Exocytosis and endocytosis of synaptic vesicles and functional roles of vesicle pools: Lessons from the Drosophila neuromuscular junction
ABSTRACT To maintain synaptic transmission during intense neuronal activities, the synaptic vesicle (SV) pool at release sites is effectively replenished by recruitment of SVs from the reserve pool and/or by endocytosis. The authors have studied dynamics of SVs using a fluorescence dye, FM1-43, which is incorporated into SVs during endocytosis and released by exocytosis. Drosophila is one of the most suitable preparations for genetic and pharmacological analyses, and this provides a useful model system. The authors found at the neuromuscular junctions of Drosophila that exocytosis and endocytosis of SVs are triggered by Ca(2+) influx through distinct routes and that selective inhibition of exocytosis or endocytosis resulted in depression of synaptic transmission with a distinct time course. They identified two SV pools in a single presynaptic bouton. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low-frequency stimulation and locates close to release sites in the periphery of boutons, whereas the reserve pool (RP) is loaded and unloaded only during high-frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the quantal content of evoked release, suggesting that SVs in the ECP are primarily involved in synaptic transmission. SVs in the RP are recruited to synaptic transmission by a process involving the cAMP/PKA cascade during high-frequency stimulation. Cytochalasin D blocked this recruitment process, suggesting involvement of filamentous actin. Endocytosed SVs replenish the ECP during stimulation and the RP after tetanic stimulation. Replenishment of the ECP depends on Ca(2+) influx from external solutions, and that of the RP is initiated by Ca(2+) release from internal stores. Thus, SV dynamics is closely involved in modulation of synaptic efficacy and influences synaptic plasticity.
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ABSTRACT: Exocytotic release of glutamate depends upon loading of the neurotransmitter into synaptic vesicles by vesicular glutamate transporters, VGLUTs. The major isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in synapses of the adult rodent brain that correlates with the probability of release and potential for plasticity. Indeed, expression of different VGLUT protein isoforms confers different properties of release probability. Expression of VGLUT1 or 2 protein also determines the kinetics of synaptic vesicle recycling. To identify molecular determinants that may be related to reported differences in VGLUT trafficking and glutamate release properties, we investigated some of the intrinsic differences between the two isoforms. VGLUT1 and 2 exhibit a high degree of sequence homology, but differ in their N- and C-termini. While the C-termini of VGLUT1 and 2 share a dileucine-like trafficking motif and a proline-, glutamate-, serine-, and threonine-rich PEST domain, only VGLUT1 contains two polyproline domains and a phosphorylation consensus sequence in a region of acidic amino acids. The interaction of a VGLUT1 polyproline domain with the endocytic protein endophilin recruits VGLUT1 to a fast recycling pathway. To identify trans-acting cellular proteins that interact with the distinct motifs found in the C-terminus of VGLUT1, we performed a series of in vitro biochemical screening assays using the region encompassing the polyproline motifs, phosphorylation consensus sites, and PEST domain. We identify interactors that belong to several classes of proteins that modulate cellular function, including actin cytoskeletal adaptors, ubiquitin ligases, and tyrosine kinases. The nature of these interactions suggests novel avenues to investigate the modulation of synaptic vesicle protein recycling.PLoS ONE 10/2014; 9(10):e109824. DOI:10.1371/journal.pone.0109824 · 3.53 Impact Factor
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ABSTRACT: We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry.PLoS ONE 01/2014; 9(1):e87174. DOI:10.1371/journal.pone.0087174 · 3.53 Impact Factor
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ABSTRACT: Distinct pools of the bone morphogenetic protein (BMP) Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, whereas muscle-derived Gbb regulates neuromuscular junction growth. Yet how cells differentiate between these ligand pools is not known. Here we present evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre- and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbb's proneurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, we propose that Gbb/Cmpy corelease from presynaptic terminals defines a neuronal protransmission signal. Copyright © 2014 Elsevier Inc. All rights reserved.