Electrophorus acetylcholinesterase. Biochemical and electron microscope characterization of low ionic strength aggregates.

The "tailed" molecules of Electrophorus (electric eel) acetylcholinesterase aggregate under conditions of low ionic strength. These aggregates have been studied by sedimentation analysis and high-resolution electron microscopy. They consist of bundles of at least half a dozen molecules, the tails of which are packed side by side, to form the core of the structure. Although aggregation is normally fully reversible, aggregates were irreversibly stabilized by methylene blue-sensitized photo-oxidation. This process was shown to consist of a singlet oxygen oxidation reaction and probably involves methionine or histidine residues. It did not modify the structural or hydrodynamic characteristics of the aggregates.

Developmental regulation of membrane traffic organization during synaptogenesis in mouse diaphragm muscle.

In innervated adult skeletal muscles, the Golgi apparatus (GA) displays a set of remarkable features in comparison with embryonic myotubes. We have previously shown by immunocytochemical techniques, that in adult innervated fibers, the GA is no longer associated with all the nuclei, but appears to be concentrated mostly in the subneural domain under the nerve endings in chick (Jasmin, B. J., J. Cartaud, M. Bornens, and J.-P. Changeux. 1989. Proc. Natl. Acad. Sci. USA. 86:7218-7222) and rat (Jasmin, B. J., C. Antony, J.-P. Changeux, and J. Cartaud. 1995. Eur. J. Neurosci. 7:470-479). In addition to such compartmentalization, biochemical modifications take place that suggest a functional specialization of the subsynaptic GA. Here, we focused on the developmental regulation of the membrane traffic organization during the early steps of synaptogenesis in mouse diaphragm muscle. We investigated by immunofluorescence microscopy on cryosections, the distribution of selected subcompartments of the exocytic pathway, and also of a representative endocytic subcompartment with respect to the junctional or extrajunctional domains of developing myofibers. We show that throughout development the RER, the intermediate compartment, and the prelysosomal compartment (mannose 6-phosphate receptor-rich compartment) are homogeneously distributed along the fibers, irrespective of the subneural or extrajunctional domains. In contrast, at embryonic day E17, thus 2-3 d after the onset of innervation, most GA markers become restricted to the subneural domain. Interestingly, some Golgi markers (e.g., alpha-mannosidase II, TGN 38, present in the embryonic myotubes) are no longer detected in the innervated fiber even in the subsynaptic GA. These data show that in innervated muscle fibers, the distal part of the biosynthetic pathway, i.e., the GA, is remodeled selectively shortly after the onset of innervation. As a consequence, in the innervated fiber, the GA exists both as an evenly distributed organelle with basic functions, and as a highly differentiated subsynaptic organelle ensuring maturation and targeting of synaptic proteins. Finally, in the adult, denervation of a hemidiaphragm causes a burst of reexpression of all Golgi markers in extrasynaptic domains of the fibers, hence showing that the particular organization of the secretory pathway is placed under nerve control.

Presence of a protein immunologically related to lamin B in the postsynaptic membrane of Torpedo marmorata electrocyte.

The Torpedo electrocyte is a flattened syncytium derived from skeletal muscle, characterized by two functionally distinct plasma membrane domains. The electrocyte is filled up with a transversal network of intermediate filaments (IF) of desmin which contact in an end-on fashion both sides of the cell. In this work, we show that polyclonal antibodies specific for lamin B recognizes a component of the plasma membrane of Torpedo electrocyte. This protein which thus shares epitopes with lamin B has a relative molecular mass of 54 kD, an acidic IP of 5.4. It is localized exclusively on the cytoplasmic side of the innervated membrane of the electrocyte at sites of IF-membrane contacts. Since our previous work showed that the noninnervated membrane contains ankyrin (Kordeli, E., J. Cartaud, H. O. Nghiêm, L. A. Pradel, C. Dubreuil, D. Paulin, and J.-P. Changeux. 1986. J. Cell Biol. 102:748-761), the present results suggest that desmin filaments may be anchored via the 54-kD protein to the innervated membrane and via ankyrin to the noninnervated membrane. These findings would represent an extension of the model proposed by Georgatos and Blobel (Georgatos, S. D., and G. Blobel. 1987a. J. Cell Biol. 105:105-115) in which type III intermediate size filaments are vectorially inserted to plasma and nuclear membranes by ankyrin and lamin B, respectively.

A protein antigenically related to nuclear lamin B mediates the association of intermediate filaments with desmosomes.

Desmosomes are specialized domains of epithelial cell plasma membranes engaged in the anchoring of intermediate filaments (IF). So far, the desmosomal component(s) responsible for this binding has not been unambiguously identified. In the present work, we have examined bovine muzzle epidermis desmosomes for the presence of protein(s) structurally and functionally related to lamin B, the major receptor for IF in the nuclear envelope (Georgatos, S. D., and G. Blobel. 1987. J. Cell Biol. 105:105-115). By using polyclonal antibodies to lamin B in immunoblotting experiments, we find that a desmosomal protein of 140-kD shares epitope(s) with lamin B. Immunoelectron microscopic and urea extraction experiments show that this protein is a peripheral protein localized at the cytoplasmic side of the desmosomes (desmosomal plaques). Furthermore, this protein binds vimentin in an in vitro assay. Since this binding is inhibited by lamin B antibodies, the epitopes common to the 140-kD protein and to lamin B may be responsible for anchoring of intermediate filaments to desmosomes. These data suggest that lamin B-related proteins (see also Cartaud, A., J. C. Courvalin, M. A. Ludosky, and J. Cartaud. 1989. J. Cell Biol. 109:1745-1752) together with lamin B, provide cells with several nucleation sites, which can account for the multiplicity of IF organization in tissues.

Asynchronous assembly of the acetylcholine receptor and of the 43-kD nu1 protein in the postsynaptic membrane of developing Torpedo marmorata electrocyte.

The assembly of the nicotinic acetylcholine receptor (AchR) and the 43-kD protein (v1), the two major components of the post synaptic membrane of the electromotor synapse, was followed in Torpedo marmorata electrocyte during embryonic development by immunocytochemical methods. At the first developmental stage investigated (45-mm embryos), accumulation of AchR at the ventral pole of the newly formed electrocyte was observed within columns before innervation could be detected. No concomitant accumulation of 43-kD immunoreactivity in AchR-rich membrane domains was observed at this stage, but a transient asymmetric distribution of the extracellular protein, laminin, which paralleled that of the AchR, was noticed. At the subsequent stage studied (80-mm embryos), codistribution of the two proteins was noticed on the ventral face of the cell. Intracellular pools of AchR and 43-kD protein were followed at the EM level in 80-mm electrocytes. AchR immunoreactivity was detected within membrane compartments, which include the perinuclear cisternae of the endoplasmic reticulum and the plasma membrane. On the other hand, 43-kD immunoreactivity was not found associated with the AchR in the intracellular compartments of the cell, but codistributed with the AchR at the level of the plasma membrane. The data reported in this study suggest that AchR clustering in vivo is not initially determined by the association of the AchR with the 43-kD protein, but rather relies on AchR interaction with extracellular components, for instance from the basement membrane, laid down in the tissue before the entry of the electromotor nerve endings.

Consequences of alkaline treatment for the ultrastructure of the acetylcholine-receptor-rich membranes from Torpedo marmorata electric organ.

After fixation with glutaraldehyde and impregnation with tannic acid, the membrane that underlies the nerve terminals in Torpedo marmorata electroplaque presents a typical asymmetric triple-layered structure with an unusual thickness; in addition, it is coated with electron-dense material on its inner, cytoplasmic face. Filamentous structures are frequently found attached to these "subsynaptic densities." The organization of the subsynaptic membrane is partly preserved after homogenization of the electric organ and purification of acetylcholine-receptor (AchR)-rich membrane fragments. In vitro treatment at pH 11 and 4 degrees C of these AchR-rich membranes releases an extrinsic protein of 43,000 mol wt and at the same time causes the complete disappearance of the cytoplasmic condensations. Freeze-etching of native membrane fragments discloses remnants of the ribbonlike organization of the AchR rosettes. This organization disappears ater alkaline treatment and is replaced by a network which is not observed after rapid freezing and, therefore, most likely results from the lateral redistribution of the AchR rosettes during condition of slow freezing. A dispersion of the AchR rosettes in the plane of the membrane also occurs after fusion of the pH 11-treated fragments with phospholipid vesicles. These results are interpreted in terms of a structural stabilization and immobilization of the AchR by the 43,000-Mr protein binding to the inner face of the subsynaptic membrane.

MAGI-1c: a synaptic MAGUK interacting with muSK at the vertebrate neuromuscular junction.

The muscle-specific receptor tyrosine kinase (MuSK) forms part of a receptor complex, activated by nerve-derived agrin, that orchestrates the differentiation of the neuromuscular junction (NMJ). The molecular events linking MuSK activation with postsynaptic differentiation are not fully understood. In an attempt to identify partners and/or effectors of MuSK, cross-linking and immunopurification experiments were performed in purified postsynaptic membranes from the Torpedo electrocyte, a model system for the NMJ. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis was conducted on both cross-link products, and on the major peptide coimmunopurified with MuSK; this analysis identified a polypeptide corresponding to the COOH-terminal fragment of membrane-associated guanylate kinase (MAGUK) with inverted domain organization (MAGI)-1c. A bona fide MAGI-1c (150 kD) was detected by Western blotting in the postsynaptic membrane of Torpedo electrocytes, and in a high molecular mass cross-link product of MuSK. Immunofluorescence experiments showed that MAGI-1c is localized specifically at the adult rat NMJ, but is absent from agrin-induced acetylcholine receptor clusters in myotubes in vitro. In the central nervous system, MAGUKs play a primary role as scaffolding proteins that organize cytoskeletal signaling complexes at excitatory synapses. Our data suggest that a protein from the MAGUK family is involved in the MuSK signaling pathway at the vertebrate NMJ.

Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes.

The subcellular distribution of the 43,000-D protein (43 kD or v1) and of some major cytoskeletal proteins was investigated in Torpedo marmorata electrocytes by immunocytochemical methods (immunofluorescence and immunogold at the electron microscope level) on frozen-fixed sections and homogenates of electric tissue. A monoclonal antibody directed against the 43-kD protein (Nghiêm, H. O., J. Cartaud, C. Dubreuil, C. Kordeli, G. Buttin, and J. P. Changeux, 1983, Proc. Natl. Acad. Sci. USA, 80:6403-6407), selectively labeled the postsynaptic membrane on its cytoplasmic face. Staining by anti-actin and anti-desmin antibodies appeared evenly distributed within the cytoplasm: anti-desmin antibodies being associated with the network of intermediate-sized filaments that spans the electrocyte, and anti-actin antibodies making scattered clusters throughout the cytoplasm without preferential labeling of the postsynaptic membrane. On the other hand, a dense coating by anti-actin antibodies became apparent on the postsynaptic membrane in homogenates of electric tissue pointing to the possible artifactual redistribution of a soluble cytoplasmic actin pool. Anti-fodrin and anti-ankyrin antibodies selectively labeled the non-innervated membrane of the cell. F actin was also detected in this membrane. Filamin and vinculin, two actin-binding proteins recently localized at the rat neuromuscular junction (Bloch, R. J., and Z. W. Hall, 1983, J. Cell Biol., 97:217-223), were detected in the electrocyte by the immunoblot technique but not by immunocytochemistry. The data are interpreted in terms of the functional polarity of the electrocyte and of the selective interaction of the cytoskeleton with the innervated and non-innervated domains of the plasma membrane.

Role of lipid rafts in agrin-elicited acetylcholine receptor clustering.

Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.

The myristoylated protein rapsyn is cotargeted with the nicotinic acetylcholine receptor to the postsynaptic membrane via the exocytic pathway.

Rapsyn, a 43 kDa protein required to cluster nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction, is tightly associated with the postsynaptic membrane via an N-terminal myristoylated site. Recent studies have shown that some acylated proteins associate with the exocytic pathway to become targeted to their correct destination. In this work, we used Torpedo electrocyte to investigate the intracellular routing of rapsyn compared to those of AChR and Na,K-ATPase, the respective components of the innervated and noninnervated membranes. We previously demonstrated that these latter two proteins are sorted and targeted to plasma membrane via distinct populations of post-Golgi vesicles (). Biochemical and immunoelectron microscopy analyses of various populations of post-Golgi vesicles immunopurified with magnetic beads led us to identify post-Golgi transport vesicles containing both rapsyn and AChR. These data suggest that rapsyn, as for AChR, specifically follows the exocytic pathway. Furthermore, immunogold-labeling experiments provided in situ evidence that AChR and rapsyn are cotransported in the same post-Golgi vesicles. Taken together, our observations suggest that rapsyn and AChR are cotargeted to the postsynaptic membrane.

Effects of temperature, lipid modification and pH on the mobility of the major proteins of the receptor-rich membranes from Torpedo marmarata.

The factors influencing the overall mobility of the major proteins of the acetylcholine receptor-rich membranes from Torpedo marmorata have been investigated by saturation transfer ESR spectroscopy and the lateral distribution of these proteins has been studied by electron microscopy. A spin-labelled derivative of maleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (MSL), was used under various conditions of incubation, enabling us to attach it mainly to either an extrinsic protein of 43 kdaltons, or an intrinsic protein (40 kdaltons) bearing the alpha-toxin-binding site. (1) The direct reaction of MSL with the membrane fragments resulted in almost exclusive labelling of the 43 kdalton protein, an extrinsic protein located on the inner face of the receptor-rich membranes. (2) After the free SH groups were blocked with N-ethylmaleimide and the disulfide bridges opened with the reducing agent dithiothreitol, MSL reacted with both the 40 and 43 kdalton proteins (6.0 +/- 0.6 MSL molecules per alpha-toxin-binding site). (3) After the latter labelling procedure membranes were exposed to pH 11, resulting in extraction of the 43 kdalton protein and leaving 2.2 +/- 0.4 MSL molecules per alpha-toxin-binding site; sodium dodecyl sulfate polyacrylamide gel electrophoresis performed with N-[14C] ethylmaleimide suggested that MSL was bound mainly to the 40 kdalton polypeptide chain of the acetylcholine receptor. The following conclusions were made with the native and alkaline-treated membranes: In the native membranes, saturation transfer ESR does not reveal any significant protein rotational diffusion (rotational correlation time tau C greater than 1 ms). Temperature variations and/or lipid modifications obtained by fusion of exogenous lipids and/or cholesterol exchange have little influence on the saturation transfer ESR spectra. Electron microscopy reveals that upon lipid addition, proteins remain in the form of clusters while areas depleted of proteins appear. On the other hand, alkaline treatment strikingly enhances the motion of the MSL-labelled proteins in the membrane (100 less than or equal to tau c less than or equal to 120 microseconds). Furthermore, the rotational diffusion of the MSL-labelled proteins (mainly the 40 kdalton protein) becomes sensitive to temperature, lipid composition and the lipid-to-proteins ratio. Electron microscopy shows that alkaline extraction does not cause large reorganization of the acetylcholine receptor in the plane of the membrane. However, when phospholipids are added to pH 11 treated membranes, a dispersion of the receptor and rosettes is observed. In contrast, cholesterol enrichment of the latter membranes induces clustering of the receptor immobilization as judged by saturation transfer ESR. Upon reassociation of the pH 11 soluble proteins with the alkaline-treated membranes, the restriction of the acetylcholine receptor rotational mobility is also restored (tau c greater than or equal to 1 ms).

Image analysis of the heavy form of the acetylcholine receptor from Torpedo marmorata.

The structure of the heavy (H) form of the acetylcholine receptor, which comprises two covalently linked 250,000 Mr oligomers, has been investigated by numerical analysis of electron microscope images. Na-cholate solubilized Torpedo marmorata H-form receptor was reintegrated into artificial lipid vesicles and negatively stained with uranyl acetate prior to imaging in a conventional transmission microscope. The reconstituted preparations exhibited the standard polypeptide composition of the purified receptor (alpha 2 beta gamma delta) and the same transmembrane arrangement as in the native subsynaptic membrane. Covalent disulfide linkage between the two oligomers took place exclusively through the delta chains. In agreement with previous work (Cartaud et al., 1980) the H-form appeared as "doublets" of two coplanar 9 nm rosettes at a center-to-center distance of 9.2 +/- 1.1 nm. The relative angular orientation of the two rosettes in a doublet was examined by correlation analysis in the real space. It exhibited a marked variability, few of the doublets featuring any kind of symmetry, suggesting that the two oligomers of a doublets are connected via an extended and flexible chain or loop. The area of contact between the two rosettes of a doublet therefore does not necessarily represent a reliable clue as to the location of the delta chain within the structure. Averaged images obtained after reorientation and summation of up to 132 rosettes revealed the three major peaks and the two grooves already observed in previous studies. Two additional smaller peaks were identified. Tentative assignment of structural details to individual subunits was deduced from an examination of alpha-bungarotoxin-labeled doublets. The alpha subunits, which carry part or all of the acetylcholine binding sites, are probably located in nonadjacent positions in the vicinity of the newly found peaks. This assignment is consistent with the image analysis of receptor-toxin complexes recently reported by Zingsheim et al. (1982b).

Acetylcholinesterase and molecular interactions at the neuromuscular junction.

The efficiency and the tight control of neurotransmission require the accumulation of synaptic proteins in discrete domains. In neuromuscular junctions, the main form of acetylcholinesterase (AChE) is a hetero-oligomer in which the catalytic subunits are associated to a specific collagen, ColQ. This structural protein is responsible for the insertion and the accumulation of AChE in the synaptic basal lamina. We have analyzed the time-course of acetylcholinesterase and acetylcholine receptors (AChR) mRNAs during mouse muscle cell differentiation in culture. In parallel, we have visualized the formation of AChE and AChR aggregates. We show that AChR clusters form first which correlates with high gamma-subunit mRNA levels. Then, AChE clusters appear with the onset of contraction and correlate with a dramatic increase in AChE, ColQ1 and ColQ1A mRNA levels in muscle cells. At that stage, AChR gamma-subunit levels drop while the expression level of epsilon-subunits increase. AChE aggregates are organized by a ternary complex, which involves direct interactions between ColQ, perlecan and MuSK.

Expression of utrophin and its mRNA in denervated mdx mouse muscle.

Utrophin is a large cytoskeletal protein which shows high homology to dystrophin. In contrast to the sarcolemmal distribution of dystrophin, utrophin accumulates at the postsynaptic membrane of the neuromuscular junction. Because of its localization within this compartment of muscle fibers, expression of utrophin may be significantly influenced by the presence of the motor nerve. We tested this hypothesis by denervating muscles of mdx mouse and monitoring levels of utrophin and its mRNA by immunofluorescence, immunoblotting and RT-PCR. A significant increase in the number of utrophin positive fibers was observed by immunofluorescence 3 to 21 days after sectioning of the sciatic nerve. Quantitative analyses of utrophin and its transcripts in hindlimb muscles denervated for two weeks showed only a moderate increase in the levels of both utrophin (approximately 2-fold) and its transcript (approximately 60 to 90%). The present data suggest that although utrophin is a component of the postsynaptic membrane, its neural regulation is distinct from that of the acetylcholine receptor.

Organization and dynamics of microtubules in Torpedo marmorata electrocyte: selective association with specialized domains of the postsynaptic membrane.

The distribution and subcellular organization of two components of the secretory pathway, the Golgi apparatus and microtubules, have been investigated in Torpedo marmorata electrocyte. This highly polarized syncytium, embryologically derived from skeletal muscle cells, displays distinct plasma membrane domains on its innervated and non-innervated faces, and it played a critical role in the identification of the acetylcholine receptor. By immunocytochemical analysis, we show that in the electrocyte, numerous focal Golgi bodies are dispersed throughout the cytoplasm in frequent association with nuclei. Under experimental conditions known to stabilize microtubules, we reveal an elaborate network composed of two populations of microtubules exhibiting different dynamic properties as evaluated by cold-stability, resistance to nocodazole and post-translational modification. This network appears organized from several nucleating centers located in the medial plane of the cell that are devoided of centrioles. The network displays an asymmetric distribution with individual microtubules converging towards the troughs of the postsynaptic membrane folds. In these particular regions, we consistently observed clusters of non-coated vesicles in association with the microtubules. The organization of the microtubules in the electrocyte may thus result in a functional polarization of the cytoplasm. In other polarized cells, the particular organization of the secretory pathway accounts for the intracellular routing of membrane proteins. The organization that we have observed in the electrocyte may thus lead to the vectorial delivery of synaptic proteins to the innervated plasma membrane. Furthermore, the abundance of synaptic proteins makes the electrocyte a unique model with which to decipher the mechanisms involved in the sorting and targeting of these glycoproteins.

The low molecular weight guanosine triphosphate-binding protein Rab6p associates with distinct post-Golgi vesicles in Torpedo marmorata electrocytes.

The Rab genes have recently been cloned and sequenced in mammals, and their products represent good candidates for low molecular weight guanosine triphosphate-binding proteins involved in the regulation of intracellular transport of vesicles in higher eukaryotes. Remarkably, each of the Rab proteins appears to be associated with a distinct step of either the exocytic or endocytic pathway. In particular, Rab6p has been localized to the outermost Golgi cisternae in normal rat kidney cells, where its function remains unclear. In this work, we have carried out a series of immunocytochemical analyses of the subcellular distribution of Rab6p in a polarized cell, the electrocyte of Torpedo marmorata, to elucidate the molecular mechanisms involved in the sorting and targeting of synaptic proteins. We report that, aside from its Golgi localization, the bulk of Rab6p associates with clusters of post-Golgi vesicles, primarily those located at the cytoplasmic face of the innervated membrane of the electrocyte. Consequently, Rab6p presents a polarized distribution in this cell. Furthermore, we show that this distribution is dependent upon the integrity of the microtubule network of the electrocyte. These data are coherent with the notion that Rab6p is involved in the regulation of membrane traffic from the trans-Golgi network to the innervated plasma membrane, delivering, by way of a microtubule-based organelle transport mechanism, synaptic proteins to their appropriate locations.

Localization of dystrophin and dystrophin-related protein at the electromotor synapse and neuromuscular junction in Torpedo marmorata.

The immunological identification of dystrophin isoforms at the neuromuscular junction and Torpedo marmorata electromotor synapse was attempted using various antibodies. A polyclonal antibody raised against electrophoretically purified dystrophin from T. marmorata electrocyte has been thoroughly investigated. This antibody recognized dystrophin in the electric tissue as well as sarcolemmal and synaptic neuromuscular junction dystrophin in all studies species (T. marmorata, rat, mice and human) at serum dilutions as high as 1:10,000. At variance, no staining of either the sarcolemma or neuromuscular junction was observed in Duchenne muscular dystrophy or mdx mice skeletal muscles. In these muscles, other members of the dystrophin superfamily, in particular the dystrophin-related protein(s) encoded by autosomal genes are present. These data thus demonstrate the specificity of our antibodies for dystrophin. Anti-dystrophin-related protein antibodies [Khurana et al. (1991) Neuromusc. Disorders 1, 185-194] which gave a strong immunostaining of the neuromuscular junction in various species, including T. marmorata, cross-reacted weakly with the postsynaptic membrane of the electrocyte. Taken together, these observations are in favor of the existence of a protein very homologous to dystrophin at the electromotor synapse in T. marmorata, whereas both dystrophin and dystrophin-related protein co-localize at the neuromuscular junction as in all species studied. The electrocyte thus offers the unique opportunity to study the interaction of dystrophin with components of the postsynaptic membrane.

Cell line-dependent release of HIV-like gag particles after infection of mammalian cells with recombinant vaccinia viruses.

We investigated the production of Gag particles by Vero, CV-1, or 1D cells infected with different vaccinia virus recombinants expressing HIV gag or gag-pol genes. Immunoblots of (centrifuged) culture media from 1D cells infected with vMM5, a vaccinia virus recombinant expressing the HIV-2 gag-pol genes, revealed the presence of abundant particles that contained (mostly processed) Gag antigens. In contrast, Gag particles were found only in low amounts in the culture medium from Vero cells infected with the same HIV gag-pol vaccinia virus recombinant; the Gag precursor remained associated with the infected Vero cells and was efficiently processed. This low excretion of Gag particles after infection of Vero cells with vMM5 was also demonstrated by assays of reverse transcriptase activity in the pellet of centrifuged culture medium. Cell fractionation showed that Gag proteins were predominantly found in the membrane fraction from both 1D and Vero cells. Electron microscopy observations of 1D or of Vero cells infected with vMM5 vaccinia virus recombinant revealed in both cases the presence of particles budding at the plasma membrane. However, the shape of the budding particles was different in the two cell lines, with immature forms present in the membrane from the infected Vero cells. An inefficient excretion of Gag particles was also observed after infection of Vero cells with different vaccinia virus recombinants expressing either an uncleaved HIV-2 Gag protein or the HIV-1 gag-pol genes, as judged both by immunoblot and reverse transcriptase activity assays.(ABSTRACT TRUNCATED AT 250 WORDS)

The torpedo electrocyte: a model system to study membrane-cytoskeleton interactions at the postsynaptic membrane.

Many aspects of the organization of the electromotor synapse of electric fish resemble the nerve-muscle junction. In particular, the postsynaptic membrane in both systems share most of their proteins. As a remarquable source of cholinergic synapses, the Torpedo electrocyte model has served to identify the most important components involved in synaptic transmission such as the nicotinic acetylcholine receptor and the enzyme acetylcholinesterase, as well as proteins associated with the subsynaptic cytoskeleton and the extracellular matrix involved in the assembly of the postsynaptic membrane, namely the 43-kDa protein-rapsyn, the dystrophin/utrophin complex, agrin, and others. This review encompasses some representative experiments that helped to clarify essential aspects of the supramolecular organization and assembly of the postsynaptic apparatus of cholinergic synapses.

Targeting of acetylcholine receptor and 43 kDa rapsyn to the postsynaptic membrane in Torpedo marmorata electrocyte.

In this study we have investigated the intracellular routing of two major components of the postsynaptic membrane in Torpedo electrocytes, the nicotinic acetylcholine receptor and the extrinsic 43 kDa protein rapsyn, and of a protein from the non-innervated membrane, the Na+,K+ ATPase. We isolated subpopulations of post-Golgi vesicles (PGVs) enriched either in AChR or in Na+,K+ ATPase. Rapsyn was associated to AChR-containing PGVs suggesting that both AChR and rapsyn are targeted to intracellular organelles in the secretory pathway before delivery to the postsynaptic membrane. In vitro assays further show that rapsyn-containing PVGs do bind more efficiently to microtubules compared to Na+,K+ ATPase-enriched PVGs. These data provide evidence in favor of the contribution of the secretory pathway to the delivery of synaptic components.