Bryan A. Stewart

Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions

Neuromuscular preparations from third instar larvae of Drosophila are not well-maintained in commonly used physiological solutions: vacuoles form in the muscle fibers, and membrane potential declines. These problems may result from the Na∶K ratio and total divalent cation content of these physiological solutions being quite different from those of haemolymph. Accordingly haemolymph-like solutions, based upon ion measurements of major cations, were developed and tested. Haemolymph-like solutions maintained the membrane potential at a relatively constant level, and prolonged the physiological life of the preparations. Synaptic transmission was well-maintained in haemolymph-like solutions, but the excitatory synaptic potentials had a slower time course and summated more effectively with repetitive stimulation, than in standard Drosophila solutions. Voltage-clamp experiments suggest that these effects are linked to more pronounced activation of muscle fiber membrane conductances in standard solutions, rather than to differences in passive muscle membrane properties or changes in postsynaptic receptor channel kinetics. Calcium dependence of transmitter release was steep in both standard and haemolymph-like solutions, but higher external calcium concentrations were required for a given level of release in haemolymph-like solutions. Thus, haemolymph-like solutions allow for prolonged, stable recording of synaptic transmission.

Quantal measurement and analysis methods compared for crayfish and Drosophila neuromuscular junctions, and rat hippocampus

Quantal content of transmission was estimated for three synaptic systems (crayfish and Drosophila neuromuscular junctions, and rat dentate gyrus neurons) with three different methods of measurement: direct counts of released quanta, amplitude measurements of evoked and spontaneous events, and charge measurements of evoked and spontaneous events. At the crayfish neuromuscular junction, comparison of the three methods showed that estimates from charge measurements were closer to estimates from direct counts, since amplitude measurements were more seriously affected by variable latency in evoked release of quantal units. Thus, charge measurements are better for estimating quantal content when direct counts cannot be made, as in crayfish at high frequency of stimulation or in the dentate gyrus neurons. At the Drosophila neuromuscular junction, there is almost no latency variation of quantal release in realistic physiological solutions, and the methods based upon amplitudes and charge give similar results. Distributions of evoked synaptic quantal events obtained by direct counts at the crayfish neuromuscular junction were compared to statistical distributions obtained by best fits. Binomial distributions with uniform or non-uniform probabilities of release generally provided good fits to the observations. From best fit distributions, the quantal parameters n (number of release sites) and p (their probability of release) can be calculated. We used two algorithms to estimate n and p: one allows for non-uniform probability of release and uses a modified chi-square (chi 2) criterion, and the second assumes uniform probability of release and derives parameters from maximum likelihood estimation (MLE). The bootstrap estimate of standard errors is used to determine the accuracy of n and p estimates.

Members of the synaptobrevin/vesicle-associated membrane protein (VAMP) family in Drosophila are functionally interchangeable in vivo for neurotransmitter release and cell viability

Synaptobrevins or VAMPs are vesicle-associated membrane proteins, often called v-SNARES, that are important for vesicle transport and fusion at the plasma membrane. Drosophila has two characterized members of this gene family: synaptobrevin (syb) and neuronal synaptobrevin (n-syb). Mutant phenotypes and gene-expression patterns indicate that n-Syb is exclusively neuronal and required only for synaptic vesicle secretion, whereas Syb is ubiquitous and, as shown here, essential for cell viability. When the eye precursor cells were made homozygous for syb−, the eye failed to develop. In contrast, n-syb− eye clones developed appropriately but failed to activate downstream neurons. To determine whether the two proteins are structurally specialized to accomplish these distinct in vivo functions, we have driven the expression of each gene in the absence of the other to look for phenotypic rescue. We find that expression of n-syb during eye development can rescue the cell lethality of the syb mutations, as can rat VAMP2 and cellubrevin. Expression of syb can restore synaptic transmission to n-syb mutants as assayed both by electroretinogram and recordings of excitatory junctional currents at the neuromuscular junction. Therefore, we find that Syb, which usually is not involved in synaptic function, can mediate Ca2+-triggered synaptic activity and that no particular specialization of the v-SNARE is required to differentiate synaptic exocytosis from other forms.

Regulated spacing of synapses and presynaptic active zones at larval neuromuscular junctions in different genotypes of the flies Drosophila and Sarcophaga

Synapses at larval neuromuscular junctions of the flies Drosophila melanogaster and Sarcophaga bullata are not distributed randomly. They have been studied in serial electron micrographs of two identified axons (axons 1 and 2) that innervate ventral longitudinal muscles 6 and 7 of the larval body wall. The following fly larvae were examined: axon 1—wild‐type Sarcophaga and Drosophila and Drosophila mutants duncem14 and fasIIe76, a hypomorphic allele of the fasciclin II gene; and axon 2—drosophila wild‐type, duncem14, and fasIIe76. These lines were selected to provide a wide range of nerve terminal phenotypes in which to study the distribution and spacing of synapses. Each terminal varicosity is applied closely to the underlying subsynaptic reticulum of the muscle fiber and has 15–40 synapses. Each synapse usually bears one or more active zones, characterized by dense bodies that are T‐shaped in cross section; they are located at the presumed sites of transmitter release. The distribution of synapses was characterized from the center‐to‐center distance of each synapse to its nearest neighbor. The mean spacing between nearest‐neighbor pairs ranged from 0.84 μm to 1.05 μm for axon 1, showing no significant difference regardless of genotype. The corresponding values for axon 2, 0.58 μm to 0.75 μm, were also statistically indistinguishable from one another in terminals of different genotype but differed significantly from the values for axon 1. Thus, the functional class of the axon provides a clear prediction of the spacing of its synapses, suggesting that spacing may be determined by the functional properties of transmission at the two types of terminals. Individual dense bodies were situated mostly at least 0.4 μm away from one another, suggesting that an interaction between neighboring active zones could prevent their final positions from being located more closely. J. Comp. Neurol. 393:482–492, 1998.

Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant

Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors). In neurons, the t‐SNARE SNAP‐25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. We have isolated a SNAP‐25 temperature‐sensitive paralytic mutant in Drosophila, SNAP‐25ts. The mutation causes a Gly50 to Glu change in SNAP‐25s first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro‐generated 70 kDa SNARE complexes containing SNAP‐25ts are thermally stable but the mutant SNARE multimers (of ∼120 kDa) rapidly dissociate at 37°C. The SNAP‐25ts mutant has two effects on neurotransmitter release depending upon temperature. At 22°C, evoked release of neurotransmitter in SNAP‐25ts larvae is greatly increased, and at 37°C, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22°C the mutation causes the SNARE complex to be more fusion competent but, at 37°C the same mutation leads to SNARE multimer instability and fusion incompetence.

Influence of semicrystalline order on the second-harmonic generation efficiency in the anisotropic bands of myocytes

The influence of semicrystalline order on the second-harmonic generation (SHG) efficiency in the anisotropic bands of Drosophila melanogaster sarcomeres from larval and adult muscle has been investigated. Differences in the semicrystalline order were obtained by using wild-type and mutant strains containing different amounts of headless myosin. The reduction in semicrystalline order without altering the chemical composition of myofibrils was achieved by observing highly stretched sarcomeres and by inducing a loss of viability in myocytes. In all cases the reduction of semicrystalline order in anisotropic bands of myocytes resulted in a substantial decrease in SHG. Second-harmonic imaging during periodic contractions of myocytes revealed higher intensities when sarcomeres were in the relaxed state compared with the contracted state. This study demonstrates that an ordered semicrystalline arrangement of anisotropic bands plays a determining role in the efficiency of SHG in myocytes.

Drosophila Amphiphysin is a Post‐Synaptic Protein Required for Normal Locomotion but Not Endocytosis

Clathrin‐mediated endocytosis is required to recycle synaptic vesicles for fast and efficient neurotransmission. Amphiphysins are thought to be multiprotein adaptors that may contribute to this process by bringing together many of the proteins required for endocytosis. Their in vivo function, however, has yet to be determined. Here, we show that the Drosophila genome encodes a single amphiphysin gene that is broadly expressed during development. We also show that, unlike its vertebrate counterparts, Drosophila Amphiphysin is enriched postsynaptically at the larval neuromuscular junction. To determine the role of Drosophila Amphiphysin, we also generated null mutants which are viable but give rise to larvae and adults with pronounced locomotory defects. Surprisingly, the locomotory defects cannot be accounted for by alterations in the morphology or physiology of the neuromuscular junction. Moreover, using stimulus protocols designed to test endocytosis under moderate and extreme vesicle cycling, we could not detect any defect in the neuromuscular junction of the amphiphysin mutant. Taken together, our findings suggest that Amphiphysin is not required for viability, nor is it absolutely required for clathrin‐mediated endocytosis. However, Drosophila Amphiphysin function is required in both larvae and adults for normal locomotion.

Nonmuscle Myosin II helps regulate synaptic vesicle mobility at the Drosophila neuromuscular junction

BackgroundAlthough the mechanistic details of the vesicle transport process from the cell body to the nerve terminal are well described, the mechanisms underlying vesicle traffic within nerve terminal boutons is relatively unknown. The actin cytoskeleton has been implicated but exactly how actin or actin-binding proteins participate in vesicle movement is not clear.ResultsIn the present study we have identified Nonmuscle Myosin II as a candidate molecule important for synaptic vesicle traffic within Drosophila larval neuromuscular boutons. Nonmuscle Myosin II was found to be localized at the Drosophila larval neuromuscular junction; genetics and pharmacology combined with the time-lapse imaging technique FRAP were used to reveal a contribution of Nonmuscle Myosin II to synaptic vesicle movement. FRAP analysis showed that vesicle dynamics were highly dependent on the expression level of Nonmuscle Myosin II.ConclusionOur results provide evidence that Nonmuscle Myosin II is present presynaptically, is important for synaptic vesicle mobility and suggests a role for Nonmuscle Myosin II in shuttling vesicles at the Drosophila neuromuscular junction. This work begins to reveal the process by which synaptic vesicles traverse within the bouton.

Regulation of Commissureless by the Ubiquitin Ligase DNedd4 Is Required for Neuromuscular Synaptogenesis in Drosophila melanogaster

ABSTRACT Muscle synaptogenesis in Drosophila melanogaster requires endocytosis of Commissureless (Comm), a binding partner for the ubiquitin ligase dNedd4. We investigated whether dNedd4 and ubiquitination mediate this process. Here we show that Comm is expressed in intracellular vesicles in the muscle, whereas Comm bearing mutations in the two PY motifs (L/PPXY) responsible for dNedd4 binding [Comm(2PY→AY)], or bearing Lys→Arg mutations in all Lys residues that serve as ubiquitin acceptor sites [Comm(10K→R)], localize to the muscle surface, suggesting they cannot endocytose. Accordingly, aberrant muscle innervation is observed in the Comm(2PY→AY) and Comm(10K→R) mutants expressed early in muscle development. Similar muscle surface accumulation of Comm and innervation defects are observed when dNedd4 is knocked down by double-stranded RNA interference in the muscle, in dNedd4 heterozygote larvae, or in muscles overexpressing catalytically inactive dNedd4. Expression of the Comm mutants fused to a single ubiquitin that cannot be polyubiquitinated and mimics monoubiquitination [Comm(2PY→AY)-monoUb or Comm(10K→R)-monoUb] prevents the defects in both Comm endocytosis and synaptogenesis, suggesting that monoubiquitination is sufficient for Comm endocytosis in muscles. Expression of the Comm mutants later in muscle development, after synaptic innervation, has no effect. These results demonstrate that dNedd4 and ubiquitination are required for Commissureless endocytosis and proper neuromuscular synaptogenesis.

Functional Roles for β1,4-N-Acetlygalactosaminyltransferase-A in Drosophila Larval Neurons and Muscles

Adult Drosophila mutant for the glycosyltransferase β1,4-N-acetlygalactosaminyltransferase-A (β4GalNAcTA) display an abnormal locomotion phenotype, indicating a role for this enzyme, and the glycan structures that it generates, in the neuromuscular system. To investigate the functional role of this enzyme in more detail, we turned to the accessible larval neuromuscular system and report here that larvae mutant for β4GalNAcTA display distinct nerve and muscle phenotypes. Mutant larvae exhibit abnormal backward crawling, reductions in nerve terminal bouton number, decreased spontaneous transmitter-release frequency, and short, wide muscles. This muscle shape change appears to result from hypercontraction since the individual sarcomeres are shorter in mutant muscles. Analysis of muscle calcium signals showed altered calcium handling in the mutant, suggesting a mechanism by which hypercontraction could occur. All of these phenotypes can be rescued by a transgene carrying the β4GalNAcTA genomic region. Tissue-specific expression, using the Gal4-UAS system, reveals that neural expression rescues the mutant crawling phenotype, while muscle expression rescues the muscle defect. Tissue-specific expression did not appear to rescue the decrease in neuromuscular junction bouton number, suggesting that this defect arises from cooperation between nerve and muscle. Altogether, these results suggest that β4GalNAcTA has at least three distinct functional roles.