Growth factor signaling in neurons controls the expansion of synaptic arbors in response to activity and external stimuli, leading to long-lasting changes in synapse strength and connectivity that underlie learning and memory. Receptor complexes regulating growth factor signal transduction are internalized via endocytosis and directed to specific subcellular membrane compartments from which they exhibit distinct signaling properties, caused by compartment-specific posttranslational modifications and degradative events, or interactions with local binding partners (Sadowski et al., 2009
). Therefore, defining the mechanisms by which the rate and direction of the flow of endosomal protein traffic are controlled is critical to determining how neuronal signal transduction pathways are tuned up and down after activation. A host of protein factors control membrane traffic through the interconnected tubules and vesicles of the endosomal system, and sorting occurs by isolation of cargo in membrane domains of defined geometry and lipid composition (Bonifacino and Rojas, 2006
). Proteins modulating membrane dynamics and organization are thus well positioned to control the sorting and activities of signaling complexes during endosomal processing.
The Drosophila melanogaster
larval neuromuscular junction (NMJ) serves as a useful model for the regulation of synaptic growth signaling as the muscle surface area expands 100-fold over 4 d of larval development, requiring increased input from its innervating motor neuron to drive contraction. NMJ synaptic arbors expand by adding matched pre- and postsynaptic specializations (termed synaptic boutons) in response to motor neuron synaptic activity, retrograde signals from the muscle to the neuron, and anterograde signals from the neuron to the muscle (Packard et al., 2002
; McCabe et al., 2003
; Yoshihara et al., 2005
). Mutants in proteins regulating endocytosis fail to properly attenuate growth factor signaling at the NMJ, resulting in abnormal overgrowth of the synaptic arbor, with the hallmark phenotype of increased arbor branching and the formation of “satellite” boutons off the main axis of the axon terminal (Dickman et al., 2006
). One such gene, nwk
), encodes a neuron-specific member of the F-BAR (Fes/CIP4 homology Bin-Amphiphysin-Rvs)/SH3 (Src homology 3) family of lipid-binding proteins that control the force-generating assembly of actin filaments at endosomal compartments by activating WASp (Wiskott–Aldrich syndrome protein)/Arp2/3 (actin-related protein 2/3)-mediated actin polymerization (Coyle et al., 2004
). Nwk attenuates the retrograde BMP (bone morphogenic protein) signal mediated by Glass Bottom Boat through its presynaptic receptor Tkv (Thickveins; O’Connor-Giles et al., 2008
), colocalizes with the recycling endosome marker Rab11, and functions together with the endosomal GTPase Cdc42 to control WASp/Arp2/3-mediated actin assembly (Rodal et al., 2008
). However, the mechanisms by which Nwk attenuates synaptic growth factor signaling at this compartment are unknown.
Here, we describe the identification of a novel Nwk-binding partner, sorting nexin 16 (SNX16). SNX16 is a member of the sorting nexin family of proteins that regulate endosomal traffic and are defined by a phosphoinositide-binding Phox homology (PX) domain (Hanson and Hong, 2003
; Choi et al., 2004
; Cullen, 2008
). We demonstrate that SNX16- and Nwk-containing compartments transiently interact in nerve terminals and that signaling at early endosomes is attenuated either through Nwk–SNX16 interactions or through endosomal sorting complex required for transport (ESCRT)–mediated internalization into the endosomal lumen. SNX16–Nwk-mediated traffic regulates synaptic growth signaling cascades, including the BMP and Wingless (Wg) pathways, and is specifically required to down-regulate activated receptors. Our results provide a mechanism by which protein traffic between endosomal intermediates at synapses controls the output of signal transduction pathways, leading to synaptic growth.