Cultured myotubes cluster achrs spontaneously and respond to agrin application with an increase in the frequency of achr clusters 18 20. A the z8 interface plays a predominant role in assembly of the agrin lrp4 complex. Neuregulin1 potentiates agrininduced acetylcholine receptor. Mechanism of acetylcholine receptor cluster formation induced. Regulation of acetylcholine receptor clustering by adfcofilin. Gst pulldown assays were performed between gst agrin variants containing a ha tag and lrp4 l23a737 containing a his tag. A the z8 interface plays a predominant role in assembly of the agrinlrp4 complex. Vdr signaling modulates the agrininduced achr clustering. Moreover, laminin induced achr clustering is impaired by the rho kinase inhibitor y27632. Achrs cluster spontaneously with a baseline frequency on c2c12 myotubes and this clustering is increased with agrin treatment 2022. At the vertebrate neuromuscular junction, agrin is one molecule presented by the presynaptic motor neuron that plays an instructive role in postsynaptic differentiation of the muscle cell, most notably in aggregation of acetylcholine. The c2c12 cell culture model has proven useful for asking fundamental questions concerned with muscle development and neuromuscular synapse formation, and is ideal for examining how plastic. Regulation of achr clustering by dishevelled interacting with. Agrin is thought to be the nervederived factor that initiates acetylcholine receptor achr clustering at the developing neuromuscularjunction.
Synaptic achr aggregation, according to the diffusionmediated trapping hypothesis, involves the establishment of a postsynaptic scaffold that traps freely diffusing receptors into. Agrin exhibits many functions in addition to nmj development. Acetylcholine receptors are required for agrininduced clustering of. During myotube formation achrs cluster spontaneously. Jan 11, 2018 inhibitors for fgfrs and mek1 block fgf18 induced achr clustering. We examined whether lrp4 is involved in wntinduced achr clustering in muscle cells. Similarly, achr clustering induced by agrin beads was also attenuated. Acetylcholine receptor achr clustering is regulated both. Agrininduced tetramerization of agrinlrp4 is critical for achr clustering and musk phosphorylation. Nano 3 decreases the frequency of agrininduced achr clusters. K downstream of musk as regulator of achr phosphorylation and clustering.
Interestingly, we found that blockade of cdk5 expression also affected agrin induced pak phosphorylation and activity data not shown. First, agrin activates pak in cultured muscle cells. Antibody to myod or myogenin decreases acetylcholine. Agrininduced achr clustering requires a myotube receptor complex that contains alpha. Skeletal muscle development is influenced by myogenic regulatory factors, including the expression of myod and myogenin. How agrin activates musk and stimulates synaptic differentiation is not known and remains a fundamental gap in our understanding of signaling at neuromuscular synapses. Role of agrinmusk signaling in crosslinkinginduced achr endocytosis. Local induction of acetylcholine receptor clustering in myotube. Regulation of acetylcholine receptor clustering by adf. Role of agrin musk signaling in crosslinking induced achr endocytosis. Treatment with motor neuron derived agrin increases the frequency of achr clusters through an agrin signaling pathway that also clusters other postsynaptic components of the neuromuscular synapse. Agrininduced achr delivery to synaptic achr clusters through clasp2mediated mt capture ensures efficient clustering of achrs at the synaptic muscle membrane. Another potential mechanism could be a direct interaction of adrb2mediated pka signalling with rapsyn.
Agrininduced achr clustering was assayed as described previously zhang et al. Musk and rapsyn suggesting that a muscle intrinsic program is able to shape the. Agrin exists in multiple isoforms, with the agrin 4,8 isoform found exclusively in neural tissue and able to cluster achrs to a much greater extent than other isoforms 21. Synaptic differentiation results from an exchange of informational molecules between synaptic partners during development. Background the formation of acetylcholine receptor achr cluster is a key event during the development of the neuromuscular junction. Plastic additives decrease agrininduced acetylcholine. Interestingly, sfk inhibition applied selectively during agrininduced achr cluster. A bsa control, agrin, fgf18, fgf18 with an inhibitor of fgf receptors fgfrs su5402, and fgf18 with an mek1 inhibitor. Mechanism of acetylcholine receptor cluster formation. Briefly, c2c12 myotubes were treated with n agrin 10 ngml for 16 h to induce achr.
We have investigated the signaling pathway in mouse c2 myotubes and report that agrin induces a rapid but transient tyrosine phosphorylation of the achr beta subunit. The formation of acetylcholine receptor clusters visualized. Neuregulin1 potentiates agrininduced acetylcholine. The agrininduced control of clasp2 phosphorylation is an important mechanism for mt capture and synaptic achr clustering. Catenin regulates acetylcholine receptor clustering in. The inhibition of agrin induced achr clustering is not a general characteristic of dominant interfering mutants of small g proteins, as indicated by the observation that rasn17, a dominant interfering mutant ras, did not block agrin induced achr aggregation fig. However, exactly how agrin mediates this effect remains unclear.
Gst pulldown assays were performed between gstagrin variants containing a ha tag and lrp4 l23a737 containing a his tag. To test this hypothesis, we used shrna specific to anxa3 to investigate whether reduced expression of anxa3 would affect achr clustering. Acetylcholine receptor achr clustering is regulated both by. Agrininduced acetylcholine receptor clustering is mediated.
Crosslinkinginduced achr endocytosis triggered by bbq labeling serves as a simple and reliable assay to investigate the cellular mechanisms underlying achr endocytosis induced by pathogenic antibodies in mg. Sodium nitrate decreases agrininduced acetylcholine receptor. Achr clustering induced by the beads was examined within a few hours of. To assay agrin induced achr clustering, transfected sol8 myotubes were treated with agrin for 5 and 24 h.
The acute potentiation of nagrininduced achr cluster formation involved. Importantly, for the purposes of this study, nsc87877, an shp2 inhibitor, has been shown to enhance agrininduced and agrinindependent achr clustering in vitro. Induction of antiagrin antibodies causes myasthenia gravis in mice. Laminin and 71 integrin regulate agrininduced clustering. The agrin induced control of clasp2 phosphorylation is an important mechanism for mt capture and synaptic achr clustering. Sodium nitrate decreases agrininduced acetylcholine. Lack of fgf18 causes abnormal clustering of motor nerve. Second, inhibition of pak signaling by dominantnegative pak or hpip1 attenuates agrin induced achr clustering. The objective of the current study was to investigate whether plastic additives interfere with agrininduced achr clustering and myotube formation. Agrininduced achr clusters formed on myotubes from scr and fyn double mutant mice dispersed more quickly than clusters from control cells when agrin was remove. Our objective was to use the c2c12 cell culture model to test the hypothesis that both myod and myogenin were required for agrininduced acetylcholine receptor achr clustering and the fusion of myoblasts into myotubes. At the vertebrate neuromuscular junction, agrin is one molecule presented by the presynaptic motor neuron that plays an instructive role in postsynaptic differentiation of the muscle cell, most notably in aggregation of acetylcholine receptors achrs. Antibody to myod or myogenin decreases acetylcholine receptor. The formation of acetylcholine receptor clusters visualized with.
On the other hand, dc electric field, a nonneuronal stimulus, is also highly effective in causing. Modulation of agrininduced acetylcholine receptor clustering. Shp2 inhibitor protects achrs from effects of myasthenia. Wnt proteins regulate acetylcholine receptor clustering in. We examined whether lrp4 is involved in wnt induced achr clustering in muscle cells. Achr clustering induced by a number of stimuli such as agrin and growth. A single pulse of agrin triggers a pathway that acts to cluster. Achr clusters are induced by agrin, a neurally released synaptogenic.
We also report that a single 5min agrin pulse, followed by extensive washing, triggered longlasting musk and achr phosphorylation and efficient achr clustering. K is localized to the postsynaptic muscle membrane consistent with a role during agrinmusk signaling. Here, we report that lrp4, a member of the ldlr family, is a receptor for agrin, forms a complex with musk, and mediates musk activation by agrin. Briefly, c2c12 myotubes were treated with nagrin 10 ngml for 16 h to induce achr clusters. Focal, clasp2mediated mt capture at agrininduced achr clusters in cultured primary myotubes requires a clasp2interaction. Data collected with a multiclamp 700b amplifier were digitized 10 khz lowpass filtered with digidata 1550a and analyzed in clampfit10. The dystroglycan complex is necessary for stabilization of. The activation is not nonspecific, because it is only elicited by neuronal, but not muscle, agrin. Jul 10, 2000 the inhibition of agrin induced achr clustering is not a general characteristic of dominant interfering mutants of small g proteins, as indicated by the observation that rasn17, a dominant interfering mutant ras, did not block agrin induced achr aggregation fig.
For cluster dispersal assays, cultures were treated with agrin for 18 h, washed, and incubated in the absence of agrin for another 6 h. The formation of complex acetylcholine receptor clusters. Agrin induced achr clustering was assayed as described previously zhang et al. Interestingly, sfk inhibition applied selectively during agrin induced achr cluster formation caused rapid cluster dispersal later upon agrin withdrawal. Decreases in myogenin and achr gene expression also correlated with decreased agrininduced achr clustering, where treatment with 1. Agrin activates the musclespecific receptor tyrosine kinase musk to initiate a. Here we report, that besides its role during agrininduced achr clustering. Agrininduced acetylcholine receptor clustering is mediated by the. Briefly, c2c12 myotubes were treated with n agrin 10 ngml for 16 h to induce achr clusters. Another potential mechanism could be a direct interaction of. Previously,trinidadandcohenshowedthatincultured myotubes agrininduced achr clustering was inhibited by cotreatment with nrg1. Pdf induction of antiagrin antibodies causes myasthenia. Clasp2dependent microtubule capture at the neuromuscular.
Agrin induced tetramerization of agrin lrp4 is critical for achr clustering and musk phosphorylation. Agrin and laminin induce acetylcholine receptor clustering by. Jan 15, 2003 synaptic differentiation results from an exchange of informational molecules between synaptic partners during development. Dec 17, 2001 when synthesis and clustering of achrs are induced by agrin, however, rapsyngfp is recruited into achr clusters. Identification of the downstream molecules of agrindok. On the other hand, dc electric field, a nonneuronal stimulus, is also highly effective in causing achrs to cluster. Laminin and 71 integrin regulate agrininduced clustering of acetylcholine receptors. Agrin regulates rapsyn interaction with surface acetylcholine. Crosslinking induced achr endocytosis triggered by bbq labeling serves as a simple and reliable assay to investigate the cellular mechanisms underlying achr endocytosis induced by pathogenic antibodies in mg. Lrp4 is a receptor for agrin and forms a complex with musk. Importantly, for the purposes of this study, nsc87877, an shp2 inhibitor, has been shown to enhance agrin induced and agrin independent achr clustering in vitro. Regulation of achr clustering by dishevelled interacting. Agrin induced achr clusters formed on myotubes from scr and fyn double mutant mice dispersed more quickly than clusters from control cells when agrin was remove.
Furthermore, in the presence of nsc87877, neuregulin1 was unable to produce any further potentiation of n agrin induced achr clustering fig. Motor innervation of skeletal muscle leads to the assembly of acetylcholine receptor achr clusters in the postsynaptic membrane at the vertebrate neuromuscular junction nmj. Furthermore, in the presence of nsc87877, neuregulin1 was unable to produce any further potentiation of nagrininduced achr clustering fig. Induction of antiagrin antibodies causes myasthenia gravis. Agrin is believed to be a factor used by motoneurons to direct acetylcholine receptor achr clustering at the neuromuscular junction. In addition, concentrations of sodium nitrate of 1. To prepare nervemuscle cocultures, spinal neurons dissociated from neural tubes were seeded onto 3day old muscle cells. Congenital myasthenic syndromeassociated agrin variants.
Mechanism of acetylcholine receptor cluster formation induced by. The formation of complex acetylcholine receptor clusters requires. Involvement of lrp4 in wnt induced achr clustering. Induction of antiagrin antibodies causes myasthenia. Like for agrin induced achr clustering an active kinase is not sufficient and specific musk sequences are necessary for cluster formation on laminin herbst and burden, 2000. C2c12 cell cultures were switched from gm to dm at 90 % confluence, 10 ngml agrin was added for the last 16 h in dm, and myotubes were examined for achr. Immunocomplexes were visualized by ecl, and signals were quantitated using a biorad laboratories imaging densitometer with molecular analyst software. Sfks initiate early phosphorylation and an achr stabilization program that. Collectively, these results provide new insight into the agrinlrp4musk signaling cascade and nmj formation and represent a novel mechanism for activation of receptor tyrosine kinases. Ethanol decreases agrininduced acetylcholine receptor. Aberrant motor axon projection, acetylcholine receptor. Clustering in response to laminin is blocked by the dominant negative mutants cdc42n17, racn17 and rhon19, as well as by the rho inhibitor c3 transferase. Lrp4 interacts not only with agrin, but also with musk, and is critical for agrin induced achr clustering and for aneural achr cluster formation and muscle fiber prepatterning 3, 4, 9.
Specifically, 10 mm ethanol decreased agrin induced achr clustering by 34%, while 100 mm ethanol decreased it by 54%. This agrininduced increase in rapsynachr interaction strongly correlates with. Oct 18, 2005 finally, the effect of cdk5 activity on the signaling implicated in agrin induced clustering of achr was examined. Lrp4 interacts not only with agrin, but also with musk, and is critical for agrininduced achr clustering and for aneural achr cluster formation and muscle fiber prepatterning 3, 4, 9. Previously,trinidadandcohenshowedthatincultured myotubes agrin induced achr clustering was inhibited by cotreatment with nrg1. Our objective was to use the c2c12 cell culture model to test the hypothesis that both myod and myogenin were required for agrin induced acetylcholine receptor achr clustering and the fusion of myoblasts into myotubes. Beta2 adrenergic receptor agonists enhance achr clustering. Agrin induced achr clustering has previously been shown to require activation of cdc42, rac and rho. Achr clustering induced by the beads was examined within a few hours of bead addition. The inhibition of agrininduced achr clustering is not a general characteristic of dominant interfering mutants of small g proteins, as indicated by the observation that rasn17, a dominant interfering mutant ras, did not block agrininduced achr aggregation fig. Interestingly, both u0126 and pd0325901 restored agrin induced achr clustering to about 120% control levels. Second, inhibition of pak signaling by dominantnegative pak or hpip1 attenuates agrininduced achr clustering.
Coincubation of agrin, pma, and a mek inhibitor completely rescued the inhibitory effects of pma on agrin induced achr clustering p 0. It is induced through the activation of musclespecific kinase musk by the heparansulfate proteoglycan agrin released from the motor axon. Crosslinkinginduced endocytosis of acetylcholine receptors. Our findings suggest that the electric fieldinduced achr clustering can be. Apr 11, 2007 agrin is believed to be a factor used by motoneurons to direct acetylcholine receptor achr clustering at the neuromuscular junction.
Feb 01, 2012 agrininduced tetramerization of agrinlrp4 is critical for achr clustering and musk phosphorylation. It is enriched in senile alzheimers disease plaques where it binds the a beta 1. The inability of laminin to induce achr clusters on dystroglycannull myotubes, in combination with the change in appearance of spontaneous and agrininduced clusters, suggested that laminindystroglycan interactions were important in the formation of dense achr clusters and that disruption of these interactions is responsible for the diffuse. May 31, 2009 similarly, achr clustering induced by agrin beads was also attenuated. Achr clustering induced by fgf18 in c2c12 myotubes is likely to be mediated by activation of fgfrs and mek1, but other pathways may also be involved in the effect of fgf18 on the nmj. Agrin induced achr delivery to synaptic achr clusters through clasp2mediated mt capture ensures efficient clustering of achrs at the synaptic muscle membrane. Agrin induces achr clustering through the activation of rho small gtpases and pak 26, 27. A key signal for achr clustering is the release of agrin by the axon terminal as the.
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