InteractiveFly: GeneBrief

Rho GTPase activating protein at 100F: Biological Overview | References

Gene name - Rho GTPase activating protein at 100F

Synonyms - Syd-1

Cytological map position - 100D2-100D3

Function - signaling

Keywords - master organizer of active zone assembly, regulates pre- and postsynaptic maturation, neuromuscular junction

Symbol - RhoGAP100F

FlyBase ID: FBgn0039883

Genetic map position - chr3R:31,812,609-31,845,244

Classification - RhoGAP_SYD1, PDZ_signaling, C2 domain

Cellular location - intracellular

NCBI link: EntrezGene
RhoGAP100F orthologs: Biolitmine
Recent literature
Spinner, M. A., Walla, D. A. and Herman, T. G. (2018). Drosophila Syd-1 has RhoGAP activity that is required for presynaptic clustering of Bruchpilot/ELKS but not Neurexin-1. Genetics 208(2): 705-716. PubMed ID: 29217522
Syd-1 proteins are required for presynaptic development in worm, fly, and mouse. Syd-1 proteins in all three species contain a Rho GTPase activating protein (GAP)-like domain of unclear significance: invertebrate Syd-1s are thought to lack GAP activity, and mouse mSYD1A has GAP activity that is thought to be dispensable for its function. This study shows that Drosophila melanogaster Syd-1 can interact with all six fly Rhos and has GAP activity toward Rac1 and Cdc42. During development, fly Syd-1 clusters multiple presynaptic proteins at the neuromuscular junction (NMJ), including the cell adhesion molecule Neurexin (Nrx-1) and the active zone (AZ) component Bruchpilot (Brp), both of which Syd-1 binds directly. A mutant form of Syd-1 that specifically lacks GAP activity localizes normally to presynaptic sites and is sufficient to recruit Nrx-1 but fails to cluster Brp normally. Evidence is provided that Syd-1 participates with Rac1 in two separate functions: (1) together with the Rac guanine exchange factor (RacGEF) Trio, GAP-active Syd-1 is required to regulate the nucleotide-bound state of Rac1, thereby promoting Brp clustering; and (2) Syd-1, independent of its GAP activity, is required for the recruitment of Nrx-1 to boutons, including the recruitment of Nrx-1 that is promoted by GTP-bound Rac1. It is concluded that, contrary to current models, the GAP domain of fly Syd-1 is active and required for presynaptic development; it is suggested that the same may be true of vertebrate Syd-1 proteins. In addition, the data provide new molecular insight into the ability of Rac1 to promote presynaptic development.
Fulterer, A., Andlauer, T. F. M., Ender, A., Maglione, M., Eyring, K., Woitkuhn, J., Lehmann, M., Matkovic-Rachid, T., Geiger, J. R. P., Walter, A. M., Nagel, K. I. and Sigrist, S. J. (2018). Active zone scaffold protein ratios tune functional diversity across brain synapses. Cell Rep 23(5): 1259-1274. PubMed ID: 29719243
High-throughput electron microscopy has started to reveal synaptic connectivity maps of single circuits and whole brain regions, for example, in the Drosophila olfactory system. However, efficacy, timing, and frequency tuning of synaptic vesicle release are also highly diversified across brain synapses. These features critically depend on the nanometer-scale coupling distance between voltage-gated Ca(2+) channels (VGCCs) and the synaptic vesicle release machinery. Combining light super resolution microscopy with in vivo electrophysiology, this study shows that two orthogonal scaffold proteins (ELKS family Bruchpilot, BRP, and Syd-1) cluster-specific (M)Unc13 release factor isoforms either close (BRP/Unc13A) or further away (Syd-1/Unc13B) from VGCCs across synapses of the Drosophila olfactory system, resulting in different synapse-characteristic forms of short-term plasticity. Moreover, BRP/Unc13A versus Syd-1/Unc13B ratios were different between synapse types. Thus, variation in tightly versus loosely coupled scaffold protein/(M)Unc13 modules can tune synapse-type-specific release features, and "nanoscopic molecular fingerprints" might identify synapses with specific temporal features.
Weiss, J. T. and Donlea, J. M. (2021). Sleep deprivation results in diverse patterns of synaptic scaling across the Drosophila mushroom bodies. Curr Biol. PubMed ID: 34107302
Sleep is essential for a variety of plastic processes, including learning and memory. However, the consequences of insufficient sleep on circuit connectivity remain poorly understood. To better appreciate the effects of sleep loss on synaptic connectivity across a memory-encoding circuit, changes were examined in the distribution of synaptic markers in the Drosophila mushroom body (MB). Protein-trap tags for active zone components indicate that recent sleep time is inversely correlated with Bruchpilot (BRP) abundance in the MB lobes; sleep loss elevates BRP while sleep induction reduces BRP across the MB. Overnight sleep deprivation also elevated levels of dSyd-1 and Cacophony, but not other pre-synaptic proteins. Cell-type-specific genetic reporters show that MB-intrinsic Kenyon cells (KCs) exhibit increased pre-synaptic BRP throughout the axonal lobes after sleep deprivation; similar increases were not detected in projections from large interneurons or dopaminergic neurons that innervate the MB. These results indicate that pre-synaptic plasticity in KCs is responsible for elevated levels of BRP in the MB lobes of sleep-deprived flies. Because KCs provide synaptic inputs to several classes of post-synaptic partners, a fluorescent reporter for synaptic contacts was used to test whether each class of KC output connections is scaled uniformly by sleep loss. The KC output synapses that were observed in this study can be divided into three classes: KCs to MB interneurons; KCs to dopaminergic neurons; and KCs to MB output neurons. No single class showed uniform scaling across each constituent member, indicating that different rules may govern plasticity during sleep loss across cell types.

Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic Neurexin/Neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to Neurexin. This study reports that the scaffold protein Spinophilin binds to the C-terminal portion of Neurexin and is needed to limit Neurexin/Neuroligin signalling by acting antagonistic to Syd-1 (RhoGAP100F). Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/Neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. It is concluded that presynaptic Spinophilin fine-tunes Neurexin/Neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis (Muhammad, 2015).

Chemical synapses release synaptic vesicles (SVs) at specialized presynaptic membranes, so-called active zones (AZs), which are characterized by electron-dense structures, reflecting the presence of extended molecular protein scaffolds. These AZ scaffolds confer stability and facilitate SV release. Importantly, at individual AZs, scaffold size is found to scale with the propensity to engage in action potential-evoked release. An evolutionarily conserved set of large multi-domain proteins operating as major building blocks for these scaffolds has been identified over the last years: Syd-2/Liprin-α, RIM, RIM-binding-protein (RBP) and ELKS family proteins (of which the the Drosophila homologue is called Bruchpilot (BRP)). However, how presynaptic scaffold assembly and maturation are controlled and coupled spatiotemporally to the postsynaptic assembly of neurotransmitter receptors remains largely unknown, although trans-synaptic signalling via Neurexin-1 (Nrx-1)-Neuroligin-1 (Nlg1) adhesion molecules is a strong candidate for a conserved 'master module' in this context, based on Nrx-Nlg signalling promoting synaptogenesis in vitro, synapses of rodents, Caenorhabditis elegans and Drosophila (Muhammad, 2015).

With respect to scaffolding proteins, Syd-1 was found to promote synapse assembly in C. elegans, Drosophila and rodents. In Drosophila, the Syd-1-PDZ domain binds the Nrx-1 C terminus and couples pre- with postsynaptic maturation at nascent synapses of glutamatergic neuromuscular junctions (NMJs) in Drosophila larvae. Syd-1 cooperates with Nrx-1/Nlg1 to stabilize newly formed AZ scaffolds, allowing them to overcome a 'threshold' for synapse formation. Additional factors tuning scaffold assembly, however, remain to be identified. This study shows that the conserved scaffold protein spinophilin (Spn) is able to fine-tune Nrx-1 function by binding the Nrx-1 C terminus with micromolar affinity via its PDZ domain. In the absence of presynaptic Spn, 'excessive seeding' of new AZs occurred over the entire NMJ due to elevated Nrx-1/Nlg1 signalling. Apart from structural changes, this study shows that Spn plays an important role in neurotransmission since it is essential to establish proper SV release probability, resulting in a changed ratio of spontaneous versus evoked release at Spn NMJ terminals. The trans-synaptic dialogue between Nrx-1 and Nlg1 aids in the initial assembly, specification and maturation of synapses, and is a key component in the modification of neuronal networks. Regulatory factors and processes that fine-tune and coordinate Nrx-1/Nlg1 signalling during synapse assembly process are currently under investigation. These data indicate that Drosophila Spn-like protein acts presynaptically to attenuate Nrx-1/Nlg1 signalling and protects from excessive seeding of new AZ scaffolds at the NMJ. In Spn mutants, excessive AZs suffered from insufficient evoked release, which may be partly explained by their reduced size, and partly by a genuine functional role of Spn (potentially mediated via Nrx-1 binding). In mice, loss of Spn (Neurabin II), one of the two Neurabin protein families present in mammals, was reported to provoke a developmental increase in synapse numbers. While Spinophilin was found to be expressed both pre- and post-synaptically, its function, so far, has only been analysed in the context of postsynaptic spines. Given the conserved Spn/Nrx-1 interaction reported in this study, Spn family proteins might execute a generic function in controlling Nrx-1/Nlg1-dependent signalling during synapse assembly (Muhammad, 2015).

This study consistently found that Spn counteracts another multi-domain synaptic regulator, Syd-1, in the control of Nrx-1/Nlg1 signalling. Previous genetic work in C. elegans identified roles of Syd-1 epistatic to Syd-2/Liprin-α in synaptogenesis. Syd-1 also operates epistatic to Syd-2/Liprin-α at Drosophila NMJs. Syd-1 immobilizes Nrx-1, positioning Nlg1 at juxtaposed postsynaptic sites, where it is needed for efficient incorporation of GluR complexes. Intravital imaging suggested an early checkpoint for synapse assembly, involving Syd-1, Nrx-1/Nlg1 signalling and oligomerization of Liprin-α in the formation of an early nucleation lattice, which is followed later by ELKS/BRP-dependent scaffolding events. As Spn promotes the diffusional motility of Nrx-1 over the terminal surface and limits Nrx-1/Nlg1 signalling, and as its phenotype is reversed by loss of a single gene copy of nrx-1, nlg1 or syd-1, Spn displays all the features of a 'negative' element mounting, which effectively sets the threshold for AZ assembly. As suggested by FRAP experiments, Spn might withdraw a population of Nrx-1 from the early assembly process, establishing an assembly threshold that ensures a 'typical' AZ design and associated postsynaptic compartments. As a negative regulatory element, Spn might allow tuning of presynaptic AZ scaffold size and function (Muhammad, 2015).

The C. elegans Spn homologue NAB-1 (NeurABin1) was previously shown to bind Syd-1 in cell culture recruitment assays. This study found consistent evidence for Syd-1/Nrx-1/Spn tripartite complexes in salivary gland experiments. Moreover, the PDZ domain containing regions of Spn and Syd-1 interacted in Y2H experiments. It would be interesting to dissect whether the interaction of Spn/Syd-1 plays a role in controlling the access of Nrx-1 to one or both factors. For C. elegans HSN synapses, a previous study showed that loss of NAB-1 results in a deficit of synaptic markers, such as Syd-1 and Syd-2/Liprin-α, while NAB-1 binding to F-actin was also found to be important for synapse assembly. Though at first glance rather contradictory to the results described in this study, differences might result from Chia (2012) studying synapse assembly executed over a short time window, when partner cells meet for the first time. In contrast, this study used a model (Drosophila larval NMJs) where an already functional neuronal terminal adds novel AZs. Despite the efforts of this study, no role of F-actin in the assembly of AZs of late larval Drosophila NMJs was demonstrated. F-actin patches might be particularly important to establish the first synaptic contacts between partner cells. Both the study by Chia et al. and this study, however, point clearly towards important regulatory roles of Spn family members in the presynaptic control of synapse assembly. Further, this study described a novel interaction between the Spn-PDZ domain and the intracellular C-term of Nrx-1 at the atomic level. Interestingly, it was found that all functions of Spn reported in this study, structural as well as functional, were strictly dependent on the ligand-binding integrity of this PDZ domain. It is noteworthy that the Spn-PDZ domain binds other ligands as well, for example, Kalirin-7 and p70S6K , and further elucidation of its role as a signal 'integrator' in synapse plasticity should be interesting. The fact that Nrx-1 levels were increased at Spn NMJs and, most importantly, that genetic removal of a single nrx-1 gene copy effectively suppressed the Spn AZ phenotype, indicates an important role of the Spn/Nrx-1 interaction in this context. Affinity of Spn-PDZ for the Nrx-1 C-term was somewhat lower than that of the Syd-1-PDZ, both in ITC and Y2H experiments. Nonetheless, overexpression of Spn was successful in reducing the targeting effect of Syd-1 on overexpressed Nrx-1GFP. It will be interesting to see whether this interaction can be differentially regulated, for example, by (de)phosphorylation. It is worth noting that apart from Syd-1 and Spn, several other proteins containing PDZ domains, including CASK, Mint1/X11, CIPP and Syntenin, were found to bind to the Nrxs C-termini. CASK was previously shown to interact genetically with Nrx-1, controlling endocytic function at Drosophila NMJs. However, when this study tested for an influence of CASK on Nrx-1GFP motility using FRAP, genetic ablation of CASK had no effect (Muhammad, 2015).

Thus, CASK function seemingly resembles neither Syd-1 nor Spn. Clearly, future work will have to address and integrate the role of other synaptic regulators converging on the Nrx-1 C-term. In particular, CASK (which displays a kinase function that phosphorylates certain motifs within the Nrx-1 C-term) might alternately control Spn- and Syd-1-dependent functions. Presynaptic Nrx-1, through binding to postsynaptic Nlg1 at developing Drosophila NMJ terminals, is important for the proper assembly of new synaptic sites. It is of note, however, that while mammalian Nrxs display robust synaptogenetic activity in cellular in vitro systems, direct genetic evidence for synaptogenetic activity of Nrxs in the mammalian CNS remained rather scarce. Triple knockout mice lacking all α-Nrxs display no gross synaptic defects at the ultrastructural level. Future analysis will have to investigate whether differences here might be explained by specific compensation mechanisms in mammals; for example, by β-Nrxs, or other parallel trans-synaptic communication modules. Genuine functional deficits in neurotransmitter release were also observed after the elimination of presynaptic Spn. Elimination of ligand binding to the PDZ domain rendered the protein completely nonfunctional, without affecting its synaptic targeting. Thus, the Spn functional defects are likely to be mediated via a lack of Nrx-1 binding. Notably, ample evidence connects Nrx-1 function with both the functional and structural maturation of Drosophila presynaptic AZs. This work now promotes the possibility that binding of Spn to Nrx-1 is important for establishing correct release probability, independent of absolute AZ scaffold size. It is noteworthy that Nrx-1 function was previously shown to be important for proper Ca2+ channel function and, as a result, properly evoked SV release. Thus, it will be interesting to investigate whether the specific functional contributions of Spn are mediated via deficits in the AZ organization of voltage-gated Ca2+ channels or Ca2+ sensors, such as synaptotagmin. Taken together, this study found an unexpected function for Spn in addition of AZs at Drosophila glutamatergic terminals, through the integration of signals from both the pre- and postsynaptic compartment. Given that the Spn/Nrx-1 interaction is found to be conserved from Drosophila to rodents, addressing similar roles of presynaptic Spn in mammalian brain physiology and pathophysiology might be informative (Muhammad, 2015).

Antagonistic interactions between two Neuroligins coordinate pre- and postsynaptic assembly

As a result of developmental synapse formation, the presynaptic neurotransmitter release machinery becomes accurately matched with postsynaptic neurotransmitter receptors. Trans-synaptic signaling is executed through cell adhesion proteins such as Neurexin::Neuroligin pairs but also through diffusible and cytoplasmic signals. How exactly pre-post coordination is ensured in vivo remains largely enigmatic. This study identified a 'molecular choreography' coordinating pre- with postsynaptic assembly during the developmental formation of Drosophila neuromuscular synapses. Two presynaptic Neurexin-binding scaffold proteins, Syd-1 and Spinophilin (Spn), spatio-temporally coordinated pre-post assembly in conjunction with two postsynaptically operating, antagonistic Neuroligin species: Nlg1 and Nlg2. The Spn/Nlg2 module promoted active zone (AZ) maturation by driving the accumulation of AZ scaffold proteins critical for synaptic vesicle release. Simultaneously, these regulators restricted postsynaptic glutamate receptor incorporation. Both functions of the Spn/Nlg2 module were directly antagonized by Syd-1/Nlg1. Nlg1 and Nlg2 also had divergent effects on Nrx-1 in vivo motility. Concerning diffusible signals, Spn and Syd-1 antagonistically controlled the levels of Munc13-family protein Unc13B at nascent AZs, whose release function facilitated glutamate receptor incorporation at assembling postsynaptic specializations. As a result, this study has provided direct in vivo evidence illustrating how a highly regulative and interleaved communication between cell adhesion protein signaling complexes and diffusible signals allows for a precise coordination of pre- with post-synaptic assembly. It will be interesting to analyze whether this logic also transfers to plasticity processes (Ramesh, 2021).

Synaptic vesicle (SV) release at chemical synapses depends on the formation of active zone (AZ) scaffolds composed of a canonical apparatus of proteins including Unc13, RIM-binding protein (RIM-BP), Liprin-α and CAST/ELKS (called Bruchpilot [BRP] in Drosophila). The size of individual AZ scaffolds scales with SV release probability. Once matured, each AZ apparently forms an integer number of release sites apposed by postsynaptic glutamate receptors (GluRs), likely spatially coordinated through a trans-synaptic micropattern ('nanocolumns'). Importantly, in the course of maturation, AZ size becomes closely matched to the size of the postsynaptic density (PSD) scaffold clustering neurotransmitter (NT) receptors (Ramesh, 2021).

How the in vivo synapse assembly process and associated regulatory steps achieve this precise pre-post matching during developmental assembly is not fully understood. Notably, trans-synaptic cell-adhesion molecules (CAMs) have the capacity to bidirectionally tune synapse assembly, and Neurexin (Nrx) and Neuroligin (Nlg) interactions represent a regulatory principle conserved across vertebrate and invertebrate synapses. Although many synaptic CAMs and cytoplasmic proteins have been studied in isolation, how different CAMs selectively engage with each other and their cytoplasmic partners to ensure pre-post matching during synapse assembly has remained enigmatic, partly because of the high genetic redundancy among mammalian CAMs. Besides CAM signaling, diffusible signals including NT release at nascent AZs might play a regulatory role in postsynaptic assembly (Ramesh, 2021).

This study characterize mechanisms that ensure pre-post matching during the assembly of individual glutamatergic synapses at the Drosophila neuromuscular junction (NMJ). In developing larvae, synapse maturation ultimately establishes a precisely defined pre-post stoichiometry over the course of several hours. To interrogate these mechanisms, the unique advantages were used of the larval NMJ system, which allows for a synergy of reduced genetic redundancy, super-resolution, and dynamic intravital microscopy and electrophysiology. Nlg1 and Nlg2, two Nlg species previously shown to functionally interact with the only Nrx family protein in Drosophila, Nrx-1. The current results reveal that these two Nlgs serve antagonistic roles and operate in conjunction with two antagonistic presynaptic proteins that bind Nrx-1: Syd-1 cooperating with Nlg1 and Spinophilin (Spn) with Nlg2. Whereas the Spn/Nlg2 functional module promoted AZ maturation (BRP/RIM-BP/Unc13A incorporation) but restrained GluRIIA-containing receptor incorporation, Syd-1/Nlg1 initiated AZ assembly and promoted GluRIIA receptor incorporation through Unc13B recruitment and its glutamate release function. Genetic interaction experiments identified a remarkable degree of crosstalk between these modules, exemplifying a regulatory principle obviously evolved to ensure precise pre-post matching, and integrating Unc13B-dependent glutamate release acting as a diffusible signal. Altogether, these data indicate that synaptic matching is not established via a trans-synaptic 'stoichiometric building principle' to continuously accumulate synaptic components, but via a regulatory crosstalk between antagonistic assembly modules (Ramesh, 2021).

Synapses form out of three interdependent molecular assemblies, each precisely crafted to execute fast and precise information transfer between two cells: the presynaptic AZ where SVs fuse at defined release sites, the synaptic cleft through which NT diffuses, and the postsynaptic compartment where the NT binds its receptors. Importantly, these compartments do not form in isolation, but the size of the AZ (and thus the number of presynaptic release sites per AZ) must closely scale with the number of postsynaptic NT receptors. Super-resolution microscopy identified presynaptic AZ protein nanoclusters to align with concentrated postsynaptic receptors and scaffolding proteins, suggesting the existence of trans-synaptic molecular 'nanocolumns'. Indeed, the exact nanometer location of vesicular release in relation to receptors might be a critical determinant of synaptic strength, which might also contribute to synaptic plasticity (Ramesh, 2021).

A central question now pertains to how trans-synaptic signaling is precisely executed in molecular terms to coordinate pre- with post-synaptic assembly. Candidate molecular scenarios include interactions that directly bridge pre- and postsynaptic membranes like trans-synaptic CAMs, which bidirectionally control synapse formation, remodeling, and elimination. This study exploited the unique features of the Drosophila NMJ system: unique accessibility to intravital imaging to accurately analyze the assembly path, a cytoarchitecture ideal for super-resolution analysis, high-resolution electrophysiological measurements, and a low level of genetic redundancy, to address how presynaptic AZs are matched to postsynaptic GluRs. Moreover, the amount of ELKS protein BRP, easily accessible for STED microscopy, directly scales with presynaptic release at AZs, making it an ideal readout to assess both structural and functional assembly (Ramesh, 2021).

In principle, a strategy of continuously accumulating stoichiometric amounts of pre- and postsynaptic material along the assembly trajectory, potentially via a single transcellular bridge connecting to nucleation processes on both sides, might appear the easiest way to establish pre-post matching. Indeed, such an idea has recently been proposed, where the age of AZs determines their size and strength at the Drosophila NMJ. However, such a solution might lack regulatory flexibility and is also not what was find in this work. Instead, this analysis identifies antagonistic regulatory inputs to be executed by two postsynaptically active Nlg species operating synergistically with their respective 'cognate' presynaptic scaffold proteins, Syd-1 and Spn, previously shown to steer synapse assembly via their Nrx-1-binding function. It here appears likely that autonomy over the presynaptic versus the postsynaptic compartment might be particularly relevant during plasticity processes, shown to involve the specific incorporation of BRP at NMJ synapses. This antagonistic operation might serve to embed contextual information while steering the assembly process and could be particularly robust when utilized in such a highly regulative scheme (Ramesh, 2021).

A model is provided for the functional relations analyzed in this study. Syd-1 and Nlg1 form new AZs in the seeding phase, whereas Spn and Nlg2 promote incorporation of BRP to appropriate levels in the maturation phase. Notably, BRP is the rate-limiting building block of the AZ scaffold, determining the size and functional strength of the AZ specialization. Overactivity of Syd-1/Nrx-1/Nlg1 signaling likely is directly responsible for the Spn AZ phenotype, given that it could be suppressed by lowering the dose of any of these molecules. The same is true for the Nlg2 phenotype as well, suggesting that the Nlg2 AZ phenotype similarly reflects Syd-1/Nlg1 axis overactivity. Furthermore, reduction of Spn efficiently suppressed the normally excessive BRP incorporation at the AZs remaining in Syd-1, Nrx-1, and Nlg-1 mutants. Mechanistically, future analysis will have to clarify whether direct physical interactions of Spn with BRP complexes, co-clustering RIM-BP and Unc13A are of relevance here. Alternatively, the Syd-1 and Spn modules might antagonistically control a downstream process such as the status of F-actin (Ramesh, 2021).

Although in the past, Spn was interpreted as functioning after Syd-1 during the AZ development process, the current data now suggest that Syd-1 and Spn in fact continuously antagonize each other throughout assembly to tune final AZ size and function. Still, intravital imaging of nascent AZs showed that the peak of Syd-1 accumulation precedes the peak of BRP accumulation by hour. The fact that the Syd-1 scaffold is favored over the Spn scaffold during the seeding phase might be explained via a 'quasi-epistatic' relation between these regulators: Syd-1 mutants show lower amounts of Spn, whereas Spn mutants show elevated amounts of Syd-1 suggesting that Syd-1 is needed for Spn accumulation at the AZ, potentially allowing Syd-1-mediated AZ seeding to precede Spn-mediated BRP accumulation. Spn and Syd-1 were shown to interact with each other in Drosophila and in C. elegans. It would be interesting to investigate whether the Spn/Syd-1 interaction plays a role in regulating access to Nrx-1, thereby contributing to define the actual 'assembly mode:' seeding or maturation. Obviously, the assembly modules must communicate to ultimately ensure a well-defined assembly product, e.g., via associated kinase and/or phosphatase activities. For example, the phosphorylation status of BRP can control transport. Furthermore, although Spn attenuation did efficiently suppress the Syd-1, Nrx-1, and Nlg1 AZ phenotypes, Nlg2 attenuation did not suppress the Nrx-1 and Nlg1 phenotypes. This suggests that the trans-synaptic signaling through Nrx-Nlgs might ensure that assembly proceeds from seeding toward maturation during development. This also opens up the possibility that Nlg2 attenuates Syd-1/Nrx-1/Nlg1 function by removing Nrx-1 from the seeding module and/or suppressing Nlg1 activity through cis-heteromerization. FRAP data also indicate that the postsynaptic binding partner identity (Nlg1 or Nlg2) has differential effects on Nrx-1 mobility. Lack of Nlg2 likely boosts the Nrx-1::Nlg1 seeding activity, directly explaining the supernumerary AZs typical for Nlg2 mutant (Ramesh, 2021).

Nlg1 promoted but Nlg2 blocked GluRIIA incorporation, which precedes BRP accumulation. Previous analysis showed that Syd-1 seemingly instructs Nrx-1 to interact with Nlg1 and promotes GluRIIA incorporation before BRP incorporation. Genetic interaction analysis showed that Syd-1/Nlg1 and Spn/Nlg2 execute a mutual regulatory counterplay here. This study now extends the understanding of GluRIIA incorporation to involve the release function of Unc13B, enriched at nascent AZs by Syd-1, a process antagonized by Spn. Spn and Nlg2 functionally cooperate to limit the amount of GluRIIA incorporation in the nascent postsynaptic specialization and match receptor amounts to the AZ size. However, although the Spn mutant phenotype was rescued by Syd-1, Nrx-1, and Nlg1 heterozygosity, the Nlg2 mutant phenotype was only rescued by Syd-1 heterozygosity, suggesting that Nlg1 and Nlg2 have an additional function in mediating GluRIIA incorporation independent of Unc13B. Mechanistically, it might well be that Nlg2 at the nascent postsynaptic compartment directly competes with Nlg1 for the binding of a critical effector, e.g., the ectodomain of the GluR complex or other membrane proteins such as Neto (Ramesh, 2021).

In mice, most synapses formed normally in the absence of NT release during development, but the synapses did not persist as they matured. Experiments in mice have shown in the past that massive local glutamate release could induce spine formation at the postsynapse. However, whether vesicular transmitter release tunes the incorporation dynamics of GluRs during developmental synapse assembly remains inconclusive (Ramesh, 2021).

Unc13B arrives early at nascent NMJ AZs. This recruitment of Unc13B is antagonistically controlled by the two complexes, given that Syd-1 mutants showed reduced but Spn mutants strongly increased synaptic Unc13B amounts. Importantly, the excessive GluRIIA incorporation in Spn mutants critically depended on Unc13B. Notably, treatment of cell cultures with BoNT-C and TNT-E previously was shown to prevent effective postsynaptic insertion of glutamatergic receptors in cultivated hippocampal neurons. However, it cannot be exclude that once Unc13A accumulates at the AZ, Unc13B might continue to mediate GluRIIA incorporation into later stages of synapse assembly (Ramesh, 2021).

Concerning the mode of Unc13B action, the data suggest that evoked Unc13B-mediated glutamate release at nascent sites attracts GluRIIA receptors, which are recruited from diffuse pools at the plasma membrane. Notably, proper gating behavior of GluRIIA in response to presynaptic glutamate release previously was shown to be essential for matching pre- with post-assembly. Unc13B-mediated release is coupled more loosely to Ca2+ channel activity compared with release mediated by the functionally dominant isoform, Unc13A. Likely, sensing glutamate at nascent sites renders GluRIIA into an active state, which allows for postsynaptic incorporation, previously shown to be nearly irreversible. Whether the GluRIIA incorporation subsequent to the glutamate sensing is truly stage dependent, e.g., via specific scaffold or cleft proteins, or whether differences in the spatio-temporal detail of glutamate release between Unc13B and Unc13A are more important here remains to be addressed (Ramesh, 2021).

Nrx-1, Nlg1, and Syd-1 mutants all show reduced NMJ area, whereas Spn and Nlg2 mutants showed normal NMJ sizes, and all of them showed reduced evoked potentials. Previous studies have shown that synaptic terminals can compensate for a change in size by adjusting NT output. A recent study showed that spontaneous neurotransmission is needed for the normal structural maturation of Drosophila NMJ synapses exclusive from the role of evoked neurotransmission. Increasing miniature events was sufficient to induce synaptic terminal growth, and this synapse maturation was locally regulated via a Trio guanine nucleotide exchange factor (GEF) and Rac1 GTPase molecular signaling pathway. Interestingly, Syd-1 was found to interact with Trio signaling. Together with the Rac guanine exchange factor (RacGEF) Trio, Syd-1 GAP activity promotes BRP clustering and independent of its GAP activity, Syd-1 recruits Nrx-1 to boutons. Additionally, mammalian Spn forms a complex with Rac1-GEF Kalirin-7 and, along with Rho-GEF Lfc, control dendritic spine morphology and function. Therefore, it will be interesting to study how Syd-1 and Spn antagonism translates into GAP/GEF signaling, which in turn might control the synapse assembly at Drosophila NMJs (Ramesh, 2021).

Serial synapse formation through filopodial competition for synaptic seeding factors

Following axon pathfinding, growth cones transition from stochastic filopodial exploration to the formation of a limited number of synapses. How the interplay of filopodia and synapse assembly ensures robust connectivity in the brain has remained a challenging problem. This study developed a new 4D analysis method for filopodial dynamics and a data-driven computational model of synapse formation for R7 photoreceptor axons in developing Drosophila brains. Live data support a 'serial synapse formation' model, where at any time point only 1-2 'synaptogenic' filopodia suppress the synaptic competence of other filopodia through competition for synaptic seeding factors. Loss of the synaptic seeding factors Syd-1 and Liprin-alpha leads to a loss of this suppression, filopodial destabilization, and reduced synapse formation. The failure to form synapses can cause the destabilization and secondary retraction of axon terminals. This model provides a filopodial 'winner-takes-all' mechanism that ensures the formation of an appropriate number of synapses (Ozel, 2019).

After pathfinding, axon growth cones transition to become terminal structures with presynaptic active zones. How axon terminals form a defined number of synaptic contacts with a specific subset of partners is a daunting problem in dense brain regions. Stochastically extending and retracting filopodial extensions occur during both pathfinding and synapse formation and are thought to facilitate interactions between synaptic partners. However, little is known about the role of stochastic filopodial dynamics for robust synapse formation (Ozel, 2019).

Presynaptic active zone assembly is a key step in synapse formation and regulated by a conserved set of proteins. An early active zone 'seeding' step has been defined through the functions of the multidomain scaffold proteins Syd-1 and Liprin-α in C. elegans and Drosophila neuromuscular junction (NMJ). Syd-1 is a RhoGAP-domain-containing protein that recruits Liprin-α to the active zone. Liprin-α is an adaptor protein named after its direct interaction with the receptor tyrosine phosphatase Leukocyte common antigen-related (LAR). The Liprin-α and LAR interaction has been directly implicated in active zone assembly across species. Downstream, Liprin-α and Syd-1 recruit core active zone components and ELKS/CAST family protein Brp. Finally, the RhoGEF Trio has been proposed to function downstream of the Lar/Liprin-α/Syd-1 and has recently been suggested to regulate active zone size (Ozel, 2019).

Remarkably, the proposed Lar-Liprin-α-Syd-1-Trio pathway has been characterized in parallel for its role in axon guidance, independent of active zone assembly. In the Drosophila visual system, mutants in all four genes have been implicated in the layer-specific targeting of photoreceptor R7 axons in the medulla neuropil. It is unclear whether any of the four mutants affect active zone assembly in R7 neurons. Dual roles in axon pathfinding and synapse formation have been shown or proposed for all four genes. Independent implications in active zone assembly and axon pathfinding raise the question what functions are primary or secondary (Ozel, 2019).

This study investigated the relationship between filopodial dynamics and synapse assembly in the presynaptic R7 terminal. The early synaptic seeding factors Liprin-α and Syd-1 accumulate in only a single filopodium per terminal at any given point in time. Consequently, only 1-2 filopodia per terminal are stabilized, suggesting that only 1-2 filopodia are synaptogenic at any time. A data-driven computational model shows that this 'serial synapse formation model' is supported by the measured dynamics and could be tested in mutants for liprin-α, syd-1, lar, and trio. Specific defects in filopodial dynamics precede all other defects, including axon terminal retractions. A quantitative 'winner-takes-all' model, from stochastic filopodial dynamics to the formation of a limited number of synapses, is presented as well as a model for axon terminal stabilization based on filopodia and synapses (Ozel, 2019).

The data link bulbous filopodia to synapse formation based on three findings: (1) in the wild type, these are the only filopodia that specifically occur during the time window of synapse formation and do not exhibit stochastic dynamics, and wild-type R7 photoreceptor axons stabilize 1-2 bulbous filopodia at a time; (2) the synaptic seeding factors Liprin-α and Syd-1 non-randomly localize to 1-2 bulbous filopodia at a time; and (3) loss of liprin-α or syd-1 selectively affects the stabilization of bulbous filopodia. Loss of the upstream receptor lar similarly selectively affects bulbous filopodia but, in addition to bulb destabilization, also strongly affects bulb initiation. Together, these findings support a model whereby stochastic filopodial exploration leads to bulb stabilization and synapse formation one at a time. In this model, restrictive synaptogenic filopodia formation 'paces' the formation of ~25 synapses over 50 h, effectively controlling synapse numbers within the available developmental time window (Ozel, 2019).

The key mechanism of this model is the inhibitory feedback of synaptogenic filopodium formation. In contrast to all other filopodia, the dynamics of bulbous filopodia are not independent events. How are synaptic seeding factors competitively distributed between these filopodia? Live-imaging data suggest that Liprin-α or Syd-1 can traffic in and out of filopodia but overexpressed proteins accumulate in the axon terminal trunk and do not enter to more than 1-2 filopodia, indicating that trafficking into filopodia is restricted. Morphologically, filopodia are very thin structures that may not provide much space for freely diffusing proteins or organelles. On the other hand, the bulbous tip provides a much larger volume that may be required for sufficient amounts of synaptic seeding factors and other building material to initiate synapse formation. Furthermore, computational tests show that 'winner-takes-all' dynamics can arise from the dynamic distribution of a limited resource that confers a competitive advantage (longer lifetime, which leads to further accumulation) without the need for active filopodial communication (Ozel, 2019).

Since Syd-1 and Liprin-α are not required for bulb initiation, it is speculated that filopodial contact with a synaptic partner may initiate the bulb and precede active zone formation. The data suggest that Lar is a good candidate for a presynaptic receptor with such a role, but it is unlikely to be the sole upstream receptor. Neurexin and PTP69D, for example, are other known candidates. In the absence of an upstream receptor or the seeding factors themselves, synapse assembly fails and bulbs destabilize. New bulb generation following loss of bulbs in the absence of seeding factors can also be explained with seeding factors as a limiting resource with a competitive advantage. This is reminiscent of other competitive processes that shape neuronal morphology, e.g., the restricting role of building material in the competitive development of dendritic branches in a motorneuron (Ryglewski, 2017). Mutant analyses suggest that stable bulbs are linked to negative feedback on other bulbs via the function of the RhoGEF Trio. While the exact mechanism is unclear, it is tempting to speculate about a role of actin-dependent signaling downstream of synaptic seeding (Ozel, 2019).

The current model only considers the presynaptic axon terminal. The main postsynaptic partner of R7 are amacrine-like Dm8 cells, whose elaborate dendritic processes are present in direct vicinity to the R7 filopodia throughout the developmental period of synapse formation. Currently the dynamics of the postsynaptic processes and whether they restrict availability or are 'easily found' as postsynaptic partners are unknown. The presynaptic model presented in this study could explain the observed slow, serial synapse formation even in the presence of abundant postsynaptic partner processes (Ozel, 2019).

Mutations in the proposed pathway components Lar, Liprin-α, Syd1, and Trio have been independently characterized for their roles in active zone assembly (mostly at the larval neuromuscular junction) and axon targeting, in large part in the visual system . It is likely that all four genes exert more than one function in different contexts. Defects in synapse formation and retraction are captured by the measured parameters and the current model. However, some differences in overall morphology, including overextensions in the syd-1 mutant, may be described by parameters not considered in the model, e.g., filopodial length, and due to some differences in their molecular function. Similarly, for Lar, independent context-dependent functions have been characterized based on different downstream adaptors (Ozel, 2019).

It was asked to what extent a primary role for lar, trio, syd-1, and liprin-α in synapse formation could explain previously observed phenotypes. All filopodial defects in the four mutants occur independently and prior to possible retraction events. Combined live imaging and computational modeling suggests that defects in the syd-1 and liprin-α mutants are consistent with a primary defect in bulb stabilization and synapse formation. These defects may in turn lead to axon destabilization or represent independent functions; lar may have an additional earlier adhesion function and trio does not play a critical role in the formation of the correct number of synapses, while its effect on general filopodial dynamics may sensitize mutant axons to other changes (Ozel, 2019).

The conclusion that Lar, Liprin-α, and Syd-1 have a primary function in synapse formation is based on three pieces of evidence: (1) all mutants initially target correctly and exhibit normal filopodial dynamics prior to synapse formation; (2) the mutants start retracting only when synaptic contacts initiate, in the order and severity from the receptor to the downstream elements; and (3) all three mutants exhibit the loss of competitive bulb stabilization. Taken together, these observations support a direct role in synapse formation following bulb stabilization, but other molecular functions cannot be excluded. For example, in both C. elegans and Drosophila, Lar has been shown to function independently in axon guidance and synapse formation. This study found that syd-1ΔRhoGAP mutants have normal terminal morphology and only a mild decrease in the number of BrpD3-puncta. This is consistent with recent findings that a RhoGAP-deficient Syd-1 fragment is sufficient to rescue early active zone seeding events at the Drosophila neuromuscular junction but not the recruitment of Brp as the active zones mature (Spinner, 2018). However, since homozygous syd-1ΔRhoGAP flies have no obvious connectivity defects, synapse numbers are apparently sufficient for axon terminal stabilization (Ozel, 2019).

Finally, these observations suggest that loss of the primary functions of these proteins in filopodial dynamics and synapse formation are sufficient to cause axon retractions. The phenotypes observed in this study for lar, liprin-α, and syd-1 are somewhat similar but in contrast to cadN only occur at or after the time of synaptic partner identification. While filopodia continuously decrease, synapses continuously increase, thereby allowing a takeover of the axon terminal stabilization function. The modeling fits the wild type, liprin-α, syd-1, and trio remarkably well. In contrast, while retractions in the lar mutant are qualitatively predicted, the model fails to explain retractions quantitatively. A partial explanation may be that the model was parameterized only based on the lar mutant axon terminals that are still unretracted at P60. These are only 30% of terminals by that time, and this study has effectively selected for terminals with dynamics that prevented retractions thus far. It is likely that earlier retractions are caused by defects in filopodial adhesion or synaptic contacts. In sum, the data and modeling support a role for synapses in the stabilization of R7 axon terminals, which can lead to probabilistic axon retractions in mutants affecting synapse formation (Ozel, 2019).

Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel-vesicle coupling

Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca2+ channel, but molecular mechanisms controlling this are unknown. This study found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins RhoGAP100F/Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca2+ channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13Anull mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. Isoform-specific Unc13-AZ scaffold interactions were identified, regulating SV-Ca2+-channel topology whose developmental tightening optimizes synaptic transmission (Bohme, 2016).

All presynaptic AZs accumulate scaffold proteins from a canonical set of few protein families, which are characterized by extended coiled-coil stretches, intrinsically unstructured regions and a few classical interaction domains, particularly PDZ and SH3 domains. These multidomain proteins collectively form a compact 'cytomatrix' often observable by electron-dense structures covering the AZ membrane, which have been found to physically contact SVs, and thus have been suggested to promote SV docking and priming as well as to recruit Ca2+ channels. Still, how the structural scaffold components (ELKS, RBP, RIM and Liprin-α) tune the functionality of the SV-release machinery has remained largely enigmatic. Liprin-α is crucial for the AZ assembly process and at Drosophila NMJ AZs, Liprin-α-Syd-1 cluster formation initializes the assembly of an 'early' scaffold complex, which subsequently guides the accumulation of a 'late' RBP-BRP scaffold complex. This study provides evidence that these scaffold complexes together operated as 'molecular rulers' that confer a remarkable degree of order, patterning AZ composition and function in space and time: the 'early' Liprin-α-Syd-1 clusters recruited Unc13B, and this scaffold served as a template to accumulate the 'late' BRP-RBP scaffold, which recruited Unc13A. Unc13 isoforms were precisely organized in the tens of nanometers range, which the data suggest to be instrumental to control SV release probability and SV-Ca2+ channel coupling. As a molecular basis of this patterning and recruitment, a multitude of molecular contacts was identified between the Unc13 N termini and the respective scaffold components using systematic Y2H analysis. As one out of several interactions, this study identified a cognate PxxP motif in the N terminus of Unc13A to interact with the second and third SH3 domains of RBP. Point mutants within the PxxP motif interfered with the binding of the RBP-SH3 domains II and III on the Y2H level but did not have a major impact on Unc13A localization and function when introduced into an Unc13 genomic transgene. Nonetheless, elimination of the scaffold components BRP and RBP on the one hand or Liprin-α on the other hand drastically impaired the accumulation of Unc13A or Unc13B. It is suggested that these results are explained by a multitude of parallel interactions that provide the avidity needed to enrich the respective Unc13 isoforms in their specific 'niches' and may cause a functional redundancy among interaction motifs, as was likely observed in the case of the Unc13A PxxP motif. Future analysis will be needed to investigate these interaction surfaces in greater detail, and address how exactly 'early' and 'late' scaffolds coordinate AZ assembly (Bohme, 2016).

Unc13 proteins have well-established functions in SV docking and priming. Accordingly, it was observed that loss of Unc13A resulted in overall reduced SV docking without affecting T-bar-tethered SVs, which is qualitatively opposite to a function of BRP in SV localization, whose C-terminal amino acids function in T-bar-tethering, but not docking. Variants lacking these residues suffer from increased synaptic depression, suggesting a role in SV replenishment. Therefore, in addition to its role in localizing Unc13A to the AZ reported here, BRP may also cooperate functionally with Unc13A by facilitating SV delivery to docking sites (Bohme, 2016).

Synapses are highly adapted to their specific features, varying widely concerning their release efficacy and short-term plasticity. These features impact information transfer and may provide neurons with the ability to detect input coherence, maintain stability and promote synchronization. Differences in the biochemical milieu of SVs can tune priming efficacy and release probability, which largely affects short-term plasticity. In the current experiments, it was found that loss of Unc13A resulted in dramatically (~90%) reduced synaptic transmission, which exceeded the (~50%) reduction in SV docking, pointing to an additional function in enhancing release efficacy. These changes were paralleled by drastically increased short-term facilitation as well as EGTA hypersensitivity and could be due to decreased Ca2+ sensitivity of the molecular release machinery, for example, mediated by different Synaptotagmin-type Ca2+ sensors, or different numbers of SNARE complexes. However, although a rightward shift was observed of the dependence of normalized release amplitudes on extracellular Ca2+ concentration at Unc13A-deficient synapses, its slope and thus Ca2+ cooperativity was unaltered, arguing against fundamentally different Ca2+-sensing mechanisms. Instead a scenario is favored in which SV Ca2+ sensing is conserved, but local Ca2+ signals at SV positions are attenuated because of their larger distances to Ca2+ channels upon loss of Unc13A. Both Unc13 isoforms were clearly segregated physically with different distances to the Ca2+ channel cluster, and loss of Unc13A selectively reduced the number of docked SVs in the AZ center. These findings are best explained by Unc13A promoting the docking and priming of SVs closer to Ca2+ channels than Unc13B. In fact, mathematical modeling reproduced the data by merely assuming release from two independent pathways with identical Ca2+ sensing and fusion mechanisms that only differed in their physical distance to the Ca2+ source in the AZ center. The distances estimated by the model were in very good agreement with the positions of the two Unc13 isoforms defined by STED microscopy. Thus, the data suggest that differences in the distance of SVs in the tens of nanometer range to the Ca2+ channels mediated by the two Unc13 isoforms likely contributed profoundly to the observed phenotypes. It is proposed that the role of the N terminus is to differentially target the isoforms into specific zones of the AZ, while the conserved C terminus confers identical docking and priming functions at both locations. Notably, recent work in Caenorhabditis elegans also characterized two Unc13 isoforms, with fast release being mediated by UNC-13L, whereas slow release required both UNC-13L and UNC-13S44. The proximity of the UNC-13L isoform to Ca2+ entry sites was mediated by the protein's N-terminal C2A-domain (not present in Drosophila) and was critical for accelerating neurotransmitter release, and for increasing/maintaining the probability of evoked release assayed by the fraction of AP- to sucrose-induced release. In contrast, the slow SV release form dominantly localized outside AZ regions. Thus it would be interesting to investigate the sub-AZ distribution of C. elegans Unc-13 isoforms and test whether the same scaffold complexes as in Drosophila mediate the localization of the different Unc-13 isoforms (Bohme, 2016).

Notable differences in short-term plasticity have been reported for mammalian Unc13 isoforms. The mammalian genome harbors five Munc13 genes. Of those, Munc13-1, -2 and -3 are expressed in the brain, and function in SV release; differential expression of Munc13 isoforms at individual synapses may represent a mechanism to control short-term plasticity. Thus, it might be warranted to analyze whether differences in the sub-active zone distribution of Munc13 isoforms contribute to these aspects of synapse diversity in the rodent brain (Bohme, 2016).

Fast and slow phases of release have recently been attributed to parallel release pathways operating in the calyx of Held of young rodents (56 nm and 135 nm) qualitatively matching the coexistence of two differentially positioned release pathways described in this study. The finding of discretely localized release pathways with distances larger than 60 nm is further in line with the recent suggestion that, at some synapses, SVs need to be positioned outside an 'exclusion zone' from the Ca2+ source (~50 nm distance to the center of the SV for the calyx of Held). At mammalian synapses, developmental changes in the coupling of SVs and Ca2+ channels have been described, which qualitatively matches the sequential arrival of loosely and tightly coupled Unc13B and Unc13A isoforms during synaptogenesis described here. Thus, this work suggests that differential positioning of Unc13 isoforms couples functional and structural maturation of AZs. To what degree modulation of this process contributes to the functional diversification of synapses is an interesting subject of future analysis (Bohme, 2016).

Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons

During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1 or Rho GTPase activating protein at 100F) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, a proteomic screen was performed of Liprin-α-interacting proteins in Drosophila brains. Drosophila protein phosphatase 2A (PP2A; see MTS, the PP2A catalytic subunit) regulatory subunit B' [Wrd (Well Rounded) or PP2A-B'] was identified as a Liprin-α-interacting protein, and it was demonstrated that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, data is provided suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot) (Li, 2014).

During presynaptic development, small synaptic vesicle (SV) precursors, dense-core vesicles (DCVs), and synaptic cytomatrix proteins are generated in the soma, transported along the axon, and eventually incorporated into the nerve terminal (Jin and Garner, 2008). Within the nerve terminal, active zones (AZs) are specialized areas of plasma membrane containing a group of evolutionarily conserved proteins, including ELKS (glutamine, leucine, lysine, and serine-rich protein)[also called CAST (cytomatrix at the active zone-associated structural protein), Drosophila homologue is BRP (Bruchpilot)], Munc13 (mammalian uncoordinated homology 13), RIM (Rab3-interacting molecule), Syd-1 (synapse defective-1), and Liprin-α, in which the releasable pool of vesicles dock and are released on stimulation (Sudhof, 2012). Despite intensive studies of the proteins localized at the presynaptic density, the assembly and maintenance of AZs remains enigmatic. Studies conducted in invertebrate model organisms suggested that Syd-1, a putative RhoGAP, and Liprin-α are two master organizers of presynaptic differentiation (Zhen, 1999; Hallam, 2002; Kaufmann, 2002; Owald, 2010). Genetic analyses in Caenorhabditis elegans demonstrated that Syd-1 works upstream of Liprin-α in synaptic assembly (Dai, 2006; Patel, 2006). Studies in Drosophila further confirmed this hierarchy by showing that Syd-1 regulates and retains proper localization of Liprin-α at the AZ (Owald, 2010; Owald 2012). However, studies also found that Syd-1 regulates Liprin-α-independent processes, such as retention of Neurexin at the presynaptic side and glutamate receptor incorporation at the postsynaptic side (Owald, 2010; Owald 2012). The morphology of the AZ is distinctly different in liprin-α and syd-1 mutants (Kaufmann, 2002; Owald, 2010). Therefore, it is unclear how Syd-1- and Liprin-α-mediated signaling collaborate to achieve the complex regulation of presynaptic differentiation. Identifying novel Liprin-α-interacting proteins at the synapse holds the key to delineating the regulatory network mediated by these two genes (Li, 2014).

This study identified protein phosphatase 2A (PP2A) as one prominent Liprin-α-interacting protein complex through an in vivo tandem affinity purification (TAP) approach. PP2A is an abundant heterotrimeric serine/threonine phosphatase that regulates a broad range of cellular processes. PP2A is highly enriched in neurons and is implicated in Tau-mediated neurodegeneration, regulation of long-term potentiation, and presynaptic and postsynaptic apposition. The diverse functions of PP2A are attributed primarily to its many interchangeable regulatory subunits (B, B', B'', or B'''), each showing specific spatial and temporal expression patterns. The Liprin-α-interacting PP2A holoenzyme that this study identified in the fly brain contains the B' regulatory subunit [also called Wrd (Well Rounded) in fly]. Wrd is highly expressed in synapses and regulates synaptic terminal growth at the Drosophila neuromuscular junction (NMJ) (Viquez, 2006). Interestingly, the Liprin-α-Wrd physical interaction may be evolutionarily conserved because PP2A B56γ, the human homolog of Wrd, can bind Liprin-α1 in HEK 293 cells. However, the function of the Liprin-α-Wrd/PP2A B56γ interaction in the nervous system is unexplored (Li, 2014).

This study shows that Syd-1, Liprin-α, and Wrd work in a linear pathway to restrain the localization of vesicles and presynaptic cytomatrix proteins at the nerve terminal. Disruption of such a pathway results in ectopic accumulation of SVs and presynaptic proteins at the distal, but not proximal, end of axons (Li, 2014).

Much progress toward understanding presynaptic differentiation has been made through unbiased forward genetic screens in invertebrates. These studies have led to the identification of several key factors for AZ formation, including two evolutionarily conserved master organizer proteins of AZ assembly: syd-1 and syd-2/liprin-α. However, how Syd-1/Liprin-α organize presynaptic sites remains unclear. This study identified a new synaptic player, the PP2A B′ regulatory subunit, that is localized to the synapse by Liprin-α and mediates Syd-1/Liprin-α signaling in stabilizing AZs and their associated vesicles at the nerve terminal (Li, 2014).

Liprin-α was first identified as a protein interacting with the LAR (leukocyte antigen-related-like) family of phosphatases. Studies during the past two decades demonstrate that Liprin-α regulates presynaptic and postsynaptic development, as well as neurotransmitter release through protein–protein interactions with a range of molecules, including CAST/ELKS/BRP, RIM, CASK (calcium/calmodulin-dependent serine protein kinase), GIT (G-protein-coupled receptor kinase-interacting ArfGAP), GRIP (glutamate receptor interacting protein), LAR, CaMKII, and Liprin-β. Proteomic data confirmed the interaction between Liprin-α and BRP/RIM in Drosophila. Another important Liprin-α binding partner was identified at the presynaptic sites, the B′ regulatory subunit of PP2A (Wrd), which depends on Liprin-α for it proper synaptic localization (Li, 2014).

Phenotypic analysis of syd-1, liprin-α, and wrd mutants demonstrate that they share a unique trafficking defect, in which SVs, DCVs, presynaptic scaffolding proteins, and voltage-gated Ca2+ channels ectopically accumulate at the distal, but not the proximal, region of the axon. Genetic rescue experiments define a linear pathway, from syd-1 to liprin-α to wrd, that works cell autonomously in the presynaptic neuron to ensure proper localization of presynaptic materials to the nerve terminal and prevents ectopic accumulation. Together, these biochemical and genetic data suggest that Wrd mediates a novel Syd-1/Liprin-α function at the presynaptic site. Such a Syd-1/Liprin-α function is likely independent of their well established roles in regulating the T-bar structure protein BRP/ELKS (Li, 2014).

Two lines of evidence suggest that a Wrd-containing PP2A mediates the function of Syd-1/Liprin-α in regulating AZ stability. First, two rounds of in vivo biochemical purification using either Liprin-α or Wrd as the bait copurified Liprin-α with Wrd and the other two core subunits of PP2A, indicating the presence of a Liprin-α/Wrd/PP2A protein complex in neurons. Second, loss of GSK-3β kinase [sgg (shaggy)] function suppresses the syd-1, liprin-α, and wrd mutant distal axon phenotype, suggesting that a Wrd/PP2A-mediated phosphatase activity normally functions to antagonize a GSK-3β kinase activity in neurons to stabilize AZ and clustering of SVs at the nerve terminal (Li, 2014).

What is the primary cause for the unique distal axon phenotype in syd-1/liprin-α/wrd mutant larvae? Liprin-α was shown to interact with KIF1A (kinesin family member 1A)/Unc-104 (Shin, 2003; Wagner, 2009), a neuron-specific kinesin motor known to transport SV precursors containing synaptophysin, Syt, and Rab1A. It was reported that Drosophila Liprin-α regulates the trafficking of SVs through its interaction with Kinesin-1 and that liprin-α mutant peripheral nerves show accumulation of clear-core vesicles similar to kinesin heavy chain (khc) mutants (Miller, 2005). However, when this study focused on the location of the phenotypes relative to the entire axonal length, liprin-α mutant accumulation of clear-core vesicles was found to be present exclusively in the distal end (the ventrolateral peripheral nerve bundles, as well as axonal regions proximal to NMJs), whereas khc mutant larvae show massive aggregation of SV-associated proteins in the proximal end (segmental nerve bundles), and very few SV precursors reach the distal of axon. The distribution pattern of the vesicle accumulation in syd-1 and wrd mutants is the same as liprin-α mutants. Such a pattern is distinct from that of typical trafficking defects induced by mutations in vesicle-transporting motors or cargos (Li, 2014).

Although a unique vesicle trafficking defect as the primary cause for the syd-1/liprin-α/wrd mutant axonal phenotype cannot be completely excluded, a number of lines of evidence suggest a plausible explanation: AZ materials at the nerve terminal become destabilized when the syd-1/liprin-α/wrd pathway is impaired, and the floating AZ materials diffuse back to the adjacent axonal regions as ectopic docking sites for vesicles. First, Syd-1, Liprin-α, and Wrd show clear synaptic localization, with little or no axonal localization detected, consistent with a collaborative function of the three at the AZs. Second, EM analysis detected floating AZ materials in the synaptic boutons and the connected axonal regions in syd-1 mutants. Some of the floating materials are very close to or touching the bouton plasma membrane, indicating a possible defect in AZ stabilization and subsequent back-diffusion of detached AZ materials to axonal regions. Third, AZ components such as BRP, RIM, and voltage-gated Ca2+ channels are identified in the mutant distal axons along with vesicles, including SVs and DCVs, but not vesicles that transport AZ scaffolding proteins, or other synaptically localized organelles, or transport machineries. This is consistent with an ectopic accumulation of vesicles attracted by ectopic floating AZ components. Fourth, live imaging analysis found that anterogradely transported DCVs accumulate at preferred spots at the mutant distal axons, consistent with the existence of static docking sites at these axonal regions. Fifth, ectopically accumulated vesicles do not participate in release or recycling, consistent with the notion that the vesicles do not dock on the axonal plasma membrane (Li, 2014).

The fact that knockdown of a kinase (GSK-3β) rescues the distal axonal defects of syd-1/liprin-α/wrd mutants indirectly suggests that a Wrd-dependent dephosphorylation event is antagonized by a phosphorylation event (mediated by GSK-3β) to regulate AZ stability. However, these data cannot exclude the possibility that PP2A-independent functions of Wrd are involved. One way to seek direct evidence that Wrd-containing PP2A is involved in regulating AZ stability is to study the loss of function of PP2A; however, this approach has its own set of complications. As a ubiquitous heterotrimetric enzyme, the substrate specificity and subcellular localization of PP2A are greatly dependent on its regulatory subunit (such as Wrd). Mutating the catalytic or structural domain blocks overall PP2A action mediated by all regulatory subunits, which precludes analysis of Wrd-specific PP2A action. For example, mutations in MTS (the PP2A catalytic subunit) cause early lethality. Overexpression of a dominant MTS protein causes massive axonal transport defects in the entire axon, as well as defects in AZ development. Therefore, identifying common substrates shared by Wrd/PP2A and GSK-3β and studying how their phosphorylation status regulates AZ stability and/or vesicle trafficking will ultimately unravel the mechanism by which a PP2A-dependent pathway regulates presynaptic development. In this context, this study set up a model to study how synapse scaffolding proteins can regulate localized phosphorylation/dephosphorylation through recruitment of specific phosphatases or kinases (Li, 2014).

A mammalian homolog of Syd-1 was identified recently as an important regulator of presynaptic differentiation at central synapses, at least partially through its interaction with mammalian Liprin-α2 (Wentzel, 2013). Given that Liprin-α1 interacts with PP2A B56γ (mammalian homolog of Wrd) in HEK 293 cells, it will be of interest to investigate whether the function of Drosophila Liprin-α in mediating the signaling from Syd-1 to the PP2A B′ subunit is also evolutionarily conserved during vertebrate synapse development (Li, 2014).

Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction

Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. This study shows that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR were used to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1 (RhoGAP100F), neurexin-1, futsch, highwire, discs large, and alpha-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. These results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. It is proposed that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function (McDermott, 2014).

Localized translation is a widespread and evolutionarily ancient strategy used to temporally and spatially restrict specific proteins to their site of function and has been extensively studied during early development and in polarized cells in a variety of model systems. It is thought to be of particular importance in the regulation of neuronal development and in the plastic changes at neuronal synapses that underlie memory and learning, allowing rapid local changes in gene expression to occur independently of new transcriptional programs. The Drosophila neuromuscular junction (NMJ) is an excellent model system for studying the general molecular principles of the regulation of synaptic development and plasticity. Genetic or activity-based manipulations of synaptic translation at the NMJ has previously been shown to affect the morphological and electrophysiological plasticity of NMJ synapses. However, neither the mRNA targets nor the molecular mechanism by which such translational regulation occurs are fully understood (McDermott, 2014).

Previously work identified CG17838, the fly homolog of the mammalian RNA binding protein SYNCRIP/hnRNPQ, which was named Syncrip (Syp). Mammalian SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules that contain CamKIIα, Arc, and IP3R1 mRNAs and is thought to regulate translation via an interaction with the noncoding RNA BC200/BC1, itself a translational repressor. Moreover, SYNCRIP/hnRNPQ competes with poly(A) binding proteins to inhibit translation in vitro and regulates dendritic morphology (Chen, 2012) via association with, and localization of, mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex. Drosophila Syp has a domain structure similar to its mammalian homolog, containing RRM RNA binding domains and nuclear localization signal(s), as well as encoding a number of protein isoforms. It was previously shown that Syp binds specifically to the gurken (grk) mRNA localization signal together with a number of factors previously shown to be required for grk mRNA localization and translational regulation (McDermott, 2012). Furthermore, syp loss-of-function alleles lead to patterning defects indicating that syp is required for grk and oskar (osk) mRNA localization and translational regulation in the Drosophila oocyte (McDermott, 2014).

This study shows that Syp is detected in the Drosophila third instar larval muscle nuclei and also postsynaptically at the NMJ. Syp is required for proper synaptic morphology at the NMJ, as syp loss-of-function mutants show a synaptic overgrowth phenotype, while overexpression of Syp in the muscle can suppress NMJ growth. Syp protein associates with a number of mRNAs encoding proteins with key roles in synaptic growth and function including, msp-300, syd-1, neurexin-1 (nrx-1), futsch, highwire (hiw), discs large 1 (dlg1), and α-spectrin (α-spec). The protein levels of a number of these mRNA targets, including msp-300 and dlg1, are significantly affected in syp null mutants. Furthermore, in addition to regulating MSP-300 protein levels, Syp is required for correct MSP-300 protein localization, and syp null mutants have defects in myonuclear distribution and morphology that resemble those observed in msp-300 mutants. It is proposed that Syp coordinates the protein levels from a number of transcripts with key roles in synaptic growth and is a mediator of synaptic morphology and growth at the Drosophila NMJ (McDermott, 2014).

The results demonstrate that Syp is required for the appropriate branching of the motoneurons and the number of synapses they make at the muscle. These observations are potentially explained by the finding that Syp is also required for the correct level of expression of msp-300, dlg1 and other mRNA targets. Given that it was previously shown that Syp regulates mRNA localization and localized translation in the Drosophila oocyte, and studies by others have shown that mammalian SYNCRIP/hnRNPQ inhibits translation initiation by competitively binding poly(A) sequences (Svitkin, 2013), these functions of Syp as occurring at the level of translational regulation of the mRNAs to which Syp binds. The data are also consistent with other work in mammals showing that SYNCRIP/hnRNPQ is a component of neuronal RNA transport granulesthat can regulate dendritic morphology via the localized expression of mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex (McDermott, 2014 and references therein).

Translation at the Drosophila NMJ is thought to provide a mechanism for the rapid assembly of synaptic components and synaptic growth during larval development, in response to rapid increases in the surface area of body wall muscles or in response to changes in larval locomotion. The phenotypes observed in this study resemble, and are comparable to, those seen when subsynaptic translation is altered genetically or by increased locomotor activity. In syp null mutants, NMJ synaptic terminals are overgrown, containing more branches and synaptic boutons. Similarly, bouton numbers are increased by knocking down Syp in the muscle using RNAi. In contrast, overexpression of Syp in the muscle has the opposite phenotype, resulting in an inhibition of synaptic growth and branching. Furthermore, expressing RNAi against syp in motoneurons alone does not result in a change in NMJ morphology, indicating that Syp acts postsynaptically in muscle, but not presynaptically at the NMJ to regulate morphology. Interestingly, pan-neuronal syp knockdown or overexpression using Elav-GAL4 also results in NMJ growth defects, revealing that some of the defects observed in the syp null mutant may be attributed to Syp function in neuronal cell types other than the motoneurons, such as glial cells, which are known to influence NMJ morphology. Finally, while Syp is not required in the motoneuron to regulate synapse growth and is not detected in the motoneuron, the possibility cannot be excluded that Syp is present at low levels in the presynapse and regulates processes independent of synapse morphology. A further detailed characterization of the cell types and developmental stages in which Syp is expressed and functions is required to better understand the complex phenotypes that were observe (McDermott, 2014).

RNA binding proteins have emerged as critical regulators of both neuronal morphology and synaptic transmision, suggesting that protein production modulates synapse efficacy. Consistent with this, it has been shown in a parallel study that Syp is required for proper synaptic transmission and vesicle release and regulates the presynapse through expression of retrograde Bone Morphogenesis Protein (BMP) signals in the postsynapse. In this role, Syp may coordinate postsynaptic translation with presynaptic neurotransmitter release. These observations provide a good explanation for how Syp influences the presynapse despite being only detectable in the postsynapse. This study has shown that Syp associates with a large number of mRNAs within third instar larvae, many of which encode proteins with key roles in synaptic growth and function. Syp mRNA targets include msp-300, syd-1, nrx-1, futsch, hiw, dlg1, and α-spec. Syp negatively regulates Syd-1, Hiw, and DLG protein levels in the larval body wall but positively regulates MSP-300 and Nrx-1 protein levels. Dysregulation of these multiple mRNA targets likely accounts for the phenotypes that were observed. Postsynaptically expressed targets with key synaptic roles that could explain the synaptic phenotypes that were observed in syp alleles include MSP-300, α-Spec, and DLG. For example, mutants in dlg1 and mutants where postsynaptic DLG is destabilized or delocalized have NMJ morphology phenotypes similar to those observed upon overexpression of Syp in the muscle. Presynaptically expressed targets include syd-1, nrx-1, and hiw. However, this study has shown that syp knockdown in presynaptic motoneurons does not result in any defects in NMJ morphology. The RIP-Seq experiments were carried out using whole larvae and will, therefore, identify Syp targets in a range of different tissues and cells, the regulation of which may or may not contribute to the phenotype that were observed in syp mutants. It is, therefore, possible that Syp associates with these presynaptic targets in other neuronal cell types such as the DA neurons of the larval peripheral nervous system. It is also possible that Nrx-1 or Hiw are expressed and required postsynaptically in the muscle, but this has not been definitively determined. syp alleles may provide useful tools to examine where key synaptic genes are expressed and how they are regulated (McDermott, 2014).

The identity of localized mRNAs and the mechanism of localized translation at the NMJ are major outstanding questions in the field. To date, studies have shown that GluRIIA mRNA aggregates are distributed throughout the muscle. The Syp targets identified in this study, such as msp-300, hiw, nrx-1, α-spec, and dlg1, are now excellent candidates for localized expression at the NMJ. Ultimately, conclusive demonstration of localized translation will involve the visualization of new protein synthesis of targets during activity-dependent synaptic plasticity. Biochemical experiments will also be required to establish the precise mode of binding of Syp to its downstream mRNA targets, the basis for interaction specificity, and the molecular mechanism by which Syp differentially regulates the protein levels of its mRNA targets at the Drosophila NMJ. Despite the fact that mammalian SYNCRIP is known to associate with poly(A) tails, this study and other published work have revealed that Syp can associate with specific transcripts. How Syp associates with specific mRNAs is unknown, and future studies are needed to uncover whether the interaction of Syp with specific transcripts is dictated by direct binding of the three Syp RRM RNA binding domains or by binding to other specific mRNA binding proteins. It is also possible that specific mRNA stem–loops, similar to the gurken localization signal, are required for Syp to bind to its mRNA targets (McDermott, 2014).

This study shows that msp-300 (also known as Nesprin) is the most significant mRNA target of Syp. MSP-300 is the Drosophila ortholog of human Nesprin proteins. These proteins have been genetically implicated in various human myopathies. For example, Nesprin/Syne-1 or Nesprin/Syne-2 is associated with Emery-Dreifuss muscular dystrophy (EDMD) as well as severe cardiomyopathies. Moreover, Syp itself is increasingly linked with factors and targets that can cause human neurodegenerative disorders. Recent work has revealed that SYNCRIP/hnRNPQ and Fragile X mental retardation protein (FMRP) are present in the same mRNP granule, and loss of expression of FMRP or the ability of FMRP to interact with mRNA and polysomes can cause cases of Fragile X syndrome. Separate studies have also shown that SYNCRIP interacts with wild-type survival of motor neuron (SMN) protein but not the truncated or mutant forms found to cause spinal muscular atrophy, and Syp genetically interacts with Smn mutations in vivo. Understanding Syp function in the regulation of such diverse and complex targets may, therefore, provide new avenues for understanding the molecular basis of complex disease phenotypes and potentially lead to future therapeutic approaches (McDermott, 2014).

Loss of syd-1 from R7 neurons disrupts two distinct phases of presynaptic development

Genetic analyses in both worm and fly have identified the RhoGAP-like protein Syd-1 (RhoGAP100F) as a key positive regulator of presynaptic assembly. In worm, loss of syd-1 can be fully rescued by overexpressing wild-type Liprin-α, suggesting that the primary function of Syd-1 in this process is to recruit Liprin-α. This study shows that loss of syd-1 from Drosophila R7 photoreceptors causes two morphological defects that occur at distinct developmental time points. First, syd-1 mutant R7 axons often fail to form terminal boutons in their normal M6 target layer. Later, those mutant axons that do contact M6 often project thin extensions beyond it. The earlier defect coincides with a failure to localize synaptic vesicles (SVs), suggesting that it reflects a failure in presynaptic assembly. The relationship between syd-1 and Liprin-α in R7s was analyzed. It was found that loss of Liprin-α causes a stronger early R7 defect and provide a possible explanation for this disparity: Liprin-α was shown to promote Kinesin-3/Unc-104/Imac-mediated axon transport independently of Syd-1 and that Kinesin-3/Unc-104/Imac is required for normal R7 bouton formation. Unlike loss of syd-1, loss of Liprin-α does not cause late R7 extensions. It was shown that overexpressing Liprin-α partly rescues the early but not the late syd-1 mutant R7 defect. It is therefore concluded that the two defects are caused by distinct molecular mechanisms. Trio overexpression was found to rescues both syd-1 defects and that trio and syd-1 have similar loss- and gain-of-function phenotypes, suggesting that the primary function of Syd-1 in R7s may be to promote Trio activity (Holbrook, 2012).

GFP-fused SV proteins, such as Syt-GFP, are classic tools for studying presynaptic development but have not been used previously to analyze R7s. This study found that, as expected, Syt-GFP within R7s is enriched at sites known by electron microscopy to contain active zones. Loss of LAR, Liprin-α, or syd-1 causes R7 terminals to fail to contact their normal, M6, target layer. This study demonstrated that this morphological defect correlates temporally with a failure to localize SVs to presynaptic sites and is therefore likely to reflect a defect in R7 presynaptic development rather than simply in target layer selection (Holbrook, 2012).

Liprin-α is not only a scaffold for the assembly and retention of presynaptic components, including SVs, at presynaptic sites but also a positive regulator of Kinesin-3/Unc-104/Imac-dependent axon transport of those components. This study shows that, unlike Liprin-α, Syd-1 is not required for normal Kinesin-3/Unc-104/Imac-mediated transport. However, SVs are similarly mislocalized in Liprin-α and syd-1 mutant R7 axons that contact M6. A simple interpretation is that this mislocalization reflects a requirement for Liprin-α and syd-1 in retaining SVs within R7 terminals; in support of this, it was found that SVs are localized normally to syd-1 mutant R7 axon terminals at 24 h APF, before synaptogenesis. It was hypothesized that the additional disruption of axon transport in Liprin-α mutant R7s is reflected in their greater inability to maintain contact with M6; in support of this, it was found that imac mutant R7 axons also lose contact with M6 (Holbrook, 2012).

Although both Liprin-α and syd-1 are required for the clustering of SVs at en passant synapses in worm, syd-1 is not required for the localization of SVs to NMJ terminals in fly. The molecular mechanisms underlying presynaptic development at NMJ and in R7s have been shown previously to differ in several respects. The current finding further highlights the importance of analyzing synapse development using multiple neuron types (Holbrook, 2012).

Although mitochondria are often enriched at synapses, it remains unclear what proportion of them might be stably associated with presynaptic sites rather than transported there in response to acute energy needs. Within at least some axons, most clusters of stationary mitochondria reside at nonsynaptic sites. In R7s, Mito-GFP was found to be enriched at presynaptic sites. Because arthropod photoreceptor neurons continuously release neurotransmitter in response to light, this enrichment might simply be caused by continuous energy needs. However, this study found that mitochondria remained enriched at R7 terminals even in the absence of light-evoked activity, indicating that either spontaneous release is sufficient for their recruitment or an activity-independent mechanism is responsible. It is speculated that the permanently high energy demands at photoreceptor synapses may have selected for the activity-independent association of mitochondria with R7 synapses and that this localization requires syd-1 and Liprin-α. Mito-GFP is mislocalized in imac mutant R7s, despite previous work indicating that Kinesin-3/Unc-104/Imac is not required for transport of mitochondria. It is therefore thought that mitochondria are normally tethered at R7 presynaptic sites and that loss of imac indirectly causes their mislocalization by disrupting transport of the components required for tethering to occur (Holbrook, 2012).

Previous work identified two different phenotypes associated with loss of the LAR/Liprin/trio pathway: loss of LAR or Liprin-α caused R7 axons to terminate before their M6 target layer, whereas loss of Liprin-β or trio caused R7 axons to project extensions beyond M6. One possibility is that these two defects are simply different manifestations of the same cellular defect: a decrease in the stability of the synaptic contact between R7s and their targets. However, this study has shown that loss of a single gene, syd-1, causes both defects and that the defects occur at distinct developmental time points, suggesting that they occur by distinct mechanisms. In support of this, Liprin-α overexpression can rescue the early but not the late syd-1 defect (Holbrook, 2012).

The earlier defect, failure to contact M6, correlates with the failure to localize SVs, suggesting, as mentioned above, that this represents a failure to assemble synapses. However, the cause of the later morphological defect and the precise nature of the extensions remain unclear. It is noted that the extensions often terminate in small varicosities that can contain Syt-GFP, and Mito-GFP, indicating that they are not simply filopodia but may instead represent sites of ectopic presynaptic assembly. One possibility is that, as at NMJ, loss of syd-1 causes ectopic accumulations of Liprin-α, Brp, Nrx-1, or other presynaptic proteins and that these might then promote ectopic, abnormal presynaptic assembly. A second possibility is that the extensions may instead be an indirect consequence of the role of syd-1 in postsynaptic development: perhaps the extensions are the response of the syd-1 mutant R7 terminal to defects in its postsynaptic target. Loss of Liprin-α causes no such postsynaptic effect, providing an explanation for why Liprin-α mutant R7s do not form extensions. A third possibility is that R7s form distinct types of synapses at different time points. Failure to assemble one type of synapse, which R7s assemble first, causes decreased contact with M6, whereas failure to assemble a second type, which occur later, results in extensions. Consistent with this model, R7s form synapses with more than one neuron type (Holbrook, 2012).

Loss of syd-1 has a significantly weaker effect on fly NMJ development than does loss of Liprin-α. Likewise, this study shows that the early phase of R7 terminal development, during which presynaptic components are localized, is less affected by loss of syd-1 than by loss of Liprin-α. A possible explanation for this difference is identified: loss of Liprin-α, but not of syd-1, significantly decreases Kinesin-3/Unc-104/Imac-mediated axon transport, and Kinesin-3/Unc-104/Imac is required for R7s to form boutons in M6 (Holbrook, 2012).

In both worm and fly, Syd-1 is required for the normal localization of Liprin-α and Brp/ELKS to presynaptic sites. In worm, loss of syd-1 can be rescued either by overexpressing full-length wild-type Liprin-α, or by overexpressing a domain of Liprin-α that promotes oligomerization of Liprin-α proteins, or by a mutation that enhances the ability of Liprin-α to bind Brp/ELKS. These results suggest that the primary function of Syd-1 is to potentiate Liprin-α activities. However, this sutyd found that Liprin-α overexpression only partially rescues the early defect that syd-1 mutant R7s have in assembling synapses. This suggests that, as in worm, Liprin-α can act partly independently of Syd-1 during presynaptic assembly but that, unlike in worm, Syd-1 also has some Liprin-α-independent function. In contrast, Liprin-α overexpression does not at all rescue the late extensions caused by loss of syd-1. As it speculated above, one possibility is that these extensions might be caused by mislocalized Liprin-α, Brp, or Nrx-1 (Holbrook, 2012).

Unlike Liprin-α, Trio overexpression fully rescues the early and partly rescues the late defect caused by loss of syd-1, suggesting that Syd-1 promotes R7 synaptic terminal development primarily by potentiating Trio activity. Consistent with this model, loss of trio phenocopies loss of syd-1 from R7s, and overexpressing Syd-1 or Trio bypasses the need for LAR to similar degrees. At fly NMJ, Trio promotes presynaptic development by acting as a GEF for Rac1. Syd-1 has a RhoGAP domain, albeit one that has not been shown to interact with GTPases. Syd-1 may act distantly upstream of Trio. However, it is also possible that Syd-1 might instead regulate one or more small GTPases in parallel with Trio. GAPs and GEFs have opposite effects on GTPases, but loss of trio or syd-1 causes similar defects at both NMJ and in R7s. One possibility, therefore, is that Syd-1 acts as a GAP not for Rac1 but for Rho, which often functions in opposition to Rac. Alternatively, Syd-1 might act as an atypical GAP for Rac1 -- perhaps lacking GAP activity but able to bind and protect Rac1-GTP from conventional GAPs -- or Syd-1 might yet act as a conventional GAP for Rac1 if it is the rate of cycling between GDP- and GTP-bound states of Rac1 (rather than simply the amount of the GTPase that is in the 'active,' GTP-bound, state) that promotes presynaptic development (Holbrook, 2012).

A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila

Active zones (AZs) are presynaptic membrane domains mediating synaptic vesicle fusion opposite postsynaptic densities (PSDs). At the Drosophila neuromuscular junction, the ELKS family member Bruchpilot (BRP) is essential for dense body formation and functional maturation of AZs. Using a proteomics approach, this study identified Drosophila Syd-1 (DSyd-1) as a BRP binding partner. In vivo imaging shows that DSyd-1 arrives early at nascent AZs together with DLiprin-alpha, and both proteins localize to the AZ edge as the AZ matures. Mutants in dsyd-1 form smaller terminals with fewer release sites, and release less neurotransmitter. The remaining AZs are often large and misshapen, and ectopic, electron-dense accumulations of BRP form in boutons and axons. Furthermore, glutamate receptor content at PSDs increases because of excessive DGluRIIA accumulation. The AZ protein DSyd-1 is needed to properly localize DLiprin-alpha at AZs, and seems to control effective nucleation of newly forming AZs together with DLiprin-alpha. DSyd-1 also organizes trans-synaptic signaling to control maturation of PSD composition independently of DLiprin-alpha (Owald, 2010).


Search PubMed for articles about Drosophila Syd-1 or RhoGAP100F

Bohme, M. A., Beis, C., Reddy-Alla, S., Reynolds, E., Mampell, M. M., Grasskamp, A. T., Lutzkendorf, J., Bergeron, D. D., Driller, J. H., Babikir, H., Gottfert, F., Robinson, I. M., O'Kane, C. J., Hell, S. W., Wahl, M. C., Stelzl, U., Loll, B., Walter, A. M. and Sigrist, S. J. (2016). Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel-vesicle coupling. Nat Neurosci [Epub ahead of print]. PubMed ID: 27526206

Dai, Y., Taru, H., Deken, S. L., Grill, B., Ackley, B., Nonet, M. L. and Jin, Y. (2006). SYD-2 Liprin-alpha organizes presynaptic active zone formation through ELKS. Nat Neurosci 9: 1479-1487. PubMed ID: 17115037

Hallam, S. J., Goncharov, A., McEwen, J., Baran, R. and Jin, Y. (2002). SYD-1, a presynaptic protein with PDZ, C2 and rhoGAP-like domains, specifies axon identity in C. elegans. Nat Neurosci 5: 1137-1146. PubMed ID: 12379863

Holbrook, S., Finley, J. K., Lyons, E. L. and Herman, T. G. (2012). Loss of syd-1 from R7 neurons disrupts two distinct phases of presynaptic development. J Neurosci 32: 18101-18111. PubMed ID: 23238725

Kaufmann, N., DeProto, J., Ranjan, R., Wan, H. and Van Vactor, D. (2002). Drosophila liprin-alpha and the receptor phosphatase Dlar control synapse morphogenesis. Neuron 34: 27-38. PubMed ID: 11931739

Li, L., Tian, X., Zhu, M., Bulgari, D., Bohme, M. A., Goettfert, F., Wichmann, C., Sigrist, S. J., Levitan, E. S. and Wu, C. (2014). Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons. J Neurosci 34: 8474-8487. PubMed ID: 24948803

McDermott, S. M., Yang, L., Halstead, J. M., Hamilton, R. S., Meignin, C. and Davis, I. (2014). Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction. RNA 20(10): 1593-606. PubMed ID: 25171822

Miller, K. E., DeProto, J., Kaufmann, N., Patel, B. N., Duckworth, A. and Van Vactor, D. (2005). Direct observation demonstrates that Liprin-alpha is required for trafficking of synaptic vesicles. Curr Biol 15: 684-689. PubMed ID: 15823543

Muhammad, K., et al. (2015). Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function. Nat Commun 6: 8362. PubMed ID: 26471740.

Owald, D., Fouquet, W., Schmidt, M., Wichmann, C., Mertel, S., Depner, H., Christiansen, F., Zube, C., Quentin, C., Korner, J., Urlaub, H., Mechtler, K. and Sigrist, S. J. (2010). A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila. J Cell Biol 188: 565-579. PubMed ID: 20176924

Owald, D., Khorramshahi, O., Gupta, V. K., Banovic, D., Depner, H., Fouquet, W., Wichmann, C., Mertel, S., Eimer, S., Reynolds, E., Holt, M., Aberle, H. and Sigrist, S. J. (2012). Cooperation of Syd-1 with Neurexin synchronizes pre- with postsynaptic assembly. Nat Neurosci 15: 1219-1226. PubMed ID: 22864612

Ozel, M. N., Kulkarni, A., Hasan, A., Brummer, J., Moldenhauer, M., Daumann, I. M., Wolfenberg, H., Dercksen, V. J., Kiral, F. R., Weiser, M., Prohaska, S., von Kleist, M. and Hiesinger, P. R. (2019). Serial synapse formation through filopodial competition for synaptic seeding factors. Dev Cell 50(4):447-461. PubMed ID: 31353313

Patel, M. R., Lehrman, E. K., Poon, V. Y., Crump, J. G., Zhen, M., Bargmann, C. I. and Shen, K. (2006). Hierarchical assembly of presynaptic components in defined C. elegans synapses. Nat Neurosci 9: 1488-1498. PubMed ID: 17115039

Ramesh, N., Escher, M. J. F., Mampell, M. M., Bohme, M. A., Gotz, T. W. B., Goel, P., Matkovic, T., Petzoldt, A. G., Dickman, D. and Sigrist, S. J. (2021). Antagonistic interactions between two Neuroligins coordinate pre- and postsynaptic assembly. Curr Biol. PubMed ID: 33651992

Ryglewski, S., Vonhoff, F., Scheckel, K. and Duch, C. (2017). Intra-neuronal competition for synaptic partners conserves the amount of dendritic building material. Neuron 93(3): 632-645. PubMed ID: 28132832

Shin, H., Wyszynski, M., Huh, K. H., Valtschanoff, J. G., Lee, J. R., Ko, J., Streuli, M., Weinberg, R. J., Sheng, M. and Kim, E. (2003). Association of the kinesin motor KIF1A with the multimodular protein liprin-alpha. J Biol Chem 278: 11393-11401. PubMed ID: 12522103

Spinner, M. A., Walla, D. A. and Herman, T. G. (2018). Drosophila Syd-1 has RhoGAP activity that is required for presynaptic clustering of Bruchpilot/ELKS but not Neurexin-1. Genetics 208(2): 705-716. PubMed ID: 29217522

Sudhof, T. C. (2012). The presynaptic active zone. Neuron 75: 11-25. PubMed ID: 22794257

Viquez, N. M., Li, C. R., Wairkar, Y. P. and DiAntonio, A. (2006). The B' protein phosphatase 2A regulatory subunit well-rounded regulates synaptic growth and cytoskeletal stability at the Drosophila neuromuscular junction. J Neurosci 26: 9293-9303. PubMed ID: 16957085

Wagner, O. I., Esposito, A., Kohler, B., Chen, C. W., Shen, C. P., Wu, G. H., Butkevich, E., Mandalapu, S., Wenzel, D., Wouters, F. S. and Klopfenstein, D. R. (2009). Synaptic scaffolding protein SYD-2 clusters and activates kinesin-3 UNC-104 in C. elegans. Proc Natl Acad Sci U S A 106: 19605-19610. PubMed ID: 19880746

Wentzel, C., Sommer, J. E., Nair, R., Stiefvater, A., Sibarita, J. B. and Scheiffele, P. (2013). mSYD1A, a mammalian synapse-defective-1 protein, regulates synaptogenic signaling and vesicle docking. Neuron 78: 1012-1023. PubMed ID: 23791195

Zhen, M. and Jin, Y. (1999). The liprin protein SYD-2 regulates the differentiation of presynaptic termini in C. elegans. Nature 401: 371-375. PubMed ID: 10517634

Biological Overview

date revised: 25 September 2021

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