CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles

A hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to the adult NMJ are not yet understood in detail. We previously showed that microtubule (MT) capture by the plus end–tracking protein CLASP2 regulates AChR density at agrin-induced AChR clusters in cultured myotubes via PI3 kinase acting through GSK3β. Here we show that knockdown of the CLASP2-interaction partner LL5β by RNAi and forced expression of a CLASP2 fragment blocking the CLASP2/LL5β interaction inhibit microtubule capture. The same treatments impair focal vesicle delivery to the clusters. Consistent with these findings, knockdown of LL5β at the NMJ in vivo reduces the density and insertion of AChRs into the postsynaptic membrane. MT capture and focal vesicle delivery to agrin-induced AChR clusters are also inhibited by microtubule- and actin-depolymerizing drugs, invoking both cytoskeletal systems in MT capture and in the fusion of AChR vesicles with the cluster membrane. Combined our data identify a transport system, organized by agrin through PI3 kinase, GSK3β, CLASP2, and LL5β, for precise delivery of AChR vesicles from the subsynaptic nuclei to the overlying synaptic membrane.     

Clustering of nicotinic acetylcholine receptors: From the neuromuscular junction to interneuronal synapses

Fast and accurate synaptic transmission requires high-density accumulation of neurotransmitter receptors in the postsynaptic membrane. During development of the neuromuscular junction, clustering of acetylcholine receptors (AChR) is one of the first signs of postsynaptic specialization and is induced by nerve-released agrin. Recent studies have revealed that different mechanisms regulate assembly vs stabilization of AChR clusters and of the postsynaptic apparatus. MuSK, a receptor tyrosine kinase and component of the agrin receptor, and rapsyn, an AChR-associated anchoring protein, play crucial roles in the postsynaptic assembly. Once formed, AChR clusters and the postsynaptic membrane are stabilized by components of the dystrophin/utrophin glycoprotein complex, some of which also direct aspects of synaptic maturation such as formation of postjunctional folds. Nicotinic receptors are also expressed across the peripheral and central nervous system (PNS/CNS). These receptors are localized not only at the pre- but also at the postsynaptic sites where they carry out major synaptic transmission. In neurons, they are found as clusters at synaptic or extrasynaptic sites, suggesting that different mechanisms might underlie this specific localization of nicotinic receptors. This review summarizes the current knowledge about formation and stabilization of the postsynaptic apparatus at the neuromuscular junction and extends this to explore the synaptic structures of interneuronal cholinergic synapses.     

Isolated primary osteocytes express functional gap junctions in vitro

The differentiation of the neuromuscular junction is a multistep process requiring coordinated interactions between nerve terminals and muscle. Although innervation is not needed for muscle production, the formation of nerve-muscle contacts, intramuscular nerve branching, and neuronal survival require reciprocal signals from nerve and muscle to regulate the formation of synapses. Following the production of muscle fibers, clusters of acetylcholine receptors (AChRs) are concentrated in the central regions of the myofibers via a process termed “prepatterning”. The postsynaptic protein MuSK is essential for this process activating possibly its own expression, in addition to the expression of AChR. AChR complexes (aggregated and stabilized by rapsyn) are thus prepatterned independently of neuronal signals in developing myofibers. ACh released by branching motor nerves causes AChR-induced postsynaptic potentials and positively regulates the localization and stabilization of developing synaptic contacts. These “active” contact sites may prevent AChRs clustering in non-contacted regions and counteract the establishment of additional contacts. ACh-induced signals also cause the dispersion of non-synaptic AChR clusters and possibly the removal of excess AChR. A further neuronal factor, agrin, stabilizes the accumulation of AChR at synaptic sites. Agrin released from the branching motor nerve may form a structural link specifically to the ACh-activated endplates, thereby enhancing MuSK kinase activity and AChR accumulation and preventing dispersion of postsynaptic specializations. The successful stabilization of prepatterned AChR clusters by agrin and the generation of singly innervated myofibers appear to require AChR-mediated postsynaptic potentials indicating that the differentiation of the nerve terminal proceeds only after postsynaptic specializations have formed.     

Neuregulin receptors, erbB3 and erbB4, are localized at neuromuscular synapses.

Neuregulin (NRG) is concentrated at synaptic sites and stimulates expression of acetylcholine receptor (AChR) genes in muscle cells grown in cell culture. These results raise the possibility that NRG is a synaptic signal that activates AChR gene expression in synaptic nuclei. Stimulation of NRG receptors, erbB3 and erbB4 initiates oligomerization between these receptors or between these receptors and other members of the epidermal growth factor (EGF) receptor family, resulting in stimulation of their associated tyrosine kinase activities. To determine which erbBs might mediate synapse-specific gene expression, we used antibodies against each erbB to study their expression in rodent skeletal muscle by immunohistochemistry. We show that erbB2, erbB3 and erbB4 are concentrated at synaptic sites in adult skeletal muscle. ErbB3 and erbB4 remain concentrated at synaptic sites following denervation, indicating that erbB3 and erbB4 are expressed in the postsynaptic membrane. In addition, we show that expression of NRG and erbBs, like AChR gene expression, increases at synaptic sites during postnatal development. The localization of erbB3 and erbB4 at synaptic sites is consistent with the idea that a NRG-stimulated signaling pathway is important for synapse-specific gene expression.     

Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction

A prominent feature of synaptic maturation at the neuromuscular junction (NMJ) is the topological transformation of the acetylcholine receptor (AChR)-rich postsynaptic membrane from an ovoid plaque into a complex array of branches. We show here that laminins play an autocrine role in promoting this transformation. Laminins containing the α4, α5, and β2 subunits are synthesized by muscle fibers and concentrated in the small portion of the basal lamina that passes through the synaptic cleft at the NMJ. Topological maturation of AChR clusters was delayed in targeted mutant mice lacking laminin α5 and arrested in mutants lacking both α4 and α5. Analysis of chimeric laminins in vivo and of mutant myotubes cultured aneurally demonstrated that the laminins act directly on muscle cells to promote postsynaptic maturation. Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation. Another synaptically concentrated laminin receptor, Bcam, is dispensable. Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation.     

A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction

The 58K protein is a peripheral membrane protein enriched in the acetylcholine receptor (AChR)-rich postsynaptic membrane of Torpedo electric organ. Because of its coexistence with AChRs in the postsynaptic membrane in both electrocytes and skeletal muscle, it is thought to be involved in the formation and maintenance of AChR clusters. Using an mAb against the 58K protein of Torpedo electric organ, we have identified a single protein band in SDS-PAGE analysis of Xenopus myotomal muscle with an apparent molecular mass of 48 kD. With this antibody, the distribution of this protein was examined in the myotomal muscle fibers with immunofluorescence techniques. We found that the 48K protein is concentrated at the myotendinous junctions (MTJs) of these muscle fibers. The MTJ is also enriched in talin and vinculin. By double labeling muscle fibers with antibodies against talin and the 48K protein, these two proteins were found to colocalize at the membrane invaginations of the MTJ. In cultured myotomal muscle cells, the 48K protein and talin are also colocalized at sites of membrane-myofibril interaction. The 48K protein is, however, not found at focal adhesion sites in nonmuscle cells, which are enriched in talin. These data suggest that the 48K protein is specifically involved in the interaction of myofibrillar actin filaments with the plasma membrane at the MTJ. In addition to the MTJ localization, 48K protein is also present at AChR clusters both in vivo and in vitro. Thus, this protein is shared by both the MTJ and the neuromuscular junction.     

Developmental increase in the amount of rapsyn per acetylcholine receptor promotes postsynaptic receptor packing and stability

Neuromuscular synaptic transmission depends upon tight packing of acetylcholine receptors (AChRs) into postsynaptic AChR aggregates, but not all postsynaptic AChRs are aggregated. Here we describe a new confocal Fluorescence Resonance Energy Transfer (FRET) assay for semi-quantitative comparison of the degree to which AChRs are aggregated at synapses. During the first month of postnatal life the mouse tibialis anterior muscle showed increases both in the number of postsynaptic AChRs and the efficiency with which AChR was aggregated (by FRET). There was a concurrent two-fold increase in immunofluorescent labeling for the AChR-associated cytoplasmic protein, rapsyn. When 1-month old muscle was denervated, postsynaptic rapsyn immunostaining was reduced, as was the efficiency of AChR aggregation. In vivo electroporation of rapsyn-EGFP into muscle fibers increased postsynaptic rapsyn levels. Those synapses with higher ratios of rapsyn-EGFP to AChR displayed a slower metabolic turnover of AChR. Conversely, the reduction of postsynaptic rapsyn after denervation was accompanied by an acceleration of AChR turnover. Thus, a developmental increase in the amount of rapsyn targeted to the postsynaptic membrane may drive enhanced postsynaptic AChRs aggregation and AChR stability within the postsynaptic membrane.     

Neural agrin increases postsynaptic ACh receptor packing by elevating rapsyn protein at the mouse neuromuscular synapse

Fluorescence resonance energy transfer (FRET) experiments at neuromuscular junctions in the mouse tibialis anterior muscle show that postsynaptic acetylcholine receptors (AChRs) become more tightly packed during the first month of postnatal development. Here, we report that the packing of AChRs into postsynaptic aggregates was reduced in 4-week postnatal mice that had reduced amounts of the AChR-associated protein, rapsyn, in the postsynaptic membrane (rapsyn(+/-) mice). We hypothesize that nerve-derived agrin increases postsynaptic expression and targeting of rapsyn, which then drives the developmental increase in AChR packing. Neural agrin treatment elevated the expression of rapsyn in C2 myotubes by a mechanism that involved slowing of rapsyn protein degradation. Similarly, exposure of synapses in postnatal muscle to exogenous agrin increased rapsyn protein levels and elevated the intensity of anti-rapsyn immunofluorescence, relative to AChR, in the postsynaptic membrane. This increase in the rapsyn-to-AChR immunofluorescence ratio was associated with tighter postsynaptic AChR packing and slowed AChR turnover. Acute blockade of synaptic AChRs with alpha-bungarotoxin lowered the rapsyn-to-AChR immunofluorescence ratio, suggesting that AChR signaling also helps regulate the assembly of extra rapsyn in the postsynaptic membrane. The results suggest that at the postnatal neuromuscular synapse agrin signaling elevates the expression and targeting of rapsyn to the postsynaptic membrane, thereby packing more AChRs into stable, functionally-important AChR aggregates.     

Cytoplasmic components of acetylcholine receptor clusters of cultured rat myotubes: the 58-kD protein

A 58-kD protein, identified in extracts of postsynaptic membrane from Torpedo electric organ, is enriched at sites where acetylcholine receptors (AChR) are concentrated in vertebrate muscle (Froehner, S. C., A. A. Murnane, M. Tobler, H. B. Peng, and R. Sealock. 1987. J. Cell Biol. 104:1633-1646). We have studied the 58-kD protein in AChR clusters isolated from cultured rat myotubes. Using immunofluorescence microscopy we show that the 58-kD protein is highly enriched at AChR clusters, but is also present in regions of the myotube membrane lacking AChR. Within clusters, the 58-kD protein codistributes with AChR, and is absent from adjacent membrane domains involved in myotube-substrate contact. Semiquantitative fluorescence measurements suggest that molecules of the 58-kD protein and AChR are present in approximately equal numbers. Differential extraction of peripheral membrane proteins from isolated AChR clusters suggests that the 58-kD protein is more tightly bound to cluster membrane than is actin or spectrin, but less tightly bound than the receptor-associated 43-kD protein. When AChR clusters are disrupted either in intact cells or after isolation, the 58-kD protein still codistributes with AChR. Clusters visualized by electron microscopy after immunogold labeling and quick-freeze, deep-etch replication show that, within AChR clusters, the 58-kD protein is sharply confined to AChR-rich domains, where it is present in a network of filaments lying on the cytoplasmic surface of the membrane. Additional actin filaments overlie, and are attached to, this network. Our results suggest that within AChR domains of clusters, the 58-kD protein lies between AChR and the receptor-associated 43-kD protein, and the membrane-skeletal proteins, beta-spectrin, and actin.     

Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells

The postsynaptic membrane from Torpedo electric organ contains, in addition to the acetylcholine receptor (AChR), a major peripheral membrane protein of approximately 43,000 mol wt (43K protein). Previous studies have shown that this protein is closely associated with AChR and may be involved in anchoring receptors to the postsynaptic membrane. In this study, binding sites for monoclonal antibodies (mabs) to the 43K protein have been compared to the distribution of AChR in Xenopus laevis muscle cells in culture. In double label immunofluorescence experiments, clusters of AChR that occur spontaneously on these cells were stained with anti-43K mabs. Newly formed receptor clusters induced with positive polypeptide-coated latex beads were also stained with anti-43K mabs as early as 12 h after the application of the beads. Exact correspondence in the distribution of the anti-43K protein binding sites and the AChR was found in both types of clusters. These results suggest that the 43K protein becomes associated with AChR clusters during a period of active postsynaptic membrane differentiation. Thus, this protein may participate in the clustering process.     

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.     

Phosphoinositide 3-kinase acts through RAC and Cdc42 during agrin-induced acetylcholine receptor clustering

The formation of the neuromuscular junction (NMJ) is regulated by the nerve-derived heparan sulfate proteoglycan agrin and the muscle-specific kinase MuSK. Agrin induces a signal transduction pathway via MuSK, which promotes the reorganization of the postsynaptic muscle membrane. Activation of MuSK leads to the phosphorylation and redistribution of acetylcholine receptors (AChRs) and other postsynaptic proteins to synaptic sites. The accumulation of high densities of AChRs at postsynaptic regions represents a hallmark of NMJ formation and is required for proper NMJ function. Here we show that phosphoinositide 3-kinase (PI3-K) represents a component of the agrin/MuSK signaling pathway. Muscle cells treated with specific PI3-K inhibitors are unable to form full-size AChR clusters in response to agrin and AChR phosphorylation is reduced. Moreover, agrin-induced activation of Rac and Cdc42 is impaired in the presence of PI3-K inhibitors. PI3-K is localized to the postsynaptic muscle membrane consistent with a role during agrin/MuSK signaling. These results put PI3-K downstream of MuSK as regulator of AChR phosphorylation and clustering. Its role during agrin-stimulated Rac and Cdc42 activation suggests a critical function during cytoskeletal reorganizations, which lead to the redistribution of actin-anchored AChRs.     

Isolation of a presynaptic plasma membrane fraction from Torpedo cholinergic synaptosomes: evidence for a specific protein

Synaptosomal plasma membranes were isolated from Torpedo cholinergic synaptosomes which had been purified as previously described or repurified by equilibrium centrifugation. The synaptosomal plasma membrane could be distinguished from postsynaptic membranes by the absence of postsynaptic specific markers (nicotinic AChR) and by its low intramembrane particle complement after freeze fracture. In addition, the presynaptic membrane fraction contained acetylcholinesterase. Gel electrophoresis permitted the identification of a major protein component of the presynaptic membrane fraction which had a molecular weight of 67,000. This protein was not found in postsynaptic membrane or synaptic vesicle fractions. Thus it appeared to be specific to the nerve terminal plasma membrane.     

A postsynaptic Mr 58,000 (58K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle

In the study of proteins that may participate in the events responsible for organization of macromolecules in the postsynaptic membrane, we have used a mAb to an Mr 58,000 protein (58K protein) found in purified acetylcholine receptor (AChR)-enriched membranes from Torpedo electrocytes. Immunogold labeling with the mAb shows that the 58K protein is located on the cytoplasmic side of Torpedo postsynaptic membranes and is most concentrated near the crests of the postjunctional folds, i.e., at sites of high AChR concentration. The mAb also recognizes a skeletal muscle protein with biochemical characteristics very similar to the electrocyte 58K protein. In immunofluorescence experiments on adult mammalian skeletal muscle, the 58K protein mAb labels endplates very intensely, but staining of extrasynaptic membrane is also seen. Endplate staining is not due entirely to membrane infoldings since a similar pattern is seen in neonatal rat diaphragm in which postjunctional folds are shallow and rudimentary, and in chicken muscle, which lacks folds entirely. Furthermore, clusters of AChR that occur spontaneously on cultured Xenopus myotomal cells and mouse muscle cells of the C2 line are also stained more intensely than the surrounding membrane with the 58K mAb. Denervation of adult rat diaphragm muscle for relatively long times causes a dramatic decrease in the endplate staining intensity. Thus, the concentration of this evolutionarily conserved protein at postsynaptic sites may be regulated by innervation or by muscle activity.     

Regulation of the interaction of nicotinic acetylcholine receptors with the cytoskeleton by agrin-activated protein tyrosine kinase

Agrin induces the accumulation of nicotinic acetylcholine receptors (AChRs) in the myofiber membrane at synaptic sites in vertebrate skeletal muscle and causes an increase in tyrosine phosphorylation of the AChR beta subunit. To examine further the mechanism of agrin- induced AChR phosphorylation and the relationship between changes in protein phosphorylation and AChR aggregation, the effect of the protein tyrosine phosphatase inhibitor sodium pervanadate was tested on chick myotubes in culture. Pervanadate caused an increase in the phosphotyrosine content of a variety of proteins, including the AChR. Pervanadate also prevented agrin-induced AChR aggregation and slowed the rate at which AChRs were extracted from intact myotubes by mild detergent treatment. The rate at which phosphorylation of the AChR beta subunit and receptor detergent extractability changed following pervanadate-induced phosphatase inhibition was increased by agrin, indicating that agrin activates a protein tyrosine kinase rather than inhibiting a protein tyrosine phosphatase. The present results, taken together with previous findings on the inhibition of agrin-induced AChR aggregation by protein kinase inhibitors, demonstrate that protein tyrosine phosphorylation regulates the formation and stability of AChR aggregates, apparently by strengthening the interaction between AChRs and the cytoskelton.     

Quantitative analysis of synaptic phosphorylation and protein expression

The postsynaptic density (PSD) signaling machinery contains proteins with diverse functions. Brain region-specific variations in PSD components mediate distinct physiological responses to synaptic activation. We have developed mass spectrometry-based methods to comprehensively compare both relative protein expression and phosphorylation status from proteins present in biochemical preparations of postsynaptic density. Using these methods, we determined the relative expression of 2159 proteins and 1564 phosphorylation sites in PSD preparations from murine cortex, midbrain, cerebellum, and hippocampus. These experiments were conducted twice using independent biological replicates, which allowed us to assess the experimental and biological variability in this system. Concerning protein expression, cluster analysis revealed that known functionally associated proteins display coordinated synaptic expression. Therefore, proteins identified as co-clustering with known protein complexes are prime candidates for assignment as previously unrecognized components. Concerning degree of phosphorylation, we observed more extensive phosphorylation sites on N-methyl-D-aspartate (NMDA) receptors than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, consistent with the central role of N-methyl-D-aspartate receptors in processing synaptic transmission patterns. Average kinase and phosphatase levels were highest in the hippocampus, correlating with a higher overall phosphopeptide abundance present in this brain region. These findings suggest that the hippocampus utilizes reversible protein phosphorylation to a greater extent than other brain regions when modifying synaptic strength.     

The alpha 5 gene product assembles with multiple acetylcholine receptor subunits to form distinctive receptor subtypes in brain.

The acetylcholine receptor (AChR) alpha 5 gene has been classified as a member of the AChR gene family based on sequence homology. Expression studies, however, have yet to identify a function for the alpha 5 gene product or even to demonstrate an interaction with known AChR subunits. We report here that the alpha 5 gene product is identical to the 49 kd protein previously found on immunoblots of AChRs purified from brain and ciliary ganglia. In brain the alpha 5 gene product is present both in alpha 3- and in alpha 4-based receptor subtypes, while in the ganglion it is found in an alpha 3-based receptor subtype concentrated in postsynaptic membrane. Immunoprecipitation experiments with subunit-specific monoclonal antibodies indicate that some native AChRs are likely to have at least three kinds of subunits, with two being of the alpha type. These findings support new views about the construction of AChRs in neurons.     

Transmembrane mechanisms in the assembly of the postsynaptic apparatus at the neuromuscular junction

The vertebrate neuromuscular junction (NMJ) is marked by molecular specializations that include postsynaptic clusters of acetylcholine receptor (AChR) and acetylcholinesterase (AChE). Whereas AChRs are aggregated in the postsynaptic muscle membrane to a density of 10,000/mum(2), AChE is concentrated, also to a high density, in the synaptic basement membrane (BM). In recent years considerable progress has been made in understanding the cellular and molecular mechanisms of AChR clustering. It is known that during the early stages of motoneuron-muscle interaction, the nerve-secreted proteoglycan agrin activates the muscle-specific kinase MuSK, which leads to the formation of a postsynaptic cytoskeletal scaffold that immobilizes and concentrates AChRs through a process generally accepted to involve diffusion-mediated trapping of the receptors. We have recently tested this diffusion-trap model at the single molecule level for the first time by using quantum-dot labeling to track individual AChRs during NMJ development. Our results showed that single AChRs exhibit Brownian-type movement, with diffusion coefficients of 10(-11) to 10(-9)cm(2)/s, until they become immobilized at "traps" assembled in response to synaptogenic stimuli. Thus, free diffusion of AChRs is an integral part of their clustering mechanism. What is the mechanism for AChE clustering? We previously showed that the A(12) asymmetric form of AChE binds to perlecan, a heparan-sulfate proteoglycan which in turn interacts with the transmembrane dystroglycan complex. Through this linkage AChE becomes bound to the muscle membrane and, like AChRs, may exhibit lateral mobility along the membrane. Consistent with this idea, pre-existent AChE at the cell surface becomes clustered together with AChRs following synaptogenic stimulation. Future studies testing diffusion-mediated trapping of AChE should provide insights into the synaptic localization of BM-bound molecules at the NMJ.