Syndapins belong to the F-BAR domain protein family whose predicted functions in membrane tubulation remain poorly studied in vivo. At Drosophila neuromuscular junctions, syndapin is associated predominantly with a tubulolamellar postsynaptic membrane system known as the subsynaptic reticulum (SSR). We show that syndapin overexpression greatly expands this postsynaptic membrane system. Syndapin can expand the SSR in the absence of dPAK and Dlg, two known regulators of SSR development. Syndapin's N-terminal F-BAR domain, required for membrane tubulation in cultured cells, is required for SSR expansion. Consistent with a model in which syndapin acts directly on postsynaptic membrane, SSR expansion requires conserved residues essential for membrane binding in vitro. However, syndapin's Src homology (SH) 3 domain, which negatively regulates membrane tubulation in cultured cells, is required for synaptic targeting and strong SSR induction. Our observations advance knowledge of syndapin protein function by 1) demonstrating the in vivo relevance of membrane remodeling mechanisms suggested by previous in vitro and structural analyses, 2) showing that SH3 domains are necessary for membrane expansion observed in vivo, and 3) confirming that F-BAR proteins control complex membrane structures.

Studies in cell-free systems and the lamprey giant synapse have implicated crucial roles for amphiphysin and endophilin in synaptic transmission. However, null mutants at the amphiphysin locus of Drosophila are viable and have no demonstrable synaptic vesicle-recycling defect. This has necessitated a re-examination of the role of Src homology 3 domain-containing proteins in synaptic vesicle recycling. In this report, we show that endophilin-deficient eye clones in Drosophila have an altered electroretinogram. A characteristic of this defect is its aggravation during heightened visual stimulation. It is shown that endophilin is primarily required in the nervous system. Decreased endophilin activity results in alterations in the neuromuscular junction structure and physiology. Immunofluorescence studies show colocalization of endophilin with dynamin consistent with a possible role in synaptic vesicle recycling.

Syndapin is a conserved dynamin-binding protein, with predicted function in synaptic-vesicle endocytosis. Here, we combine genetic mutational analysis with in vivo cell biological assays to ask whether Drosophila syndapin (Synd) is an essential component of synaptic-vesicle recycling. The only isoform of Drosophila syndapin (synd) is broadly expressed and at high levels in the nervous system. synd mutants are late-larval lethals, but fertile adult “escapers” frequently emerge. Contrary to expectation, we report that the Synd protein is predominantly postsynaptic, undetectable at presynaptic varicosities at Drosophila third-instar larval neuromuscular junctions. Electrophysiological and synaptopHluorin imaging in control, synd-deficient or synd-overexpressing motor neurons reveals that synd is dispensable for synaptic-vesicle endocytosis. Our work in Drosophila leads to the suggestion that syndapin may not be a general or essential component in dynamin-dependent synaptic-vesicle endocytosis.

The functional requirement of adapter protein 2 (AP2) complex in synaptic membrane retrieval by clathrin-mediated endocytosis is not fully understood. Here we isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction (NMJ) synapse, we named this mutation angur (a Hindi word meaning "grapes"). Loss-of-function alleles of angur show more than twofold overgrowth in bouton numbers and a dramatic decrease in bouton size. We mapped the angur mutation to s 2 -adaptin, the smallest subunit of the AP2 complex. Reducing the neuronal level of any of the subunits of the AP2 complex or disrupting AP2 complex assembly in neurons phenocopied the s 2 -adaptin mutation. Genetic perturbation of s 2 -adaptin in neurons leads to a reversible temperature-sensitive paralysis at 38°. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high-frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic levels of subunits of the AP2 complex and clathrin, but not other endocytic proteins, were reduced in the mutants. Moreover, bone morphogenetic protein (BMP)/transforming growth factor b (TGFb) signaling was altered in these mutants and was restored by normalizing s 2 -adaptin in neurons. Thus, our data suggest that (1) while s 2 -adaptin facilitates synaptic vesicle (SV) recycling for basal synaptic transmission, its activity is also required for regenerating SVs during highfrequency nerve stimulation, and (2) s 2 -adaptin regulates NMJ morphology by attenuating TGFb signaling. KEYWORDS Drosophila; angur; synapse; physiology; pMAD S YNAPTIC transmission requires fusion of synaptic vesicles (SVs) at the active zones followed by their efficient retrieval and recycling through endocytic mechanisms (Heuser and Reese 1973;Jahn and Sudhof 1994). Retrieval and sorting of membrane lipids and vesicular proteins at the synapse are mediated by a well-orchestrated and coordinated action of several adapter and endocytic proteins (Stimson et al. 2001;Rikhy et al. 2002;Verstreken et al. 2002;Koh et al. 2004;Marie et al. 2004). Clathrin-mediated endocytosis (CME) is the primary pathway operative at the synapses for membrane retrieval (Granseth et al. 2006(Granseth et al. , 2007Heerssen et al. 2008;Dittman and Ryan 2009;McMahon and Boucrot 2011;Saheki and De Camilli 2012). Genetic analysis of the components of the CME pathway in Caenorhabditis elegans and Drosophila has revealed that this pathway is required for SV re-formation, and in many cases, blocking CME at synapses results in temperature-sensitive paralysis (Gonzalez-Gaitan and Jackle 1997;Zhang et al. 1998;Stimson et al. 2001;Koh et al. 2004Koh et al. , 2007Sato et al. 2009). Additionally, CME plays a crucial role in regulating synaptic morphology (Rikhy et al. 2002;Koh et al. 2004Koh et al. , 2007Dickman et al. 2006). At Drosophila NMJs, blocking CME results in enhanced bone morphogenetic protein (BMP) si...

In all nervous systems, short-term enhancement of transmitter release is achieved by increasing the weights of unitary synapses; in contrast, long-term enhancement, which requires nuclear gene expression, is generally thought to be mediated by the addition of new synaptic vesicle release sites. In Drosophila motor neurons, induction of AP-1, a heterodimer of Fos and Jun, induces cAMP-and CREB-dependent forms of presynaptic enhancement. Light and electron microscopic studies indicate that this synaptic enhancement is caused by increasing the weight of unitary synapses and not through the insertion of additional release sites. Electrophysiological and optical measurements of vesicle dynamics demonstrate that enhanced neurotransmitter release is accompanied by an increase in the actively cycling synaptic vesicle pool at the expense of the reserve pool. Finally, the observation that AP-1 mediated enhancement eliminates tetanus-induced forms of presynaptic potentiation suggests: (i) that reserve-pool mobilization is required for tetanus-induced short-term synaptic plasticity; and (ii) that long-term synaptic plasticity may, in some instances, be accomplished by stable recruitment of mechanisms that normally underlie short-term synaptic change. neuromuscular junction ͉ facilitation ͉ augmentation ͉ posttetanic potentiation