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RNA-binding proteins play an important role in the regulation of post-transcriptional gene expression throughout the nervous system. This is underscored by the prevalence of mutations in genes encoding RNA splicing factors and other RNA-binding proteins in a number of neurodegenerative and neurodevelopmental disorders. The highly conserved alternative splicing factor Caper is widely expressed throughout the developing embryo and functions in the development of various sensory neural subtypes in the Drosophila peripheral nervous system. Here we find that caper dysfunction leads to aberrant neuromuscular junction morphogenesis, as well as aberrant locomotor behavior during larval and adult stages. Despite its widespread expression, our results indicate that caper function is required to a greater extent within the nervous system, as opposed to muscle, for neuromuscular junction development and for the regulation of adult locomotor behavior. Moreover, we find that Caper interacts with the RNA-binding protein Fmrp to regulate adult locomotor behavior. Finally, we show that caper dysfunction leads to various phenotypes that have both a sex and age bias, both of which are commonly seen in neurodegenerative disorders in humans., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Following nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments (aspartate to asparagine mutations, sytD-N) had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca2+ binding. Thus, rather than modulating an asynchronous sensor, sytD-N may be mimicking one. To directly test the C2A inhibition hypothesis, we utilized an alternate C2A mutation that we designed to block Ca2+ binding without mimicking it (an aspartate to glutamate mutation, sytD-E). Analysis of both the original sytD-N mutation and our alternate sytD-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the sytD-E mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo., Competing Interests: The authors have declared that no competing interests exist.

Synaptic transmission relies on the fast, synchronous fusion of neurotransmitter filled vesicles with the presynaptic membrane. Synaptotagmin is the Ca 2+ sensor that couples the Ca 2+ influx into nerve terminals following an action potential with this fast, synchronous vesicle fusion. Over two decades of synaptotagmin research has provided many clues as to how Ca 2+ binding by synaptotagmin may lead to vesicle fusion. In vitro studies of molecular binding interactions are essential for elucidating potential mechanisms. However, an in vivo system to evaluate the postulated mechanisms is required to determine functional significance. The neuromuscular junction (NMJ) has long been an indispensable tool for synaptic research and studies at the NMJ will undoubtedly continue to provide key insights into synaptotagmin function., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2020. Published by Elsevier B.V.)

The synaptic vesicle protein, synaptotagmin, is the principle Ca2+ sensor for synaptic transmission. Ca2+ influx into active nerve terminals is translated into neurotransmitter release by Ca2+ binding to synaptotagmin's tandem C2 domains, triggering the fast, synchronous fusion of multiple synaptic vesicles. Two hydrophobic residues, shown to mediate Ca2+-dependent membrane insertion of these C2 domains, are required for this process. Previous research suggested that one of its tandem C2 domains (C2B) is critical for fusion, while the other domain (C2A) plays only a facilitatory role. However, the function of the two hydrophobic residues in C2A have not been adequately tested in vivo. Here we show that these two hydrophobic residues are absolutely required for synaptotagmin to trigger vesicle fusion. Using in vivo electrophysiological recording at the Drosophila larval neuromuscular junction, we found that mutation of these two key C2A hydrophobic residues almost completely abolished neurotransmitter release. Significantly, mutation of both hydrophobic residues resulted in more severe deficits than those seen in synaptotagmin null mutants. Thus, we report the most severe phenotype of a C2A mutation to date, demonstrating that the C2A domain is absolutely essential for synaptotagmin's function as the electrostatic switch., Competing Interests: The authors have declared that no competing interests exist.

During chemical transmission, the function of synaptic proteins must be coordinated to efficiently release neurotransmitter. Synaptotagmin 2, the Ca2+ sensor for fast, synchronized neurotransmitter release at the human neuromuscular junction, has recently been implicated in a dominantly inherited congenital myasthenic syndrome associated with a non-progressive motor neuropathy. In one family, a proline residue within the C2B Ca2+-binding pocket of synaptotagmin is replaced by a leucine. The functional significance of this residue has not been investigated previously. Here we show that in silico modeling predicts disruption of the C2B Ca2+-binding pocket, and we examine the in vivo effects of the homologous mutation in Drosophila. When expressed in the absence of native synaptotagmin, this mutation is lethal, demonstrating for the first time that this residue plays a critical role in synaptotagmin function. To achieve expression similar to human patients, the mutation is expressed in flies carrying one copy of the wild type synaptotagmin gene. We now show that Drosophila carrying this mutation developed neurological and behavioral manifestations similar to those of human patients and provide insight into the mechanisms underlying these deficits. Our Drosophila studies support a role for this synaptotagmin point mutation in disease etiology.

Bone morphogenetic protein (BMP) retrograde signaling is crucial for neuronal development and synaptic plasticity. However, how the BMP effector phospho-Mother against decapentaplegic (pMad) is processed following receptor activation remains poorly understood. Here we show that Drosophila Epsin1/Liquid facets (Lqf) positively regulates synaptic growth through post-endocytotic processing of pMad signaling complex. Lqf and the BMP receptor Wishful thinking (Wit) interact genetically and biochemically. lqf loss of function (LOF) reduces bouton number whereas overexpression of lqf stimulates bouton growth. Lqf-stimulated synaptic overgrowth is suppressed by genetic reduction of wit. Further, synaptic pMad fails to accumulate inside the motoneuron nuclei in lqf mutants and lqf suppresses synaptic overgrowth in spinster (spin) mutants with enhanced BMP signaling by reducing accumulation of nuclear pMad. Interestingly, lqf mutations reduce nuclear pMad levels without causing an apparent blockage of axonal transport itself. Finally, overexpression of Lqf significantly increases the number of multivesicular bodies (MVBs) in the synapse whereas lqf LOF reduces MVB formation, indicating that Lqf may function in signaling endosome recycling or maturation. Based on these observations, we propose that Lqf plays a novel endosomal role to ensure efficient retrograde transport of BMP signaling endosomes into motoneuron nuclei.

Synaptotagmin is the major calcium sensor for fast synaptic transmission that requires the synchronous fusion of synaptic vesicles. Synaptotagmin contains two calcium-binding domains: C2A and C2B. Mutation of a positively charged residue (R233Q in rat) showed that Ca2+-dependent interactions between the C2A domain and membranes play a role in the electrostatic switch that initiates fusion. Surprisingly, aspartate-to-asparagine mutations in C2A that inhibit Ca2+ binding support efficient synaptic transmission, suggesting that Ca2+ binding by C2A is not required for triggering synchronous fusion. Based on a structural analysis, we generated a novel mutation of a single Ca2+-binding residue in C2A (D229E in Drosophila) that inhibited Ca2+ binding but maintained the negative charge of the pocket. This C2A aspartate-to-glutamate mutation resulted in ∼80% decrease in synchronous transmitter release and a decrease in the apparent Ca2+ affinity of release. Previous aspartate-to-asparagine mutations in C2A partially mimicked Ca2+ binding by decreasing the negative charge of the pocket. We now show that the major function of Ca2+ binding to C2A is to neutralize the negative charge of the pocket, thereby unleashing the fusion-stimulating activity of synaptotagmin. Our results demonstrate that Ca2+ binding by C2A is a critical component of the electrostatic switch that triggers synchronous fusion. Thus, Ca2+ binding by C2B is necessary and sufficient to regulate the precise timing required for coupling vesicle fusion to Ca2+ influx, but Ca2+ binding by both C2 domains is required to flip the electrostatic switch that triggers efficient synchronous synaptic transmission.

The vesicle protein synaptotagmin I is the Ca(2+) sensor that triggers fast, synchronous release of neurotransmitter. Specifically, Ca(2+) binding by the C(2)B domain of synaptotagmin is required at intact synapses, yet the mechanism whereby Ca(2+) binding results in vesicle fusion remains controversial. Ca(2+)-dependent interactions between synaptotagmin and SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment receptor) complexes and/or anionic membranes are possible effector interactions. However, no effector-interaction mutations to date impact synaptic transmission as severely as mutation of the C(2)B Ca(2+)-binding motif, suggesting that these interactions are facilitatory rather than essential. Here we use Drosophila to show the functional role of a highly conserved, hydrophobic residue located at the tip of each of the two Ca(2+)-binding pockets of synaptotagmin. Mutation of this residue in the C(2)A domain (F286) resulted in a ∼50% decrease in evoked transmitter release at an intact synapse, again indicative of a facilitatory role. Mutation of this hydrophobic residue in the C(2)B domain (I420), on the other hand, blocked all locomotion, was embryonic lethal even in syt I heterozygotes, and resulted in less evoked transmitter release than that in syt(null) mutants, which is more severe than the phenotype of C(2)B Ca(2+)-binding mutants. Thus, mutation of a single, C(2)B hydrophobic residue required for Ca(2+)-dependent penetration of anionic membranes results in the most severe disruption of synaptotagmin function in vivo to date. Our results provide direct support for the hypothesis that plasma membrane penetration, specifically by the C(2)B domain of synaptotagmin, is the critical effector interaction for coupling Ca(2+) binding with vesicle fusion.

The synaptic vesicle protein, synaptotagmin I, is a multifunctional protein required for several steps in the synaptic vesicle cycle. It is primarily composed of two calcium-binding domains, C(2)A and C(2)B. Within each of these domains, a polylysine motif has been identified that is proposed to mediate specific functions within the synaptic vesicle cycle. While the C(2)B polylysine motif plays an important role in synaptic transmission in vivo, the C(2)A polylysine motif has not previously been analyzed at an intact synapse. Here, we show that mutation of the C(2)A polylysine motif increases the frequency of spontaneous transmitter release in vivo. The increased frequency is not a developmental consequence of disrupted synaptic transmission, as evoked transmitter release is unimpaired in the mutants. Our results demonstrate that synaptotagmin I plays a direct role in regulating spontaneous transmitter release, indicative of an active role in synaptic vesicle stabilization mediated by the C(2)A polylysine motif., (2009 Wiley-Liss, Inc.)

The ubiquitin-proteasome system plays an important role in synaptic development and function. However, many components of this system, and how they act to affect synapses, are still not well understood. In this study, we use the Drosophila neuromuscular junction to study the in vivo function of Liquid facets (Lqf), a homolog of mammalian epsin 1. Our data show that Lqf plays a novel role in synapse development and function. Contrary to prior models, Lqf is not required for clathrin-mediated endocytosis of synaptic vesicles. Lqf is required to maintain bouton size and shape and to sustain synapse growth by acting as a specific substrate of the deubiquitinating enzyme Fat facets. However, Lqf is not a substrate of the Highwire (Hiw) E3 ubiquitin ligase; neither is it required for synapse overgrowth in hiw mutants. Interestingly, Lqf converges on the Hiw pathway by negatively regulating transmitter release in the hiw mutant. These observations demonstrate that Lqf plays distinct roles in two ubiquitin pathways to regulate structural and functional plasticity of the synapse.

Synaptotagmin I is the Ca(2+) sensor for fast, synchronous release of neurotransmitter; however, the molecular interactions that couple Ca(2+) binding to membrane fusion remain unclear. The structure of synaptotagmin is dominated by two C(2) domains that interact with negatively charged membranes after binding Ca(2+). In vitro work has implicated a conserved basic residue at the tip of loop 3 of the Ca(2+)-binding pocket in both C(2) domains in coordinating this electrostatic interaction with anionic membranes. Although results from cultured cells suggest that the basic residue of the C(2)A domain is functionally significant, such studies provide contradictory results regarding the importance of the C(2)B basic residue during vesicle fusion. To directly test the functional significance of each of these residues at an intact synapse in vivo, we neutralized either the C(2)A or the C(2)B basic residue and assessed synaptic transmission at the Drosophila neuromuscular junction. The conserved basic residues at the tip of the Ca(2+)-binding pocket of both the C(2)A and C(2)B domains mediate Ca(2+)-dependent interactions with anionic membranes and are required for efficient evoked transmitter release. Our results directly support the hypothesis that the interactions between synaptotagmin and the presynaptic membrane, which are mediated by the basic residues at the tip of both the C(2)A and C(2)B Ca(2+)-binding pockets, are critical for coupling Ca(2+) influx with vesicle fusion during synaptic transmission in vivo. Our model for synaptotagmin's direct role in coupling Ca(2+) binding to vesicle fusion incorporates this finding with results from multiple in vitro and in vivo studies.

The distal Ca(2+)-binding domain of synaptotagmin I (Syt I), C2B, has two Ca(2+)-binding sites. To study their function in Drosophila, pairs of aspartates were mutated to asparagines and the mutated syt I was expressed in the syt I-null background (P[syt I(B-D1,2N)] and P[syt I(B-D3,4N)]). We examined the effects of these mutations on nerve-evoked synchronous synaptic transmission and high K(+)-induced quantal events at embryonic neuromuscular junctions. The P[syt I(B-D1,2N)] mutation virtually abolished synaptic transmission, whereas the P[syt I(B-D3,4N)] mutation strongly reduced but did not abolish it. The quantal content in P[syt I(B-D3,4N)] increased with the external Ca(2+) concentration, [Ca(2+)](e), with a slope of 1.86 in double-logarithmic plot, whereas that of control was 2.88. In high K(+) solutions the quantal event frequency in P[syt I(B-D3,4N)] increased progressively with [Ca(2+)](e) between 0 and 0.15 mM as in control. In contrast, in P[syt I(B-D1,2N)] the event frequency did not increase progressively between 0 and 0.15 mM and was significantly lower at 0.15 than at 0.05 mM [Ca(2+)](e). The P[syt I(B-D1,2N)] mutation inhibits high K(+)-induced quantal release in a narrow range of [Ca(2+)](e) (negative regulatory function). When Sr(2+) substituted for Ca(2+), nerve-evoked synchronous synaptic transmission was severely depressed and delayed asynchronous release was appreciably increased in control embryos. In high K(+) solutions with Sr(2+), the quantal event frequency was higher than that in Ca(2+) and increased progressively with [Sr(2+)](e) in control and in both mutants. Sr(2+) partially substitutes for Ca(2+) in synchronous release but does not support the negative regulatory function of Syt I.

Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca2+ affinity of release, and, at low Ca2+, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca2+ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt(null) mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca2+-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca2+-independent docking and/or priming of synaptic vesicles in vivo.

Synaptotagmin I is a synaptic vesicle protein postulated to mediate vesicle docking, vesicle recycling, and the Ca(2+) sensing required to trigger vesicle fusion. Analysis of synaptotagmin I knockouts (sytI(NULL) mutants) in both Drosophila and mice led to these hypotheses. Although much research on the mechanisms of synaptic transmission in Drosophila is performed at the third instar neuromuscular junction, the ultrastructure of this synapse has never been analyzed in sytI(NULL) mutants. Here we report severe synaptic vesicle depletion, an accumulation of large vesicles, and decreased vesicle docking at sytI(NULL) third instar neuromuscular junctions. Mutations in synaptotagmin I's C(2)B Ca(2+)-binding motif nearly abolish synaptic transmission and decrease the apparent Ca(2+) affinity of neurotransmitter release. Although this result is consistent with disruption of the Ca(2+) sensor, synaptic vesicle depletion and/or redistribution away from the site of Ca(2+) influx could produce a similar phenotype. To address this question, we examined vesicle distributions at neuromuscular junctions from third instar C(2)B Ca(2+)-binding motif mutants and transgenic wild-type controls. The number of docked vesicles and the overall number of synaptic vesicles in the vicinity of active zones was unchanged in the mutants. We conclude that the near elimination of synaptic transmission and the decrease in the Ca(2+) affinity of release observed in C(2)B Ca(2+)-binding motif mutants is not due to altered synaptic vesicle distribution but rather is a direct result of disrupting synaptotagmin I's ability to bind Ca(2+). Thus, Ca(2+) binding by the C(2)B domain mediates a post-docking step in fusion.

Synaptotagmin is a synaptic vesicle protein that is postulated to be the Ca(2+) sensor for fast, evoked neurotransmitter release. Deleting the gene for synaptotagmin (syt(null)) strongly suppresses synaptic transmission in every species examined, showing that synaptotagmin is central in the synaptic vesicle cycle. The cytoplasmic region of synaptotagmin contains two C(2) domains, C(2)A and C(2)B. Five, highly conserved, acidic residues in both the C(2)A and C(2)B domains of synaptotagmin coordinate the binding of Ca(2+) ions, and biochemical studies have characterized several in vitro Ca(2+)-dependent interactions between synaptotagmin and other nerve terminal molecules. But there has been no direct evidence that any of the Ca(2+)-binding sites within synaptotagmin are required in vivo. Here we show that mutating two of the Ca(2+)-binding aspartate residues in the C(2)B domain (D(416,418)N in Drosophila) decreased evoked transmitter release by >95%, and decreased the apparent Ca(2+) affinity of evoked transmitter release. These studies show that the Ca(2+)-binding motif of the C(2)B domain of synaptotagmin is essential for synaptic transmission.

Synaptotagmin is a synaptic vesicle protein required for efficient neurotransmitter release, yet its exact role in the synaptic vesicle cycle is unclear. Drosophila presents an ideal organism for studies aimed at determining the in vivo functions of proteins. However, synaptotagmin studies have been limited by the early (embryonic or first instar) lethality previously reported for Drosophila synaptotagmin I null (syt(null)) mutants. Here we report a new culturing technique that enhances survival of severely uncoordinated mutants thereby permitting Drosophila syt(null) mutants to survive through early adulthood. We examined synapses in syt(null) third instar larvae by electrophysiology and found that they exhibit severely decreased and asynchronous evoked neurotransmitter release, as well as an increased rate of spontaneous neurotransmitter release, as previously seen in first instar syt(null) larvae. The ability to examine severe synaptotagmin mutants as third instar larvae, a stage where electrophysiological and morphological analyses are more easily accomplished, will facilitate structure/function studies., (Copyright 2001 Wiley-Liss, Inc.)

The synaptic vesicle protein, synaptotagmin, has been hypothesized to mediate several functions in neurotransmitter release including calcium sensing, vesicle recycling, and synaptic vesicle docking. These hypotheses are based on evidence from in vitro binding assays, peptide and antibody injection experiments, and genetic knockout studies. Synaptotagmin contains two domains that are homologous to the calcium ion (Ca(2+))-binding C2 domain of protein kinase C. The two C2 domains of synaptotagmin have broadly differing ligand-binding properties. We have focused on the second C2 domain (C2B) of synaptotagmin I, in particular, on a series of conserved lysine residues on beta-strand 4 of C2B. This polylysine motif binds clathrin-adapter protein AP-2, neuronal calcium channels, and inositol high polyphosphates. It also mediates Ca(2+)-dependent oligomerization. To investigate the importance of these lysine residues in synaptic transmission, we have introduced synaptotagmin I (syt) transgenes harboring specific polylysine motif mutations into flies otherwise lacking the synaptotagmin I protein (syt(null)). Electrophysiological analyses of these mutants revealed that evoked transmitter release is decreased by approximately 36% and that spontaneous release is increased approximately twofold relative to syt(null) flies that express a wild type syt transgene. Synaptotagmin expression in both the mutant and the wild type transgenic lines was equivalent, as measured by semiquantitative Western blot analysis. Thus, the alteration in synaptic transmission was due to the mutation and not to the level of synaptotagmin expression. We conclude that synaptotagmin interactions mediated by the C2 B polylysine motif are required to attain full synaptotagmin function in vivo.

Nerve terminal specializations include mechanisms for maintaining a subpopulation of vesicles in a docked, fusion-ready state. We have investigated the relationship between synaptotagmin and the number of morphologically docked vesicles by an electron microscopic analysis of Drosophila synaptotagmin (syt) mutants. The overall number of synaptic vesicles in a terminal was reduced, although each active zone continued to have a cluster of vesicles in its vicinity. In addition, there was an increase in the number of large vesicles near synapses. Examining the clusters, we found that the pool of synaptic vesicles immediately adjacent to the presynaptic membrane, the pool that includes the docked population, was reduced to 24 +/- 5% (means +/- SEM) of control in the sytnull mutation. To separate contributions of overall vesicle depletion and increased spontaneous release from direct effects of synaptotagmin on morphological docking, we examined syt mutants in an altered genetic background. Recombining syt alleles onto a second chromosome bearing an as yet uncharacterized mutation resulted in the expected decrease in evoked release but suppressed the increase in spontaneous release frequency. Motor nerve terminals in this genotype contained more synaptic vesicles than control, yet the number of vesicles immediately adjacent to the presynaptic membrane near active zones was still reduced (33 +/- 4% of control). Our findings demonstrate that there is a decrease in the number of morphologically docked vesicles seen in syt mutants. The decreases in docking and evoked release are independent of the increase in spontaneous release. These results support the hypothesis that synaptotagmin stabilizes the docked state.

We examined the effects of motor-nerve stimulation on the intracellular Ca2+ levels of Schwann cells, the glial cells at the frog neuromuscular junction. Schwann cells, which were loaded with the fluorescent Ca2+ indicator fluo-3 and examined by confocal microscopy, showed a transient increase in free Ca2+ within a few seconds of the onset of tetanic stimulation of the motor nerve. The Ca2+ response was specific to the synapse in that it was found in the terminal Schwann cells at the junction but not in the myelinating Schwann cells along the axon. The Ca2+ transients occurred in the presence of d-tubocurare, indicating that they were not mediated by nicotinic acetylcholine receptors and recurred when the stimulus was repeated. The Ca2+ response persisted after degeneration of the postsynaptic muscle fiber, demonstrating that the terminal Schwann cell was stimulated directly by presynaptic activity. The finding that terminal Schwann cells at the neuromuscular junction respond to presynaptic activity suggests that glial-cell function is modulated by synaptic transmission.

To test the hypothesis that agrin mediates motor neuron-induced aggregation of acetylcholine receptors (AChRs) in skeletal muscle fibers and to determine whether the agrin active in this process is released by motor neurons, we raised polyclonal antibodies to purified ray agrin that blocked its receptor aggregating activity. When the antibodies were applied to chick motor neuron--chick myotube cocultures, they inhibited the formation of AChR aggregates at and near neuromuscular contacts, demonstrating that agrin plays a role in the induction of the aggregates. Rat motor neurons, like chick motor neurons, induce AChR aggregates on chick myotubes. This effect was not inhibited by our antibodies, indicating that, although the antibodies inhibited the activity of chick agrin, they did not have a similar effect on rat agrin. We conclude that agrin released by rat motor neurons induced the chick myotubes to aggregate AChRs.

Several lines of evidence have led to the hypothesis that agrin, a protein extracted from the electric organ of Torpedo, is similar to the molecules in the synaptic cleft basal lamina at the neuromuscular junction that direct the formation of acetylcholine receptor and acetylcholinesterase aggregates on regenerating myofibers. One such finding is that monoclonal antibodies against agrin stain molecules concentrated in the synaptic cleft of neuromuscular junctions in rays. In the studies described here we made additional monoclonal antibodies against agrin and used them to extend our knowledge of agrin-like molecules at the neuromuscular junction. We found that anti-agrin antibodies intensely stained the synaptic cleft of frog and chicken as well as that of rays, that denervation of frog muscle resulted in a reduction in staining at the neuromuscular junction, and that the synaptic basal lamina in frog could be stained weeks after degeneration of all cellular components of the neuromuscular junction. We also describe anti-agrin staining in nonjunctional regions of muscle. We conclude the following: (a) agrin-like molecules are likely to be common to all vertebrate neuromuscular junctions; (b) the long-term maintenance of such molecules at the junction is nerve dependent; (c) the molecules are, indeed, a component of the synaptic basal lamina; and (d) they, like the molecules that direct the formation of receptor and esterase aggregates on regenerating myofibers, remain associated with the synaptic basal lamina after muscle damage.

Basal lamina-rich extracts of Torpedo californica electric organ contain a factor that causes acetylcholine receptors (AChRs) on cultured myotubes to aggregate into patches. Our previous studies have indicated that the active component of these extracts is similar to the molecules in the basal lamina which direct the aggregation of AChRs in the muscle fibre plasma membrane at regenerating neuromuscular junctions in vivo. Because it can be obtained in large amounts and assayed in controlled conditions in cell culture, the AChR-aggregating factor from electric organ may be especially useful for examining in detail how the postsynaptic apparatus of regenerating muscle is assembled. Here we demonstrate that the electric organ factor causes not only the formation of AChR aggregates on cultured myotubes, but also the formation of patches of acetylcholinesterase (AChE). This finding, together with the observation that basal lamina directs the formation of both AChR and AChE aggregates at regenerating neuromuscular junctions in vivo, leads us to hypothesize that a single component of the synaptic basal lamina causes the formation of both these synaptic specializations on regenerating myofibres.

The basal lamina in the synaptic cleft of the vertebrate skeletal neuromuscular junction contains molecules that direct the formation of synaptic specializations in regenerating axons and muscle fibres. We have undertaken a series of experiments aimed at identifying and characterizing the molecules responsible for the formation of one of these specializations, the aggregates of acetylcholine receptors (AChRs) in the muscle fibre plasma membrane. We began by preparing an insoluble, basal lamina-containing fraction from Torpedo californica electric organ, a tissue which has a far higher concentration of cholinergic synapses than muscle, and showing that this fraction caused AChRs on cultured chick myotubes to aggregate. A critical step is learning whether or not the electric organ factor is similar to the receptor-aggregating molecule in the basal lamina at the neuromuscular junction. The importance of this problem is emphasized by reports that clearly non-physiological agents, such as positively charged latex beads, can cause AChR aggregation on cultured muscle cells. We have already shown that Torpedo muscle contains an AChR-aggregating factor similar to that of electric organ, although in much lower amounts. Here we demonstrate, using monoclonal antibodies, that the AChR-aggregating factor in our extracts of electric organ is, in fact, antigenically related to molecules concentrated in the synaptic cleft at the neuromuscular junction.

The portion of the muscle fibre's basal lamina that occupies the synaptic cleft at the neuromuscular junction contains molecules that cause the aggregation of acetylcholine receptors and acetylcholinesterase on regenerating muscle fibres. Agrin, which is extracted from basal lamina-containing fractions of the Torpedo electric organ and causes the formation of acetylcholine receptor and acetylcholinesterase aggregates on cultured myotubes, may be similar, if not identical, to the acetylcholine receptor- and acetylcholinesterase-aggregating molecules at the neuro-muscular junction. Here we summarize experiments which led to the identification of agrin and established that the basal lamina at the neuromuscular junction contains molecules antigenically similar to agrin. We also discuss results which raise the possibility that agrin-like molecules at the neuromuscular junction are produced by motor neurones.