InteractiveFly: GeneBrief

mmenage a trois: Biological Overview | References

Structural plasticity of synaptic junctions is a prerequisite to achieve and modulate connectivity within nervous systems, e.g., during learning and memory formation. It demands adequate backup systems that allow remodeling while retaining sufficient stability to prevent unwanted synaptic disintegration. The strength of submembranous scaffold complexes, which are fundamental to the architecture of synaptic junctions, likely constitutes a crucial determinant of synaptic stability. Postsynaptic density protein-95 (PSD-95)/ Discs-large (Dlg)-like membrane-associated guanylate kinases (DLG-MAGUKs) are principal scaffold proteins at both vertebrate and invertebrate synapses. At Drosophila larval glutamatergic neuromuscular junctions (NMJs) DlgA and DlgS97 exert pleiotropic functions, probably reflecting a few known and a number of yet-unknown binding partners. This study has identified Metro, a novel p55/MPP-like Drosophila MAGUK as a major binding partner of perisynaptic DlgS97 at larval NMJs. Based on homotypic LIN-2,-7 (L27) domain interactions, Metro stabilizes junctional DlgS97 in a complex with the highly conserved adaptor protein DLin-7. In a remarkably interdependent manner, Metro and DLin-7 act downstream of DlgS97 to control NMJ expansion and proper establishment of synaptic boutons. Using quantitative 3D-imaging it was further demonstrated that the complex controls the size of postsynaptic glutamate receptor fields. These findings accentuate the importance of perisynaptic scaffold complexes for synaptic stabilization and organization (Bachmann, 2010).

To identify Drosophila proteins, which carry two L27 domains and hence might interact with DlgS97 and DLin-7 simultaneously, sequences of L27 domain proteins were used for BLAST search screening. Four MAGUKs were found to meet this criterion. Stardust (Sdt), a homolog of PALS-1/MPP5, interacts with DLin-7 at epithelial membranes but not at NMJs (Bachmann, 2004). Similarly, Varicose (Vari), an MPP2-like protein, binds to both DlgS97 and DLin-7 in vitro but does not colocalize with them at NMJs. Thus, the CASK homolog Camguk (Cmg) and predicted translational products of a gene annotated as CG30021 (CG30021-P) remained as candidate linker proteins. Regarding its involvement in a ménage à trois with DlgS97 and DLin-7, CG30021-P was referred to as 'Metro' (Bachmann, 2010).

All predicted Metro isoforms comprise two L27 domains followed by one PDZ, one SH3, and one carboxy terminal GUK domain. This domain organization is typical for p55/MPP-like MAGUKs, among which the vertebrate members MPP3, MPP4, and MPP7 share highest similarity with Metro. Northern blot analyses on mRNA from embryos, third-instar larvae, and adult flies revealed a single metro-specific band of 3 kb. As this assay may fail to resolve subtle splice variations, RT-PCR was conducted on mRNA from third-instar larval body walls. Using primers designed to amplify the complete coding region, just one cDNA encoding a 556 aa protein was repeatedly detected. Hence this variant is considered as the prevailing muscle isoform and is referred to as Metro-B in accordance to its annotation as variant B in Flybase. In situ hybridizations revealed that during embryogenesis metro is mainly expressed in the developing CNS, in which it is detectable from around stage 11 onwards. Both the temporal and spatial expression patterns of metro mRNA are consistent with a role for Metro as a neural scaffold protein (Bachmann, 2010).

The establishment of neural networks involves mechanisms that coordinate the assembly and selective stabilization of synapses. Multivalent scaffold molecules that link transmembrane proteins to the cytoskeleton are candidate determinants of synapse stability. Recent studies imply a stabilizing role for Postsynaptic density protein-95 (PSD-95), a principal vertebrate synaptic scaffold protein, during activity-dependent maturation of glutamatergic synapses. The linkage of PSD-95 to ionotropic glutamate receptors (GluRs), however, makes it difficult to assess a direct involvement in synaptic structural integrity independent from activity-related effects. Moreover, the existence of additional PSD-95/Discs-large (Dlg)-like membrane-associated guanylate kinases (DLG-MAGUKs) accounts for partial redundancy and functional diversification including perisynaptic and extrasynaptic activities (Bachmann, 2010 and references therein).

In Drosophila a single gene, discs-large (dlg), encodes DlgA and DlgS97. The latter is specified by an N-terminal L27 domain (Mendoza, 2003) and thus corresponds to the predominant isoform of vertebrate SAP97 (Schlüter, 2006). Both isoforms, collectively referred to as Dlg, are present at glutamatergic larval neuromuscular junctions (NMJs) (Ataman, 2006). Strikingly, Dlg omits GluR-containing PSDs but is enriched within the subsynaptic reticulum (SSR), a postsynaptic membrane specialization commonly categorized as perisynaptic. Strong dlg mutants display aberrant motornerve terminal morphology and severely reduced SSR complexity. Dlg further controls the size of synaptic contacts (i.e., active zones and PSDs), possibly involving the perisynaptic cell adhesion molecule Fasciclin II (FasII) as binding partner. Similar to various dlg alleles, strong fasII alleles display enlarged active zones. Mutations that specifically abolish DlgS97, however, result in a similar phenotype while leaving junctional FasII largely unaffected (Mendoza-Topaz, 2008), suggesting that DlgS97 acts in a FasII-independent pathway to restrict synaptic contacts (Bachmann, 2010).

To specify the role of DlgS97 isoform-specific interactions have been analyzed and it has been shown that DlgS97 is crucial for proper NMJ localization of the PDZ domain protein DLin-7. It is further predicted that this interaction relies on a linker protein expressed in muscles but not in epithelia (Bachmann, 2004). Proteins bearing a tandem of L27 domains such as the MAGUK CASK/mLin-2 or members of the p55 subfamily of MAGUKs have emerged as primary candidates to serve the linkage between vertebrate Lin-7 (Veli) and SAP97 in epithelial cells. This study introduces Metro, a novel Drosophila MAGUK, as the missing link between DlgS97 and DLin-7 at NMJs. Genetic analyses reveal that the three scaffold proteins control each other. NMJs lacking Metro display reduced growth and are predestined to structural abnormalities. Notably, Metro and DLin-7 are involved in the dimensioning of glutamate receptor fields. These findings show that Metro and DLin-7 augment the complexity of the perisynaptic scaffold system and thereby control the synaptic organization of the NMJ (Bachmann, 2010).

This study has focused on a complex formed by Drosophila SAP97β alias DlgS97, the MPP-like MAGUK Metro, and the Veli/MALS homolog DLin-7. Related scaffold complexes exist at vertebrate epithelial junctions and at presynaptic photoreceptor terminals. The existence of the respective complexes in vertebrate CNS neurons and their synaptic or extrasynaptic roles therein remain elusive. Using larval NMJs as an in vivo model, this study now shows that DlgS97-Metro-DLin-7-type complexes indeed control the proper organization of a synaptic junction (Bachmann, 2010).

Genetic analyses implied that Metro constitutes the exclusive link between DlgS97 and DLin-7 at NMJs. In vitro binding assays, however, revealed that the interaction between DlgS97 and Metro is fairly weak. This issue was eventually clarified by two observations: (1) DLin-7 is absolutely required for DlgS97-dependent localization of Metro-B at NMJs, and (2) biochemical studies by Bohl (2007) revealed that binding of the Metro homolog MPP7 to Veli promotes its binding to hDlg. This allosteric mechanism is thus evolutionary conserved and distinguishes Metro and its closest mammalian homologs from other MPP-like MAGUKs and Cmg/Cask. In this way a one-to-one ratio of Metro and DLin-7 can be maintained, possibly translating into a balance of yet-to-be-identified junctional binding partners of the PDZ domains of either protein. Moreover, it was found that the spectrin-based cytoskeleton to some extent assures the junctional anchorage of Metro and DLin-7 in the absence of DlgS97. This link might contribute to a regular positioning of DlgS97-based scaffold complexes dictated by a spectrin-defined network (Bachmann, 2010).

The knock-out of metro leads to a considerable reduction of DlgS97. Such chronic downregulation of DlgS97 may be partially compensated by recruitment of DlgA. Stabilization of DlgS97 by Metro and DLin-7, however, involves the formation of higher-order complexes driven by (1) the ability of L27 domains to form dimers of dimers and (2) the tandem arrangement of L27 domains in Metro. Formally, such complexes are unlimited. In addition to the reduced abundance of junctional DlgS97, the breakdown of its multimeric context is consider as a crucial consequence of lacking Metro and DLin-7 (Bachmann, 2010).

The formation of new boutons strongly correlates with a temporally restricted downregulation of the Dlg-based scaffold at the respective site. Normally, Dlg reassociates with the nascent bouton shortly thereafter and recent studies suggest that GluRs and the actin regulators dPix and Pak act upstream of Dlg in this process. The occurrence of relatively large boutons with only a few GluR clusters and very little Dlg in both metro and DLin-7 mutants suggests that the formation of the postsynaptic scaffold is disturbed. Interestingly, a knockdown of MPP7 was found to cause a significant delay in the establishment of epithelial tight junctions. Considering the temporal aspect, it is proposed that Metro and DLin-7 are required to synchronize junctional expansion and scaffold assembly. Indeed various other abnormalities were observed that are consistent with a role for Metro and DLin-7 in balancing NMJ growth and stability. Reduced proliferation of boutons was accompanied by an overall enlargement of boutons, a reciprocal correlation, observed in several instances, possibly indicative of a disturbed linkage between submembranous scaffold and cytoskeletal elements. Notably, enlarged boutons may harbor more active zones, explaining the virtual invariance in the overall number of synaptic contacts at mutant versus wild-type NMJs (Bachmann, 2010).

The data indicate a stabilizing role for Metro and DLin-7, possibly as part of a back-up system to cope with operational demands such as junctional plasticity. It remains elusive, however, whether both proteins are regulated to weaken or strengthen the Dlg-based scaffold. Weakening may be involved in defining sites for bouton formation, but might also be a prerequisite for the de novo formation of synaptic contact sites on preexisting boutons. The striking protein instability of Metro in the absence of DLin-7 is suggestive in this regard, as it implies that upon breakdown, the complex can only be reassembled based on newly synthesized Metro. This would result in latency, which in turn could contribute to the temporal fidelity of the processes (Bachmann, 2010).

Given the strong dependency of Metro on DlgS97 it seemed plausible that loss of Metro would affect the size of receptor fields. A novel method was used that, once established, allowed measurement of the size of a high number of receptor fields. In this way it was found that the receptor fields were indeed enlarged at metro mutant NMJs. The specificity of this phenotype was further confirmed by transgenic rescue, which, however, remained incomplete. Although Metro-B clearly occurred as the principal isoform at NMJs, it is possible that the A- and/or C-variants are required at a low level or just temporarily to cover metro function at NMJs entirely. Compared with the enormous expansion of synaptic contacts associated with simultaneous depletion of DlgA and DlgS97, the effects on receptor field size detected in this study appear moderate. It is conceivable, however, that the reduced spacing between synaptic contacts that were frequently observed in the mutants represents a pre-stage toward the fusion of neighboring contacts (Bachmann, 2010).

While the size of receptor fields differed markedly between metro mutants and controls, no striking differences occurred in local GluRIID-specific fluorescence intensities. Moreover, despite the structural abnormalities, metro mutants displayed a rather normal profile of electrophysiological parameters. In particular, quantal currents were not significantly altered, consistent with the assumption that the composition and local density of GluRs remained normal and that transmitter release from a single vesicle does not saturate a normal-sized receptor field. Notably, normal mEJC amplitudes have been measured in case of a pronounced enlargement of synaptic contacts and increased mEJC amplitudes in strong dlg alleles have been assorted to enlarged synaptic vesicles rather than the size increment of synaptic contacts. The fact that the frequency of spontaneous release events and the evoked transmission remained unaffected is consistent with the virtual invariance in the number of active zones facing GluR fields at mutant NMJs and further implies that the presynaptic release machinery is largely intact in the metro mutants (Bachmann, 2010).

To date there is little evidence for the enrichment of Metro-like MAGUKs at synapses in the mammalian CNS, whereas mammalian homologs of Dlg and DLin-7 are prominent presynaptic and postsynaptic components of excitatory synapses. Reminiscent of the current observations, depletion of Veli in mice was found to cause a moderate increase in synaptic size, and yet this effect was assigned to its presynaptic interaction with liprin-α via Cask (Olsen, 2005). Notably, there is no previous report on a close link between DLG-MAGUKs and Veli at synapses of CNS neurons, despite the presence of Cask as a potential linker protein. Nevertheless an association of MPP3 with SAP97 and Veli is implied by coimmunoprecipitations from rat brain. Moreover, MPP3 was found to bind to a serotonin receptor and to the CAM Necl-1/SynCAM3 at extrasynaptic sites. The current results thus lead to a proposal that the perisynaptic interplay of Metro, DlgS97, and DLin-7 represents a conserved mechanism that confers structural fidelity and stability onsynaptic systems during development and plasticity (Bachmann, 2010).

Search PubMed for articles about Drosophila Metro

Ataman, B., Budnik, V. and Thomas, U. (2006). Scaffolding proteins at the Drosophila neuromuscular junction. Int. Rev. Neurobiol. 75: 181-216. PubMed ID: 17137929

Bachmann, A, et al. (2004). Cell type-specific recruitment of Drosophila Lin-7 to distinct MAGUK-based protein complexes defines novel roles for Sdt and Dlg-S97. J. Cell Sci. 117: 1899-1909. PubMed ID: 15039455

Bachmann, A., (2010). A perisynaptic ménage à trois between Dlg, DLin-7, and Metro controls proper organization of Drosophila synaptic junctions. J. Neurosci. 30(17): 5811-24. PubMed ID: 20427642

Bohl, J., Brimer, N., Lyons, C. and Vande Pol, S. B. (2007). The stardust family protein MPP7 forms a tripartite complex with LIN7 and DLG1 that regulates the stability and localization of DLG1 to cell junctions. J. Biol. Chem. 282: 9392-9400. PubMed ID: 17237226

Mendoza, C., et al. (2003). Novel isoforms of Dlg are fundamental for neuronal development in Drosophila. J. Neurosci. 23: 2093-2101. PubMed ID: 12657668

Mendoza-Topaz, C., et al. (2008) DLGS97/SAP97 is developmentally upregulated and is required for complex adult behaviors and synapse morphology and function. J Neurosci 28: 304-314. PubMed ID: 18171947

Olsen, O., et al. (2005). Neurotransmitter release regulated by a MALS-liprin-alpha presynaptic complex. J. Cell Biol. 170: 1127-1134. PubMed ID: 16186258

Schlüter, O. M., Xu, W., and Malenka, R. C. (2006). Alternative N-terminal domains of PSD-95 and SAP97 govern activity-dependent regulation of synaptic AMPA receptor function. Neuron 51: 99-111. PubMed ID: 16815335

date revised: 12 October 2011

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