Academic journal article Genetics

[Sigma]^sub 2^-Adaptin Facilitates Basal Synaptic Transmission and Is Required for Regenerating Endo-Exo Cycling Pool under High-Frequency Nerve Stimulation in Drosophila

Academic journal article Genetics

[Sigma]^sub 2^-Adaptin Facilitates Basal Synaptic Transmission and Is Required for Regenerating Endo-Exo Cycling Pool under High-Frequency Nerve Stimulation in Drosophila

Article excerpt

SYNAPTIC 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, 2007; Heerssen 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. 2004, 2007; Sato et al. 2009). Additionally, CME plays a crucial role in regulating synaptic morphology (Rikhy et al. 2002; Koh et al. 2004, 2007; Dickman et al. 2006). At Drosophila NMJs, blocking CME results in enhanced bone morphogenetic protein (BMP) signaling and affects synaptic growth (Coyle et al. 2004; O'Connor-Giles et al. 2008).

The heterotetrameric adapter protein 2 (AP2) complex is a major effector of the CME pathway. AP2 serves as a major hub for a large number of molecular interactions and links plasma membrane, cargo/signaling molecules, clathrin, and accessory proteins in the CME pathway (Traub 2003; Schmid and McMahon 2007) and hence can directly influence synaptic signaling. The AP2 complex is pseudo-asymmetric and contains four subunits-one each of large a and b2 subunits, one medium m2 subunit, and a small s2 subunit (Matsui and Kirchhausen 1990; Collins et al. 2002; Traub 2003). Depletion of clathrin or its major adapter, AP2, in either Drosophila or mammalian central synapses results in accumulation of endosome-like vacuoles and reduction of SVs, suggesting that CME may not be essential for membrane retrieval (Heerssen et al. 2008; Gu et al. 2013; Kononenko et al. 2014). Similarly, genetic perturbation of m2-adaptin or a-adaptin shows only mild defects in vesicle biogenesis at C. elegans synapses, but simultaneous loss of both adaptins leads to severely compromised SV biogenesis and accumulation of large vacuoles at nerve terminals (Kim and Ryan 2009; Gu et al. 2013). While Drosophila loss-of-function mutations in a-adaptin are embryonic lethal, hypomorphic mutants exhibit reduced FM1-43 uptake, suggesting a compromised endocytosis in these mutants (Gonzalez-Gaitan and Jackle 1997). Whether reduced endocytosis reflects a defect in membrane retrieval or a defect in SV biogenesis remains unclear. Moreover, the consequences of AP2 reduction on synaptic morphology and physiology remain unknown.

Here we present an analysis of Drosophila s2-adaptin in the context of regulating NMJ morphological plasticity and physiology. We first identified a mutation that dramatically altered NMJ morphology. Next, we mapped this mutation to s2-adaptin by deficiency mapping. We show that AP2dependent vesicle endocytosis regulates both synaptic growth and transmitter release. The AP2 complex is a heterotetramer, and our studies in Drosophila show that the four subunits are obligate partners of each other and are required for a functional AP2 complex (Collins et al. 2002). This finding is in contrast to the hemicomplex model in C. elegans,inwhicha/s2 and b2/m2 can sustain the function, if any one of the subunits is mutated (Gu et al. 2013). We find that loss of AP2 disrupts stable microtubule loops of the presynaptic cytoskeleton and exacerbates growth signaling through the phosphorylated Mothers Against Decapentaplegic (pMAD) pathway, suggesting that normal AP2 constrains the TGFb signaling module. …

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