Agrin triggers the clustering of raft-associated acetylcholine receptors through actin cytoskeleton reorganization.

Background Information: Cholesterol/sphingolipid-rich membrane microdomains or membrane rafts have been implicated in various aspects of receptor function such as activation, trafficking and synapse localization. More specifically in muscle, membrane rafts are involved in AChR (acetylcholine receptor) clustering triggered by the neural factor agrin, a mechanism considered integral to NMJ (neuromuscular junction) formation. In addition, actin polymerization is required for the formation and stabilization of AChR clusters in muscle fibres.

COPI coat assembly occurs on liquid-disordered domains and the associated membrane deformations are limited by membrane tension.

Cytoplasmic coat proteins are required for cargo selection and budding of tubulovesicular transport intermediates that shuttle between intracellular compartments. To better understand the physical parameters governing coat assembly and coat-induced membrane deformation, we have reconstituted the Arf1-dependent assembly of the COPI coat on giant unilamellar vesicles by using fluorescently labeled Arf1 and coatomer. Membrane recruitment of Arf1-GTP occurs exclusively on disordered lipid domains and does not induce optically visible membrane deformation.

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.

Rafts are required for acetylcholine receptor clustering.

Cholesterol-sphingolipid microdomains, or lipid rafts, are major regulators of molecular interactions in membrane organization. Because lipid rafts can move laterally and cluster into larger patches, they have been proposed to play a role in the redistribution of specific molecules to specialized cellular structures. Rafts have been shown to favor formation and maintenance of synaptic receptor clusters in neurons of the central nervous system.

A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family.

Ligand-gated ion channels (LGICs) mediate excitatory and inhibitory transmission in the nervous system. Among them, the pentameric or 'Cys-loop' receptors (pLGICs) compose a family that until recently was found in only eukaryotes. Yet a recent genome search identified putative homologues of these proteins in several bacterial species.

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes.

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line.

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.

The synaptic muscle-specific kinase (MuSK) complex: new partners, new functions.

The muscle-specific kinase MuSK is part of an agrin receptor complex that stimulates tyrosine phosphorylation and drives clustering of acetylcholine receptors (AChRs) in the postsynaptic membrane at the vertebrate neuromuscular junction (NMJ). MuSK also regulates synaptic gene transcription in subsynaptic nuclei. Over the past few years, decisive progress has been made in the identification of MuSK effectors, helping to understand its function in the formation of the NMJ.

Differential recruitment of p160 coactivators by glucocorticoid receptor between Schwann cells and astrocytes.

In the nervous system, glucocorticoids can exert beneficial or noxious effects, depending on their concentration and the duration of hormonal stimulation. They exert their effects on neuronal and glial cells by means of their cognate receptor, the glucocorticoid receptor (GR), which recruits the p160 coactivator family members SRC-1 (steroid receptor coactivator 1), SRC-2, and SRC-3 after hormone binding. In this study, we investigated the molecular pathways used by the GR in cultured glial cells of the central and the peripheral nervous systems, astrocytes and Schwann cells (MSC80 cells), respectively.

14-3-3 gamma associates with muscle specific kinase and regulates synaptic gene transcription at vertebrate neuromuscular synapse.

The muscle-specific receptor tyrosine kinase (MuSK) is part of a receptor complex, activated by neural agrin, that orchestrates the differentiation of the neuromuscular junction (NMJ). To gain insight into the function of the MuSK complex, we have developed a proteomic approach to identify new MuSK partners. MS analysis of MuSK crosslink products from postsynaptic membranes of the Torpedo electrocytes identified the adaptor protein 14-3-3 gamma.

MuSK is required for anchoring acetylcholinesterase at the neuromuscular junction.

At the neuromuscular junction, acetylcholinesterase (AChE) is mainly present as asymmetric forms in which tetramers of catalytic subunits are associated to a specific collagen, collagen Q (ColQ). The accumulation of the enzyme in the synaptic basal lamina strictly relies on ColQ. This has been shown to be mediated by interaction between ColQ and perlecan, which itself binds dystroglycan.

Electron microscopic evidence for nucleation and growth of 3D acetylcholine receptor microcrystals in structured lipid-detergent matrices.

Nicotinic acetylcholine receptors (AChRs) belong to a superfamily of oligomeric proteins that transduce electric signals across the cell membrane on binding of neurotransmitters. These receptors harbor a large extracellular ligand-binding domain directly linked to an ion-conducting channel-forming domain that spans the cell membrane 20 times and considerably extends into the cytoplasm. Thus far, none of these receptor channels has been crystallized in three dimensions.

Expression of mutant Ets protein at the neuromuscular synapse causes alterations in morphology and gene expression.

The localized transcription of several muscle genes at the motor endplate is controlled by the Ets transcription factor GABP. To evaluate directly its contribution to the formation of the neuromuscular junction, we generated transgenic mice expressing a general Ets dominant-negative mutant specifically in skeletal muscle. Quantitative RT-PCR analysis demonstrated that the expression of genes containing an Ets-binding site was severely affected in the mutant mice.

Targeted trafficking of neurotransmitter receptors to synaptic sites.

Emerging data are sheding light on the critical task for synapses to locally control the production of neurotransmitter receptors ultimately leading to receptor accumulation and modulation at postsynaptic sites. By analogy with the epithelial-cell paradigm, the postsynaptic compartment may be regarded as a polarized domain favoring the selective recruitment and retention of newly delivered receptors at synaptic sites. Targeted delivery of receptors to synaptic sites is facilitated by a local organization of the exocytic pathway, likely resulting from spatial cues triggered by the nerve.

Rapsyn escorts the nicotinic acetylcholine receptor along the exocytic pathway via association with lipid rafts.

The 43 kDa receptor-associated protein rapsyn is a myristoylated peripheral protein that plays a central role in nicotinic acetylcholine receptor (AChR) clustering at the neuromuscular junction. In a previous study, we demonstrated that rapsyn is specifically cotransported with AChR via post-Golgi vesicles targeted to the innervated surface of the Torpedo electrocyte (Marchand et al., 2000).

A minimal system allowing tubulation with molecular motors pulling on giant liposomes.

The elucidation of physical and molecular mechanisms by which a membrane tube is generated from a membrane reservoir is central to the understanding of the structure and dynamics of intracellular organelles and of transport intermediates in eukaryotic cells. Compelling evidence exists that molecular motors of the dynein and kinesin families are involved in the tubulation of organelles. Here, we show that lipid giant unilamellar vesicles (GUVs), to which kinesin molecules have been attached by means of small polystyrene beads, give rise to membrane tubes and to complex tubular networks when incubated in vitro with microtubules and ATP.

Identification of Ank(G107), a muscle-specific ankyrin-G isoform.

We previously showed that alternatively spliced ankyrins-G, the Ank3 gene products, are expressed in skeletal muscle and localize to the postsynaptic folds and to the sarcoplasmic reticulum. Here we report the molecular cloning, tissue expression, and subcellular targeting of Ank(G107), a novel ankyrin-G from rat skeletal muscle. Ank(G107) lacks the entire ANK repeat domain and contains a 76-residue sequence near the COOH terminus.

Biochemical characterization of nuclear pore complex protein gp210 oligomers.

The membrane-spanning glycoprotein gp210 is a major component of the nuclear pore complex. This nucleoporin contains a large cisternal N-terminal domain, a short C-terminal cytoplasmic tail, and a single transmembrane segment. We show here that dimers of native gp210 can be isolated from cell extracts by immunoprecipitation, and from purified rat liver nuclear envelopes by velocity sedimentation and gel filtration.

Lack of desmin results in abortive muscle regeneration and modifications in synaptic structure.

Desmin, a muscle-specific intermediate filament protein, is expressed in all muscle tissues. Its absence leads to a multisystemic disorder involving cardiac, skeletal, and smooth muscles. In skeletal muscle, structural abnormalities include lack of alignment of myofibrils, Z disk streaming, and focal muscle degeneration.

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.

Differential targeting of components of the dystrophin complex to the postsynaptic membrane.

Accumulating evidence points to the participation of dystroglycan in the clustering of nicotinic acetylcholine receptors at the neuromuscular junction [Côté et al. (1999) Nature Genet., 3, 338--342].

Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and 31P-nuclear magnetic resonance.

It has been reported that repetitive freeze-thaw cycles of aqueous suspensions of dioleoylphosphatidylcholine form vesicles with a diameter smaller than 200 nm. We have applied the same treatment to a series of phospholipid suspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoylphosphatidic acid (DOPC/DOPA) mixtures. Freeze-fracture electron microscopy revealed that these unsaturated lipids form unilamellar vesicles after 10 cycles of freeze-thawing.

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.