AP2 regulates Thickveins trafficking through Rab11 to attenuate NMJ growth signaling inDrosophila

Abstract

Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remains poorly understood. Here we show that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with BMP type I receptor, Thickveins (Tkv), and Daughter against decapentaplegic (Dad), two of the components of BMP signaling. We found that mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the NMJ and results in a significant reduction in Tkv-positive early endosomes in the presynaptic terminals. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. Consistent with the role of σ2-adaptin and Rab11 in the regulation of the same signaling pathway, a mutation in Rab11 or overexpression of a GDP-locked form of Rab11 (Rab11S25N) phenocopies the morphological and signaling defects of the σ2-adaptin mutants. Finally, we demonstrate that σ2-adaptin mutants show an accumulation of large vesicles and massive membranous structures, akin to endosomes at the synapse. Thus, we propose a model in which AP2 regulates Tkv internalization and recycling through a process that requires Rab11 activity to control the synaptic growth.

Local regulation of extracellular vesicle traffic by the synaptic endocytic machinery

“…However, we find that mutants with more severe defects in synaptic vesicle recycling (e.g., AP-2α ; Gonzalez-Gaitan and Jackle, 1997 ) exhibit very mild EV phenotypes, while mutants with mild synaptic vesicle cycling phenotypes (e.g., nwk and dap160 ; Del Signore et al, 2021 ; Koh et al, 2004 ; Marie et al, 2004 ) have severe EV defects. In a similar vein, AP-2α , AP-2μ , and dap160 mutants exhibit severe synaptic growth phenotypes ( Choudhury et al, 2016 ; Dwivedi et al, 2021 ; Koh et al, 2004 ; Marie et al, 2004 ), likely due to the failure to downregulate BMP receptor signaling ( Deshpande and Rodal, 2015 ). Among these, only loss of dap160 has a severe EV defect.…”

“…The canonical function of endocytic proteins is to control clathrin-mediated endocytosis, which is a primary mechanism for synaptic vesicle recycling at the Drosophila larval NMJ ( Heerssen et al, 2008 ; Kasprowicz et al, 2008 ) and is required for synaptic morphogenesis via endocytic traffic of growth factor receptors ( Choudhury et al, 2016 ; Dwivedi et al, 2021 ). We asked if clathrin and its heterotetrameric AP-2 adaptor complex were similarly involved in synaptic EV cargo traffic.…”

Local regulation of extracellular vesicle traffic by the synaptic endocytic machinery

“…The canonical function of endocytic proteins is to control clathrin-mediated endocytosis, which is the primary mechanism for synaptic vesicle recycling at the Drosophila larval NMJ (Heerssen et al, 2008; Kasprowicz et al, 2008), and is required for synaptic morphogenesis via endocytic traffic of growth factor receptors (Choudhury et al, 2016; Dwivedi et al, 2021). We asked if clathrin and its heterotetrameric AP-2 adaptor complex were similarly involved in synaptic EV cargo traffic.…”

“…nwk and dap160 (Del Signore et al, 2021;Koh et al, 2004;Marie et al, 2004)) have severe EV defects. In a similar vein, AP-2α, AP-2µ and dap160 mutants exhibit severe synaptic growth phenotypes (Choudhury et al, 2016;Dwivedi et al, 2021;Koh et al, 2004;Marie et al, 2004), likely due to failure to downregulate BMP (Bone Morphogenetic Protein) receptor signaling (Deshpande and Rodal, 2015). Among these, only dap160 has a severe EV defect.…”