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. Since membrane rafts are platforms sustaining actin nucleation, we hypothesize that these microdomains provide the suitable microenvironment favouring agrin/MuSK (muscle-specific kinase) signalling, eliciting in turn actin cytoskeleton reorganization and AChR clustering. However, the identity of the signalling pathways operating through these microdomains still remains unclear. RESULTS: In this work, we attempted to identify the interactions between membrane raft components and cortical skeleton that regulate, upon signalling by agrin, the assembly and stabilization of synaptic proteins of the postsynaptic membrane domain at the NMJ. We provide evidence that in C2C12 myotubes, agrin triggers the association of a subset of membrane rafts enriched in AChR, the -MuSK and Cdc42 (cell division cycle 42) to the actin cytoskeleton. Disruption of the liquid-ordered phase by methyl-ß-cyclodextrin abolished this association. We further show that actin and the actin-nucleation factors, N-WASP (neuronal Wiscott-Aldrich syndrome protein) and Arp2/3 (actin-related protein 2/3) are transiently associated with rafts on agrin engagement. Consistent with these observations, pharmacological inhibition of N-WASP activity perturbed agrin-elicited AChR clustering. Finally, immunoelectron microscopic analyses of myotube membrane uncovered that AChRs were constitutively associated with raft nanodomains at steady state that progressively coalesced on agrin activation. These rearrangements of membrane domains correlated with the reorganization of cortical actin cytoskeleton through concomitant and transient recruitment of the Arp2/3 complex to AChR-enriched rafts. CONCLUSIONS: The present observations support the notion that membrane rafts are involved in AChR clustering by promoting local actin cytoskeleton reorganization through the recruitment of effectors of the agrin/MuSK signalling cascade. These mechanisms are believed to play an important role in vivo in the formation of the NMJ.

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. In the presence of Arf1-GTP, coatomer self-assembles into weakly curved coats on membranes under high tension, while it induces extensive membrane deformation at low membrane tension. These deformations appear to have a composition different from the parental membrane because they are protected from phase transition. These findings suggest that the COPI coat is adapted to liquid disordered membrane domains where it could promote lipid sorting and that its mechanical effects can be tuned by membrane tension.

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. Here we report the cloning, expression and functional identification of one of these putative homologues from the cyanobacterium Gloeobacter violaceus. It was expressed as a homo-oligomer in HEK 293 cells and Xenopus oocytes, generating a transmembrane cationic channel that is opened by extracellular protons and shows slow kinetics of activation, no desensitization and a single channel conductance of 8 pS. Electron microscopy and cross-linking experiments of the protein fused to the maltose-binding protein and expressed in Escherichia coli are consistent with a homo-pentameric organization. Sequence comparison shows that it possesses a compact structure, with the absence of the amino-terminal helix, the canonical disulphide bridge and the large cytoplasmic domain found in eukaryotic pLGICs. Therefore it embodies a minimal structure required for signal transduction. These data establish the prokaryotic origin of the family. Because Gloeobacter violaceus carries out photosynthesis and proton transport at the cytoplasmic membrane, this new proton-gated ion channel might contribute to adaptation to pH change.

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. However, little is known about their role in formation of the neuromuscular junction (NMJ). To determine whether lipid rafts are involved in acetylcholine receptor (AChR) cluster formation and stabilization in myogenic cells, two standard tools were employed: (1) Perturbation of lipid rafts by drugs that deplete membrane cholesterol was carried out to verify that cholesterol is required for AChR clustering in agrin-treated C2C12 myotubes; and (2) detergent resistance of lipid-ordered domains was also used to demonstrate that AChRs, as well as key components of the postsynaptic membrane of the NMJ, are associated with rafts.

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. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.

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. We performed functional studies based on transient transfection of a minimal glucocorticoid-sensitive reporter gene into the glial cells to test the influence of overexpression or selective inhibition by short interfering RNA of the three p160 coactivator family members on GR transactivation. We demonstrate that, depending on the glial cell type, GR differentially recruits p160 family members: in Schwann cells, GR recruited SRC-1a, SRC-1e, or SRC-3, whereas in astrocytes, SRC-1e and SRC-2, and to a lesser extent SRC-3, were active toward GR signaling. The C-terminal nuclear receptor-interacting domain of SRC-1a participates in its exclusion from the GR transcriptional complex in astrocytes. Immunolocalization experiments revealed a cell-specific intracellular distribution of the p160s, which was dependent on the duration of the hormonal induction. For example, within astrocytes, SRC-1 and SRC-2 were mainly nuclear, whereas SRC-3 unexpectedly localized to the lumen of the Golgi apparatus. In contrast, in Schwann cells, SRC-1 showed a nucleocytoplasmic shuttling depending on hormonal stimulation, whereas SRC-2 remained strictly nuclear and SRC-3 remained predominantly cytoplasmic. Altogether, these results highlight the cell specificity and the time dependence of p160s recruitment by the activated GR in glial cells, revealing the complexity of GR-p160 assembly in the nervous system.

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. Similarly to AChR, MuSK and several of its partners are the target of mutations responsible for diseases of the NMJ, such as congenital myasthenic syndromes. This minireview will focus on the multiple MuSK effectors so far identified that place MuSK at the center of a multifunctional signaling complex involved in the organization of the NMJ and associated disorders.

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. The 14-3-3 gamma protein was found localized at the adult rat NMJ. Cotransfection experiments in COS-7 cells showed that MuSK codistributed with the 14-3-3 gamma protein at the plasma membrane. Furthermore, 14-3-3 gamma was copurified by affinity chromatography with MuSK from transfected COS-7 cells and myotubes. The 14-3-3 gamma protein did not colocalize with agrin-elicited acetylcholine receptor (AChR) aggregates in cultured myotubes, suggesting that it is not involved in AChR clustering. Expression of 14-3-3 gamma specifically repressed the transcription of several synaptic reporter genes in cultured myotubes. This repression was potentiated by MuSK expression. Moreover, the expression of 14-3-3 gamma in muscle fibers in vivo caused both the repression of synaptic genes transcription and morphological perturbations of the NMJ. Our data extend the notion that, apart from its well documented role in AChR clustering, the MuSK complex might also be involved in the regulation of synaptic gene expression at the NMJ.

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. Here, using transfected mutants of ColQ in a ColQ-deficient muscle cell line or COS-7 cells, we report that ColQ clusterizes through a more complex mechanism. This process requires two heparin-binding sites contained in the collagen domain as well as the COOH terminus of ColQ. Cross-linking and immunoprecipitation experiments in Torpedo postsynaptic membranes together with transfection experiments with muscle-specific kinase (MuSK) constructs in MuSK-deficient myotubes or COS-7 cells provide the first evidence that ColQ binds MuSK. Together, our data suggest that a ternary complex containing ColQ, perlecan, and MuSK is required for AChE clustering and support the notion that MuSK dictates AChE synaptic localization at the neuromuscular junction.

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. The crystallization of the AChR from Torpedo marmorata electric organs is challenged here in lipidic-detergent matrices. Detergent-soluble AChR complexed with alpha-bungarotoxin (alphaBTx), a polypeptidic competitive antagonist, was purified. The AChR-alphaBTx complex was reconstituted in a lipidic matrix composed of monoolein bilayers that are structured in three dimensions. The alphaBTx was conjugated to a photo-stable fluorophore, enabling us to monitor the physical behavior of the receptor-toxin complex in the lipidic matrix under light stereomicroscope, and to freeze fracture regions containing the receptor-toxin complex for visualization under a transmission electron microscope. Conditions were established for forming 2D receptor-toxin lattices that are stacked in the third dimension. 3D AChR nanocrystals were thereby grown inside the highly viscous lipidic 3D matrix. Slow emulsification of the lipidic matrix converted these nanocrystals into 3D elongated thin crystal plates of micrometer size. The latter are stable in detergent-containing aqueous solutions and can currently be used for seeding and epitaxial growth, en route to crystals of appropriate dimensions for x-ray diffraction studies.

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. This review focuses on the various mechanisms responsible for regulation of receptor assembly and trafficking. A particular emphasis is given to the role of synaptic anchoring and scaffolding proteins in the sorting and routing of their receptor companion along the exocytic pathway. Other cellular components such as lipidic microdomains, the docking and fusion machinery, and the cytoskeleton also contribute to the dynamics of receptor trafficking at the synapse.

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). In the present study, to further elucidate the mechanisms for sorting and assembly of postsynaptic proteins, we analyzed the dynamics of the intracellular trafficking of fluorescently labeled rapsyn in the transient-expressing COS-7 cell system. Our approach was based on fluorescence, time-lapse imaging, and immunoelectron microscopies, as well as biochemical analyses. We report that newly synthesized rapsyn associates with the trans-Golgi network compartment and traffics via vesiculotubular organelles toward the cell surface of COS-7 cells. The targeting of rapsyn organelles appeared to be mediated by a microtubule-dependent transport. Using cotransfection experiments of rapsyn and AChR, we observed that these two molecules codistribute within distal exocytic routes and at the plasma membrane. Triton X-100 extraction on ice and flotation gradient centrifugation demonstrated that rapsyn and AChR are recovered in low-density fractions enriched in two rafts markers: caveolin-1 and flotillin-1. We propose that sorting and targeting of these two companion molecules are mediated by association with cholesterol-sphingolipid-enriched raft microdomains. Collectively, these data highlight rapsyn as an itinerant vesicular protein that may play a dynamic role in the sorting and targeting of its companion receptor to the postsynaptic membrane. These data also raise the interesting hypothesis of the participation of the raft machinery in the targeting of signaling molecules to synaptic sites.

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. Conversely, the expression of other synaptic genes, including MuSK and Rapsyn, was unchanged. In these animals, muscles expressing the mutant transcription factor developed normally, but examination of the post-synaptic morphology revealed marked alterations of both the primary gutters and secondary folds of the neuromuscular junction. Our results demonstrate that Ets transcription factors are crucial for the normal formation of the neuromuscular junction. They further show that Ets-independent mechanisms control the synaptic expression of a distinct set of synaptic genes.

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. Similar tubes and networks are obtained with GUVs made of purified Golgi lipids, as well as with Golgi membranes. No tube formation was observed when kinesins were directly bound to the GUV membrane, suggesting that it is critical to distribute the load on both lipids and motors by means of beads. A kinetic analysis shows that network growth occurs in two phases: a phase in which membrane-bound beads move at the same velocity than free beads, followed by a phase in which the tube growth rate decreases and strongly fluctuates. Our work demonstrates that the action of motors bound to a lipid bilayer is sufficient to generate membrane tubes and opens the way to well controlled experiments aimed at the understanding of basic mechanisms in intracellular transport.

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. This sequence shares homology with COOH-terminal sequences of ankyrins-R and ankyrins-B, including the muscle-specific skAnk1. Despite widespread tissue expression of Ank3, the 76-residue sequence is predominantly detected in transcripts of skeletal muscle and heart, including both major 8- and 5.6-kb mRNAs of skeletal muscle. In 15-day-old rat skeletal muscle, antibodies against the 76-residue sequence localized to the sarcolemma and to the postsynaptic membrane and cross-reacted with three endogenous ankyrins-G, including one 130-kDa polypeptide that comigrated with in vitro translated Ank(G107). In adult muscle, these polypeptides appeared significantly decreased, and immunofluorescence labeling was no more detectable. Green fluorescent protein-tagged Ank(G107) transfected in primary cultures of rat myotubes was targeted to the plasma membrane. Deletion of the 76-residue insert resulted in additional cytoplasmic labeling suggestive of a reduced stability of Ank(G107) at the membrane. Recruitment of the COOH-terminal domain to the membrane was much less efficient but still possible only in the presence of the 76-residue insert. We conclude that the 76-residue sequence contributes to the localization and is essential to the stabilization of Ank(G107) at the membrane. These results suggest that tissue-dependent and developmentally regulated alternative processing of ankyrins generates isoforms with distinct sequences, potentially involved in specific protein-protein interactions during differentiation of the sarcolemma and, in particular, of the postsynaptic membrane.