Drosophila studies support a role for a presynaptic synaptotagmin mutation in a human congenital myasthenic syndrome.

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

Epsin 1 Promotes Synaptic Growth by Enhancing BMP Signal Levels in Motoneuron Nuclei

We thank Carl-Henrik Heldin (Uppsala University, Sweden) for his generous gift of the PS1 pMad antibody, Hugo Bellen, Corey Goodman, Janis Fischer, Graeme Davis, Guillermo Marques, Michael O'Connor, Kate O'Connor-Giles, and the Bloomington Drosophila Stock Center for flies strains, the Developmental Studies Hybridoma Bank at the University of Iowa for antibodies to Wit and CSP; Marie Phillips for advice on membrane fractionation; Avital Rodal, Kate O'Connor-Giles, Ela Serpe, Kristi Wharton, Mojgan Padash-Barmchi for discussions or comments on the manuscript. We also thank Jody Summers at OUHSC for her generosity in letting us to use her confocal microscope.Conceived and designed the experiments: PAV TRF LRC BZ. Performed the experiments: PAV TRF LRC SMR HB NER BZ. Analyzed the data: PAV TRF LRC SMR HB NER BZ. Wrote the paper: PAV TRF BZ.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.Yeshttp://www.plosone.org/static/editorial#pee

The C2A domain of synaptotagmin is an essential component of the calcium sensor for synaptic transmission.

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

The role of the C2A domain of synaptotagmin 1 in asynchronous neurotransmitter release.

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

Membrane Penetration by Synaptotagmin Is Required for Coupling Calcium Binding to Vesicle Fusion In Vivo

The vesicle protein synaptotagmin I is the Ca2+ sensor that triggers fast, synchronous release of neurotransmitter. Specifically, Ca2+ binding by the C2B domain of synaptotagmin is required at intact synapses, yet the mechanism whereby Ca2+ binding results in vesicle fusion remains controversial. Ca2+ -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 effectorinteraction mutations to date impact synaptic transmission as severely as mutation of the C2B Ca2+ -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 Ca2+ -binding pockets of synaptotagmin. Mutation of this residue in the C2A 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 C2B 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 sytnull mutants, which is more severe than the phenotype of C2BCa2+ -binding mutants. Thus, mutation of a single, C2B hydrophobic residue required for Ca2+ -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 C2B domain of synaptotagmin, is the critical effector interaction for coupling Ca2+ binding with vesicle fusion

pMad fails to accumulate in the nucleus of motoneurons in <i>lqf</i> mutants.

<p>(A–E) Representative images of motoneuron nuclei in <i>Drosophila</i> 3^rd instar larval ventral nerve cord (VNC). pMad signal alone is shown on the left, whereas neuronal membrane (marked by HRP, green) and pMad (red) are shown on the right. (B) In <i>lqf</i> mutants, nuclear pMad is significantly reduced relative to control (CS, A), whereas neuronal overexpression of Lqf results in an increase in nuclear localized pMad (C). Similarly, two other endocytotic mutants <i>endo</i> (D) and <i>nwk</i> (E) have increased levels of nuclear pMad. (F) Quantification of nuclear pMad levels in A–E. Error bars represent SEM. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Tukey's Multiple Comparison Post test. (G–H') Dissected and fixed 3^rd Instar larvae were stained with anti-CSP, and segmental nerves (which contain hundreds axons of sensory and motoneurons) were examined for general traffic defects, which would be apparent as CSP accumulations. There are no significant CSP accumulations in either the wild type or <i>lqf</i> mutants. Scale bars are 10 µm in all images. n values denote number of nuclei for each genotype, quantified from three animals per genotype. Control (CS), <i>nwk</i> (<i>nwk¹/nwk¹</i>), <i>endo</i> (<i>endo^A/endo^Δ4</i>), <i>lqf</i> (<i>lqf^ARI/lqf^FDD9</i>), Lqf^O/E (Elav^C155-Gal4/+; UAS-Lqf/+).</p

Lqf is required for synaptic overgrowth and pMad retrograde transport in <i>spinster</i> mutants.

<p>(A–F) Representative images of bouton morphology and pMad levels at the NMJ (A, C, E) and motoneuron nuclei (B, D, F) in <i>Drosophila</i> 3^rd instar larval NMJs from control larvae (CS, A–B), <i>spin</i> mutants (C–D) and <i>spin;lqf</i> double mutants (E–F). Synaptic boutons are overgrown in <i>spin</i> mutants (C), and this overgrowth is suppressed in the <i>spin;lqf</i> double mutants (E). (G) Quantification of synaptic bouton number in <i>spin</i> and <i>spin;lqf</i> mutants. Taken from three different animals for each genotype, n values represent the number of NMJs quantified. (H) Quantification of pMad intensity in boutons (white bars) and motoneuron nuclei (black bars). n = 5 NMJs from three larvae and 20 nuclei from five different larvae. Error bars represent SEM. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Tukey's Multiple Comparison Post test. Control (CS), <i>spin</i> (<i>spin⁴/spin⁵</i>), <i>spin, lqf</i> (<i>spin⁴/spin⁵; lqf^ARI/lqf Df</i>).</p

Lqf interacts with Wishful thinking (Wit) to regulate synapse growth.

<p>(A–G) Representative images of synaptic bouton morphology in <i>Drosophila</i> 3^rd instar larval NMJs from the indicated genotypes. Compared to control boutons (A), both <i>lqf</i> (<i>lqf^ARI</i>/<i>lqf</i>^FDD9) and <i>wit</i> mutants (<i>wit</i>^B11/<i>wit</i>^A12) (B and C, respectively), have fewer boutons, as does the <i>wit, lqf</i> double mutant (<i>wit^A12</i>/<i>wit^B11</i>; <i>lqf^ARI</i>/<i>lqf</i> Df; D). Neuronal overexpression of Lqf induces synaptic growth with increased number of branches and small satellite boutons and the presence of abnormally large ‘growth cone’-like boutons (Elav^C155-Gal4/+; UAS-Lqf/+; E), which is suppressed partially by removal of a single copy of <i>wit</i> (Elav^C155-Gal4/+; UAS-Lqf/+; <i>wit^A12/+</i>; F), or suppressed completely by removal of both copies of <i>wit</i> (Elav^C155-Gal4/+; UAS-Lqf/+; <i>wit^A12/wit^B11</i>; G). H). Quantification of A–G. Error bars represent SEM, n values represent the number of animals per genotype. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Tukey's Multiple Comparison Post test. (I) Co-immunoprecipitation of Flag-tagged Lqf from adult brain lysates from either control flies (containing no Flag), or flies expressing Flag-tagged Lqf, shows Wit co-immunoprecipitates with Lqf. Abbreviations are as follows: Immunoprecipitation (IP); Immunoblot (IB); Input (I); Supernatant (S); Immunoprecipitate (IP).</p