Plasticity and structural alterations of mitochondria and sarcoplasmic organelles in muscles of mice deficient in α-dystrobrevin, a component of the dystrophin-glycoprotein complex.

The dystrophin-glycoprotein complex (DGC) plays a crucial role in maintaining the structural integrity of the plasma membrane and the neuromuscular junction. In this study, we investigated the impact of the deficiency of α-dystrobrevin (αdbn), a component of the DGC, on the homeostasis of intracellular organelles, specifically mitochondria and the sarcoplasmic reticulum (SR). In αdbn deficient muscles, we observed a significant increase in the membrane-bound ATP synthase complex levels, a marker for mitochondria in oxidative muscle fiber types compared to wild-type. Furthermore, examination of muscle fibers deficient in αdbn using electron microscopy revealed profound alterations in the organization of mitochondria and the SR within certain myofibrils of muscle fibers. This included the formation of hyper-branched intermyofibrillar mitochondria with extended connections, an extensive network spanning several myofibrils, and a substantial increase in the number/density of subsarcolemmal mitochondria. Concurrently, in some cases, we observed significant structural alterations in mitochondria, such as cristae loss, fragmentation, swelling, and the formation of vacuoles and inclusions within the mitochondrial matrix cristae. Muscles deficient in αdbn also displayed notable alterations in the morphology of the SR, along with the formation of distinct anomalous concentric SR structures known as whorls. These whorls were prevalent in αdbn-deficient mice but were absent in wild-type muscles. These results suggest a crucial role of the DGC αdbn in regulating intracellular organelles, particularly mitochondria and the SR, within muscle cells. The remodeling of the SR and the formation of whorls may represent a novel mechanism of the unfolded protein response (UPR) in muscle cells.

Distinct roles of the dystrophin-glycoprotein complex: α-dystrobrevin and α-syntrophin in the maintenance of the postsynaptic apparatus of the neuromuscular synapse.

α-syntrophin (α-syn) and α-dystrobrevin (α-dbn), two components of the dystrophin-glycoprotein complex, are essential for the maturation and maintenance of the neuromuscular junction (NMJ) and mice deficient in either α-syn or α-dbn exhibit similar synaptic defects. However, the functional link between these two proteins and whether they exert distinct or redundant functions in the postsynaptic organization of the NMJ remain largely unknown. We generated and analyzed the synaptic phenotype of double heterozygote (α-dbn+/-, α-syn+/-), and double homozygote knockout (α-dbn-/-; α-syn-/-) mice and examined the ability of individual molecules to restore their defects in the synaptic phenotype. We showed that in double heterozygote mice, NMJs have normal synaptic phenotypes and no signs of muscular dystrophy. However, in double knockout mice (α-dbn-/-; α-syn-/-), the synaptic phenotype (the density, the turnover and the distribution of AChRs within synaptic branches) is more severely impaired than in single α-dbn-/- or α-syn-/- mutants. Furthermore, double mutant and single α-dbn-/- mutant mice showed more severe exercise-induced fatigue and more significant reductions in grip strength than single α-syn-/- mutant and wild-type. Finally, we showed that the overexpression of the transgene α-syn-GFP in muscles of double mutant restores primarily the abnormal extensions of membrane containing AChRs that extend beyond synaptic gutters and lack synaptic folds, whereas the overexpression of α-dbn essentially restores the abnormal dispersion of patchy AChR aggregates in the crests of synaptic folds. Altogether, these data suggest that α-syn and α-dbn act in parallel pathways and exert distinct functions on the postsynaptic structural organization of NMJs.

Phosphorylation of α-dystrobrevin is essential for αkap accumulation and acetylcholine receptor stability.

The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein (αkap) encoded within the calcium/calmodulin-dependent protein kinase IIα (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which αkap is maintained and regulated remains unknown. Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that α-dystrobrevin (α-dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of αkap in a concentration-dependent manner. Mechanistically, we found that the phosphorylatable tyrosine residues of α-dbn are essential for the stability of α-dbn itself and its interaction with αkap, with substitution of three tyrosine residues in the α-dbn C terminus with phenylalanine compromising the αkap-α-dbn interaction and significantly reducing both αkap and α-dbn accumulation. Moreover, the αkap-α-dbn interaction was critical for αkap accumulation and stability. We also found that the absence of either αkap or α-dbn markedly reduces AChRα accumulation and that overexpression of α-dbn or αkap in cultured muscle cells promotes the formation of large agrin-induced AChR clusters. Collectively, these results indicate that the stability of αkap and α-dbn complex plays an important role in the maintenance of high-level expression of AChRs.

Spatial distribution and molecular dynamics of dystrophin glycoprotein components at the neuromuscular junction in vivo.

A bimolecular fluorescence complementation (BiFC) approach was used to study the molecular interactions between different components of the postsynaptic protein complex at the neuromuscular junction of living mice. We show that rapsyn forms complex with both α-dystrobrevin and α-syntrophin at the crests of junctional folds. The linkage of rapsyn to α-syntrophin and/or α-dystrobrevin is mediated by utrophin, a protein localized at acetylcholine receptor (AChR)-rich domains. In mice deficient in α-syntrophin, in which utrophin is no longer present at the synapse, rapsyn interaction with α-dystrobrevin was completely abolished. This interaction was completely restored when either utrophin or α-syntrophin was introduced into muscles deficient in α-syntrophin. However, in neuromuscular junctions deficient in α-dystrobrevin, in which utrophin is retained, complex formation between rapsyn and α-syntrophin was unaffected. Using fluorescence recovery after photobleaching, we found that α-syntrophin turnover is 5-7 times faster than that of AChRs, and loss of α-dystrobrevin has no effect on rapsyn and α-syntrophin half-life, whereas the half-life of AChR was significantly altered. Altogether, these results provide new insights into the spatial distribution of dystrophin glycoprotein components and their dynamics in living mice.

AChRs Are Essential for the Targeting of Rapsyn to the Postsynaptic Membrane of NMJs in Living Mice.

UNLABELLED: Rapsyn, a 43 kDa scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at synaptic sites between mammalian motor neurons and muscle cells. However, the mechanism by which rapsyn is inserted and retained at postsynaptic sites at the neuromuscular junction (NMJ) in vivo remains largely unknown. We found that neither the N-terminal myristoylation nor the cysteine-rich RING H2 domain of rapsyn is required for its stable association with the postsynaptic membrane of NMJs. When N-myristoylation-defective rapsyn-EGFP mutant (G2A) and RING-H2 domain truncated rapsyn-EGFP were electroporated into sternomastoid muscles, a strong rapsyn fluorescent signal was observed selectively at synapses, similar to WT rapsyn-EGFP. The targeting of rapsyn-EGFP (WT and mutants) is independent of synaptic activity because they were inserted at denervated NMJs. However, when the coiled-coil domain (the AChR-binding domain of rapsyn) is deleted, rapsyn fails to associate with AChRs at NMJs of living mice. In cultured myoblasts (in which AChRs are absent), myristoylated WT rapsyn mostly localizes to lysosomes and is not associated with the plasma membrane. However, in the presence of AChR subunits, rapsyn molecules were targeted to the cell surface and formed aggregates with AChRs. The targeting of AChRs to the cell membrane, in contrast, does not require rapsyn because expressed AChRs are visible on the cell membranes of rapsyn-deficient myoblasts. These results provide evidence for an active role of AChRs in the targeting of rapsyn to the NMJ in vivo SIGNIFICANCE STATEMENT: Rapsyn is required for the clustering of acetylcholine receptors (AChRs) at postsynaptic sites. However, the mechanism by which rapsyn is targeted to synaptic sites at the vertebrate neuromuscular junction remains unclear. In this study, we showed that the coiled-coil domain of rapsyn is required for its targeting to the cell surface via its interaction with AChRs. In contrast, the targeting of AChRs to the cell membrane does not require rapsyn. These results indicate that AChRs play a critical role in the insertion and/or association of rapsyn with the plasma membrane of synaptic sites.

Failure of lysosome clustering and positioning in the juxtanuclear region in cells deficient in rapsyn.

Rapsyn, a scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at contacts between motor neurons and differentiating muscle cells. Rapsyn is also expressed in cells that do not express AChRs. However, its function in these cells remains unknown. Here, we show that rapsyn plays an AChR-independent role in organizing the distribution and mobility of lysosomes. In cells devoid of AChRs, rapsyn selectively induces the clustering of lysosomes at high density in the juxtanuclear region without affecting the distribution of other intracellular organelles. However, when the same cells overexpress AChRs, rapsyn is recruited away from lysosomes to colocalize with AChR clusters on the cell surface. In rapsyn-deficient (Rapsn(-/-)) myoblasts or cells overexpressing rapsyn mutants, lysosomes are scattered within the cell and highly dynamic. The increased mobility of lysosomes in Rapsn(-/-) cells is associated with a significant increase in lysosomal exocytosis, as evidenced by increased release of lysosomal enzymes and plasma membrane damage when cells were challenged with the bacterial pore-forming toxin streptolysin-O. These findings uncover a new link between rapsyn, lysosome positioning, exocytosis and plasma membrane integrity.

The knockdown of αkap alters the postsynaptic apparatus of neuromuscular junctions in living mice.

A muscle-specific nonkinase anchoring protein (αkap), encoded within the calcium/calmodulin kinase II (camk2) α gene, was recently found to control the stability of acetylcholine receptor (AChR) clusters on the surface of cultured myotubes. However, it remains unknown whether this protein has any effect on receptor stability and the maintenance of the structural integrity of neuromuscular synapses in vivo. By knocking down the endogenous expression of αkap in mouse sternomastoid muscles with shRNA, we found that the postsynaptic receptor density was dramatically reduced, the turnover rate of receptors at synaptic sites was significantly increased, and the insertion rates of both newly synthesized and recycled receptors into the postsynaptic membrane were depressed. Moreover, we found that αkap shRNA knockdown impaired synaptic structure as postsynaptic AChR clusters and their associated postsynaptic scaffold proteins within the neuromuscular junction were completely eliminated. These results provide new mechanistic insight into the role of αkap in regulating the stability of the postsynaptic apparatus of neuromuscular synapses.

Recycling of acetylcholine receptors at ectopic postsynaptic clusters induced by exogenous agrin in living rats.

During the development of the neuromuscular junction, motor axons induce the clustering of acetylcholine receptors (AChRs) and increase their metabolic stability in the muscle membrane. Here, we asked whether the synaptic organizer agrin might regulate the metabolic stability and density of AChRs by promoting the recycling of internalized AChRs, which would otherwise be destined for degradation, into synaptic sites. We show that at nerve-free AChR clusters induced by agrin in extrasynaptic membrane, internalized AChRs are driven back into the ectopic synaptic clusters where they intermingle with pre-existing and new receptors. The extent of AChR recycling depended on the strength of the agrin stimulus, but not on the development of junctional folds, another hallmark of mature postsynaptic membranes. In chronically denervated muscles, in which both AChR stability and recycling are significantly decreased by muscle inactivity, agrin maintained the amount of recycled AChRs at agrin-induced clusters at a level similar to that at denervated original endplates. In contrast, AChRs did not recycle at agrin-induced clusters in C2C12 or primary myotubes. Thus, in muscles in vivo, but not in cultured myotubes, neural agrin promotes the recycling of AChRs and thereby increases their metabolic stability.

A role for the calmodulin kinase II-related anchoring protein (αkap) in maintaining the stability of nicotinic acetylcholine receptors.

αkap, a muscle specific anchoring protein encoded within the Camk2a gene, is thought to play a role in targeting multiple calcium/calmodulin kinase II isoforms to specific subcellular locations. Here we demonstrate a novel function of αkap in stabilizing nicotinic acetylcholine receptors (AChRs). Knockdown of αkap expression with shRNA significantly enhanced the degradation of AChR α-subunits (AChRα), leading to fewer and smaller AChR clusters on the surface of differentiated C2C12 myotubes. Mutagenesis and biochemical studies in HEK293T cells revealed that αkap promoted AChRα stability by a ubiquitin-dependent mechanism. In the absence of αkap, AChRα was heavily ubiquitinated, and the number of AChRα was increased by proteasome inhibitors. However, in the presence of αkap, AChRα was less ubiquitinated and proteasome inhibitors had almost no effect on AChRα accumulation. The major sites of AChRα ubiquitination reside within the large intracellular loop and mutations of critical lysine residues in this loop to arginine increased AChRα stability in the absence of αkap. These results provide an unexpected mechanism by which αkap controls receptor trafficking onto the surface of muscle cells and thus the maintenance of postsynaptic receptor density and synaptic function.

Nicotinic acetylcholine receptor stability at the NMJ deficient in α-syntrophin in vivo.

α-Syntrophin (α-syn), a scaffold protein, links signaling molecules to the dystrophin-glycoprotein complex. Absence of α-syn from the DGC is known to lead to structurally aberrant neuromuscular junctions (NMJs) with few acetylcholine receptors (AChRs) clustered at synaptic sites. Using α-syn knock-out mice, we show that during the first postnatal week, α-syn is not required for synapse formation. However, at postnatal day 6 (P6)-P7, the structural integrity of the postsynaptic apparatus is altered, the turnover rate of AChRs increases significantly, and the number/density of AChRs is impaired. At the adult α-syn(-/-) NMJ, the turnover rate of AChRs is ∼ 4 times faster than wild-type synapses, and most removed receptors are targeted to degradation as few AChRs recycled to synaptic sites. Biochemical analyses show that in muscle cells of adult knock-out α-syn mice, total AChRs and scaffold protein rapsyn are significantly reduced, the 89 kDa and 75 kDa isoforms of tyrosine phosphorylated α-dystrobrevin (α-dbn) 1 (which are required for the maintenance and stability of AChR in α-dbn(-/-) synapses) are barely detectable. Electroporation of GFP-α-dbn1 in α-syn(-/-) muscle cells partially restored receptor density, turnover rate, and the structural integrity of the postsynaptic apparatus, whereas expression of rapsyn-GFP failed to rescue the α-syn(-/-) synaptic phenotype. These results demonstrate that α-syn is required for the maturation and stability of the postsynaptic apparatus and suggest that α-syn may act via α-dbn1.

Dynamics of the rapsyn scaffolding protein at the neuromuscular junction of live mice.

The efficacy of synaptic transmission depends on the maintenance of a high density of neurotransmitter receptors and their associated scaffold proteins in the postsynaptic membrane. While the dynamics of receptors has been extensively studied, the dynamics of the intracellular scaffold proteins that make up the postsynaptic density are largely unknown in vivo. Here, we focused on the dynamics of rapsyn, a protein required for the clustering and maintenance of acetylcholine receptor (AChR) density at postsynaptic sites. Using time-lapse imaging, we demonstrated that rapsyn is remarkably dynamic compared to AChRs at functional synapses, turning over 4-6 times more rapidly than AChRs. In addition we found that the rapid turnover of rapsyn is insensitive to alterations in synaptic activity, whereas AChR turnover is profoundly affected, illustrating that rapsyn and receptor dynamics are controlled by distinct mechanisms. These data indicate that individual postsynaptic components are in permanent exchange despite the overall stability of synaptic structure, which may play a role in synaptic plasticity.

Calcium/calmodulin kinase II-dependent acetylcholine receptor cycling at the mammalian neuromuscular junction in vivo.

At the mammalian skeletal neuromuscular junction, cycling of nicotinic ACh receptors (nAChRs) is critical for the maintenance of a high postsynaptic receptor density. However, the mechanisms that regulate nAChRs recycling in living animals remain unknown. Using in vivo time-lapse imaging, fluorescence recovery after photobleaching, and biochemical pull down assays, we demonstrated that recycling of internalized nAChRs into fully functional and denervated synapses was promoted by both direct muscle stimulation and pharmacologically induced intracellular calcium elevations. Most of internalized nAChRs are recycled directly into synaptic sites. Chelating of intracellular calcium below resting level drastically decreased cycling of nAChRs. Furthermore we found that calcium-dependent AChR recycling is mediated by Ca(2+)/calmodulin-dependent kinase II (CaMKII). Inhibition of CaMKII selectively blocked recycling and caused intracellular accumulation of internalized nAChRs, whereas internalization of surface receptors remained unaffected. Electroporation of CaMKII-GFP isoforms into the sternomastoid muscle showed that muscle-specific CaMKIIßm isoform is highly expressed at the neuromuscular junction (NMJ) and precisely colocalized with nAChRs at crests of synaptic folds while the CaMKIIγ and δ isoforms are poorly expressed in synaptic sites. These results indicate that Ca(2+) along with CaMKII activity are critical for receptor recycling and may provide a mechanism by which the postsynaptic AChR density is maintained at the NMJ in vivo.

Acetylcholine receptor clustering is required for the accumulation and maintenance of scaffolding proteins.

The maintenance of a high density of postsynaptic receptors is essential for proper synaptic function. At the neuromuscular junction, acetylcholine receptor (AChR) aggregation is induced by nerve-clustering factors and mediated by scaffolding proteins. Although the mechanisms underlying AChR clustering have been extensively studied, the role that the receptors themselves play in the clustering process and how they are organized with scaffolding proteins is not well understood. Here, we report that the exposure of AChRs labeled with Alexa 594 conjugates to relatively low-powered laser light caused an effect similar to chromaphore-assisted light inactivation (CALI) , which resulted in the unexpected dissipation of the illuminated AChRs from clusters on cultured myotubes. This technique enabled us to demonstrate that AChR removal from illuminated regions induced the removal of scaffolding proteins and prevented the accumulation of new AChRs and associated scaffolding proteins. Further, the dissipation of clustered AChRs and scaffold was spatially restricted to the illuminated region and had no effect on neighboring nonilluminated AChRs. These results provide direct evidence that AChRs are essential for the local maintenance and accumulation of intracellular scaffolding proteins and suggest that the scaffold is organized into distinct modular units at AChR clusters.

The Metabolic Stability of the Nicotinic Acetylcholine Receptor at the Neuromuscular Junction.

The clustering and maintenance of nicotinic acetylcholine receptors (AChRs) at high density in the postsynaptic membrane is a hallmark of the mammalian neuromuscular junction (NMJ). The regulation of receptor density/turnover rate at synapses is one of the main thrusts of neurobiology because it plays an important role in synaptic development and synaptic plasticity. The state-of-the-art imaging revealed that AChRs are highly dynamic despite the overall structural stability of the NMJ over the lifetime of the animal. This review highlights the work on the metabolic stability of AChRs at developing and mature NMJs and discusses the role of synaptic activity and the regulatory signaling pathways involved in the dynamics of AChRs.

Receptor-associated proteins and synaptic plasticity.

Changes in synaptic strength are important for synaptic development and synaptic plasticity. Most directly responsible for these synaptic changes are alterations in synaptic receptor number and density. Although alterations in receptor density mediated by the insertion, lateral mobility, removal, and recycling of receptors have been extensively studied, the dynamics and regulators of intracellular scaffolding proteins have only recently begun to be illuminated. In particular, a closer look at the receptor-associated proteins, which bind to receptors and are necessary for their synaptic localization and clustering, has revealed broader functions than previously thought and some rather unexpected thematic similarities. More than just "placeholders" or members of a passive protein "scaffold," receptor-associated proteins in every synapse studied have been shown to provide a number of signaling roles. In addition, the most recent state-of-the-art imaging has revealed that receptor-associated proteins are highly dynamic and are involved in regulating synaptic receptor density. Together, these results challenge the view that receptor-associated proteins are members of a static and stable scaffold and argue that their dynamic mobility may be essential for regulating activity-dependent changes in synaptic strength.

Acetylcholinesterase mobility and stability at the neuromuscular junction of living mice.

Acetylcholinesterase (AChE) is an enzyme that terminates acetylcholine neurotransmitter function at the synaptic cleft of cholinergic synapses. However, the mechanism by which AChE number and density are maintained at the synaptic cleft is poorly understood. In this work, we used fluorescence recovery after photobleaching, photo-unbinding, and quantitative fluorescence imaging to investigate the surface mobility and stability of AChE at the adult innervated neuromuscular junction of living mice. In wild-type synapses, we found that nonsynaptic (perisynaptic and extrasynaptic) AChEs are mobile and gradually recruited into synaptic sites and that most of the trapped AChEs come from the perijunctional pool. Selective labeling of a subset of synaptic AChEs within the synapse by using sequential unbinding and relabeling with different colors of streptavidin followed by time-lapse imaging showed that synaptic AChEs are nearly immobile. At neuromuscular junctions of mice deficient in alpha-dystrobrevin, a component of the dystrophin glycoprotein complex, we found that the density and distribution of synaptic AChEs are profoundly altered and that the loss rate of AChE significantly increased. These results demonstrate that nonsynaptic AChEs are mobile, whereas synaptic AChEs are more stable, and that alpha-dystrobrevin is important for controlling the density and stability of AChEs at neuromuscular synapses.

The role of the dystrophin glycoprotein complex on the neuromuscular system.

The Dystrophin Glycoprotein Complex (DGC) is a large multi-protein complex that links cytoskeleton actin to the extracellular matrix. This complex is critical in maintaining the structural integrity of muscle fibers and the stability of the neuromuscular synapse. The DGC consists of dystrophin and its utrophin homolog, as well as dystroglycans, sarcoglycans, sarcospan, syntrophins, and dystrobrevins. Deficiencies in DGC proteins result in several forms of muscular dystrophy with varying symptoms and degrees of severity in addition to structurally abnormal neuromuscular junctions (NMJs). This mini-review highlights current knowledge regarding the role of the DGC on the molecular dynamics of acetylcholine receptors (AChRs) as it relates to the formation and maintenance of the mammalian NMJ.

The disassembly of the neuromuscular synapse in high-fat diet-induced obese male mice.

OBJECTIVE: A sustained high fat diet in mice mimics many features of human obesity. We used male and female Non-Swiss albino mice to investigate the impact of short and long-term high-fat diet-(HFD)-induced obesity on the peripheral neuromuscular junction (NMJ) and whether obesity-related synaptic structural alterations were reversible after switching obese mice from HFD to a standard fat diet (SD). METHODS: HFD-induced obese and age-matched control mice fed SD were used. We carried out in vivo time lapse imaging to monitor changes of synapses over time, quantitative fluorescence imaging to study the regulation of acetylcholine receptor number and density at neuromuscular junctions, and high resolution confocal microscope to study structural alterations in both the pre- and postsynaptic apparatus. RESULTS: Time-lapse imaging in vivo over a 9 month period revealed that NMJs of HFD obese male mice display a variety of obesity-related structural alterations, including the disappearance of large synaptic areas, significant reduction in the density/number of nicotinic acetylcholine receptor (AChRs), abnormal distribution of AChRs, high turnover rate of AChRs, retraction of axons from lost postsynaptic sites, and partially denervated synapses. The severity of these synaptic alterations is associated with the duration of obesity. However, no substantial alterations were observed at NMJs of age-matched HFD obese female mice or male mice fed with a standard or low fat diet. Intriguingly, when obese male mice were switched from HFD to a standard diet, receptor density and the abnormal pattern of AChR distribution were completely reversed to normal, whereas lost synaptic structures were not restored. CONCLUSIONS: These results show that the obese male mice are more vulnerable than female mice to the impacts of long-term HFD on the NMJ damage and provide evidence that diet restriction can partially reverse obesity-related synaptic changes.

Tyrosine-phosphorylated and nonphosphorylated isoforms of alpha-dystrobrevin: roles in skeletal muscle and its neuromuscular and myotendinous junctions.

alpha-Dystrobrevin (DB), a cytoplasmic component of the dystrophin-glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking alphaDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main alphaDB isoforms, alphaDB1 and alphaDB2, with common NH2-terminal but distinct COOH-terminal domains. alphaDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. alphaDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in alphaDB-/- mice prevented muscle fiber degeneration; however, only alphaDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of alphaDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, alphaDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, alphaDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.

Neurotransmitter receptor dynamics studied in vivo by reversible photo-unbinding of fluorescent ligands.

We show that fluorescently tagged ligands with high affinity for their targets can be reversibly unbound by focused laser excitation. By sequential unbinding and relabeling with different colors of alpha-bungarotoxin, we selectively labeled adjacent pools of acetylcholine receptors (AChRs) at neuromuscular junctions of adult mice. Timelapse imaging in vivo revealed that synaptic AChRs completely intermingle over approximately 4 days and many extrasynaptic AChRs are incorporated into the synapse each day. In mice that lacked alpha-dystrobrevin, a component of the dystrophin-glycoprotein complex, rates of AChR turnover, and intermingling were increased approximately 4- to 5-fold. These results demonstrate remarkable molecular dynamism underlying macroscopic stability of the postsynaptic membrane, and establish alpha-dystrobrevin as a key control point for regulation of mobility and turnover.