Dap160/intersectin acts as a stabilizing scaffold required for synaptic development and vesicle endocytosis

We describe the isolation of mutations in dynamin-associated protein 160 kDa (dap160), the Drosophila homolog of intersectin, a putative adaptor for proteins involved in endocytosis, cytoskeletal regulation, and signaling. We show that partial loss-of-function mutants display temperature-sensitive (ts) paralysis, whereas null mutants show ts defects in endocytosis. Loss-of-function mutants exhibit bouton overgrowth at larval neuromuscular junctions (NMJs), but evoked neurotransmission is normal. Mutant NMJs show a mild endocytic defect at 22 degrees C, which is strongly enhanced at 34 degrees C. The levels of dynamin, synaptojanin and endophilin are severely reduced in dap160 mutant NMJs, suggesting that Dap160 serves to stabilize an endocytic macromolecular complex. Electron microscopy reveals fewer vesicles, aberrant large vesicles, and an accumulation of endocytic intermediates at active and periactive zones in mutant terminals. Our data suggest that Dap160, like dynamin, is involved in synaptic vesicle retrieval at active and periactive zones.     

Endophilin is required for synaptic vesicle endocytosis by localizing synaptojanin

Endophilin is a membrane-associated protein required for endocytosis of synaptic vesicles. Two models have been proposed for endophilin: that it alters lipid composition in order to shape membranes during endocytosis, or that it binds the polyphosphoinositide phosphatase synaptojanin and recruits this phosphatase to membranes. In this study, we demonstrate that the unc-57 gene encodes the Caenorhabditis elegans ortholog of endophilin A. We demonstrate that endophilin is required in C. elegans for synaptic vesicle recycling. Furthermore, the defects observed in endophilin mutants closely resemble those observed in synaptojanin mutants. The electrophysiological phenotype of endophilin and synaptojanin double mutants are virtually identical to the single mutants, demonstrating that endophilin and synaptojanin function in the same pathway. Finally, endophilin is required to stabilize expression of synaptojanin at the synapse. These data suggest that endophilin is an adaptor protein required to localize and stabilize synaptojanin at membranes during synaptic vesicle recycling.     

Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin

Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.     

Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.     

Mutations in synaptojanin disrupt synaptic vesicle recycling

Synaptojanin is a polyphosphoinositide phosphatase that is found at synapses and binds to proteins implicated in endocytosis. For these reasons, it has been proposed that synaptojanin is involved in the recycling of synaptic vesicles. Here, we demonstrate that the unc-26 gene encodes the Caenorhabditis elegans ortholog of synaptojanin. unc-26 mutants exhibit defects in vesicle trafficking in several tissues, but most defects are found at synaptic termini. Specifically, we observed defects in the budding of synaptic vesicles from the plasma membrane, in the uncoating of vesicles after fission, in the recovery of vesicles from endosomes, and in the tethering of vesicles to the cytoskeleton. Thus, these results confirm studies of the mouse synaptojanin 1 mutants, which exhibit defects in the uncoating of synaptic vesicles (Cremona, O., G. Di Paolo, M.R. Wenk, A. Luthi, W.T. Kim, K. Takei, L. Daniell, Y. Nemoto, S.B. Shears, R.A. Flavell, D.A. McCormick, and P. De Camilli. 1999. Cell. 99:179-188), and further demonstrate that synaptojanin facilitates multiple steps of synaptic vesicle recycling.     

Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission

Endophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.     

ITSN-1 controls vesicle recycling at the neuromuscular junction and functions in parallel with DAB-1

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 membrane-associated 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.     

Endophilin functions as a membrane-bending molecule and is delivered to endocytic zones by exocytosis

Two models have been proposed for endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that endophilin's SH3 domain recruits essential endocytic proteins, whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, whereas those disrupting membrane bending cause significant defects. By anchoring endophilin to the plasma membrane, we show that endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This endophilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.     

Polyunsaturated fatty acids influence synaptojanin localization to regulate synaptic vesicle recycling

The lipid polyunsaturated fatty acids are highly enriched in synaptic membranes, including synaptic vesicles, but their precise function there is unknown. Caenorhabditis elegans fat-3 mutants lack long-chain polyunsaturated fatty acids (LC-PUFAs); they release abnormally low levels of serotonin and acetylcholine and are depleted of synaptic vesicles, but the mechanistic basis of these defects is unclear. Here we demonstrate that synaptic vesicle endocytosis is impaired in the mutants: the synaptic vesicle protein synaptobrevin is not efficiently retrieved after synaptic vesicles fuse with the presynaptic membrane, and the presynaptic terminals contain abnormally large endosomal-like compartments and synaptic vesicles. Moreover, the mutants have abnormally low levels of the phosphoinositide phosphatase synaptojanin at release sites and accumulate the main synaptojanin substrate phosphatidylinositol 4,5-bisphosphate at these sites. Both synaptobrevin and synaptojanin mislocalization can be rescued by providing exogenous arachidonic acid, an LC-PUFA, suggesting that the endocytosis defect is caused by LC-PUFA depletion. By showing that the genes fat-3 and synaptojanin act in the same endocytic pathway at synapses, our findings suggest that LC-PUFAs are required for efficient synaptic vesicle recycling, probably by modulating synaptojanin localization at synapses.     

Characterization of two distinct modes of endophilin in clathrin-mediated endocytosis

Endophilin, one of the main accessory proteins involved in clathrin-mediated endocytosis, interacts with other endocytic proteins, such as dynamin, by its SH3 domain. We previously reported that voltage-gated Ca(2+) channels are an integral part of the synaptic vesicle (SV) endocytosis machinery through their interaction with endophilin. Formation of the endophilin-channel complex is Ca(2+) dependent. A glutamate residue, E264, in endophilin is part of the primary Ca(2+) sensor for Ca(2+)-dependent formation of the channel-endophilin complex. We proposed that endophilin exists in two distinct modes (conformations), an open mode in the absence of Ca(2+), and a closed mode in the presence of Ca(2+). Binding of Ca(2+) switches endophilin from its open mode to the closed mode, resulting in dissociation of endophilin from other proteins. The present study is aimed at understanding the functional roles of endophilin in its two different modes, by creating two endophilin mutants, E264A and E264R, to mimic endophilin in its permanent open mode and permanent closed mode respectively. Here, we show that these two modes of endophilin have different effects on how endophilin interacts with other proteins, such as dynamin or β1-adrenergic receptors. In living cells, endophilin in its permanent closed mode does not show obvious effects on agonist-induced internalization of β1-adrenergic receptors. Endophilin, when in its permanent open mode, enhances the short-term synaptic depression in cultured hippocampal neurons, due partly to its failure to dissociate from Ca(2+) channels in the presence of Ca(2+). Our results show that modal switching by Ca(2+) allows endophilin to regulate, more effectively, the clathrin-mediated endocytosis of SV at the nerve terminal.     

MAN1 Restricts BMP Signaling During Synaptic Growth in <Emphasis Type="Italic">Drosophila</Emphasis>

Bone morphogenic protein (BMP) signaling is crucial for coordinated synaptic growth and plasticity. Here, we show that the nuclear LEM-domain protein MAN1 is a negative regulator of synaptic growth at Drosophila larval and adult neuromuscular junctions (NMJs). Loss of MAN1 is associated with synaptic structural defects, including floating T-bars, membrane attachment defects, and accumulation of vesicles between perisynaptic membranes and membranes of the subsynaptic reticulum. In addition, MAN1 mutants accumulate more heterogeneously sized vesicles and multivesicular bodies in larval and adult synapses, the latter indicating that MAN1 may function in synaptic vesicle recycling and endosome-to-lysosome trafficking. Synaptic overgrowth in MAN1 is sensitive to BMP signaling levels, and loss of key BMP components attenuate BMP-induced synaptic overgrowth. Based on these observations, we propose that MAN1 negatively regulates accumulation and distribution of BMP signaling components to ensure proper synaptic growth and integrity at larval and adult NMJs.     

Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse

Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.     

Drosophila Amphiphysin is a Post-Synaptic Protein Required for Normal Locomotion but Not Endocytosis

Clathrin-mediated endocytosis is required to recycle synaptic vesicles for fast and efficient neurotransmission. Amphiphysins are thought to be multiprotein adaptors that may contribute to this process by bringing together many of the proteins required for endocytosis. Their in vivo function, however, has yet to be determined. Here, we show that the Drosophila genome encodes a single amphiphysin gene that is broadly expressed during development. We also show that, unlike its vertebrate counterparts, Drosophila Amphiphysin is enriched postsynaptically at the larval neuromuscular junction. To determine the role of Drosophila Amphiphysin, we also generated null mutants which are viable but give rise to larvae and adults with pronounced locomotory defects. Surprisingly, the locomotory defects cannot be accounted for by alterations in the morphology or physiology of the neuromuscular junction. Moreover, using stimulus protocols designed to test endocytosis under moderate and extreme vesicle cycling, we could not detect any defect in the neuromuscular junction of the amphiphysin mutant. Taken together, our findings suggest that Amphiphysin is not required for viability, nor is it absolutely required for clathrin-mediated endocytosis. However, Drosophila Amphiphysin function is required in both larvae and adults for normal locomotion.     

Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling

Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.     

Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis

Endophilin/SH3p4 is a protein highly enriched in nerve terminals that binds the GTPase dynamin and the polyphosphoinositide phosphatase synaptojanin, two proteins implicated in synaptic vesicle endocytosis. We show here that antibody-mediated disruption of endophilin function in a tonically stimulated synapse leads to a block in the invagination of clathrin-coated pits adjacent to the active zone and therefore to a block of synaptic vesicle recycling. We also show that in a cell-free system, endophilin is not associated with clathrin coats and is a functional partner of dynamin. Our findings suggest that endophilin is part of a biochemical machinery that acts in trans to the clathrin coat from early stages to vesicle fission.     

Formation of an endophilin-Ca2+ channel complex is critical for clathrin-mediated synaptic vesicle endocytosis

A tight balance between synaptic vesicle exocytosis and endocytosis is fundamental to maintaining synaptic structure and function. Calcium influx through voltage-gated Ca2+ channels is crucial in regulating synaptic vesicle exocytosis. However, much less is known about how Ca2+ regulates vesicle endocytosis or how the endocytic machinery becomes enriched at the nerve terminal. We report here a direct interaction between voltage-gated Ca2+ channels and endophilin, a key regulator of clathrin-mediated synaptic vesicle endocytosis. Formation of the endophlin-Ca2+ channel complex is Ca2+ dependent. The primary Ca2+ binding domain resides within endophilin and regulates both endophilin-Ca2+ channel and endophilin-dynamin complexes. Introduction into hippocampal neurons of a dominant-negative endophilin construct, which constitutively binds to Ca2+ channels, significantly reduces endocytosis-mediated uptake of FM 4-64 dye without abolishing exocytosis. These results suggest an important role for Ca2+ channels in coordinating synaptic vesicle recycling by directly coupling to both exocytotic and endocytic machineries.     

A slowed classical pathway rather than kiss-and-run mediates endocytosis at synapses lacking synaptojanin and endophilin

The extent to which a "kiss-and-run" mode of endocytosis contributes to synaptic-vesicle recycling remains controversial. The only genetic evidence for kiss-and-run at the synapse comes from mutations in the genes encoding synaptojanin and endophilin, proteins that together function to uncoat vesicles in classical clathrin-mediated endocytosis. Here we have characterized the endocytosis that persists in null alleles of Drosophila synaptojanin and endophilin. In response to high-frequency stimulation, the synaptic-vesicle pool can be reversibly depleted in these mutants. Recovery from this depletion is slow and indicates the persistence of an impaired form of classical endocytosis. Steady-state exocytosis rates reveal that endocytosis saturates in mutant neuromuscular terminals at approximately 80 vesicles/s, 10%-20% of the wild-type rate. Analyses of quantal size, FM1-43 loading, and dynamin function further demonstrate that, even in the absence of synaptojanin or endophilin, vesicles undergo full fusion and re-formation. Therefore, no genetic evidence remains to indicate that synaptic vesicles undergo kiss-and-run.     

Lipids, lipid modification and lipid-protein interaction in membrane budding and fission--insights from the roles of endophilin A1 and synaptophysin in synaptic vesicle endocytosis

Studies on the endocytosis of synaptic vesicles have provided two novel insights into the mechanism of vesicle formation from donor membranes, both of which concern lipids. One is the essential role of endophilin, a cytosolic protein converting lysophosphatidic acid by addition of the fatty acid arachidonate into phosphatidic acid. The other is the essential role of membrane cholesterol, which specifically interacts with synaptophysin, the major transmembrane protein of synaptic vesicles. These findings reveal novel modes of membrane lipid modification and lipid-protein interaction in vesicle biogenesis.     

The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin

Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.     

The synaptic vesicle cycle: exocytosis and endocytosis in Drosophila and C. elegans

Advances in the study of Drosophila melanogaster and Caenorhabditis elegans have provided key insights into the processes of neurotransmission and neuromodulation. Work in the past year has revealed that Unc-13 and Rab3a-interacting molecule regulate the conformational state of syntaxin to prime synaptic vesicle fusion. Analyses of synaptotagmin support its role as a putative calcium sensor triggering vesicular fusion and highlight the possible role of SNARE complex oligomerization in the fusion mechanism. Characterization of endophilin mutants demonstrates that kiss-and-run endocytosis is a major component of synaptic vesicle recycling. In neuromodulation, dcaps mutants provide the first genetic insight into possible roles of the CAPS protein in mediating dense core vesicle fusion and modulating synaptic vesicle fusion.