postsynaptic submembraneous cytomatrix. Here, we present SAP102, a novel 102kDa SAP detected in dendritic shafts and spines of asymmetric type 1 synapses. SAP102 is enriched in preparations of synaptic junctions, where it biochemically behaves as a component of the cortical cytoskeleton. Antibodies directed against NMDA receptors coimmunoprecipitate SAP102 from rat brain synaptosomes. Recombinant proteins containing the carboxy-terminal tail of NMDA receptor subunit NR2B interact with SAP102 from rat brain homogenates. All three PDZ domains in SAP102 bind the cytoplasmic tail of NR2B in vitro. These data represent direct evidence that in vivo SAP102 is involved in linking NMDA receptors to the submembraneous cytomatrix associated with postsynaptic densities at excitatory synapses.

Amino Acid Sequence↗
PubMed1996

Piccolo, a novel 420 kDa protein associated with the presynaptic cytomatrix.

In this study, we describe a novel 420 kDa protein, called Piccolo, found at a wide variety of adult rat brain synapses. High protein levels in the cerebellum, the olfactory bulb and the hippocampus were frequently observed to be associated with asymmetric type 1 synapses. Piccolo is selectively enriched in presynaptic terminals, but is not a component of synaptic vesicles (SVs). Immunogold electron microscopy revealed that Piccolo localizes to the amorphous material among SVs at the presynaptic plasma membrane. Biochemical studies showed that it is very tightly bound to this structure. Thus, we speculate that Piccolo is a structural component of the presynaptic cytomatrix which anchors SVs to the presynaptic plasmalemma.

Animals↗
PubMed1995

Spatial and sub-cellular localization of the membrane cytoskeleton-associated protein alpha-adducin in the rat brain.

Studies on the identification and characterization of constituents of rat brain synaptic junctions have lead to the isolation of cDNA clones encoding segments of alpha-adducin. These and other studies suggest that adducin, a protein involved in promoting the assembly of actin and spectrin filaments at the plasma membrane, may play a role in dynamic assembly-disassembly processes underlying synaptic plasticity. In order to verify that brain alpha-adducin is indeed a constituent of synaptic structures, we have generated monoclonal antibodies against epitopes in the C-terminal region of alpha-adducin and have determined its spatial and sub-cellular distribution in postnatal day-30 rat brain. Alpha-adducin is found to be highly enriched in regions with high synapse densities of the hippocampus, corpus striatum, cerebral cortex and cerebellum. Immuno-electron microscopic analysis of peroxidase stained sections of the hippocampus and the cerebellum revealed that alpha-adducin is localized at distinct sub-cellular structures. In the CA1 and CA3 regions of the hippocampus alpha-adducin immunoreactivity is found in a distinct subset of dendrites and dendritic spines. In the molecular layer of the cerebellum, a distinct fraction of pre-synaptic terminals of parallel fiber terminals is labeled. In both cases the majority of synaptic structures does not contain adducin. Significant immunoreactivity is also detected in processes of glial cells both in the hippocampus and the cerebellum.

Animals↗
PubMed1995

Brevican, a chondroitin sulfate proteoglycan of rat brain, occurs as secreted and cell surface glycosylphosphatidylinositol-anchored isoforms.

cDNA clones encoding proteins related to the aggrecan/versican family of proteoglycan core proteins have been isolated with antisera against rat brain synaptic junctions. Two sets of overlapping cDNAs have been characterized that differ in their 3'-terminal regions. Northern analyses with probes derived from unique regions of each set were found to hybridize with two brain-specific transcripts of 3.3 and 3.6 kilobases (kb). The 3.6-kb transcript encodes a polypeptide that exhibits 82% sequence identity with bovine brevican and is thought to be the rat ortholog of brevican. Interestingly, the polypeptide deduced from the open reading frame of the 3.3-kb transcript is truncated just carboxyl-terminal of the central domain of brevican and instead contains a putative glypiation signal. Antibodies raised against a bacterially expressed glutathione S-transferase-brevican fusion protein have been used to show that both soluble and membrane-bound brevican isoforms exist. Treatment of the crude membrane fraction and purified synaptic plasma membranes with phosphatidylinositol-specific phospholipase C revealed that isoforms of brevican are indeed glycosylphosphatidylinositol-anchored to the plasma membrane. Moreover, digestions with chondroitinase ABC have indicated that rat brevican, like its bovine ortholog, is a conditional chondroitin sulfate proteoglycan. Immunohistochemical studies have shown that brevican is widely distributed in the brain and is localized extracellularly. During postnatal development, amounts of both soluble and phosphatidylinositol-specific phospholipase C-sensitive isoforms increase, suggesting a role for brevican in the terminally differentiating and the adult nervous system.

Amino Acid Sequence↗
PubMed1995

Distribution pattern of three neural calcium-binding proteins (NCS-1, VILIP and recoverin) in chicken, bovine and rat retina.

Neural Ca(2+)-binding proteins (NCaPs) constitute a subfamily of 4-EF-hand proteins, and display a histological and structural dichotomy: the A-type NCaPs are selectively expressed by the retina and pineal organ and display two canonical EF-hands, whereas the B-type NCaPs are found in the entire brain and present three regular EF-hands. In this study, antisera were raised against the A-type NCaP recoverin (26 kDa) and the B-type NCaPs VILIP and NCS-1 (22 kDa). Since the sequence identity among NCaPs is high, specific polyclonal antibodies were purified by double cross-immunoaffinity chromatography; both ELISA and immunoblot analyses determined that the resulting antibodies showed selectivity ratios inferior to 1/363 for the two other related NCaPs. Besides, the anti-VILIP antibodies displayed some affinity toward neurocalcin delta, and the antirecoverin antibodies recognized a 24 kDa protein, which is most likely visinin. Thus, immunohistochemical studies on the chicken, rat and cow retina revealed that anti-recoverin antibodies recognized the vertebrate photoreceptors and a small number of mammalian bipolar cells. Anti-VILIP antibodies exclusively labelled the inner retina, i.e. the amacrine and ganglion cells. NCS-1 was mainly present in the photoreceptor inner segments, the inner plexiform layer and the ganglion cells. NCS-1 showed the highest species disparity. The retinal localization of NCS-1 and VILIP offered an important morphological basis for the understanding of their function. Furthermore, specific antibodies against the NCaPs may enable the identification of cell populations in more complex neural tissues, such as the brain.

Animals↗
PubMed1995

Molecular characterization and spatial distribution of SAP97, a novel presynaptic protein homologous to SAP90 and the Drosophila discs-large tumor suppressor protein.

Synapses are highly specialized sites of cell-cell contact involved in signal transfer. The molecular mechanisms modulating the assembly and stability of synapses are unknown. We previously reported the identification of a 90 kDa synapse-associated protein, SAP90, that is localized at the presynaptic termini of inhibitory GABAergic synapses. SAP90 is a mosaic protein composed of three 90 amino acid residue repeats, an SH3 domain and a region homologous to guanylate kinases. SAP90 shares domain specific homology with a family of proteins involved in the assembly and possibly stability of sites of cell contact. These include the product of the lethal(1) discs-large-1 (dlgA) tumor suppressor gene and the zonula occludens proteins ZO-1, ZO-2. The further characterization of cDNA clones encoding components of synaptic junctions has lead to the identification of a 97 kDa protein, called SAP97, that exhibits a strong overall sequence similarity to SAP90. The present study was undertaken to determine the spatial distribution of SAP97, and to reveal further clues to the possible roles of these proteins in synapses. Light and immunoelectron microscopic analysis of the rat hippocampal formation revealed that SAP97 is localized in the presynaptic nerve termini of excitatory synapses. In other brain regions, SAP97 is found in and along bundles of unmyelinated axons. SAP97 is not restricted to the CNS, but is also present at the basal lateral membrane between a variety of epithelial cells. In cultured T84 cells, it is restricted to the cytoplasmic surface of the plasma membranes between adjacent cells, but not at the edges of cells lacking cell-cell contact suggesting a role for SAP97 in cell adhesion. These data suggest that members of the SAP90/SAP97 subfamily may be involved in the site specific assembly, stability or functions of membrane specialization at sites of cell-cell contact.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Cation binding and conformational changes in VILIP and NCS-1, two neuron-specific calcium-binding proteins.

VILIP and NCS-1, neural-specific, 22-kDa Ca(2+)-binding proteins possessing four EF-hands, were expressed in Escherichia coli to study their divalent cation properties. Flow dialysis (Ca2+ binding) and equilibrium gel filtration (Mg2+ binding) revealed that both recombinant proteins possess only two active metal-binding sites, which can accommodate either Ca2+ or Mg2+. VILIP binds cations without cooperativity with intrinsic affinity constants K'Ca of 1.0 x 10(6) M-1 and K'Mg of 4.8 x 10(3) M-1.Mg2+ antagonizes Ca2+ binding by shifting the isotherms to higher free Ca2+ concentrations without changing their shape. The competition equation yields a K'Mg, comp value of 180 M-1 for both sites. NCS-1 binds two Mg2+ without cooperativity with K'Mg of 8.3 x 10(4) M-1 and two Ca2+ with very strong positive cooperativity (nH = 1.96). In the absence of Mg2+ the K'Ca1 and K'Ca2 values are 8.9 x 10(4) and 1.4 x 10(8) M-1, respectively, which represent an allosteric increase of 1600-fold. Mg2+ shifts the Ca(2+)-binding isotherms to higher Ca2+ concentrations, yielding a K'Mg, comp value of 800 M-1 for both sites. Thus VILIP and NCS-1 show three remarkable differences in the Ca2+/Mg2+ binding parameters: 1) VILIP binds Ca2+ with much lower affinity than NCS-1; 2) VILIP binds Ca2+ in a noncooperative way, whereas NCS-1 shows maximal positive cooperativity; 3) in VILIP the Mg2+/Ca2+ antagonism is much weaker than in NCS-1. Conformational changes monitored by Trp fluorescence indicate that the metal-free forms already are highly structured. Ca2+ binding promotes a 20-30% increase of fluorescence in both proteins, but whereas the Mg2+ form of VILIP has the same fluorescence properties as the metal-free form, Mg(2+)-saturated NCS-1 has those of the Ca2+ form. Near UV difference spectra confirmed that in VILIP the Mg2+ form is very similar to the metal-free form; in NCS-1 it is different, especially in the Tyr region. NCS-1 possesses one unique Cys-38 in EF-hand site I. Its reactivity (kSH) toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) is the same for the Ca(2+)- and Mg(2+)-loaded protein, but kSH is 4-fold higher in metal-free NCS-1. VILIP possesses two additional thiols, one of which is inaccessible to DTNB in the native protein. The reactivity of the two accessible thiols is identical in the metal-free and Mg2+ forms and 5-fold higher than in the Ca2+ form.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Acetylcholine receptors of the Drosophila brain: a 900 bp promoter fragment contains the essential information for specific expression of the ard gene in vivo.

The ard gene encodes a beta-subunit of Drosophila nicotinic acetylcholine receptors specifically expressed in a subset of neurons. To identify the cis-regulatory region responsible for this cell-specific expression, various 5' fragments of the ard gene were fused to a lacZ reporter gene and introduced into the Drosophila genome. A DNA fragment spanning approximately 760 bp upstream and approximately 140 bp downstream of a cluster of putative transcription start sites produced a pattern of beta-galactosidase activity that resembles the distribution of ARD transcripts. Both in embryos and adults the levels of lacZ RNA were similar to those of endogenous ARD transcripts, suggesting that the 900 bp fragment harbors all essential elements for proper expression of the ard gene.

Animals↗
PubMed1994

Physiological properties of neuronal nicotinic receptors reconstituted from the vertebrate beta 2 subunit and Drosophila alpha subunits.

Three cDNAs (ALS, D alpha 2 and ARD) isolated from the nervous system of Drosophila and encoding putative nicotinic acetylcholine receptor subunits were expressed in Xenopus oocytes in order to study their functional properties. Functional receptors could not be reconstituted from any of these subunits taken singly or in twos and threes. In contrast, large evoked currents (in the microA range) were consistently observed upon agonist application on oocytes co-injected with ALS or D alpha 2 in combination with the chick beta 2 structural subunit. The ALS/beta 2 and D alpha 2/beta 2 receptors are highly sensitive to acetylcholine and nicotine, and their physiological properties resemble those of native or reconstituted receptors from vertebrates. Although the physiological properties of ALS/beta 2 and D alpha 2/beta 2 receptors are quite similar, clear differences appear in their pharmacological profiles. The ALS/beta 2 receptor is highly sensitive to alpha-bungarotoxin while the D alpha 2/beta 2 receptor is totally insensitive to this agent. These results demonstrate that the Drosophila ALS and D alpha 2 cDNAs encode neuronal nicotinic subunits responding to physiological concentrations of the agonists acetylcholine and nicotine.

Animals↗
PubMed1994

Expression of the ligand-binding nicotinic acetylcholine receptor subunit D alpha 2 in the Drosophila central nervous system.

The D alpha 2 gene encodes a ligand-binding subunit of nicotinic acetylcholine receptors (nAChRs) from Drosophila melanogaster. We have studied the distribution of D alpha 2 transcripts and protein by in situ hybridization and immunohistochemistry, respectively, as well as the regulation of D alpha 2 gene expression in vivo using D alpha 2 promoter fragments fused to the Escherichia coli lacZ gene. Transcripts and protein from the D alpha 2 gene were detected exclusively in the central nervous system. Both in late embryos and adults D alpha 2-like immunoreactivity is widely but not uniformly distributed in the synaptic neuropil, suggesting that the D alpha 2 protein is a subunit of a synaptic nicotinic receptor. Its distribution resembles that of ALS and ARD proteins, two other nAChR subunits of the fly. Five different D alpha 2-lacZ fusion gene constructs were introduced into the Drosophila genome by P-element-mediated gene transfer to identity functional elements of the D alpha 2 promoter. All constructs produce a basic lacZ expression pattern that is compatible with the distribution of D alpha 2 transcripts and protein. A 880 bp upstream fragment harbors the cis elements for the expression of a weak but specific basic D alpha 2 pattern. The next 350 bp further upstream significantly enhance beta-galactosidase expression without influencing the pattern of expression. Between 1.7 and 7.3 kb upstream of the transcription start site one or more elements that are required for D alpha 2 expression in optic lobe tangential cells are located.

Animals↗
PubMed1994

Sequence of a Drosophila ligand-gated ion-channel polypeptide with an unusual amino-terminal extracellular domain.

We report the isolation of a full-length clone from a Drosophila melanogaster head cDNA library that encodes a 614-residue polypeptide that exhibits all of the features of a ligand-gated chloride-channel/receptor subunit. This polypeptide, which has been named GRD (denoting that the polypeptide is a GABAA and glycine receptor-like subunit of Drosophila), displays between 33 and 44% identity to vertebrate GABAA and glycine receptor subunits and 32-37% identity to the GABAA receptor-like polypeptides from Drosophila and Lymnaea. It is interesting that the large amino-terminal, presumed extracellular domain of the GRD protein contains an insertion, between the dicysteine loop and the first putative membrane-spanning domain, of 75 amino acids that is not found in any other ligand-gated chloride-channel subunit. Analysis of cDNA and genomic DNA reveals that these residues are encoded by an extension of an exon that is equivalent to exon 6 of vertebrate GABAA and glycine receptor genes. The gene (named Grd) that encodes the Drosophila polypeptide has been mapped, by in situ hybridization, to position 75A on the left arm of chromosome 3.

Amino Acid Sequence↗