InteractiveFly: GeneBrief

Spinophilin: Biological Overview | References

BIOLOGICAL OVERVIEW

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 (Feng, 2000). While Spinophilin was found to be expressed both pre- and post-synaptically (Muly, 2004a; Muly, 2004b), its function, so far, has only been analysed in the context of postsynaptic spines (Feng, 2000; Terry-Lorenzo 2005; Allen, 2006; Sarrouilhe, 2005). 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 (Chia, 2012). 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 p70^S6K , 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-1^GFP. 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-1^GFP 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).

Spinophilin facilitates dephosphorylation of doublecortin by PP1 to mediate microtubule bundling at the axonal wrist

The axonal shafts of neurons contain bundled microtubules, whereas extending growth cones contain unbundled microtubule filaments, suggesting that localized activation of microtubule-associated proteins (MAP) at the transition zone may bundle these filaments during axonal growth. Dephosphorylation is thought to lead to MAP activation, but specific molecular pathways have remained elusive. This study found that Spinophilin, a Protein-phosphatase 1 (PP1) targeting protein, is responsible for the dephosphorylation of the MAP Doublecortin (Dcx) Ser 297 selectively at the "wrist" of growing axons, leading to activation. Loss of activity at the 'wrist' is evident as an impaired microtubule cytoskeleton along the shaft. These findings suggest that spatially restricted adaptor-specific MAP reactivation through dephosphorylation is important in organization of the neuronal cytoskeleton (Bielas, 2007).

The 62E early-late puff of Drosophila contains D-spinophilin, an ecdysone-inducible PDZ-domain protein dynamically expressed during metamorphosis

At the onset of metamorphosis in Drosophila melanogaster, the steroid hormone 20-OH ecdysone induces a small number of early and early-late puffs in the polytene chromosomes of the third-instar larval salivary gland whose activity is required for regulating the activity of a larger set of late puffs. Most of the corresponding early and early-late genes have been found to encode transcription factors that regulate a much larger set of late genes. In contrast, this study describes the identification of an ecdysone-regulated gene in the 62E early-late puff, denoted D-spinophilin, that encodes a protein similar to the mammalian protein spinophilin/neurabin II. The D-spinophilin protein is predicted to contain a highly conserved PP1-binding domain and adjacent PDZ domain, as well as a coiled-coil domain and SAM domain, and belongs to a family of related proteins from diverse organisms. Transcription of D-spinophilin is correlated with 62E puff activity during the early stages of metamorphosis and is ecdysone-dependent, making this the first member of this gene family shown to be regulated by a steroid hormone. Examination of the dynamic patterns of D-spinophilin expression during the early stages of metamorphosis are consistent with a role in central nervous system metamorphosis as well as a more general role in other tissues. As D-spinophilin appears to be the only member of this gene family in Drosophila, its study provides an excellent opportunity to elucidate the role of an important adaptor protein in a genetic model organism (Keegan, 2001).

Search PubMed for articles about Drosophila Spinophilin

Allen, P. B., Zachariou, V., Svenningsson, P., Lepore, A. C., Centonze, D., Costa, C., Rossi, S., Bender, G., Chen, G., Feng, J., Snyder, G. L., Bernardi, G., Nestler, E. J., Yan, Z., Calabresi, P. and Greengard, P. (2006). Distinct roles for spinophilin and neurabin in dopamine-mediated plasticity. Neuroscience 140: 897-911. PubMed ID: 16600521

Bielas, S. L., Serneo, F. F., Chechlacz, M., Deerinck, T. J., Perkins, G. A., Allen, P. B., Ellisman, M. H. and Gleeson, J. G. (2007). Spinophilin facilitates dephosphorylation of doublecortin by PP1 to mediate microtubule bundling at the axonal wrist. Cell 129: 579-591. PubMed ID: 17482550

Chia, P. H., Patel, M. R. and Shen, K. (2012). NAB-1 instructs synapse assembly by linking adhesion molecules and F-actin to active zone proteins. Nat Neurosci 15: 234-242. PubMed ID: 22231427

Feng, J., Yan, Z., Ferreira, A., Tomizawa, K., Liauw, J. A., Zhuo, M., Allen, P. B., Ouimet, C. C. and Greengard, P. (2000). Spinophilin regulates the formation and function of dendritic spines. Proc Natl Acad Sci U S A 97: 9287-9292. PubMed ID: 10922077

Keegan, J., Schmerer, M., Ring, B. and Garza, D. (2001). The 62E early-late puff of Drosophila contains D-spinophilin, an ecdysone-inducible PDZ-domain protein dynamically expressed during metamorphosis. Genet Res 77: 27-39. PubMed ID: 11279828

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

Muly, E. C., Allen, P., Mazloom, M., Aranbayeva, Z., Greenfield, A. T. and Greengard, P. (2004a). Subcellular distribution of neurabin immunolabeling in primate prefrontal cortex: comparison with spinophilin. Cereb Cortex 14: 1398-1407. PubMed ID: 15217898

Muly, E. C., Smith, Y., Allen, P. and Greengard, P. (2004b). Subcellular distribution of spinophilin immunolabeling in primate prefrontal cortex: localization to and within dendritic spines. J Comp Neurol 469: 185-197. PubMed ID: 14694533

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

Sarrouilhe, D., di Tommaso, A., Metaye, T. and Ladeveze, V. (2006). Spinophilin: from partners to functions. Biochimie 88: 1099-1113. PubMed ID: 16737766

Terry-Lorenzo, R. T., Roadcap, D. W., Otsuka, T., Blanpied, T. A., Zamorano, P. L., Garner, C. C., Shenolikar, S. and Ehlers, M. D. (2005). Neurabin/protein phosphatase-1 complex regulates dendritic spine morphogenesis and maturation. Mol Biol Cell 16: 2349-2362. PubMed ID: 15743906

date revised: 27 December 2015

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