Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development

Members of the DISABLED (DAB) family of proteins are known to play a conserved role in endocytic trafficking of cell surface receptors by functioning as monomeric CLATHRIN-associated sorting proteins that recruit cargo proteins into endocytic vesicles. Here, we report a Drosophila disabled mutant revealing a novel role for DAB proteins in chemical synaptic transmission. This mutant exhibits impaired synaptic function, including a rapid activitydependent reduction in neurotransmitter release and disruption of synaptic vesicle endocytosis. In presynaptic boutons, Drosophila DAB and CLATHRIN were highly colocalized within two distinct classes of puncta, including relatively dim puncta that were located at active zones and may reflect endocytic mechanisms operating at neurotransmitter release sites. Finally, broader analysis of endocytic proteins, including DYNAMIN, supported a general role for CLATHRIN-mediated endocytic mechanisms in rapid clearance of neurotransmitter release sites for subsequent vesicle priming and refilling of the release-ready vesicle pool.I ntensive study of chemical synaptic transmission has led to detailed molecular models of synaptic vesicle endocytosis (1-4). Ongoing efforts seek to define the underlying molecular components and interactions further as well as their spatial organization with respect to neurotransmitter release sites. The present study reveals novel molecular mechanisms and interactions in synaptic vesicle endocytosis involving a member of the DISABLED family of Phosphotyrosine Binding (PTB) domain proteins. In Drosophila, the disabled (dab) gene, the founding member of the dab gene family, was first identified in a screen for genetic modifiers of abelson (5). Although putative dab mutations were subsequently mapped to a different gene (6), recent studies have established functional interactions of Drosophila DISABLED (dDAB) and ABELSON (7). Proteins closely related to dDAB, including mouse DAB-2 (mDAB-2) and the single Caenorhabditis elegans family member ceDAB-1, function as CLATHRIN-associated adaptors in endocytic trafficking of cell surface receptors (8-10). These DABs have been shown to interact with CLATHRIN, the α-ADAPTIN (AP-2) adaptor complex, and other components of the endocytic machinery through conserved binding motifs (8-10) (Fig. 1A). Notably, mDAB-2, ceDAB-1, and dDAB share a PTB/DAB Homology (DH) domain near the N terminus (Fig. 1B), which interacts with specific vesicle cargo proteins and phosphatidylinositol-4,5-diphosphate (11, 12). Neither DAB proteins nor PTB domain interactions have been previously implicated in synaptic vesicle trafficking.The present study reveals a role for DAB proteins in synaptic vesicle endocytosis and extends previous work on interactions of endocytic and exocytic mechanisms in neurotransmitter release. Here, we use a glutamatergic neuromuscular synapse of the Drosophila adult as a model for genetic analysis of molecular mechanisms determining conserved properties of glutamatergic synapse function (13,14). Previous analysis i...

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“…refs. [26][27][28][29][30][31]. First, synaptic vesicle size was increased in both mutants with respect to WT ( Fig.…”

Section: Resultsmentioning

confidence: 99%

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

Kawasaki

Iyer

Posey

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…S1B). In contrast to the essential roles of the FCHO proteins, Eps15, and intersectin in other species (7,8,25), null mutations in each gene failed to substantially impact C. elegans viability or embryonic development (Table S1). …”

Section: The Fei Adaptor Complex Regulates Protein Sorting In the Endmentioning

confidence: 99%

Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane

Mayers¹,

Wang²,

Pramanik³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Endocytic protein trafficking is directed by sorting signals on cargo molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during endocytosis remain poorly defined. Using Caenorhabditis elegans, we demonstrate that multiple plasma membrane endocytic adaptors function redundantly to regulate clathrin-mediated endocytosis and to recruit components of the endosomal sorting complex required for transport (ESCRT) machinery to the cell surface to direct the sorting of ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the endolysosomal system. In the absence of a heterooligomeric adaptor complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of its clathrin-mediated internalization. Consistent with a role for the ESCRT machinery during cargo endocytosis, we further show that the ESCRT-0 complex accumulates at a subset of clathrin-coated pits on the surface of human cells. Our findings suggest a unique mechanism by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway.clathrin | multivesicular endosome

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“…Within the periactive zone, exocytosed synaptic vesicle proteins are translocated and then recaptured by endocytic sorting adaptors, including stonin 2 (also known as stoned B) and perhaps AP180. It is likely that these adaptor proteins in turn are spatiotemporally organized by large multidomain scaffolds such as intersectin and EPS15 (REFS 62,72). The periactive zone also contains cytoskeletal elements, in particular actin and perhaps septin filaments.…”

Section: Box 1 | Presynaptic Organization -Exocytic and Endocytic Zonesmentioning

confidence: 99%

“…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 .…”

Section: Box 1 | Presynaptic Organization -Exocytic and Endocytic Zonesmentioning

confidence: 99%

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis

2011

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Communication between nerve cells largely occurs at chemical synapses -specialized sites of cell-cell contact where electrical signals trigger the exocytic release of neurotransmitter, which in turn activates postsynaptic receptor channels. The efficacy with which such chemical signals are transmitted is crucial for the functioning of the nervous system. Modulation of neurotransmission enables nerve cells to respond to a vast range of stimulus patterns. Synaptic activity can also be tremendously diverse; from the fast synapses of the hippocampus, to slow neuropeptide-containing synapses. Indeed, some signalling pathways are active in the absence of stimulation, such as those involved in the processing of sensory information, which transmit tonically at high rates of up to 100 Hz or more 1,2 .Intriguing questions arise from these facts, including how high rates of neurotransmission can be maintained over long periods of time, and what limits the ability of a given synapse to release neurotransmitter during sustained periods of activity. Recent data indicate that in many synapses, exocytosis of neurotransmitter is coupled to endocytosis, and that synapses have evolved a specialized apparatus of scaffolding proteins to comply with these demands. Such scaffolds aid the temporal and spatial coupling of exocytosis and endocytosis, and are crucial for maintaining rapid neurotransmission during sustained activity 3 .Exocytosis of neurotransmitter is triggered by stimulusinduced calcium influx into the nerve terminal 4,5 and the subsequent fusion of synaptic vesicles with the presynaptic plasma membrane at specialized regions called active zones (BOX 1). Exocytosed synaptic vesicle membrane proteins and lipids in turn are recycled at the endocytic or periactive zone (BOX 1) that surrounds the release site, to restore functional synaptic vesicle pools for reuse and to ensure long-term functionality of the synapse 6-8 (FIG. 1). Depending on the type of synapse and the stimulation frequency, several modes of endocytosis with different time constants -for example, fast and slow endocytosisseem to operate 7,9,10 . Clathrin-mediated endocytosis arguably represents the main pathway of synaptic vesicle endocytosis 6,8,11 , although other modes -for example, fast kiss-and-run exocytosis and endocytosis -could operate in parallel.Although the machineries for exocytic membrane fusion 12,13 and for endocytic retrieval of synaptic vesicle membranes have been studied separately in some detail 6,14 , comparatively little is known about the coupling between exocytosis and endocytosis. Here we synthesize recent data from physiological, morphological and biochemical studies in several model systems into hypothetical models for scaffold-based mechanisms underlying exocytic-endocytic coupling.The synaptic vesicle cycle Synaptic vesicle pools. Some defining features of chemical synapses are the presence of one or several clusters of synaptic vesicles in the presynaptic nerve terminal, and ultrastructural specializations at the pre-and postsy...

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Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development

Cited by 120 publications

References 87 publications

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis

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