Dap160/Intersectin Scaffolds the Periactive Zone to Achieve High-Fidelity Endocytosis and Normal Synaptic Growth

Abstract

Dap160/Intersectin is a multidomain adaptor protein that colocalizes with endocytic machinery in the periactive zone at the Drosophila NMJ. We have generated severe loss-of-function mutations that eliminate Dap160 protein from the NMJ. dap160 mutant synapses have decreased levels of essential endocytic proteins, including dynamin, endophilin, synaptojanin, and AP180, while other markers of the active zone and periactive zone are generally unaltered. Functional analyses demonstrate that dap160 mutant synapses are unable to sustain high-frequency transmitter release, show impaired FM4-64 loading, and show a dramatic increase in presynaptic quantal size consistent with defects in synaptic vesicle recycling. The dap160 mutant synapse is grossly malformed with abundant, highly ramified, small synaptic boutons. We present a model in which Dap160 scaffolds both endocytic machinery and essential synaptic signaling systems to the periactive zone to coordinately control structural and functional synapse development.

RAB-10 Regulates Glutamate Receptor Recycling in a Cholesterol-dependent Endocytosis Pathway

“…Intersectin proteins contain multiple protein-protein interaction domains, and they have been shown to bind proteins involved in processes such as actin remodeling, signal transduction, exocytosis, and endocytosis (Roos and Kelly, 1998;Koh et al, 2004;Marie et al, 2004;Martin et al, 2006;Evergren et al, 2007). The Drosophila orthologue of Intersectin, Dap160, is essential for viability, and it functions as an endocytic scaffolding protein at neuromuscular junctions (Koh et al, 2004;Marie et al, 2004). In mammals, which have two Intersectin homologues, there is evidence suggesting that an interaction between Intersectin and the E3 ubiquitin ligase Cbl is important for endocytosis and degradation of epidermal growth factor receptor (Martin et al, 2006).…”

“…The Intersectin adaptor protein is thought to function in clathrin-dependent endocytosis by recruiting endocytosis machinery to clathrin-coated pits (Yamabhai et al, 1998;Sengar et al, 1999;Simpson et al, 1999;Koh et al, 2004;Marie et al, 2004). The C. elegans genome contains a single Intersectin orthologue, called ITSN-1, which is most similar to Drosophila dynamin-associated protein of 160 kDa (Dap160) and the short isoform of human ITSN1 ( Figure 5A).…”

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis

“…A crucial part in synaptic vesicle endocytosis is played by the GTPase dynamin (FIG. 2), a mechanochemical enzyme recruited to endocytic sites by interaction with SH3 domain proteins 58,59 , including intersectin [60][61][62] , amphiphysin 63 , endophilin 64,65 and syndapin 66 , suggesting a tight interplay between BAR-SH3 protein-mediated membrane deformation (FIG. 2) and dynamin-catalyzed fission 58,67 .…”

“…Genetic studies have identified the multidomain endocytic scaffolding proteins intersectin [60][61][62]71 and epidermal growth factor receptor substrate 15 (EPS15) 72 as crucial for synaptic vesicle membrane retrieval and synapse development in Drosophila melanogaster and Caenorhabditis elegans. Expression levels of intersectin and EPS15 are interdependent, and the phenotypes observed on loss of either protein are nearly identical, indicating a close functional relationship between both proteins 72 .…”

“…Consistent with these observations EPS15 and intersectin, together with FCHo proteins, serve as nucleators for clathrin-coated pit assembly 73 . Drosophila melanogaster intersectin mutants display severe defects in synaptic vesicle recycling, an accumulation of endocytic intermediates at active and periactive zones, and reduced levels of endocytic proteins 60,61 . As intersectin interacts with several endocytic proteins, including AP2, stonin 2, dynamin, the exocytic SnARE protein SnAP25, and with the actin regulatory proteins neural wiskott-Aldrich syndrome protein (nwASP) and Evidence for exocytic-endocytic coupling Fusion of synaptic vesicles with the membrane of the nerve terminal implies that the nerve terminal expands.…”