ITSN‐1 Controls Vesicle Recycling at the Neuromuscular Junction and Functions in Parallel with DAB‐1

Abstract

Intersectins (Itsn) are conserved EH and SH3 domain containing adaptor proteins. In Drosophila melanogaster, ITSN is required to regulate synaptic morphology, to facilitate efficient synaptic vesicle recycling and for viability. Here, we report our genetic analysis of Caenorhabditis elegans intersectin. In contrast to Drosophila, C. elegans itsn-1 protein null mutants are viable and display grossly normal locomotion and development. However, motor neurons in these mutants show a dramatic increase in large irregular vesicles and accumulate membraneassociated vesicles at putative endocytic hotspots, approximately 300 nm from the presynaptic density. This defect occurs precisely where endogenous ITSN-1 protein localizes in wild-type animals and is associated with a significant reduction in synaptic vesicle number and reduced frequency of endogenous synaptic events at neuromuscular junctions (NMJs). ITSN-1 forms a stable complex with EHS-1 (Eps15) and is expressed at reduced levels in ehs-1 mutants. Thus, ITSN-1 together with EHS-1, coordinate vesicle recycling at C. elegans NMJs. We also found that both itsn-1 and ehs-1 mutants show poor viability and growth in a Disabled (dab-1) null mutant background. These results show for the first time that intersectin and Eps15 proteins function in the same genetic pathway, and appear to function synergistically with the clathrin-coat-associated sorting protein, Disabled, for viability.

Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5

“…NPF repeats commonly interact with the EH-domain and regulate endocytosis and endocytic recycling (41, 64 -66). Furthermore, intersectins, EH-domaincontaining proteins that were reported to bind to the NPF repeats of SCAMP (39), regulate recycling of synaptic vesicles in Drosophila and Caenorhabditis elegans (67)(68)(69)(70). Thus, we hypothesize that SCAMP2 recruits cytosolic EH-domain proteins to recycling endosomes via its N-terminal NPF repeats and promotes vesicle formation and the plasma membrane targeting of NHE5.…”

Cargo Recognition in Clathrin-Mediated Endocytosis

“…By binding to multiple adaptors or CLASPs, cargoes participate directly in the network, and thus modulate the coat assembly process (Mettlen et al 2010). A corollary of this densely wired redundant network is that gene silencing or RNAi of numerous individual CLASPs does not have a dramatic penetrant effect on endocytosis (Garcia et al 2001;Kang-Decker et al 2001;Morris et al 2002;Kamikura and Cooper 2003;Holmes et al 2007;Koh et al 2007;Wang et al 2008;Chen et al 2009;Mullen et al 2012;Pozzi et al 2012;Scotland et al 2012;Suzuki et al 2012;Umasankar et al 2012;Kononenko et al 2013;Tsushima et al 2013). Rather, subtle or tissue-specific defects are manifest linked to im- proper signal relay by the cargo affected.…”

Intersectin Regulates Dendritic Spine Development and Somatodendritic Endocytosis but Not Synaptic Vesicle Recycling in Hippocampal Neurons

“…Intersectin is a multimodule scaffolding protein that interacts with a wide range of proteins, including several involved in CME (16). Intersectin has two N-terminal Eps15 homology domains that are responsible for binding to epsin, SCAMP1, and numb (17)(18)(19), a central coilcoiled domain that interacts with Eps15 and SNAP-23 and -25 (17,20,21), and five SH3 domains in its C-terminal region that interact with multiple proline-rich domain proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS (16,(22)(23)(24)(25). The rich binding capability of intersectin has linked it to various functions from CME (17,26,27) and signaling (22,28,29) to mitogenesis (30,31) and regulation of the actin cytoskeleton (23).…”

“…Intersectin functions in SV recycling at the neuromuscular junction of Drosophila and C. elegans where it acts as a scaffold, regulating the synaptic levels of endocytic accessory proteins (21,(32)(33)(34). In vertebrates, the intersectin gene is subject to alternative splicing, and a longer isoform (intersectin-l) is generated that is expressed exclusively in neurons (26,28,35,36).…”