Maintenance Of Acetylcholine Receptor Clusters Research Articles

Balanced Rac1 activity controls formation and maintenance of neuromuscular acetylcholine receptor clusters.

Rac1, an important Rho GTPase that regulates the actin cytoskeleton, has long been suggested to participate in acetylcholine receptor (AChR) clustering at the postsynaptic neuromuscular junction. However, how Rac1 is regulated and how it influences AChR clusters have remained unexplored. This study shows that breaking the balance of Rac1 regulation, by either increasing or decreasing its activity, led to impaired formation and maintenance of AChR clusters. By manipulating Rac1 activity at different stages of AChR clustering in cultured myotubes, we show that Rac1 activation was required for the initial formation of AChR clusters, but its persistent activation led to AChR destabilization, and uncontrolled hyperactivation of Rac1 even caused excessive myotube fusion. Both AChR dispersal and myotube fusion induced by Rac1 were dependent on its downstream effector Pak1. Two Rac1 GAPs and six Rac1 GEFs were screened and found to be important for normal AChR clustering. This study reveals that, although general Rac1 activity remains at low levels during terminal differentiation of myotubes and AChR cluster maintenance, tightly regulated Rac1 activity controls normal AChR clustering.

Nestin Is Not Essential for Development of the CNS But Required for Dispersion of Acetylcholine Receptor Clusters at the Area of Neuromuscular Junctions

Nestin is expressed in many different progenitors during development including those of the CNS, heart, skeletal muscle, and kidney. The adult expression is mainly restricted to the subependymal zone and dentate gyrus of the brain, the neuromuscular junction, and renal podocytes. In addition, this intermediate filament protein has served as a marker of neural stem/progenitor cells for close to 20 years. Therefore it is surprising that its function in development and adult physiology is still poorly understood. Here we report that nestin deficiency is compatible with normal development of the CNS. The mutant mice, however, show impaired motor coordination. Furthermore, we found that the number of acetylcholine receptor clusters, the nerve length, and the endplate bandwidth are significantly increased in neuromuscular junction area of nestin-deficient mice. This is similar to the phenotype described for deficiency of cyclin-dependent kinase 5 (Cdk5), a candidate downstream affecter of nestin. Moreover, we demonstrate that nestin deficiency can rescue maintenance of acetylcholine receptor clusters in the absence of agrin, similar to Cdk5/agrin double knock-outs, suggesting that the observed nestin deficiency phenotype is the consequence of aberrant Cdk5 activity.

Cholesterol Modulates the Rate and Mechanism of Acetylcholine Receptor Internalization

Stability of the nicotinic acetylcholine receptor (AChR) at the cell surface is key to the correct functioning of the cholinergic synapse. Cholesterol (Chol) is necessary for homeostasis of AChR levels at the plasmalemma and for ion translocation. Here we characterize the endocytic pathway followed by muscle-type AChR in Chol-depleted cells (Chol(-)). Under such conditions, the AChR is internalized by a ligand-, clathrin-, and dynamin-independent mechanism. Expression of a dominant negative form of the small GTPase Rac1, Rac1N17, abolishes receptor endocytosis. Unlike the endocytic pathway in control CHO cells (1), accelerated AChR internalization proceeds even upon disruption of the actin cytoskeleton. Under Chol(-) conditions, AChR internalization is furthermore found to require the activity of Arf6 and its effectors Rac1 and phospholipase D. The Arf6-dependent mechanism may constitute the default endocytic pathway followed by the AChR in the absence of external ligands, membrane Chol levels acting as a key homeostatic regulator of cell surface receptor levels.

α-Dystrobrevin isoforms differ in their colocalization with and stabilization of agrin-induced acetylcholine receptor clusters

In skeletal muscle, alpha-dystrobrevin (alphaDB) is expressed throughout the sarcolemma with high concentrations at the neuromuscular junction. Mice lacking alphaDB display a mild muscular dystrophy and perturbations at the neuromuscular junction that include disruptions to acetylcholine receptor (AChR) cluster stability and patterning. In adult skeletal muscle, three alternatively spliced isoforms (alphaDB1, alphaDB2, alphaDB3) are expressed, while two other splice variants (alphaDB1(-) and alphaDB2(-)) are expressed only during early development. alphaDB is clearly important in AChR stabilization; however, the degree to which individual alphaDB isoforms and their specific functional domains contribute to AChR cluster stability is not fully understood. To investigate this, we established a primary muscle cell culture system from alphaDB knockout mice and stably expressed individual alphaDB isoforms using retroviral infection. A comparison between wild-type and alphaDB knockout muscle cells showed that in the absence of alphaDB, fewer AChR clusters formed in response to agrin treatment, and these AChR clusters were very unstable. Retroviral expression studies revealed that the largest isoforms (alphaDB1, alphaDB1(-), alphaDB2, alphaDB2(-)) colocalized with agrin-induced AChR clusters and rescued the AChR cluster formation defects back to wild-type levels, while only the first three isoforms fully rescued AChR cluster stability back to wild-type levels. alphaDB2(-) conferred an intermediate level of stability to the AChR clusters. In contrast, alphaDB3 showed no specific colocalization with AChR clusters and little effect on AChR cluster formation or stabilization. Twice as much syntrophin was found associated with alphaDB2 compared with alphaDB2(-) in myotubes suggesting that increased recruitment of syntrophin by alphaDB may enhance the stability of AChR clusters. Taken together, these data demonstrate that different alphaDB isoforms have different functional capabilities in the formation and maintenance of AChR clusters in muscle cells, and that these differences are likely due to the presence of different functional domains in each isoform.

α-Dystroglycan Functions in Acetylcholine Receptor Aggregation But Is Not a Coreceptor for Agrin-MuSK Signaling

alpha-dystroglycan (alpha-DG) is an agrin-binding protein that has been implicated in acetylcholine receptor (AChR) clustering, but it is unclear whether it acts as a coreceptor involved in initial agrin signaling or as a component involved in later events. To investigate its role, we have generated antisense derivatives of the C2 mouse muscle cell line, which have reduced alpha-DG expression. When compared with wild-type cells, the alpha-DG-deficient myotubes have a dramatic reduction in the number of spontaneous and agrin-induced AChR clusters. Several findings suggest that this decrease in AChR clustering is likely not because of a defect in agrin signaling through the MuSK receptor tyrosine kinase. Compared with wild-type cells, the alpha-DG-deficient cell lines showed only a transient reduction in the level of agrin-induced MuSK tyrosine phosphorylation and no reduction in AChR beta-subunit tyrosine phosphorylation. Additionally, agrin-induced phosphorylation of MuSK in wild-type myotubes was not decreased using agrin fragments that lack the domain primarily responsible for binding to alpha-DG. Finally, neural agrin-induced phosphorylation of MuSK was unaffected by treatments such as excess muscle agrin or anti-alpha-DG antibodies, both of which block agrin-alpha-DG binding. Together, these results suggest that alpha-DG is not required for agrin-MuSK signaling but rather that it may play a role elsewhere in the clustering pathway, such as in the downstream consolidation or maintenance of AChR clusters.

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.

Effects of denervation and direct electrical stimulation upon acetylcholine receptors and acetylcholinesterase accumulation in developing latissimus dorsi muscle of the chick.

The effects of denervation and of direct electrical stimulation of denervated muscle upon the acetylcholine receptor (AChR) clusters and acetylcholinesterase (AChE) spots in the fast avian muscle posterior latissimus dorsi have been investigated. Denervation at day 2 after hatching leads to a disappearance of the junctional AChR clusters and to a marked decrease of AChE spots. Direct electrical stimulation of denervated muscle allows the maintenance of AChR clusters and partly prevents the loss of AChE spots. When AChR cluster and post-synaptic AChE have disappeared in a denervated muscle, muscle activity induced by direct stimulation is unable to induce their accumulation.