UNC-41/Stonin Functions with AP2 to Recycle Synaptic Vesicles in Caenorhabditis elegans

Abstract

The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.

Cargo Recognition in Clathrin-Mediated Endocytosis

“…Worms with both snt-1 and unc-41 genes disrupted have a more severe phenotype than with either single mutation alone (Mullen et al 2012). And forced SNT-1 overexpression does not rescue the unc-41 phenotype, unlike in Drosophila (Fergestad and Broadie 2001).…”

“…The stonins are ubiquitously expressed in chordate tissues but seem most critical in neurons; mutant alleles or silencing produces neurological phenotypes in flies, worms, and mice (Stimson et al 2001;Estes et al 2003;Mullen et al 2012;Kononenko et al 2013). At the synapse, tight exo-endocytic coupling maintains a pool of neurotransmitter-filled synaptic vesicles.…”

“…Biochemical and functional evidence indicates that the C2 domains of synaptotagmin 1 bind physically to the stonin 2 mHD (Martina et al 2001;Walther et al 2001;Diril et al 2006;Jung et al 2007). In neurons, the two proteins colocalize in puncta, and proper subcellular localization of SNT-1 depends on UNC-41 (the respective nematode orthologs) but not vice versa (Mullen et al 2012). Because synaptotagmin (snt-1)-null worms phenocopy stonin (stn-1/ unc-41) mutants (Mullen et al 2012), a principal task of stonins is likely to promote uptake of synaptotagmin at nerve terminals.…”

“…In neurons, the two proteins colocalize in puncta, and proper subcellular localization of SNT-1 depends on UNC-41 (the respective nematode orthologs) but not vice versa (Mullen et al 2012). Because synaptotagmin (snt-1)-null worms phenocopy stonin (stn-1/ unc-41) mutants (Mullen et al 2012), a principal task of stonins is likely to promote uptake of synaptotagmin at nerve terminals.…”

“…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.…”