InteractiveFly: GeneBrief

lin-7/veli: Biological Overview | References


Gene name - veli

Synonyms - Lin-7

Cytological map position -

Function - scaffolding protein

Keywords - synaptic stabilization and organization, acts downstream of DlgS97 to control NMJ expansion and proper establishment of synaptic boutons - present both presynaptically and postsynaptically

Symbol - veli

FlyBase ID: FBgn0039269

Genetic map position - chr3R:20,890,140-20,891,317

Classification - . PubMed ID: L27 domain, PDZ domain

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

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 (Menage a trois), 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).

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 prestage 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).

Multiple domains of Stardust differentially mediate localisation of the Crumbs-Stardust complex during photoreceptor development in Drosophila

Drosophila Stardust (Sdt), a member of the MAGUK family of scaffolding proteins, is a constituent of the evolutionarily conserved Crumbs-Stardust (Crb-Sdt) complex that controls epithelial cell polarity in the embryo and morphogenesis of photoreceptor cells (PRCs). Although apical localisation is a hallmark of the complex in all cell types and in all organisms analysed, only little is known about how individual components are targeted to the apical membrane. A structure-function analysis of Sdt was performed by constructing transgenic flies that express altered forms of Sdt to determine the roles of individual domains for localisation and function in photoreceptor cells. The results corroborate the observation that the organisation of the Crb-Sdt complex is differentially regulated in pupal and adult photoreceptors. In pupal photoreceptors, only the PDZ domain of Sdt - the binding site of Crb - is required for apical targeting. In adult photoreceptors, by contrast, targeting of Sdt to the stalk membrane, a distinct compartment of the apical membrane between the rhabdomere and the zonula adherens, depends on several domains, and seems to be a two-step process. The N-terminus, including the two ECR domains and a divergent N-terminal L27 domain that binds the multi-PDZ domain protein PATJ in vitro, is necessary for targeting the protein to the apical pole of the cell. The PDZ-, the SH3- and the GUK-domains are required to restrict the protein to the stalk membrane. Drosophila PATJ or Drosophila Lin-7 are stabilised whenever a Sdt variant that contains the respective binding site is present, independently of where the variant is localised. By contrast, only full-length Sdt, confined to the stalk membrane, stabilises and localises Crb, although only in reduced amounts. The amount of Crumbs recruited to the stalk membrane correlates with its length. These results highlight the importance of the different Sdt domains and point to a more intricate regulation of the Crb-Sdt complex in adult photoreceptor cells (Bulgakova, 2008).

Data presented in this study corroborate the view that distinct mechanisms control localisation of the Crb-Sdt complex in PRCs at different developmental stages. This conclusion is further supported by the observation that a truncated PATJ protein, consisting of only L27 and the first PDZ domain, is localised correctly during the first half of pupal development, but is delocalised in adult PRCs. The stability of the complex at pupal stages seems to depend only on Crb. In pupae, all core components of the complex are mislocalised in crb-mutant PRCs, whereas the absence of sdt, PATJ or Lin-7 does not affect apical localisation of the others. Accordingly, Sdt localisation at this stage only depends on its PDZ domain that binds the cytoplasmic tail of Crb. Neither the non-canonical L27N domain of Sdt, which is responsible for binding PATJ, nor the other protein-protein interaction domains are required for Sdt localisation in pupal PRCs (Bulgakova, 2008).

In the adult Drosophila eye, localisation of Crb-Sdt-complex core proteins to the stalk membrane is mutually dependent, with the exception of Lin-7, which is not required to localise other components. Similarly, in zebrafish the levels of the Crb orthologous proteins require the function of the Sdt orthologue Nagie oko. In the fly eye, changes observed at different developmental stages point to a transition in the mechanisms regulating the building and stability of the complex. This transition occurs gradually in the second half of pupal development. At the same time, Bazooka, which is associated with the adherens junctions in the first half of pupal development, accumulates in the cytoplasm. The transition also correlates with the formation of stalk membrane, which initiates around 55% pupal development and ultimately separates the apical plasma domain into two distinct compartments. This process seems to require additional, more complex control mechanisms, as reflected by the fact that several Sdt domains are required for its proper localisation at later stages. It is very possible that other, yet unknown components contribute to the stability and/or restriction of Sdt at the stalk membrane (Bulgakova, 2008).

Results presented in this study also suggest that in the adult Drosophila eye, localisation of Sdt occurs in several steps that rely on different domains. In the first step, Sdt is brought close to the apical membrane. This function is mediated by the N-terminus, including the two ECR domains and the N-terminal L27 domain. Since Par-6, a known binding partner of the ECR motifs, is localised basolaterally in adult PRCs, PATJ binding is more likely to be crucial for apical recruitment of Sdt. In fact, no localised Sdt is detected in PATJ-mutant adult PRCs. In the absence of all other domains besides the N-terminus (with the exception of L27C), Sdt proteins accumulate at the rhabdomere base, a specialised region that seems to have an important role in PRCs. Many proteins involved in morphogenesis, phototransduction or endocytosis, such as Drosophila moesin, TRPL (transient receptor potential-like) and Rab11, to mention just a few, are enriched there. The final step, recruitment of Sdt to the stalk membrane, requires the PDZ-, the SH3- and the GUK-domain. Whereas the PDZ-domain binds Crb, no binding partners for the SH3- and the GUK-domain are known. It was shown that these two domains can bind each other in vitro. Similar interactions between corresponding domains of the human MAGUK CASK were reported to occur either intramolecularly or intermolecularly between the GUK domain of human CASK and the SH3 domain of hDLG. In the MAGUK PSD-93, binding of a ligand to the PDZ domain releases intramolecular inhibition of the GUK domain by the SH3 domain. This possible complexity currently does not distinguish whether the failure to recruit Sdt to the stalk membrane upon removal of one of these domains is due to either the lack of binding additional partner(s) or the lack of intramolecular interactions, or both (Bulgakova, 2008).

Whereas Sdt is not required to restrict components of the Crb-Sdt complex to the apical membrane in pupal PRCs, the apical localisation of Par-6, a member of the Par-protein network, depends on Sdt at this developmental stage. Recently, several studies suggested a direct interaction between the Crb-Sdt and the PAR complex, but the proposed interactions differ with respect to the partners mediating the link. Results obtained from in vitro analysis have suggested a number of interactions: aPKC with both PATJ and the intracellular domain of Crb; the PDZ domain of Par-6 with either the N-terminus of Sdt and/or Pals1 or the C-terminus of CRB1 or CRB3; and the N-terminus of Par-6 with the third PDZ domain of PATJ. The observations that neither Crb nor PATJ localisation is affected in sdt-mutant pupal PRCs and that expression of Sdt-B2 in sdt-mutant PRCs completely restores Par-6 apical localisation, strongly suggests that in pupal PRCs the interaction between the Crb complex and Par-6 is mediated by the ECR motifs of Sdt. Sdt-A, which carries an additional 433 amino-acid-long stretch between ECR1 and ECR2, only partially restored apical recruitment of Par-6, suggesting that separation of ECR1 from ECR2 interferes with efficient interactions between the two proteins (Bulgakova, 2008).

The results show that in adult PRCs, sdt controls localisation and stability of Crb, PATJ and Lin-7 but the mechanisms differ. Whenever a Sdt protein is expressed that contains binding domains for PATJ or Lin-7, the amount of the latter is, independently of localisation, restored to wild-type levels. By contrast, Crb protein is stabilised only when Sdt is associated with the stalk membrane (expression of Sdt-A, Sdt-B2, Sdt-βL27C and Sdt-βN). Interestingly, none of the constructs used, including the two full-length variants, rescued Crb protein to wild-type levels. One possible explanation is that other, yet uncharacterised Sdt isoforms are expressed in the eye, which, together with Sdt-B2 and/or unknown interaction partners of the Crb-Sdt complex, regulate the amount of Crb at the stalk membrane. Additional Sdt isoforms are predicted by Flybase to exist. They mainly differ from the known forms in their N-termini, which suggests alternative interaction partners (Bulgakova, 2008).

One striking phenotype observed in PRCs mutant for crb, sdt or PATJ is the reduction of stalk-membrane length. This raises questions about how the Crb-Sdt complex regulates the size of this distinct apical membrane compartment. The results provide evidence that the amount of Crb protein is a crucial determinant of stalk-membrane length. This agrees with the observation that Crb overexpression increases stalk-membrane length. Interestingly, overexpression of a Crb protein that lacks the cytoplasmic domain and, hence, the binding site for Sdt, is sufficient to cause this increase. This suggests that either the transmembrane and/or extracellular domain of Crb regulates stalk-membrane growth. Sdt contributes to the stabilisation of Crb at the stalk and, hence, is indirectly involved in the control of stalk-membrane length. It will be interesting to explore the mechanism by which Crb regulates stalk-membrane length (Bulgakova, 2008).

The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth

Disruption of epithelial polarity is a key event in the acquisition of neoplastic growth. JNK signalling is known to play an important part in driving the malignant progression of many epithelial tumours, although the link between loss of polarity and JNK signalling remains elusive. In a Drosophila genome-wide genetic screen designed to identify molecules implicated in neoplastic growth, this study identified grindelwald (grnd; CG10176), a gene encoding a transmembrane protein with homology to members of the tumour necrosis factor receptor (TNFR) superfamily. This study shows that Grnd mediates the pro-apoptotic functions of Eiger (Egr), the unique Drosophila TNF, and that overexpression of an active form of Grnd lacking the extracellular domain is sufficient to activate JNK signalling in vivo. Grnd also promotes the invasiveness of RasV12/scrib-/- tumours through Egr-dependent Matrix metalloprotease-1 (Mmp1) expression. Grnd localizes to the subapical membrane domain with the cell polarity determinant Crumbs (Crb) and couples Crb-induced loss of polarity with JNK activation and neoplastic growth through physical interaction with Veli (also known as Lin-7). Therefore, Grnd represents the first example of a TNFR that integrates signals from both Egr and apical polarity determinants to induce JNK-dependent cell death or tumour growth (Andersen, 2015).

A genome-wide screen was carried to identify molecules that are required for neoplastic growth. The condition used for this screen was the disc-specific knockdown of avalanche, also known as syntaxin 7), a gene encoding a syntaxin that functions in the early step of endocytosis2. avl-RNAi results in ectopic Wingless (Wg) expression, neoplastic disc overgrowth, and a 2-day delay in larva-to-pupa transition. A collection of 10,100 transgenic RNA interference (RNAi) lines were screened for their ability to rescue the pupariation delay, and 121 candidate genes were identified. Interestingly, only eight candidate genes also rescued ectopic Wg expression and neoplastic overgrowth. These included five lines targeting core components of the JNK pathway (Bendless, Tab2, Tak1, Hemipterous and Basket. Using a puckered enhancer trap (puc-lacZ) as a readout for JNK activity, it was confirmed that JNK signalling is highly upregulated in avl-RNAi discs. One of the remaining lines targets CG10176, a gene encoding a transmembrane protein. Reducing expression of CG10176 by using two different RNAi lines was as efficient as tak1 silencing to restore normal Wg pattern and suppresses JNK signalling and neoplastic growth in the avl-RNAi background. Sequence analysis of GC10176 identified a cysteine-rich domain (CRD) in the extracellular part with homology to vertebrate TNFRs harbouring a glycosphingolipid-binding motif (GBM) characteristic of many TNFRs including Fas. CG10176 was named grindelwald (grnd) , after a village at the foot of Eiger, a Swiss mountain that lent its name to the unique Drosophila TNF, Egr. Immunostaining and subcellular fractionation of disc extracts confirmed that Grnd localizes to the membrane. Moreover, co-immunoprecipitation experiments showed that both Grnd full-length and Grnd-intra, a form lacking its extracellular domain, directly associate with Traf2, the most upstream component of the JNK pathway. This interaction is disrupted by a single amino acid substitution within a conserved Traf6-binding motif (human TRAF6 is the closest homologue to Traf2. Overexpression of Grnd-intra, but not full-length Grnd, is sufficient to induce JNK signalling, ectopic Wg expression and apoptosis, and Grnd-intra-induced apoptosis is efficiently suppressed in a hep (JNKK) mutant background, confirming that Grnd acts upstream of the JNK signalling cascade (Andersen, 2015).

The Drosophila TNF Egr activates JNK signalling and triggers cell death or proliferation, depending on the cellular context. Therefore tests were performed to see whether Grnd is required for the small-eye phenotype generated by Egr-induced apoptosis in the retinal epithelium (via Egr overexpression). Inhibition of JNK signalling by reducing tak1 or traf2 expression, or by overexpressing puckered, blocks Egr-induced apoptosis and rescues the small-eye phenotype. In contrast to a previous report, RNAi silencing of wengen (wgn) , a gene encoding a presumptive receptor for Egr, does not rescue the small-eye phenotype. Furthermore, the small-eye phenotype is not modified in a wgn-null mutant background, confirming that Wgn is not required for Egr-induced apoptosis in the eye. By contrast, reducing grnd levels partially rescues the Egr-induced small-eye phenotype, producing a 'hanging-eye' phenotype that is not further rescued in a wgn-knockout mutant background. A similar phenotype was previously reported as a result of non-autonomous cell death induced by a diffusible form of Egr. This suggests that Grnd prevents Egr from diffusing outside of its expression domain. Co-immunoprecipitation experiments show that both full-length Grnd and Grnd-extra, a truncated form of Grnd lacking the cytoplasmic domain, associate with Egr through its TNF-homology domain. Although Grnd-extra can bind Egr, it cannot activate JNK signalling. Therefore, it was reasoned that Grnd-extra expression might prevent both cell-autonomous and non-autonomous apoptosis by trapping Egr and preventing its diffusion and binding to endogenous Grnd. Indeed, GMR-Gal4-mediated expression of grnd-extra fully rescues the Egr small-eye phenotype. To confirm that the removal of Grnd induces Egr-mediated non-autonomous cell death, wing disc clones were generated expressing egr alone, egr + tak1 RNAi, or egr + grnd RNAi. As expected, reducing tak1 levels in egr-expressing clones prevents their elimination by apoptosis. Similarly, reducing grnd levels prevents autonomous cell death, but also induces non-autonomous apoptosis. This suggests that Egr, like its mammalian counterpart TNF-α, can be processed into a diffusible form in vivo whose interaction with Grnd limits the potential to act at a distance. Flies carrying homozygous (grndMinos/Minos) or transheterozygous (grndMinos/Df) combinations of a transposon inserted in the grnd locus express no detectable levels of Grnd protein and are equally resistant to Egr-induced cell death. In addition, grndMinos/Minos mutant flies are viable and display no obvious phenotype, suggesting that Grnd, like Egr, participates in a stress response to limit organismal damage. Collectively, these data demonstrate that Grnd is a new Drosophila TNF receptor that mediates most, if not all, Egr-induced apoptosis (Andersen, 2015).

TNFs probably represent a danger signal produced in response to tissue damage to rid the organism of premalignant tissue or to facilitate wound healing. Disc clones mutant for the polarity gene scribbled (scrib) induce an Egr-dependent response resulting in the elimination of scrib mutant cells by JNK-mediated apoptosis. To test the requirement for Grnd in this process, scrib-RNAi and scrib-RNAi + grnd-RNAi clones obtained 72 h after heat shock induction were compared. As expected, scrib-RNAi cells undergo apoptosis and detach from the epithelium. By contrast, scrib-RNAi clones with reduced grnd expression survive, indicating that Grnd is required for Egr-dependent elimination of scrib-RNAi cells. Similar results were obtained by generating scrib mutant clones in the eye disc (Andersen, 2015).

In both mammals and flies, TNFs are double-edged swords that also have the capacity to promote tumorigenesis in specific cellular contexts. Indeed, scrib minus eye disc cells expressing an activated form of Ras (RasV12) exhibit a dramatic tumour-like overgrowth and metastatic behaviour, a process that critically relies on Egr. RasV12/scrib-/- metastatic cells show a strong accumulation of Grnd and Mmp1, and invade the ventral nerve cord. Primary tumour cells reach peripheral tissues such as the fat body and the gut, where they form micro-metastases expressing high levels of Grnd. Reducing grnd levels in RasV12/scrib-/- clones is sufficient to restore normal levels of Mmp1 and abolish invasiveness in a way similar to that observed in an egr mutant background. Therefore, Grnd is required for the Egr-induced metastatic behaviour of RasV12/scrib-/- tumorous cells. Similarly, reducing grnd, but not wgn levels, strongly suppresses Mmp1 expression in RasV12/dlg-RNAi cells and limits tumour invasion, indicating that Wgn does not have a major role in the progression of these tumours (Andersen, 2015).

Perturbation of cell polarity is an early hallmark of tumour progression in epithelial cells. In contrast to small patches of polarity-deficient cells, for example, scrib mutant clones, organ compartments or animals fully composed of polarity-deficient cells become refractory to Egr-induced cell death and develop epithelial tumours. The formation of these tumours requires JNK/MAPK signalling, but not Egr, suggesting Egr-independent coupling between loss of polarity and JNK/MAPK-dependent tumour growth. In line with these observations, it was noticed that, in contrast to Grnd, Egr is not required to drive neoplastic growth in avl-RNAi conditions. This suggests that, in addition to its role in promoting Egr-dependent functions, Grnd couples loss of polarity with JNK-dependent growth independently of Egr. Disc immunostainings revealed that Grnd co-localizes with the apical determinant Crb in the marginal zone, apical to the adherens junction protein E-cadherin (E-cad) and the atypical protein kinase C (aPKC). In avl-RNAi discs, Grnd and Crb accumulate in a wider apical domain. Apical accumulation of Crb is proposed to be partly responsible for the neoplastic growth induced by avl knockdown, since overexpression of Crb or a membrane-bound cytoplasmic tail of Crb (Crb-intra) mimics the avl-RNAi phenotype. Therefore whether Grnd might couple the activity of the Crb complex with JNK-mediated neoplastic growth was examined. Indeed, reducing grnd levels, but not wgn, in ectopic crb-intra discs suppresses neoplastic growth as efficiently as inhibiting the activity of the JNK pathway. Notably, Yki activation is not rescued in these conditions, illustrating the ability of Crb-intra to promote growth independently of Grnd by inhibiting Hippo signalling through its FERM-binding motif (FBM). Indeed, neoplastic growth and polarity defects induced by a form of Crb-intra lacking its FBM (CrbΔFBM-intra) are both rescued by Grnd silencing. As expected, the size of ectopic crbΔFBM-intra;grnd-RNAi discs is reduced compared to the size of ectopic crb-intra; grnd-RNAi discs (Andersen, 2015).

Crb, Stardust (Sdt; PALS1 in humans), and Pals1-associated tight junction protein (Patj) make up the core Crb complex, which recruits the adaptor protein Veli (MALS1-3 in humans). In agreement with previous yeast two-hybrid data, this study found that Grnd binds directly and specifically to the PDZ domain of Veli through a membrane-proximal stretch of 28 amino acids in its intracellular domain. Grnd localization is unaffected in crb and veli RNAi mutant clones. However, reducing veli expression rescues the patterning defects and disc morphology of ectopic crb-intra mutant cells, suggesting that Grnd couples Crb activity with JNK signalling through its interaction with Veli. Interestingly, aPKC-dependent activation of JNK signalling also depends on Grnd. aPKC is capable of directly binding and phosphorylating Crb, which is important for Crb function. This suggests that aPKC, either directly or through Crb phosphorylation, activates Grnd-dependent JNK signalling in response to perturbation of apico-basal polarity (Andersen, 2015).

These data are consistent with a model whereby Grnd integrates signals from Egr, the unique fly TNF, and apical polarity determinants to induce JNK-dependent neoplastic growth or apoptosis in a context-dependent manner. Recent work reveals a correlation between mammalian Crb3 expression and tumorigenic potential in mouse kidney epithelial cells. The conserved nature of the Grnd receptor suggests that specific TNFRs might carry out similar functions in vertebrates, in which the link between apical cell polarity and tumour progression remains elusive (Andersen, 2015).

Cell type-specific recruitment of Drosophila Lin-7 to distinct MAGUK-based protein complexes defines novel roles for Sdt and Dlg-S97

Stardust (Sdt) and Discs-Large (Dlg) are membrane-associated guanylate kinases (MAGUKs) involved in the organization of supramolecular protein complexes at distinct epithelial membrane compartments in Drosophila. Loss of either Sdt or Dlg affects epithelial development with severe effects on apico-basal polarity. Moreover, Dlg is required for the structural and functional integrity of synaptic junctions. Recent biochemical and cell culture studies have revealed that various mammalian MAGUKs can interact with mLin-7/Veli/MALS, a small PDZ-domain protein. To substantiate these findings for their in vivo significance with regard to Sdt- and Dlg-based protein complexes, the subcellular distribution of Drosophila Lin-7 (DLin-7) was analyzed and genetic and biochemical assays were performed to characterize its interaction with either of the two MAGUKs. In epithelia, Sdt mediates the recruitment of DLin-7 to the subapical region, while at larval neuromuscular junctions, a particular isoform of Dlg, Dlg-S97, is required for postsynaptic localization of DLin-7. Ectopic expression of Dlg-S97 in epithelia, however, was not sufficient to induce a redistribution of DLin-7. These results imply that the recruitment of DLin-7 to MAGUK-based protein complexes is defined by cell-type specific mechanisms and that DLin-7 acts downstream of Sdt in epithelia and downstream of Dlg at synapses (Bachmann, 2004).

Searching the Drosophila genome database revealed that a single gene annotated as CG7662 encodes two conceptual splice isoforms representing fly homologues of Lin-7. RT-PCR analyses performed on mRNA samples from different developmental stages and northern blot analysis of embryonic mRNA, however, indicated that only one major gene product is expressed throughout development. An embryonic RT-PCR product was subcloned and sequenced. The deduced 195 aa gene product comprises an N-terminal L27-type domain and a C-terminal PDZ domain. DLin-7 exhibits 70% sequence identity with the corresponding region of LIN-7 in C. elegans and is even more closely related to the three paralogous isoforms of mammals (collectively referred to as mLin-7). Western blot analyses using an antibody raised against DLin-7 yielded a prominent immunoreactive band at ~27 kDa. As expected, a slightly increased molecular weight was apparent for a transgenically expressed, Flag epitope-tagged version of DLin-7 (Bachmann, 2004).

Tests were performed to see whether the sequence homology between DLin-7 and its orthologues is significant with regard to protein-protein interactions. Since the PDZ domains of DLin-7 and mLin-7 are almost identical, it may be assumed that they exhibit identical binding specificities. A couple of binding partners of the PDZ domain have been identified in other species; however, their putative fly homologues carry no C-terminal PDZ-binding motifs. Therefore focus was placed on potential interactions corresponding to those mediated by the L27 domain of mLin-7. Using a yeast two-hybrid assay it was demonstrated that DLin-7 binds to L27 domains in the fly homologues of mLin-2 (DLin-2/CamGUK/CAKI) and Pals1 (Sdt). Whereas in a previous study a direct interaction between mLin-7 and SAP97 was not detectable, a weak interaction between DLin-7 and the N-terminal region of Dlg-S97 was noticed. Similarly to mLin-7, DLin-7 did not display homophilic interactions. It is thus concluded that, for DLin-7 and mLin-7, the in vitro binding properties of their L27 domains with members of distinct MAGUK subfamilies are very similar (Bachmann, 2004).

To assess the subcellular localization of DLin-7, crossreacting antibodies raised against mLin-7 as well as antisera raised against DLin-7 were used. Both antisera yielded virtually identical results in western blot and immunofluorescence analyses. The specificity of the antisera was further evident from increased or decreased immunoreactivities upon transgenic overexpression or reduction of DLin-7, respectively (Bachmann, 2004).

In the embryonic epidermis and in imaginal disc epithelia, DLin-7-specific immunofluorescence was found highly enriched at the apical membrane. Accordingly, confocal microscopy on double-stained imaginal discs revealed that the immunoreactivities for DLin-7 and Dlg, which localizes to septate junctions, were totally disjunct, whereas a striking colocalization of DLin-7 with Sdt was evident in these epithelia. Considering the in vitro interaction between DLin-7 and Sdt it is assumed that DLin-7 is a component of the previously described subapical Crb/Sdt complex (Bachmann, 2004).

The single fly homologue of Lin-7 is a component of different MAGUK-based protein complexes in epithelia and at synaptic junctions. This finding is in line with the previously reported association of LIN-7 and mLin-7 with various membrane specializations in worms or mammals, respectively (Simske, 1996; Butz, 1998; Jo, 1999; Perego, 2000; Straight, 2000). Nonetheless, the results deviate from these earlier reports in several regards and thereby imply novel roles for Sdt and Dlg-S97. Most notably, the requirement for either MAGUK to recruit DLin-7 to distinct membrane domains was not simply predictable from studies on their homologues in other species (Bachmann, 2004).

Pals1, a putative mammalian homologue of Sdt, binds mLin-7 in vitro (Kamberov, 2000). The physiological significance of this finding remains unclear since Pals1 localizes to tight junctions of epithelial cells, whereas a basolateral localization for mLin-7 was emphasized in several other reports (Perego, 1999; Straight, 2000; Straight, 2001). In Madin-Darby canine kidney cells, however, mLin-7 has also been detected at tight junctions (Irie, 1999; Perego, 2000). The current results now indicate that the interaction between the fly orthologues of Pals1 and mLin-7 is employed in epithelia for the recruitment of DLin-7 to the Crb-Sdt complex within the SAR (Bachmann, 2004).

The virtual absence of DLin-7 from basolateral plasma membrane compartments in Drosophila imaginal disc epithelia is in striking contrast to the situation in both mammals and nematodes. Differences in the expression, subcellular localization and binding capacity of potential interaction partners may account for this discrepancy. Two types of evolutionary conserved proteins have been implicated in the basolateral membrane recruitment of LIN-7 and mLin-7: the MAGUKs LIN-2/mLin-2 (CASK) and β-catenin. The latter was found to recruit mLin-7 to cadherin-based epithelial junctions via its C-terminal PDZ-binding motif (Perego, 2000). Although this motif (tDTDL) is conserved in C. elegans β-catenin, it is aberrant in the fly orthologue, Armadillo (tDTDC). In fact, a direct interaction between DLin-7 and Armadillo was not detectable in a yeast two-hybrid assay. Hence it appears unlikely that DLin-7 and Armadillo exhibit a mode of interaction similar to that of their counterparts in mammals. In contrast, DLin-2 (Caki/CamGUK) and DLin-7 displayed strong interaction in the yeast two-hybrid assay. Therefore it would be expected that DLin-2 competes with Sdt for binding to the L27 domain of DLin-7 when expressed in epithelia. An epithelial expression of DLin-2, however, has not yet been documented and, instead, both immunostainings and mRNA analyses revealed that DLin-2 is predominantly expressed in the CNS (Bachmann, 2004).

Sdt is not expressed at detectable levels at larval NMJs and thus cannot contribute to the postsynaptic enrichment of DLin-7 at these junctions. Instead it was demonstrated that Dlg-S97 is required for the recruitment of DLin-7 to scaffolding complexes within the SSR around type I boutons. Severe mutations in dlg cause a decrease in the length of the SSR to about 40% [relative to bouton size. In immunofluorescence analyses, however, the reduction of both endogeneous or Flag-tagged DLin-7 at dlgXI-2 mutant NMJs appeared clearly more dramatic, suggesting that impaired recruitment of DLin-7 is not simply due to reduced SSR complexity. This reasoning is supported by co-immunoprecipitation experiments that revealed a physical linkage between DLin-7 and Dlg-S97. This linkage is most likely indirect, as implied by the failure of EGFP-Dlg-S97 to recruit cytosolic DLin-7 in epithelia. Although an interaction could be monitored between DLin-7 and the N-terminal domain of Dlg-S97 in yeast, it was also noted that this interaction is much weaker compared with those displayed by DLin-7 in combination with DLin-2 or Sdt. In accordance with recent biochemical studies and cell culture assays, which imply the coupling of SAP97 and mLin-7 via MAGUKs such as mLin-2 or MPP3, it is therefore proposed that Dlg-S97 and DLin-7 are linked via an intermediate protein factor. In fact, both the N-terminal domain of Dlg-S97 and DLin-7 can bind to L27 domains of DLin-2 in vitro. The presence of DLin-2 at larval NMJs, however, remains questionable. Unfortunately an antibody against DLin-2 could not be deployed to address this issue in further detail. Third instar larvae that are homo- or hemizygous for the DLin-2 mutant allele cakix-307 exhibit normal levels of both DLin7- and Dlg-S97-specific immunofluorescence. This allele has been characterized as a deletion that removes large portions of the gene including the region encoding the PDZ-, SH3- and GUK domains of DLin-2. Nonetheless some residual function might be displayed by a truncated DLin-2x-307 mutant isoform. Thus, the observations strongly argue against, but do not completely rule out, an involvement of DLin-2 in the recruitment of DLin-7 to NMJs. It should be noted, however, that Dlg-S97, DLin-2 and DLin-7 could co-assemble into synaptic protein complexes in the CNS where they are found equally enriched within the neuropil regions (Bachmann, 2004).

In light of recent work, which has revealed complex intramolecular interactions displayed by SAP97, one should also consider the possibility that the SAP97-type N-terminus is only accessible upon binding of tissue- or compartment-specific factors to other domains within Dlg-S97. This mode of regulation, however, would not apply to Dlg-S97N-EGFP and thus cannot explain its inability to induce nuclear targeting of DLin-7 in epithelia (Fig. 9H-J) (Bachmann, 2004).

It has been proposed that the targeting of SAP97 to epithelial membranes depends on mLin-2. This hypothesis was based on the finding that the expression of truncated mLin-2 exerts a dominant-negative effect on the localization of SAP97 in cultured epithelial cells (Lee, 2002). It is stressed that this hierarchical mode does not apply to the respective fly homologues, since Dlg-A, which lacks the SAP97-type N-terminus, is efficiently targeted to epithelial septate junctions and to NMJs. Moreover, the recruitment of Dlg-S97 by a DLin-7 binding MAGUK could hardly explain the Dlg-S97-dependent recruitment of DLin-7 (Bachmann, 2004).

These analyses strongly suggest that the interactions between DLin-7 and Sdt or Dlg-S97 take place within the respective submembraneous target regions. In addition, these interactions could play a role during the trafficking of DLin-7. In mammalian neurons mLin-7 was found in a complex with mLin-2, mLin-10 and the NMDA-type glutamate receptor subunit NR2B on dendritic vesicles, which are transported along microtubules (Setou, 2000). Likewise, the subcellular targeting of Dlg-like MAGUKs involves the association with vesicles and/or intracellular membrane compartments and depends on microtubular transport (Bachmann, 2004).

In vertebrates, mLin-7 isoforms have been detected in axonal and dendritic compartments (Butz, 1998; Jo, 1999). The postsynaptic colocalization of DLin-7 and Dlg-S97 is reminiscent of the association of mLin-7 with PSD-95/SAP90, a prominent Dlg-like MAGUK present in postsynaptic densities of vertebrate neurons (Jo, 1999). Interestingly, a recently discovered isoform of PSD-95 (PSD-95β) exhibits a SAP97-type N-terminus with conserved binding properties. In light of these findings it is speculated that PSD-95β, as opposed to conventional PSD-95, is involved in the postsynaptic recruitment of mLin-7. SAP97 could also serve this role, although a physical association of SAP97 and mLin-7 at synaptic junctions has not yet been reported. A possible association of DLin-7 with the presynaptic membrane of synaptic boutons can hardly be resolved by confocal microscopy in the presence of strong postsynaptic immunoreactivity. Targeted expression of Flag-DLin-7 in motorneurons did not yield considerable immunofluorescence signals at NMJs, suggesting that DLin-7 is barely targeted to presynaptic nerve terminals. It should be noted, however, that the relative pre-versus postsynaptic abundance of a protein does not necessarily reflect its functional impact on either side of the synaptic cleft. For instance, while Dlg, Scrib and D-VAP-33A are clearly enriched postsynaptically at larval NMJs, genetic rescue and gain-of-function experiments have highlighted the importance of the minor presynaptic component in all three cases (Bachmann, 2004).

The roles of DLin-7 within the SAR and at synapses remain elusive. Overexpression of Flag-DLin-7 did not result in easily detectable phenotypes within epithelia or at NMJs. It is predicted that DLin-7 acts downstream of Sdt or Dlg-S97. Accordingly, loss-of-function alleles of DLin-7 are expected to mimic previously described or yet concealed phenotypical aspects of sdt and dlg mutants. The partial reduction of DLin-7 as achieved by transgenic expression of dsRNA had no obvious effect on the shape of boutons or on epithelial polarity. Current studies in are therefore aimed at both the generation of complete loss-of-function alleles and monitoring more subtle phenotypes. In accordance with previous studies in other species, it is expected that DLin-7 binds at least one ligand via its single PDZ domain. Thereby it may help to retain this ligand within the respective compartment and/or to regulate its endosomal sorting (Bachmann, 2004).


REFERENCES

Search PubMed for articles about Drosophila Lin-7

Andersen, D. S., Colombani, J., Palmerini, V., Chakrabandhu, K., Boone, E., Rothlisberger, M., Toggweiler, J., Basler, K., Mapelli, M., Hueber, A. O. and Leopold, P. (2015). The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth. Nature. PubMed ID: 25874673

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

Bulgakova, N. A., Kempkens, O. and Knust, E. (2008). Multiple domains of Stardust differentially mediate localisation of the Crumbs-Stardust complex during photoreceptor development in Drosophila. J. Cell Sci. 121: 2018-2026. PubMed ID: 18495840

Butz, S., Okamoto, M. and Südhof, T. C. (1998). A tripartite protein complex with the potential to couple synaptic vesicle exocytosis to cell adhesion in brain. Cell 94: 773-782. PubMed ID: 9753324

Irie, M., Hata, Y., Deguchi, M., Ide, N., Hirao, K., Yao, I., Nishioka, H. and Takai, Y. (1999). Isolation and characterization of mammalian homologues of Caenorhabditis elegans lin-7: localization at cell-cell junctions. Oncogene 18: 2811-2817. PubMed ID: 10362251

Jo, K., Derin, R., Li, M. and Bredt, D. S. (1999). Characterization of MALS/Velis-1, -2, and -3: a family of mammalian Lin-7 homologs enriched at brain synapses in association with the Postsynaptic Density-95/NMDA receptor postsynaptic complex. J. Neurosci. 19: 4189-4199. PubMed ID: 10341223

Kamberov, E., et al. (2000). Molecular cloning and characterization of Pals, proteins associated with mLin-7. J. Biol. Chem. 275(15): 11425-31. PubMed ID: 10753959

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

Perego, C., Vanoni, C., Villa, A., Longhi, R., Kaech, S. M., Frohli, E., Hajnai, A., Kim, S. K. and Pietrini, G. (1999). PDZ-mediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial cells. EMBO J. 18: 2384-2393. PubMed ID: 10228153

Perego, C., Vanoni, C., Massari, S., Longhi, R. and Pietrini, G. (2000). Mammalian LIN-7 PDZ proteins associate with β-catenin at the cell-cell junctions of epithelia and neurons. EMBO J. 19: 3978-3989. PubMed ID: 10921879

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

Setou, M., Nakagawa, T., Seog, D.-H. and Hirokawa, N. (2000). Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport. Science 288: 1796-1802. PubMed ID: 10846156

Simske, J. S., Kaech, S. M., Harp, S. A. and Kim, S. K. (1996). LET-23 receptor localization by the cell junction protein LIN-7 during C. elegans vulval induction. Cell 85: 195-204. PubMed ID: 8612272

Straight, S. W., Chen, L., Karnak, D. and Margolis, B. (2001). Interaction with mLin-7 alters the targeting of endocytosed transmembrane proteins in mammalian epithelial cells. Mol. Biol. Cell 12: 1329-1340. PubMed ID: 11359925


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