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).
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 UAStkvACT 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 TkvACT-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 TkvACT 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 TkvACT 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).
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).
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).
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 (nwk2-5) 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.
Reference names in red indicate recommended papers.
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
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
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
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
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
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 February 2009
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