BIOLOGICAL OVERVIEW

nwk (nervous wreck), a temperature-sensitive paralytic mutant, causes excessive growth of larval neuromuscular junctions (NMJs), resulting in increased synaptic bouton number and branch formation. Ultrastructurally, mutant boutons have reduced size and fewer active zones, associated with a reduction in synaptic transmission. nwk encodes an FCH and SH3 domain-containing adaptor protein that localizes to the periactive zone of presynaptic terminals and binds to the Drosophila ortholog of Wasp (Wsp), a key regulator of actin polymerization. wsp null mutants display synaptic overgrowth similar to nwk and enhance the nwk morphological phenotype in a dose-dependent manner. Evolutionarily, Nwk belongs to a previously undescribed family of adaptor proteins that includes the human srGAPs, which regulate Rho activity downstream of Robo receptors. It is proposed that Nwk controls synapse morphology by regulating actin dynamics downstream of growth signals in presynaptic terminals (Coyle, 2004).

SH3 adaptor proteins, characterized by the presence of SRC homology 3 (SH3) domains, commonly link transmembrane signaling molecules with cytoskeleton-associated effectors in the cytoplasm. They play a major role in axon pathfinding by recruiting proteins that affect actin polymerization downstream of guidance cue receptors to generate acute changes in growth cone movement. For example, Dreadlocks (Dock), which contains three SH3 domains, facilitates Slit-Robo repulsion in commissural axons of the Drosophila CNS and also mediates projection of retinal axons into optic lobes. Dock recruits proteins that affect Rho GTPase activation, as well as the Rho-dependent kinase, Pak, downstream of transmembrane receptors such as Robo and Dscam (Coyle, 2004 and references therein).

In organisms ranging from yeast to humans, cytoskeletal growth is controlled by the Rho GTPases, including RhoA, Rac, and Cdc42. They act as binary switches that are active in the GTP bound form and inactive in the GDP bound form. Rho signaling is modulated by GTPase activating proteins (GAPs) that accelerate hydrolysis of GTP and guanine nucleotide exchange factors (GEFs) that promote GTP for GDP exchange. Rho proteins and their effectors are highly expressed in nerve terminals and are known to regulate neurite extension, guidance, branching, and stability. Genetic analysis in Drosophila has demonstrated roles for Rho effectors in synapse formation. For example, loss-of-function mutations in p190RhoGAP and the RhoGEF, still life (sif), affect axon growth in mushroom body neurons and motor neurons, respectively. Also, the RhoGEF Trio combines with Dock and Pak to activate Rac during photoreceptor axon guidance (Coyle, 2004 and references therein).

Activated Rho proteins can activate members of the Wiscott-Aldrich Syndrome Protein (Wasp) family. Wasp and its homologs, N-Wasp and the Wave/Scar proteins, promote F-actin assembly at the leading edge of motile cells by activating the ARP2/3 (actin-related protein) complex. This regulation is essential for the formation of lamellipodia and filopodia during axonal and dendritic growth in mammalian neurons. Wasp and N-Wasp contain a CRIB/PBD domain that binds activated Cdc42, an interaction that is required for efficient stimulation of ARP2/3. The Wave/Scars do not directly bind Rho GTPases, but can be activated through binding to Rac-associated intermediaries like IRSp53. All Wasp/N-Wasp and Wave/Scar relatives contain a central proline-rich domain that is recognized by SH3 adaptor proteins in numerous signal transduction pathways. Including Dock and its mammalian homolog, Nck, these SH3 adaptors control the spatial and temporal activation of the Wasps (Coyle, 2004 and references therein).

In addition to growth cone motility, Wasp-dependent F-actin polymerization also plays a role in some types of vesicle transport and endocytosis. Although Wasp and its homologs are not yet implicated in synaptic transmission, functional analysis has revealed a requirement for F-actin in synaptic vesicle recycling and for the induction of long-term potentiation, during which rapid formation and retraction of actin-rich filopodia and dendritic spines are observed both pre- and post-synaptically. Despite the pervasive role of actin in neuronal growth and function, the molecular pathways involving Rho and Wasp at synapses are not well understood (Coyle, 2004).

Nwk, a novel synaptic adaptor protein, binds the Drosophila ortholog of Wasp (Wsp) and regulates both NMJ growth and activity. Nwk has a distinct domain organization, including an N-terminal FCH and two SH3 domains, that is conserved throughout evolution. Mutation of a human Nwk homolog, srGAP3 (Slit-Robo GTPase Activating Protein), is associated with severe mental retardation (Endris, 2002), suggesting that Nwk proteins regulate the development of both vertebrate and invertebrate nervous systems (Coyle, 2004).

Thus nwk encodes an SH3 adaptor protein that interacts with Wsp, a principal regulator of ARP2/3-dependent actin polymerization. Regulated actin polymerization via WASP and its homologs is thought to underlie neurite extension, cell junction formation, receptor-mediated endocytosis, and other processes relevant to synaptic growth and function. These results reveal a novel molecular pathway that potentially links signal transduction and actin dynamics in synapses (Coyle, 2004).

Nwk negatively regulates larval NMJ growth, since null mutants exhibit increased bouton number, branch formation, and total length. In addition, nwk adults undergo rapid temperature-sensitive (ts) paralysis at 38°C, suggesting that Nwk continues to have an important function(s) in the adult nervous system that may include a direct role in neurotransmission. The molecular mechanism of ts paralysis is not understood at present, and further studies are underway to determine how this behavioral defect is related to the morphological changes in nwk synapses. Nevertheless, it has been verified by several means that all of these phenotypes arise from mutations of the same gene. (1) The same abnormalities are observed when nwk is homozygous as when it is hemizygous over Df(3L)Rdl2. (2) Multiple independent alleles of nwk isolated by failure to complement the adult ts paralytic phenotype all display identical changes in larval NMJ development. (3) Each of these phenotypes is partially or completely rescued by panneural expression of a transgene that contains a complete nwk ORF (Coyle, 2004).

While panneural expression of Nwk rescues both larval and adult phenotypes, expression in muscle does not rescue either, indicating that Nwk does not function postsynaptically. In agreement with these observations, it was found that Nwk expression in wild-type larvae is restricted to neurons and is localized to a distinct presynaptic, subcellular region of synaptic boutons termed the periactive zone. It has been proposed that the periactive zone is specialized for regulating synaptic development. It encompasses the area immediately surrounding synaptic densities and contains several known regulators of synaptic growth and plasticity, including the cell adhesion molecule, FasII, the RhoGEF, Sif, and the microtubule-associated protein, DVap-33A. Thus, Nwk is positioned at a critical juncture between transmembrane receptors, cytoskeletal proteins, and active zones (Coyle, 2004).

Nwk contains an N-terminal FCH domain, a novel ARNEY domain, two SH3 domains, and many polyproline sequences. This particular domain arrangement is distinct, found only in Nwk and 11 other homologs in organisms ranging from yeast to humans. Therefore, Nwk is a member of a small subfamily of evolutionarily conserved SH3 adaptor proteins that includes other potential regulators of synaptic development and function. The only substantial difference in domain structure among these 12 proteins is that 7 of them have a RhoGAP domain in place of the SH3a domain. Among the six human or three mouse Nwk homologs, there are proteins of both structural types. In contrast, the D. melanogaster and S. cerevisiae genomes each contain a single Nwk ortholog lacking the RhoGAP domain but containing a second SH3 domain. Nevertheless, the Nwk to Rho signaling pathway may be conserved in these organisms because the S. cerevisiae ortholog of Nwk, Bzz1p, was found to coprecipitate with the RhoGAP protein, ECM25 (Gavin, 2002). Similar to Dock, Nwk is likely to interact with multiple targets in a variety of cellular contexts by virtue of its modular protein binding domains (Coyle, 2004).

It is proposed that Nwk and Wsp interact directly as components of a signal transduction pathway in neurons controlling NMJ growth and bouton branching on the basis of several lines of evidence. (1) The SH3a domain of Nwk binds Wsp in vitro, and Wsp interacts directly with the SH3 domains of Nwk in the yeast two-hybrid system. (2) Nwk and Wsp immunoreactivity overlap in discrete regions of periactive zones. (3) nwk and wsp null mutations increase synaptic growth and bouton branching in identical fashion. (4) Each mutant can be rescued by presynaptic expression of the respective wild-type gene. (5) The severity of the nwk and wsp mutant phenotypes are enhanced by removal of the other gene in a dose-dependent manner. (6) The proposed interaction between Nwk and Wsp is supported by studies in yeast, demonstrating that the ortholog of Nwk, Bzz1p, binds to the ortholog of Wasp, Las17p, promoting actin polymerization and regulating yeast cell growth (Soulard, 2002; Coyle, 2004 and references therein).

Wasp and its relatives such as Wave/Scar stimulate the ARP2/3 complex, which nucleates and crosslinks F-actin. Other neuronally expressed proteins have distinct effects on the actin cytoskeleton. For example, Cortactin stablizes F-actin branches and promotes ARP2/3 activity independently of Wasp; Ena/Vasp increases the rate of actin filament extension; and Cofilin severs existing filaments to accelerate F-actin turnover. The Drosophila genome contains homologs of scar, cortactin, ena, cofilin, and others that may function either in parallel or antagonistically to wsp. Consequently, loss of Wsp may only partially or selectively disrupt F-actin polymerization at the synapse. In support of this possibility, phalloidin staining was not abolished in wsp mutant NMJs, although the precise arrangement of individual actin filaments cannot be resolved (Coyle, 2004).

Adaptor proteins such as Nwk may specify which combinations of actin regulators are recruited in response to specific cues. Different combinations of actin regulators may contribute to distinct cellular mechanisms such as adhesion, axon extension, endocytosis, and vesicle transport. It is proposed that the morphological abnormalities observed in nwk and wsp single and double mutants result from altered activity in a Nwk/Wsp/ARP2/3-dependent pathway that regulates one or more of these mechanisms within periactive zones (Coyle, 2004).

The involvement of ARP2/3 is inferred from the extensively documented role of Wsp in various cell types including neurons. However, the possibility cannot be excluded that the Nwk/Wasp interaction in boutons may involve other, novel functions (Coyle, 2004).

Recent analysis of three human Nwk homologs, the Slit-Robo GAPs (srGAPs 1-3), supports the proposal that Nwk family members link transmembrane signals with cytoskeletal effectors. Robo receptors bind secreted ligands, Slits, to control neuronal migration and axon guidance in mammals and insects. The srGAPs share the same domain organization with Nwk, except they contain one SH3 domain and a RhoGAP domain instead of two SH3 domains. The SH3 domain of the srGAPs binds to the cytoplasmic CC3 domain of activated Robo1, and the neighboring RhoGAP domain locally inactivates Cdc42 (Wong, 2001). As a result, ARP2/3-dependent F-actin assembly is decreased downstream of activated Robo and migrating neurons are repulsed from Slit. This activity is important for CNS development, since mutations of srGAP3 are associated with severe mental retardation (Endris, 2002). Thus, Nwk and its relatives potentially regulate F-actin polymerization and synaptic growth in both vertebrates and invertebrates (Coyle, 2004).

In Drosophila, mutations in nwk lead not only to increased NMJ growth, but also to decreased synaptic transmission. At nwk NMJs, quantal content is reduced by more than half, indicating that presynaptic vesicle release is curtailed. However, it is not believed that the overgrowth observed in nwk NMJs is a secondary, compensatory effect of reduced synaptic transmission for several reasons: (1) mutations that directly decrease quantal content at larval NMJs, such as in synaptotagmin and methuselah, do not cause compensatory overgrowth in presynaptic terminals; (2) Nwk localizes to the periactive zone and is therefore unlikely to have a primary effect on synaptic vesicle exocytosis in active zones; (3) no decreases were detected in the distribution or size of synaptic vesicles in the ultrastructural analysis of synaptic boutons or in functional assays of quantal size; (4) normal endocytosis of synaptic vesicles was observed in nwk. These results indicate that Nwk is not directly involved with synaptic vesicle dynamics. Instead, ultrastructural analysis shows that synaptic contacts are fewer and smaller in nwk boutons: this may account for the reduced synaptic currents. Future experiments will address whether this phenotype is also mediated via Wsp or through other effectors (Coyle, 2004).

Regulation of synaptic architecture and synaptic vesicle pools by Nervous wreck at Drosophila Type 1b glutamatergic synapses

Nervous wreck (Nwk), a protein that is present at Type 1 glutamatergic synapses that contains an SH3 domain and an FCH motif, is a Drosophila homolog of the human srGAP3/MEGAP protein, which is associated with mental retardation. Confocal microscopy revealed that circles in Nwk reticulum enclosed T-shaped active zones (T-AZs) and partially colocalized with synaptic vesicle (SV) markers and both exocytosis and endocytosis components. Results from an electron microscopic (EM) analysis showed that Nwk proteins localized at synaptic edges and in SV pools. Both the synaptic areas and the number of SVs in the readily releasable (RRPs) and reserve (RPs) SV pools in nwk² were significantly reduced. Synergistic, morphological phenotypes observed from eag1;;nwk² neuromuscular junctions suggested that Nwk may regulate synaptic plasticity differently from activity-dependent Hebbian plasticity. Although the synaptic areas in eag1;;nwk² boutons were not significantly different from those of nwk², the number of SVs in the RRPs was similar to those of Canton-S. In addition, three-dimensional, high-voltage EM tomographic analysis demonstrated that significantly fewer enlarged SVs were present in nwk² RRPs. Furthermore, Nwk formed protein complexes with Drosophila Synapsin and Synaptotagmin 1 (DSypt1). Taken together, these findings suggest that Nwk is able to maintain synaptic architecture and both SV size and distribution at T-AZs by interacting with Synapsin and DSypt1 (Hur, 2018).

In order to investigate the unknown in vivo functions of proteins containing SH3 and F-BAR domains in synaptic plasticity and transmission, this study took advantage of Drosophila NMJs and available nwk mutants. Nwk is a Drosophila homolog of MEGAP, one of the proteins responsible for mental retardation in humans; Nwk was initially discovered by temperature-sensitive paralysis behavior of adult flies. Although genetic and physical interactions between Nwk and components of endocytosis, such as Dap160 and Dynamin, and actin cytoskeleton regulators, such as Wiskott-Aldrich syndrome protein and CDC42, have been reported, the localization patterns of Nwk at synaptic boutons appear to be different from localization patterns of the above-mentioned proteins. Thus it was hypothesized that Nwk may directly or indirectly interact with other synaptic or cytoskeletal components. In addition, by performing immunohistochemistry to confirm partial colocalization of Nwk with DAP160. Confocal microscopy also revealed that Nwk partially colocalized and formed protein complexes with Synapsin and DSypt1. Furthermore, Synapsin immunoreactivities at nwk synaptic boutons were significantly reduced. These findings are highly intriguing because recent studies have shown that Synapsin, the most abundant presynaptic phosphoprotein in Drosophila, has a pivotal role in regulating SV trafficking at synaptic terminals by forming protein complexes with Dap16050 in glutamatergic NMJs. The defects of readily releasable pool (RRP) and RP in nwk mutant synapses were further confirmed by performing transmission EM analysis and HVEM 3D tomography. Thus it is possible that Nwk is actively participating in SV trafficking at RRP and RP via interactions with Synapsin, DSypt1, Dap160,50, Dynamin48 and other interacting proteins that have yet to be identified (Hur, 2018).

Another phenotype in nwk loss-of-function mutants that was able to be identified by this study's use of HVEM 3D tomography was that of enlarged SVs. Because 1 μm thick sections for examining size and distribution of SVs at nwk mutant and control synapses and as 20 nm gold particles were used to create a list of fiducial points and size standards for SVs, the HVEM 3D analysis results did not contain biased data originating from measuring diameters of sectioned SVs when 50-100 nm of thin sections were used for conventional transmission EM analysis. Recently, it has been shown that an F-BAR domain in Nwk binds to and remodels membranes to form concave membrane structures. Therefore, it is possible that Nwk may regulate SV recycling both directly, by interacting with synaptic membranes, and indirectly, through its binding partners, such as Synapsin, DSypt1, Dap160 and Dynamin. Thus loss of Nwk at synapses might induce enlarged SVs compared with control synapses (Hur, 2018).

In addition to regulating SV size and trafficking, the NMJ and synapse morphometric analysis results provided several lines of evidence supporting that Nwk regulates synaptic plasticity of glutamatergic NMJs in a manner different from activity-dependent Hebbian plasticity. Previous studies have shown one of the classical examples of Hebbian plasticity, in which increased neuronal activity in NMJs reduces FasII localization at synaptic boutons, resulting in both an increase in the number of synaptic boutons and branch elongation. However, the findings of this study, which supported that genetically increased neuronal activity in nwk² mutant NMJs resulted in significantly more synaptic boutons and branch elongations and that the levels of FasII in nwk² mutants were significantly higher than those of CS, suggested that Nwk may exist in a different signaling pathway from that of Hebbian plasticity (Hur, 2018).

The evidence presented in this study is important because most psychiatric and neurological diseases, ranging from autism and mental retardation to dementia, are reported to be accompanied by altered synaptic structures and functions. The association of MEGAP and mental retardation has been revealed in the process of identifying genes that are linked with distal chromosome 3p deletion syndrome (3p-syndrome) patients with severe mental retardation. Similar to Nwk, MEGAP expression was observed in the embryonic central nervous systems of mice and high expression was found in the hippocampus, amygdala, piriform cortex, posterior parietal associative area and the dentate gyrus of adult mouse brains. In addition, similar to Nwk, MEGAP binds strongly to the phosphoinositide-rich membrane and is involved in the formation of filopodia. In the synaptogenesis stage, MEGAP has an important role in synapse selection, and consequently, the loss of MEGAP function results in a decrease in the number of neuron spines. Thus, although it has not been elucidated to date whether MEGAP expresses at presynaptic or postsynaptic terminals or both, it is possible that MEGAP may regulate SV size and distribution in neuron spines at glutamatergic synapses of the mouse central nervous system, especially at the hippocampus, resulting in mental retardation phenotypes in human patients3 and mouse knockout models. Thus further studies in Nwk and MEGAP will reveal unknown molecular and cellular functions of SH3 and F-BAR domain proteins in synaptic plasticity and mental retardation (Hur, 2018).

PROTEIN STRUCTURE

Amino Acids - 1001

Structural Domains

nwk was identified by positional cloning. Recombination mapping placed nwk 1 map unit to the right of hairy (3-26.5); this corresponds approximately to cytological interval 66E-67A. Therefore, Df(3L)29A6,, which is deleted for 66F5-67B1, was tested; it failed to complement the ts paralysis of nwk. Df(3L)Rdl2, which contains a small deletion within 66F5 that includes the GABA-A receptor gene Rdl, also uncovered nwk. Complementation tests indicated that nwk and Rdl null mutants are not allelic (Coyle, 2004).

The breakpoints of Df(3L)Rdl2 were mapped by Southern blot analysis using overlapping cosmids that spanned 66F. The Drosophila genome database lists 20 ORFs within this interval, representing candidates for the nwk locus. Sequence analysis revealed that one of these candidates, CG4684, a predicted ORF with homology to Dock, contained a unique polymorphism in each of three alleles (nwk^2,4,5). The remaining alleles, nwk¹ and nwk³, had molecular lesions downstream of CG4684 but within an overlapping EST (SD0455), suggesting that the predicted ORF in the database was incomplete. Therefore, the 5' end of CG4684 was isolated via RACE and this sequence was used to probe an adult Drosophila cDNA library. Several cDNAs were recovedred that linked CG4684 and SD04555 into a single full-length ORF encoding a 1001 amino acid protein. All five nwk alleles contained unique molecular lesions within this ORF (Coyle, 2004).

nwk encodes a predicted 110 kDa protein with several recognizable functional domains. An Fes/CIP4 Homology (FCH) domain is present at the N terminus. The function of this domain is unknown, but it has been implicated in actin binding. Following the FCH domain are two canonical SH3 domains, modular protein binding domains that recognize proline-rich consensus sequences, often found in signal transduction pathways linking the membrane and cytoskeleton. In addition, the C terminus of Nwk contains a proline-rich segment with at least five potential SH3 binding sites (Coyle, 2004).

Database searches identified 11 related proteins in yeast, worms, and mammals, including the three human srGAPs. Nwk and these 11 homologs share a highly conserved 60 amino acid sequence adjacent to the FCH domain that is not present in any other proteins. This is referred to as the ARNEY domain based on its central consensus sequence ARNEYLL. There are six Nwk relatives in humans and three in mice. Drosophila, C. elegans, and S. cerevisiae each contain a single Nwk protein. Seven of the twelve Nwk family members differ in that the first SH3 domain (SH3a) is replaced by a RhoGAP domain. The presence of FCH, ARNEY, RhoGAP, and SH3 domains suggests that Nwk and its homologs compose a distinct family of adaptor proteins that interact with cytoskeletal regulators (Coyle, 2004).

Protein Interactions

Several lines of evidence suggested that Nwk might interact with Wasp: (1) the SH3a domain of Nwk is between 35% and 45% identical to the three SH3 domains of Dock. Nck, a mammalian homolog of Dock, binds to Wasp via its SH3 domains. (2) Bzz1p, the yeast ortholog of Nwk, binds to Las17p, the yeast ortholog of Wasp (Soulard, 2002), via its SH3 domains. (3) wsp mutants have a morphological phenotype similar to nwk at larval NMJs (Coyle, 2004).

To evaluate whether Nwk and Wsp interact biochemically, affinity chromatography was performed using GST-fusion proteins containing each of the two Nwk SH3 domains. The construct containing the SH3a domain, but not the one containing the SH3b domain, precipitated Wsp immunoreactivity from Drosophila head homogenates with high affinity (Coyle, 2004).

A yeast two-hybrid protein binding assay further indicated that Nwk and Wsp can interact directly. Two-bait constructs expressing either full-length Nwk or a subfragment consisting of the two SH3 domains plus the C terminus stimulates transcription of reporter genes in yeast cells cotransformed with a full-length Wsp prey. Colonies grew rapidly on selectable media and expressed β-gal. In contrast, bait constructs containing the FCH and ARNEY domains of Nwk but lacking the SH3 domains did not activate reporter genes when cotransformed with Wsp prey, indicating that the SH3 domains are required for binding with Wsp. Single transformation of any Nwk bait or Wsp prey alone did not stimulate transcription of reporter genes at substantial levels (Coyle, 2004).

A previous study of Drosophila wsp mutants revealed defects in sensory cell proliferation during PNS development, but a role at NMJs had not been investigated (Ben-Yaacov, 2001). Therefore, to determine whether endogenous Nwk and Wsp are capable of interacting in vivo, the distribution of Wsp at the NMJ was examined by immunocytochemistry. Wsp was found to be localized diffusely at synaptic boutons in irregular patches. Postsynaptically, Wsp is associated with the subsynaptic reticulum (ssr), which was revealed by colocalization with the ssr marker, Dlg, and by reduction of immunoreactivity in pak mutants that reduce the ssr. There is also a significant presynaptic component of Wsp immunoreactivity that is most evident in thin (0.25 μm) optical sections through the center of boutons. Wsp is clearly present both inside the bouton as well as in association with the surrounding ssr. Although the presynaptic distribution of Wsp is not continuous throughout the periactive zone, regions of overlap with Nwk can be observed in thin optical confocal sections through double-labeled boutons. Thus, Nwk and Wsp colocalize in spatially restricted regions of the periactive zone. Nwk is not required for Wsp localization since Wsp immunoreactivity is not obviously disrupted in nwk mutants (Coyle, 2004).

Wasp promotes the nucleation and branching of F-actin. Phalloidin staining revealed that F-actin is enriched around synaptic boutons and generally colocalizes with Wsp. The overall abundance and distribution of F-actin appear normal in wsp mutants, indicating that Wsp is not the only factor promoting actin polymerization at the synapse. The Drosophila genome contains one other wsp family member, scar, whose function may overlap with wsp, although its role at synapses has not been investigated. Thus, loss of Wsp may have only a limited, localized effect on actin dynamics and architecture without completely abolishing the cortical cytoskeleton (Coyle, 2004).

Nervous wreck interacts with thickveins and the endocytic machinery to attenuate retrograde BMP signaling during synaptic growth

Regulation of synaptic growth is fundamental to the formation and plasticity of neural circuits. This study demonstrates that Nervous wreck (Nwk), a negative regulator of synaptic growth at Drosophila NMJs, interacts functionally and physically with components of the endocytic machinery, including dynamin and Dap160/intersectin, and negatively regulates retrograde BMP growth signaling through a direct interaction with the BMP receptor, Thickveins. Synaptic overgrowth in nwk is sensitive to BMP signaling levels, and loss of Nwk facilitates BMP-induced overgrowth. Conversely, Nwk overexpression suppresses BMP-induced synaptic overgrowth. Analogous genetic interactions were observed between dap160 and the BMP pathway, confirming that endocytosis regulates BMP signaling at NMJs. Finally, a correlation exists between synaptic growth and pMAD levels and Nwk regulates these levels. It is proposed that Nwk functions at the interface of endocytosis and BMP signaling to ensure proper synaptic growth by negatively regulating Tkv to set limits on this positive growth signal (O'Connor-Giles, 2008).

Nwk interacts functionally and physically with a number of known endocytic proteins, notably dynamin and Dap160. Uptake experiments showed that Nwk does not function in an internalization step of synaptic vesicle endocytosis, suggesting that Nwk affects a later step in endocytic trafficking. In agreement, colocalization was observed between Nwk and the recycling endosome-associated Rab GTPase Rab11, but not between Nwk and Rab proteins associated with either early or late endosomes. Consistent with this observation, it has been found that hypomorphic mutations in Drosophila rab11 result in a synaptic overgrowth phenotype that very closely resembles nwk (O'Connor-Giles, 2008).

Importantly, Nwk, Dap160, and dynamin are all linked to regulation of actin assembly. Recent experiments demonstrate that dynamin-mediated vesicle fission requires actin polymerization. For example, inhibiting actin polymerization blocks fission. Nwk likely facilitates a critical interaction between the endocytic machinery and actin polymerization at NMJs because it directly binds both dynamin and Wasp. Nwk also contains an F-BAR domain, which promotes membrane invagination. These domains are found almost exclusively in adaptor proteins that associate both with actin regulators and the endocytic machinery, highlighting the important links between these cellular processes (O'Connor-Giles, 2008).

The critical role of endocytic accessory proteins in linking the core endocytic machinery to cell signaling molecules is becoming increasingly clear. In addition to attenuating signaling by targeting receptors for degradation, endocytic adaptor proteins play key roles in spatial and temporal regulation of signal transduction from ligand-activated receptors. Recent work in other systems also suggests a critical role for endocytic trafficking during TGF-β/BMP signal transduction. For example, Smad phosphorylation and nuclear translocation in vertebrates depend on localization of the endosomal protein Smad anchor for receptor activation (SARA) and activated type-I and -II receptors to EEA1-positive endosomal compartments. Similarly, in the Drosophila wing, targeting of Sara, Tkv, and the ligand decapentaplegic (Dpp) to early endosomes is required for productive signaling. Nwk is ideally situated to bridge endocytosis and growth signal regulation at presynaptic terminals because of its links to actin assembly and endocytosis as well as its capacity for binding a number of proteins (including Tkv) through its multiple protein-protein interaction domains (O'Connor-Giles, 2008).

Loss of endocytic proteins results in the specific morphological phenotype of excessive satellite bouton formation, as do mutations in actin-associated proteins, including Nwk, Wasp, and components of the Scar complex. These observations suggest that satellite bouton formation may result from impairment of an actin-dependent step in endocytosis. Misregulation of a signaling pathway responsible for bouton growth and morphology in endocytic mutants may occur and, because satellite bouton formation had not been linked with any known pathway, the existence of either an unidentified positive growth signal downregulated by endocytosis or an endocytosis-dependent negative growth signal has been postulated (O'Connor-Giles, 2008).

This study show that BMP signaling regulates satellite bouton formation. Increasing levels of BMP signaling, either by expressing UAStkv^ACT or by reducing endogenous negative regulation of the pathway, generates a significant increase in satellite bouton formation. Further, overexpression of Nwk or Dap160 suppresses BMP-induced overgrowth, including satellite bouton formation. Finally, a direct correlation was observed between pMAD levels and satellite bouton formation in each of the genetic backgrounds analyzed. Together, these results indicate that impaired endocytic regulation of retrograde BMP signaling results in generation of satellite boutons at NMJs. In the case of endocytic and Dad mutants, downregulation of endogenous BMP signaling is impaired, while in Tkv^ACT-expressing larvae, ectopic BMP signaling apparently overwhelms the usual mechanisms of negative regulation (O'Connor-Giles, 2008).

Although BMP signaling is required for NMJ growth, it has remained unclear whether the signal acts merely as a switch to initiate or permit growth or instead plays a more instructive role in regulating and coordinating synaptic growth. This study demonstrates a direct relationship between levels of BMP signaling and extent of synaptic growth. Neuronal expression of a single copy of Tkv^ACT results in a modest increase in pMAD and no significant increase in synaptic growth, whereas expression of two copies induces a dramatic increase in pMAD levels and extensive synaptic overgrowth, both of which are suppressed by overexpression of Nwk. Further, it was found that mutations in the endogenous negative regulator Dad also cause increased synaptic growth. These data indicate that the level of BMP signaling has an instructive role in governing synaptic size and complexity and reveal the importance of interactions between positive and negative regulators that modulate the growth signal in response to internal and external cues (O'Connor-Giles, 2008).

The results demonstrate that endocytosis is an important regulatory mechanism for attenuating BMP signaling at synapses. Previous work suggested that Hiw, an E3 ubiquitin ligase and negative regulator of synaptic growth, also acted to limit BMP signaling. However, subsequent work demonstrated that pMAD levels are not increased in hiw, and no effects of Hiw overexpression on BMP signaling have been described. In addition, hiw synapses are extremely expansive, elaborately branched, and contain numerous small boutons, but no satellite boutons. This phenotype is distinct from that associated with Tkv^ACT overexpression or other genotypes believed to elevate BMP signaling, including Dad, nwk, and known endocytic genes -- all of which exhibit satellite boutons. Together with the recent finding that Hiw regulates MAPKKK-dependent Fos activity, these observations suggest that Hiw and BMP signaling may regulate different aspects of synaptic growth (O'Connor-Giles, 2008).

In their recent study of Spict, Wang (2007) demonstrated the localization of Spict to early endosomes along with data suggesting that Spict plays a role in BMP receptor trafficking (Wang, 2007). For example, Spict overexpression in S2 cells caused Wit to relocalize to early endosomes, suggesting negative regulation of BMP signaling by sequestering Wit receptor and/or Wit-Gbb signaling complexes. However, studies of Wit trafficking at spict NMJs, which are limited by the small size of boutons and the lack of reliable antibodies, did not uncover this trafficking defect but instead revealed increased Wit levels consistent with a role for Spict in the degradation of Wit. Nonetheless, the results from the analysis of Spict support the idea that endocytic regulation of BMP signaling is required for proper synaptic growth. It will be interesting to determine whether Spict interacts with Nwk or plays a distinct role in the regulation of BMP signaling at synapses (O'Connor-Giles, 2008).

Overall, the current findings support a model in which Nwk constrains synaptic growth by regulating endocytic trafficking of Tkv to attenuate positive retrograde growth signaling. While the simplest model is that Nwk targets Tkv for degradation, gross differences were not observed in levels of ectopically expressed Tkv-GFP in otherwise wild-type, nwk, and C155GAL4; UAS-nwk larvae (unpublished data). A caveat is that, without the necessary antibody reagents, it was not possible to look at endogenous Tkv. Thus, these observations might not accurately reflect normal receptor trafficking but rather the fact that high levels of Tkv can override endogenous regulation. Nonetheless, together with the observation that Nwk colocalizes with Rab11, this finding is consistent with a role for Nwk in BMP receptor recycling. For example, Nwk might attenuate BMP signaling levels by regulating the rate at which vacant Tkv receptors are recycled back to the plasma membrane following activation and internalization. Nwk might also regulate the trafficking of unbound receptors from the plasma membrane. Previous work in vertebrates demonstrates that TGF-β receptors are recycled through a Rab11-dependent mechanism independent of ligand binding, possibly as a means of rapidly and dynamically regulating surface receptor number and, thus, sensitivity to TGF-β. Interestingly, the relocalization of Nwk from a more uniform to a more punctate expression pattern was observed upon overexpression of Tkv, consistent with recruitment of Nwk to regulate trafficking of ectopic Tkv. Nwk might also have localized effects within boutons, for example, by restricting sites of BMP signaling through spatial regulation of receptor recycling. It is intriguing to speculate that disruption of spatial constraints on BMP signaling results in ectopic bouton division and, thus, satellite bouton formation. Such a mechanism could provide a critical means for effecting localized changes to existing synapses that underlie neural plasticity. A conceptually similar Rab11-dependent process for the asymmetric activation of Notch signaling in the developing nervous system has recently been described. A future challenge will be to further dissect the regulatory mechanisms that control the levels, timing, and localization of BMP signaling at synapses. These studies will advance understanding of the dynamic regulation of synaptic growth and plasticity and likely provide additional general insights into the intricate role of endocytosis in signal transduction (O'Connor-Giles, 2008).

Nervous wreck and Cdc42 cooperate to regulate endocytic actin assembly during synaptic growth

Regulation of synaptic morphology depends on endocytosis of activated growth signal receptors, but the mechanisms regulating this membrane-trafficking event are unclear. Actin polymerization mediated by Wiskott-Aldrich syndrome protein (WASp) and the actin-related protein 2/3 complex generates forces at multiple stages of endocytosis. FCH-BIN amphiphysin RVS (F-BAR)/SH3 domain proteins play key roles in this process by coordinating membrane deformation with WASp-dependent actin polymerization. However, it is not known how other WASp ligands, such as the small GTPase Cdc42, coordinate with F-BAR/SH3 proteins to regulate actin polymerization at membranes. Nervous Wreck (Nwk) is a conserved neuronal F-BAR/SH3 protein that localizes to periactive zones at the Drosophila larval neuromuscular junction (NMJ) and is required for regulation of synaptic growth via bone morphogenic protein signaling. This study shows that Nwk interacts with the endocytic proteins dynamin and Dynamin associated protein 160 (Dap160) and functions together with Cdc42 to promote WASp-mediated actin polymerization in vitro and to regulate synaptic growth in vivo. Cdc42 function is associated with Rab11-dependent recycling endosomes, and this study shows that Rab11 colocalizes with Nwk at the NMJ. Together, these results suggest that synaptic growth activated by growth factor signaling is controlled at an endosomal compartment via coordinated Nwk and Cdc42-dependent actin assembly (Rodal, 2008).

Nwk interacts with the endocytic machinery and activates Wsp/Arp2/3 actin polymerization together with Cdc42 to regulate synaptic growth upstream of growth factor signaling. Mapping these interactions and activities provides a critical framework for determining the mechanism by which endocytic accessory proteins and the cytoskeleton control membrane deformation during endocytosis (Rodal, 2008).

Nwk activates Wsp/Arp2/3 actin polymerization via its SH3a domain, and Nwk-SH3b is not required for Wsp binding or activation, but is required for the residual Wsp-inhibitory activity of Nwk when SH3a function is abolished. This activity may be more pronounced on endogenous Wsp, which is more tightly autoinhibited than recombinant WASp, raising the possibility that Nwk-SH3b could potently regulate Nwk-SH3a-dependent activation of Wsp. Thus, ligands of Nwk-SH3b are in a position to serve as activators of Nwk and Wsp/Arp2/3 actin polymerization. Nwk-SH3b is required for interactions between Nwk and Dap160, which is an excellent candidate for acting upstream of Nwk, because dap160 mutants exhibit synaptic overgrowth and temperature-sensitive seizures like those of nwk mutants, and Nwk is mislocalized in dap160 NMJs. Recently, it was reported that the fragment of Dap160 containing its last two SH3 domains is required for interaction with full-length Nwk in Drosophila extracts, leading to the hypothesis that the C terminal proline-rich region of Nwk mediates these interactions (O'Connor-Giles, 2008). The current results show instead that interactions between purified Nwk{Delta}C (i.e., Nwk lacking the C terminus) and both endogenous full-length Dap160 as well as purified Dap160 SH3 domain-containing fragment depend on Nwk SH3b. Two possible interpretations can reconcile these results. Nwk SH3b may interact with a noncanonical SH3 domain-binding site in the intervening sequences between the Dap160 SH3 domains. Alternatively, Nwk SH3b may function in an intramolecular interaction within Nwk that is required to expose one of several proline-rich sequences in the N-terminal region Nwk for interaction with Dap160 SH3 domains. Thus, it is concluded that Nwk SH3b is important for Dap160-Nwk interactions via an indirect or noncanonical mechanism. Further experiments will be needed to identify the Nwk-binding site on Dap160 and to confirm activity of Dap160 on Nwk in vitro (Rodal, 2008).

Nwk-SH3a is required for interactions of Nwk with both dynamin and Wsp. Other F-BAR/SH3 family members have been postulated to link dynamin and Wsp by multimerization via their F-BAR domains (Itoh, 2006; Tsujita, 2006; Shimada, 2007), but endogenous complexes containing Wsp and dynamin have only been demonstrated for the F-BAR/SH3 protein syndapin (Kessels, 2006). Nwk could thus be in a position to bring dynamin and Wsp together. It has not been possible to coimmunoprecipitate endogenous Wsp and Nwk using the available antibodies. However, dynamin immunoprecipitates contain Nwk but not Wsp, suggesting that Nwk-SH3a may switch associations between dynamin and Wsp. Another interpretation is that Wsp and dynamin binding are restricted to separate populations of Nwk molecules, and that the SH3a domain thus acts in two parallel biochemical pathways (Rodal, 2008).

In vivo analysis reflects the complexity of these SH3 domain interactions. SH3a and SH3b of Nwk have both separate and overlapping functions in regulating synaptic growth, perhaps reflecting the multivalent nature of interactions in the Nwk network. [In addition to binding Nwk, Dap160 binds to both dynamin and to Wsp.] Furthermore, the fact that mutation of both SH3 domains together (Nwk-SH3a*b*) produces additional dominant effects suggests that a non-SH3 ligand of Nwk is inappropriately titrated away from its function after mutation of Nwk SH3 domains. An excellent candidate ligand is the membrane itself, because the Nwk F-BAR domain has the potential to bind to and tubulate phospholipid bilayers. Determining the specific order and regulation of F-BAR/SH3 domain protein interactions with competing SH3 domain ligands and with the membrane will be important for uncovering the molecular mechanisms of these proteins during endocytosis (Rodal, 2008).

NMJ overgrowth with an excess of satellite boutons is a hallmark of endocytic mutants. Nwk interacts with the endocytic machinery and cdc42 and nwk mutants exhibit overproliferation of satellite boutons. A prominent function of endocytosis in nerve terminals is the recycling of synaptic vesicles. However, nwk single mutants and cdc42; nwk double mutants show no detectable defect in endocytosis of synaptic vesicles. One interpretation of this result is that receptor endocytosis is more sensitive to perturbation than synaptic vesicle recycling. However, given the documented function of Cdc42 and Wsp in endosomes, it is more likely that Nwk functions in a later step of endocytic traffic. Importantly, although the synaptic vesicle endocytosis defects in shi (dynamin) and dap160 reflect the function of these molecules in the internalization step of endocytosis, synaptic overgrowth in these mutants could arise from defects at later steps of endocytic traffic, because dynamin functions in a variety of membrane-trafficking events, ranging from Golgi traffic to endosome traffic (van Dam and Stoorvogel, 2002Go; Kessels et al., 2006Go) (Rodal, 2008).

The endosomal system is organized into subdomains defined by specific members of the Rab GTPase family and adopts distinct morphology and ultrastructure in different cell types. Thus, functionally conserved Rab subdomains provide a unifying approach to understanding structurally diverse membrane systems. Rab11 controls the function of the recycling endosome in directing traffic to the cell surface and colocalizes with Nwk in periactive zones at the Drosophila NMJ [although it can occasionally be observed in larger puncta]. Like cdc42 and nwk mutants, rab11 mutants have a profound defect in synaptic growth, exhibiting excessive satellite boutons. Cdc42 and WASp have recently been implicated in recycling endosome function. Thus, periactive zones may be the synaptic representation of the recycling endosome, with Cdc42 and Nwk controlling actin polymerization-dependent traffic of signaling complexes at this Rab11-positive compartment. Whether Cdc42 functions as a signal-responsive element in this compartment or forms part of the constitutive machinery for membrane traffic remains uncertain (Rodal, 2008).

The TGF-β/BMP family member Gbb activates downstream signals that may be the critical targets of Nwk/Cdc42-mediated endocytosis in synaptic growth. Indeed, recent work has shown that Gbb signaling is required for synaptic overgrowth in nwk mutants, phosphorylation of the Gbb signaling target Mothers against decapentaplegic (Mad) is upregulated in nwk mutants, and Nwk biochemically interacts with the intracellular domain of the Gbb receptor Tkv. However, other signaling pathways could equally be regulated by Nwk/Cdc42-mediated endocytosis, lead to upregulation of phosphorylated Mad, and contribute to the synaptic overgrowth in cdc42; nwk mutants. One candidate pathway is the presynaptic component of the Wnt/Wg cascade, which may converge on Gbb/Mad regulation in the synapse as observed in other tissues. It has not been possible to detect any change in the steady-state localization of candidate cargoes in synaptic boutons in nwk or cdc42 mutants, suggesting that Nwk and Cdc42 are not required for the gross morphology of endosomes, but instead contribute to the rate of cargo trafficking through this compartment. Determining the specific signaling pathways, receptors, and their activation states in recycling endosomes will require tools to measure the activity and rates of traffic of specific receptors in situ (Rodal, 2008).

Nwk is conserved from insects to higher vertebrates, and the mammalian genome encodes two Nwk homologs, which have not yet been characterized. However, Cdc42 and WASp-induced actin polymerization have been implicated in synapse formation in Aplysia sensory neurons and in mammalian hippocampal cultures. These reports suggest that the direct consequence of activating these proteins was the formation of filopodia that mature into synapses. An alternative hypothesis, consistent with the established function of Cdc42 and WASp family members in generating force for intracellular membrane traffic rather than in filopodial formation, is that synaptic growth regulatory functions of Cdc42 and WASp depend on endosomal traffic of signaling complexes by a similar mechanism to Drosophila Nwk-Wsp-induced synapse formation (Rodal, 2008).

A presynaptic endosomal trafficking pathway controls synaptic growth signaling

Structural remodeling of synapses in response to growth signals leads to long-lasting alterations in neuronal function in many systems. Synaptic growth factor receptors alter their signaling properties during transit through the endocytic pathway, but the mechanisms controlling cargo traffic between endocytic compartments remain unclear. Nwk (Nervous Wreck) is a presynaptic F-BAR/SH3 protein that regulates synaptic growth signaling in Drosophila. This study shows that Nwk acts through a physical interaction with sorting nexin 16 (SNX16). SNX16 promotes synaptic growth signaling by activated bone morphogenic protein receptors, and live imaging in neurons reveals that SNX16-positive early endosomes undergo transient interactions with Nwk-containing recycling endosomes. An alternative signal termination pathway was identified in the absence of Snx16 that is controlled by endosomal sorting complex required for transport (ESCRT)-mediated internalization of receptors into the endosomal lumen. These results define a presynaptic trafficking pathway mediated by SNX16, NWK, and the ESCRT complex that functions to control synaptic growth signaling at the interface between endosomal compartments (Rodal, 2011).

Endocytic membrane traffic regulates signaling by synaptic growth factor receptors and may be used as a point of control in tuning receptor traffic in response to neuronal activity. The neuronal F-BAR/SH3 protein Nwk is involved in endocytic membrane traffic that attenuates synaptic growth signaling, but the mechanism by which it acts has been unknown. This study has identified a physical interaction between Nwk and the early endosomal protein SNX16 that is critical for down-regulating the synaptic growth-promoting activity of SNX16. This interaction may bring together the actin-polymerizing activity of the first SH3 domain of Nwk with the potential lipid-binding/tubulating activities of the F-BAR domain of Nwk and the PX domain of SNX16, driving tubule-based membrane flux from early to recycling endosomes (Rodal, 2011).

Sorting nexins form a large family of proteins that share a common phosphoinositide-binding PX domain and are involved in diverse aspects of membrane traffic (Cullen, 2008). The best-characterized members of this family are the sorting nexin-BAR family, including Snx1 (tied to endosome-to-Golgi traffic) and Snx9 (tied to the internalization step of endocytosis), which each contains both a lipid-tubulating BAR domain and a PX domain. Crystal structures and functional studies of Snx9 have shown that its BAR and PX domains form a single lipid-binding and -deforming module with combined specificities that neither domain exhibits alone. As such, SNX16-Nwk interactions may form an analogous F-BAR/PX module via an intermolecular rather than intramolecular interaction. It has not been possible to purify sufficient amounts of SNX16 to directly test its effects on lipid binding by Nwk; therefore, further analysis of their interaction will require an approach to isolate SNX16 in vitro. Interestingly, the SNX16-Nwk interaction depends on a region of Nwk surrounding its second SH3 domain, raising the possibility that this region of Nwk may exert intramolecular effects on the amino-terminal F-BAR domain, as has previously been shown for other F-BAR proteins (Rao, 2010). Furthermore, Nwk binds to the coiled-coil region of SNX16, which is involved in SNX16 dimerization, suggesting that Nwk may act on SNX16 by affecting its dimerization state (Rodal, 2011).

Mammalian SNX16 has been implicated in the trafficking of the EGF receptor from early to late endosomes, which may mediate down-regulation of receptor signaling. However, the mechanism by which SNX16 promotes this trafficking step is not understood and no loss-of-function studies on SNX16 have been reported. A mutant of mammalian SNX16 that lacks 60 aa corresponding to the Drosophila SNX16 Nwk-binding site blocks trafficking of SNX16 and EGF to late endosomes, leading to increased EGF signaling. Interestingly, the F-BAR protein FBP17 has been reported to interact with SNX2. These results raise the possibility that BAR family-sorting nexin interactions may be broadly used to control membrane traffic in cells (Rodal, 2011).

Drosophila SNX16 localizes to an early endosomal compartment at the NMJ defined by the small GTPases Rab4 and Rab5. This compartment accumulates signaling receptors, such as the BMP receptors Tkv and Wit, and signaling is amplified when receptors are stalled in this compartment, suggesting that the SNX16 compartment is an active site of signaling. By identifying specific Snx16 mutants that disrupt interactions with Nwk, it was possible to separate the requirements for receptor down-regulation at early endosomes and show that activated receptors specifically require SNX16-Nwk-mediated traffic to recycle to the plasma membrane (Rodal, 2011).

Previous studies have shown that Nwk colocalizes with the recycling endosome marker Rab11 in fixed tissue and cooperates with Cdc42, which is thought to function at the recycling endosome, to activate WASp/Arp2/3-mediated actin polymerization. Together with the result that rab11 mutants exhibit synaptic overgrowth similar to nwk mutants (Khodosh, 2006), it is concluded that Nwk functions at recycling endosomes, which are downstream of early endosomes. This study shows that Nwk tagged with fluorescent proteins localizes to novel punctate structures in heterologous cells and nerve terminals. These puncta had not been observed using anti-Nwk antibodies against either endogenous or overexpressed Nwk in fixed tissue, as they are poorly preserved upon fixation. In living synapses, Rab11 colocalizes with Nwk to mobile puncta, SNX16-containing early endosomes interact transiently with Nwk puncta, and inhibition of the GTPase dynamin blocks separation of these compartments. Dynamin inhibition has previously been shown to disrupt endosomal function in the fly NMJ, it cannot be certain that the collapse of SNX16-Nwk compartments under these conditions is caused by the specific interactions of these proteins with dynamin. However, because SNX16-Nwk interactions lead to the down-regulation of synaptic growth signals, the data are consistent with a model in which the exchange of receptors from the SNX16 endosome to the Nwk/Rab11 endosome leads to the attenuation of receptor signaling. Binding of Nwk to the cytoplasmic tail of the BMP receptor Tkv may mediate this event (O'Connor-Giles, 2008), but further experiments will be required to directly examine cargo transfer in the future. Because there are significant cytoplasmic pools of both Nwk and SNX16, the contribution of these soluble proteins to membrane traffic in the nerve terminal or the possibility that overexpression of Nwk and Snx16 to visualize live trafficking events does not faithfully recapitulate the behaviors of endogenous proteins cannot be excluded. However, the transient interaction of Nwk and SNX16 puncta correlates well with genetic results showing that nwk attenuates a synaptic growth-promoting activity of Snx16 at early endosomes (Rodal, 2011).

Because Nwk- and SNX16-labeled compartments transiently interact in nerve terminals, temporal control of the interaction between their lipid-binding domains may provide a mechanism to acutely drive membrane tubulation in a regulated fashion. Furthermore, the association of SNX16 with endosomes depends on phosphoinositides, as the phosphatidylinositol 3-kinase inhibitor wortmannin disrupts localization of SNX16 in cultured cells, indicating that regulation of phospholipid composition may also contribute to the membrane-deforming activities of SNX16 and Nwk (Rodal, 2011).

This study found that Snx16 loss-of-function mutants suppress synaptic overgrowth resulting from loss of Nwk-mediated traffic as well as from activation of Wg and BMP signaling pathways. These data suggest that SNX16 plays an active role in promoting synaptic growth at the endosome, aside from its function in signal attenuation through Nwk. Snx16 was found to be required to restrict synaptic growth when MVB formation is hampered in hrs mutants, suggesting that receptor entry into the endosomal lumen is an alternative signal attenuation pathway. Furthermore, it was found that overexpression of a mutant Snx16 that cannot bind Nwk promotes the accumulation of endosomal structures at the NMJ and drives excess synaptic growth, suggesting that Snx16 may play a role in MVB maturation and acts at the branch point between endosomal sorting pathways. Defining the mechanism by which SNX16 promotes synaptic growth will require further structure-function analysis of the active domains of the protein aside from its Nwk-binding coiled coil (Rodal, 2011).

Traffic through endocytic compartments has proven to be a critical point of regulation in the nervous system. Synaptic vesicle endocytosis is controlled by synaptic activity through calcium-dependent dephosphorylation of endocytic proteins, and postsynaptic trafficking of AMPA receptors through the recycling endosome is increased in response to activity via the calcium-dependent motor myosin V. Rab5-positive compartments at the Drosophila NMJ have been previously characterized for their role in the synaptic vesicle cycle, and it will be interesting to determine how receptor-mediated endocytosis and synaptic vesicle endocytosis are coordinately or separately regulated in response to activity. Synaptic growth at the Drosophila NMJ is positively regulated by calcium influx through voltage-gated calcium channels, and endosome number increases in response to activity. It is tempting to speculate that calcium influx acts through conserved mechanisms for modulating membrane dynamics to delay the attenuation of receptor signaling by SNX16-Nwk-mediated traffic, leading to increased synapse growth in response to activity. A key future goal will be to determine specific points of activity-dependent regulation of membrane traffic in presynaptic endosomes (Rodal, 2011).

In situ hybridization of embryos has shown that nwk is abundantly transcribed throughout the CNS and PNS, whereas its expression was not detected in other tissues, including muscles. A polyclonal anti-Nwk antiserum stained motorneuron axons and terminals, but not muscles. Confocal microscopy revealed a distinctive distribution of Nwk within synaptic boutons. Nwk is distributed in a patchwork of rings in the plane of the bouton membrane. Pak, a marker of synaptic densities, has a complementary punctate distribution, such that each Nwk ring surrounds one Pak punctum. This distribution of Nwk corresponds to the periactive zone, described by for Highwire. These proteins also regulate NMJ morphology: this led to the proposal that the periactive zone is specialized for synaptic growth regulation. Thus, the localization of Nwk to this region is consistent with the morphological defects in nwk mutants (Coyle, 2004).

Effects of Mutation or Deletion

To identify mutations that perturb function and development of the nervous system in Drosophila, a large collection of temperature-sensitive (ts) paralytic mutants was isolated following mutagenesis by ethylmethane sulfonate (EMS). The ts paralytic phenotype often results from the absence of an affected protein and not from a thermolabile variant. Such mutants can have defective synaptic morphology or function even at nominally permissive temperatures. Therefore, ts mutants were screened to identify those with abnormal morphology at the larval NMJ. nwk is the first of these lines to be so characterized (Coyle, 2004).

nwk adults behave normally at room temperature, but rapidly (10-40 s) lose coordination at 38°C and undergo seizure-like spasms that gradually diminish until the flies become paralyzed within 3 min. When returned to room temperature after a 5 min exposure to 38°C, nwk mutants gradually recover, and normal behavior is fully restored after 5 to 10 min. Wild-type flies exhibit no locomotor defects at 38°C for at least 20 min (Coyle, 2004).

The glutamatergic synapses of the Drosophila larval NMJ have become a favored genetic model for studies of synaptic development and plasticity. Each motorneuron forms distinctive synaptic junctions on its target muscle(s), containing stereotypic numbers of branches and boutons. Initial observations revealed that NMJs in nwk larvae were more extensive than in wild-type and that this phenotype was 100% penetrant. No defects in axon pathfinding were observed (Coyle, 2004).

To quantify the nwk phenotype, bouton number, NMJ length, branch number, and branch complexity at NMJ 6/7 and NMJ 4 were measured. Compared with controls, the number of boutons at NMJ 6/7 in nwk¹ larvae is increased by 50%. A similar result was also observed at NMJ 4. Because NMJ growth varies in proportion to muscle size, measurements were normalized to muscle surface area. At NMJ 6/7, normalized bouton number remains 50% larger in nwk¹ compared with control larvae, whereas at NMJ 4, it becomes 22% larger. The increase in bouton number was accompanied by an expansion in NMJ length as measured by summing the lengths of all axon branches within NMJ 6/7. After normalization to muscle area, NMJ length was increased by 30% (Coyle, 2004).

nwk mutants also display increased frequency and complexity of branch formation. Branches form from pre-existing boutons in a process that superficially resembles budding or asymmetric division in yeast cells. Typically, a branching bouton bifurcates to yield just two new branches. In agreement with previous studies, only a small percentage of boutons in wild-type larvae contained branch points at NMJ 6/7 and NMJ 4 and virtually all of these branches were bifurcations. Among all 36 wild-type NMJs examined, only a single instance was found of a bouton extending more than two new branches. In nwk¹, the proportion of branching boutons was increased by 50% at NMJ 6/7 and by 125% at NMJ 4. Moreover, there was a striking increase in the incidence of hyperbranched boutons, those extending three or more new branches. At least one such hyperbranched bouton was observed at all NMJs 6/7 and among about half of all NMJs 4 examined in nwk¹ larvae. Most of these hyperbranched boutons extended three new branches but 10% extended even four, suggesting that the underlying mechanism of bouton division is misregulated in nwk mutants (Coyle, 2004).

Four additional nwk alleles (nwk^2-5) were obtained following EMS and γ ray mutagenesis in screens for failure to complement the ts paralytic phenotype of nwk. All of these mutants are thought to be functional nulls, and all display similar NMJ overgrowth phenotypes, confirming that nwk is responsible for both morphological defects in larvae and ts paralysis in adults. To further verify that loss of Nwk is responsible for the observed phenotypes, the phenotype of nwk² heterozygous were evaluated with the deficiency Df(3L)Rdl2, which uncovers nwk. These nwk² hemizygotes had synaptic defects that were nearly identical with those observed in nwk² homozygotes (Coyle, 2004).

To investigate the effects of nwk on synaptic growth at the ultrastructural level, electron microscopic analysis of serially sectioned type Ib boutons from NMJ 6/7 in nwk² and wild-type larvae was performed. General bouton anatomy in nwk² appeared normal. Postsynaptically, boutons were surrounded by an extensive subsynaptic reticulum (ssr) that resembled wild-type with respect to thickness and complexity. Individual synaptic contacts (synaptic densities), which are identified by dark electron-dense areas in the cell membrane, were present in mutant boutons and contained active zones, marked by the presence of T bars surrounded by clouds of synaptic vesicles. The size and abundance of these vesicles appeared normal. In addition, the distribution of T bars (ranging from 0 to 3) per synaptic contact was similar in mutant and wild-type boutons. In wild-type, 25% of synaptic contacts had no T bars, 63% had one T bar, 10% had two T bars, and 2% had three T bars. In nwk², 20.6% had no T bars, 63.7% had one T bar, 13.7% had two T bars, and 2% had three T bars. Comparable results were obtained for nwk²/Df (Coyle, 2004).

Nonetheless, the general shape of nwk boutons was abnormal. Midline cross-sections of wild-type boutons were circular in outline, but those of nwk² were consistently elliptical. Morphometric analysis indicated that the surface area and volume of nwk² boutons were reduced by 42% and 60%, respectively. These changes were accompanied by a reduction in the size and number of individual synaptic contacts. Each wild-type bouton contained an average of 20 synaptic contacts, with an average surface area of 0.3673 ± 0.0202 μm² each. In contrast, nwk² boutons contained an average of only 10 synaptic contacts, 50% percent fewer, with an average surface area of 0.2817 ± 0.0181 μm² (p < 0.005), 23% smaller (Coyle, 2004).

It is concluded that the total area of synaptic contact is reduced in each nwk NMJ. Because type Ib boutons contain the majority of the synaptic contacts within a given NMJ 6/7, having almost 3 times more than type I boutons per μm length, the 50% reduction in synaptic contact number observed in nwk² indicates a large reduction in the total synaptic complement. This reduction would not be rectified by the increased synaptic bouton number in nwk mutants. Although there are 50% more boutons at NMJ 6/7 in nwk larvae, at least a 100% increase would be required to compensate for the 50% reduction in synaptic contacts per bouton. Since T bars (active zones) are distributed normally among nwk synapses, nwk NMJs contain fewer total active zones. Furthermore, the average surface area of each synaptic contact is reduced in nwk. Taken together, these data demonstrate that motorneuron terminals in nwk NMJs contain less total surface area of synaptic contact with the postsynaptic muscle (Coyle, 2004).

Nerve-evoked excitatory junctional currents (EJCs) were recorded from NMJ 6/7 to determine if nwk causes defects in synaptic function that parallel the morphological defects described above. Both nwk¹ and nwk² reduced mean EJC amplitude by >50% compared with wild-type. Although EJC amplitudes were reduced, the amplitude of miniature EJCs (mEJCs), or quantal size, was slightly increased in nwk¹ and nwk² compared with wild-type. mEJC frequency was not significantly different in nwk², although it was slightly elevated in nwk¹. Taken together, these results demonstrate that synaptic transmission is impaired in nwk mutants. The large reduction in quantal content, despite a small increase in quantal size, suggests that presynaptic vesicle release is reduced (Coyle, 2004).

Transgenic flies were generated expressing a cDNA that encodes a complete nwk ORF plus 5' and 3' untranslated sequences (UTRs) linked to a yeast UAS promoter (UAS-nwk⁺). Neural-specific expression of UAS-nwk⁺ using an elaV-Gal4 driver in a homozygous nwk² background restored the presence of Nwk at motor terminals with normal periactive zone localization. The ts paralytic phenotype of nwk² adults was also rescued: after 15 min at 38°C, more than 90% of the transgenic flies continued to walk and climb normally (Coyle, 2004).

NMJ overgrowth phenotypes were fully or partially rescued by neural-specific expression of UAS-nwk⁺. The normalized numbers of boutons and branches were fully rescued at NMJ 6/7 and NMJ 4. The incidence of hyperbranching boutons per NMJ was not significantly different between the rescue and control lines. Total synapse length of NMJ 6/7 was substantially reduced compared with nwk². At the electrophysiological level, EJC amplitude was fully rescued by neural-specific expression of the UAS-nwk⁺ transgene, and mEJC amplitude was partially rescued (Coyle, 2004).

The inability to achieve perfect rescue could result from failure of the transgene to exactly recapitulate the timing and amplitude of nwk expression, or from the lack of particular splice variants. Nonetheless, all nwk phenotypes were at least partially rescued by neural-specific expression of UAS-nwk⁺. In contrast, postsynaptic expression of UAS-nwk⁺ using a muscle driver did not rescue the ts or morphological phenotypes. In summary, these results demonstrate that the behavioral, developmental, and electrophysiological phenotypes associated with nwk all result from mutation of a single gene that acts presynaptically (Coyle, 2004).

The physical interaction between Nwk and Wsp suggest that they might participate in a common regulatory pathway affecting F-actin dynamics in synapses. If so, then wsp loss-of-function mutations should cause increased NMJ growth and branching similar to nwk. Indeed, it was found that bouton number, branch number, total NMJ length, and hyperbranching are all increased in wsp¹/Df and wsp¹. wsp¹ is a putative null allele, with occasional escapers surviving to the pharate adult stage. Although there is maternal contribution of Wsp in embryos, no Wsp immunoreactivity was detected at wsp NMJs. Thus, Wsp and Nwk have a similar function in the regulation of synaptic growth and bouton branching (Coyle, 2004).

To further investigate the functional relationship between Nwk and Wsp in vivo, double mutants were examined. The phenotype of homozygous nwk wsp double mutants is more severe than that of either single mutant with respect to bouton number, branch formation, and NMJ length. Also, hyperbranch complexity was dramatically increased, producing distinct synaptic morphologies. At NMJ 6/7 in double mutants, 3.2 ± 0.5 hyperbranched boutons were observed per NMJ, compared with only 0.56 ± 0.16 hyperbranches per NMJ in nwk² or 0.86 ± 0.35 in wsp¹/Df (p < 0.05). On average, each double mutant NMJ contained one bouton that emitted four new branches, while this type of hyperbranch was observed in only 10% of NMJs in single mutants. Boutons extending 5 or 6 new branches are found in more than 10% of the double mutant larvae (Coyle, 2004).

These results suggest that Nwk and Wsp interact presynaptically to regulate NMJ growth. Consistent with this interpretation, the morphological phenotype of wsp mutants was partially rescued by driving expression of a UAS-wsp⁺ transgene using a panneuronal Gal4 driver. Normalized bouton number, NMJ length, and branch number were significantly reduced toward wild-type values. Thus, presynaptic expression of Wsp, like Nwk, normally suppresses NMJ growth and bouton branching (Coyle, 2004).

In summary, the striking similarity in NMJ phenotypes exhibited by nwk and wsp mutants and the enhanced phenotypes observed in double mutants strongly support the idea that Nwk and Wsp act in a common regulatory pathway. The more extreme phenotype observed in double mutants compared with either single mutant most likely indicates that either mutation alone does not completely abolish the pathway as would be expected if there is some functional redundancy. For example, Wsp is known to be activated by many different binding partners and Nwk may act in part via other effectors besides Wsp (Coyle, 2004).

These data above suggest that nwk and wsp contribute to a common regulatory mechanism in a gene dosage-dependent manner, as do several other cytoskeletal effectors required for axon growth. To verify this, focus was placed on the incidence of hyperbranching, which is the most distinctive trait observed in these mutants. An increase was observed in the frequency and complexity of these structures; the number of wild-type nwk and wsp alleles decreased. Wild-type larvae exhibited no hyperbranches. Heterozygotes for either nwk or wsp alone displayed a low incidence of hyperbranching, but boutons with four or more new branches were never observed. In contrast, double heterozygotes (i.e., nwk² wsp¹/+ +) were intermediate between wild-type and the respective homozygotes, including the appearance of boutons that gave rise to four new branches. In hemizygotes for nwk or wsp, loss of one wild-type allele of the other gene caused a further enhancement of the mutant phenotype. Double homozygotes displayed the highest incidence of hyperbranch formation with boutons emitting up to six new branches. Similar trends were observed for bouton number and NMJ length. These data provide additional evidence that Nwk and Wsp interact within a common regulatory complex to regulate bouton proliferation and branching (Coyle, 2004).

The mechanism of bouton branching is unresolved at present, but it involves the reorganization of cytoskeletal components including microtubules (MTs). Overall MT organization appeared normal in nwk and wsp mutants, including the presence of microtubule loops within a subset of terminal boutons. In wild-type larvae, these loops are associated with stable boutons and disintegrate during branch formation. In addition, the patterns of MTs within branching boutons in wild-type and double mutants were indistinguishable. Thus, there is no major disorganization of MT structure in nwk and wsp mutants. Rather, abnormal proliferation and branching of boutons in these mutants is likely caused by the disruption of some other pathway (Coyle, 2004).

EVOLUTIONARY HOMOLOGS

The Slit protein guides neuronal and leukocyte migration through the transmembrane receptor Roundabout (Robo). The intracellular domain of Robo interacts with a novel family of Rho GTPase activating proteins (GAPs). Two of the Slit-Robo GAPs (srGAPs) are expressed in regions responsive to Slit. Slit increases srGAP1-Robo1 interaction and inactivates Cdc42. A dominant negative srGAP1 blocks Slit inactivation of Cdc42 and Slit repulsion of migratory cells from the anterior subventricular zone (SVZa) of the forebrain. A constitutively active Cdc42 blocks the repulsive effect of Slit. These results have demonstrated important roles for GAPs and Cdc42 in neuronal migration. A signal transduction pathway is proposed, from the extracellular guidance cue to intracellular actin polymerization (Wong, 2001).

In Saccharomyces cerevisiae, the WASP (Wiskott-Aldrich syndrome protein) homolog Las17p (also called Bee1p) is an important component of cortical actin patches. Las17p is part of a high-molecular-weight protein complex that regulates Arp2/3 complex-dependent actin polymerization at the cell cortex and that includes the type I myosins Myo3p and Myo5p and verprolin (Vrp1p). To identify other factors implicated with this complex in actin regulation, proteins were isolated that bind to Las17p by two-hybrid screening and affinity chromatography. Lsb7/Bzz1p (for Las seventeen binding protein 7) is an Src homology 3 (SH3) domain protein that interacts directly with Las17p via a polyproline-SH3 interaction. Bzz1p coimmunoprecipitates in a complex with Las17p, Vrp1p, Myo3/5p, Bbc1p, Hsp70p, and actin. It colocalizes with cortical actin patches and with Las17p. This localization is dependent on Las17p, but not on F-actin. Bzz1p interacts physically and genetically with type I myosins. While deletion of BZZ1 shows no obvious phenotype, simultaneous deletion of the BZZ1, MYO3, and MYO5 genes is lethal. Overexpression of Bzz1p inhibits cell growth, and a bzz1Delta myo5Delta double mutant is unable to restore actin polarity after NaCl stress. Finally, Bzz1p in vitro is able to recruit a functional actin polymerization machinery through its SH3 domains. Its interactions with Las17p, Vrp1p, and the type I myosins are essential for this process. This suggests that Bzz1p could be implicated in the regulation of actin polymerization (Soulard, 2002).

In the last few years, several genes involved in X-specific mental retardation (MR) have been identified by using genetic analysis. Although it is likely that additional genes responsible for idiopathic MR are also localized on the autosomes, cloning and characterization of such genes have been elusive so far. A previously uncharacterized gene, MEGAP, has been isolated that is disrupted and functionally inactivated by a translocation breakpoint in a patient who shares some characteristic clinical features, such as hypotonia and severe MR, with the 3p(-) syndrome. By fluorescence in situ hybridization and loss of heterozygosity analysis, it has been demonstrated that this gene resides on chromosome 3p25 and is deleted in 3p(-) patients that present MR. MEGAP/srGAP3 mRNA is predominantly and highly expressed in fetal and adult brain, specifically in the neurons of the hippocampus and cortex, structures known to play a pivotal role in higher cognitive function, learning, and memory. Several MEGAP/srGAP3 transcript isoforms are described; MEGAP/srGAP3a and -b represent functional GTPase-activating proteins (GAP) by an in vitro GAP assay. MEGAP/srGAP3 has recently been shown to be part of the Slit-Robo pathway regulating neuronal migration and axonal branching, highlighting the important role of MEGAP/srGAP3 in mental development. It is proposed that haploinsufficiency of MEGAP/srGAP3 leads to the abnormal development of neuronal structures that are important for normal cognitive function (Endris, 2002).

Formation of membrane ridges and scallops by the F-BAR protein Nervous Wreck

Eukaryotic cells are defined by extensive intracellular compartmentalization, which requires dynamic membrane remodeling. FER/Cip4 homology-Bin/amphiphysin/Rvs (F-BAR) domain family proteins form crescent-shaped dimers, which can bend membranes into buds and tubules of defined geometry and lipid composition. However, these proteins exhibit an unexplained wide diversity of membrane-deforming activities in vitro and functions in vivo. This study found that the F-BAR domain of the neuronal protein Nervous Wreck (Nwk) has a novel higher-order structure and membrane-deforming activity that distinguishes it from previously described F-BAR proteins. The Nwk F-BAR domain assembles into zigzags, creating ridges and periodic scallops on membranes in vitro. This activity depends on structural determinants at the tips of the F-BAR dimer and on electrostatic interactions of the membrane with the F-BAR concave surface. In cells, Nwk-induced scallops can be extended by cytoskeletal forces to produce protrusions at the plasma membrane. These results define a new F-BAR membrane-deforming activity and illustrate a molecular mechanism by which positively curved F-BAR domains can produce a variety of membrane curvatures. These findings expand the repertoire of F-BAR domain mediated membrane deformation and suggest that unique modes of higher-order assembly can define how these proteins sculpt the membrane (Becalska, 2013).

Becalska, A. N., Kelley, C. F., Berciu, C., Stanishneva-Konovalova, T. B., Fu, X., Wang, S., Sokolova, O. S., Nicastro, D. and Rodal, A. A. (2013). Formation of membrane ridges and scallops by the F-BAR protein Nervous Wreck. Mol Biol Cell 24: 2406-2418. PubMed ID: 23761074

Ben-Yaacov, S., Le Borgne, R., Abramson, I., Schweisguth, F. and Schejter, E.D. (2001). Wasp, the Drosophila Wiskott-Aldrich syndrome gene homolog, is required for cell fate decisions mediated by Notch signaling. J. Cell Biol. 152: 1-13. 11149916

Coyle, I. P., et al. (2004). Nervous wreck, an SH3 adaptor protein that interacts with Wsp, regulates synaptic growth in Drosophila. Neuron 41: 521-534. 14980202

Cullen, P. J. (2008). Endosomal sorting and signalling: an emerging role for sorting nexins. Nat. Rev. Mol. Cell Biol. 9: 574-582. PubMed Citation: 18523436

Endris, V., et al. (2002). The novel Rho-GTPase activating gene MEGAP/ srGAP3 has a putative role in severe mental retardation. Proc. Natl. Acad. Sci. 99: 11754-11759. 12195014

Gavin, A.C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A., Schultz, J., Rick, J. M., Michon, A. M. and Cruciat, C. M., et al. (2002). Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415: 141-147. 11805826

Hur, J. H., Lee, S. H., Kim, A. Y., Koh, Y. H. (2018). Regulation of synaptic architecture and synaptic vesicle pools by Nervous wreck at Drosophila Type 1b glutamatergic synapses. Exp Mol Med, 50(8):1 PubMed ID: 30127363

Itoh, T., et al. (2005). Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev. Cell 9: 791-804. PubMed Citation: 16326391

Kessels, M. M. and Qualmann, B. (2006). Syndapin oligomers interconnect the machineries for endocytic vesicle formation and actin polymerization. J. Biol. Chem. 281: 13285-13299. PubMed Citation: 16540475

Khodosh R., Augsburger A., Schwarz T. L. and Garrity P. A. (2006). Bchs, a BEACH domain protein, antagonizes Rab11 in synapse morphogenesis and other developmental events. Development 133: 4655-4665. PubMed Citation: 17079274

O'Connor-Giles, K. M., Ho, L. L. and Ganetzky, B. (2008). Nervous wreck interacts with thickveins and the endocytic machinery to attenuate retrograde BMP signaling during synaptic growth. Neuron 58(4): 507-18. PubMed Citation: 18498733

Rao, Y., et al. (2010). Molecular basis for SH3 domain regulation of F-BAR-mediated membrane deformation. Proc. Natl. Acad. Sci. 107: 8213-8218. PubMed Citation: 20404169

Rodal, A. A., Motola-Barnes, R. N. and Littleton J. T. (2008). Nervous wreck and Cdc42 cooperate to regulate endocytic actin assembly during synaptic growth. J. Neurosci. 28(33): 8316-25. PubMed Citation: 18701694

Rodal, A. A., et al. (2011). A presynaptic endosomal trafficking pathway controls synaptic growth signaling. J. Cell Biol. 193(1): 201-17. PubMed Citation: 21464232

Shimada, A., et al. (2007). Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis. Cell 129: 761-772. PubMed Citation: 17512409

Soulard, A., Lechler, T., Spiridonov, V., Shevchenko, A., Shevchenko, A., Li, R., and Winsor, B. (2002). Saccharomyces cerevisiae Bzz1p is implicated with type I myosins in actin patch polarization and is able to recruit actin-polymerizing machinery in vitro. Mol. Cell. Biol. 22: 7889-7906. 12391157

Tsujita, K., et al. (2006). Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. J. Cell Biol. 172: 269-279. PubMed Citation: 16418535

Wang, K., et al. (2008). Drosophila spichthyin inhibits BMP signaling and regulates synaptic growth and axonal microtubules. Nat. Neurosci. 10: 177-185. PubMed Citation: 17220882

Wong, K., Ren, X. R., Huang, Y.Z., Xie, Y., Liu, G., Saito, H., Tang, H., Wen, L., Brady-Kalnay, S. M. and Mei, L. et al. (2001). Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell 107: 209-221. 11672528

date revised: 25 March 2014

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