Protein Interactions

Membrane-associated guanylate kinases (MAGUKs), such as Discs-large (Dlg), play critical roles in synapse maturation by regulating the assembly of synaptic multiprotein complexes. Previous studies have revealed a genetic interaction between Dlg and another PDZ scaffolding protein, Scribble (Scrib), during the establishment of cell polarity in developing epithelia. The biochemical nature of this interaction has remained elusive, raising questions regarding the mechanisms by which the actions of both proteins are coordinated. This study reports a new Dlg-interacting protein, GUK-holder (GUKh), that interacts with the GUK domain of Dlg and that is dynamically expressed during synaptic bouton budding. At Drosophila synapses Dlg colocalizes with Scrib and this colocalization is likely to be mediated by direct interactions between GUKh and the PDZ2 domain of Scrib. Dlg, GUKh, and Scrib form a tripartite complex at synapses, in which Dlg and GUKh are required for the proper synaptic localization of Scrib. These results provide a mechanism by which developmentally important PDZ-mediated complexes are associated at the synapse (Mathew, 2002).

In Drosophila, dlg mutants in which the GUK domain is absent exhibit abnormalities in synapse structure. Moreover, transgenic Dlg lacking the GUK domain fails to localize at synapses when expressed in a dlg mutant background. These findings imply that the GUK domain is required for a synaptic function and targeting of Dlg. To gain further insight on how the GUK domain of DLG exerts its various functions, proteins interacting with this domain were sought. GUK-holder, a novel synaptic protein contains a WH1/EVH1-like domain in its N-terminal half and a PDZ binding motif at its C terminus; the PDZ binding motiif has been identified as a GUK interactor. GUKh is expressed in a dynamic fashion during synaptic bouton formation. In addition, it also binds to a PDZ domain of Scribble (Scrib; a tumor suppressor protein interacts genetically with Dlg in developing epithelia) thus physically linking Dlg to Scrib. Indeed, coimmunoprecipitation analyses together with immunocytochemical studies on wild-type and mutant larvae provide strong evidence that Dlg, GUKh, and Scrib exist in a tripartite complex at the NMJ. Most notably, normal GUKh function is required for the synaptic localization of Scrib (Mathew, 2002).

Together, these yeast two-hybrid, coimmunoprecipitation, and colocalization studies provide compelling evidence that GUKh interacts with Dlg in vivo. This interaction is mediated by a region near the C terminus of GUKh. However, as revealed by genetic analysis, the synaptic localization of GUKh does not depend on Dlg. This suggests that domains other than the Dlg interacting motif may mediate its synaptic localization. For instance, the single WH1-like domain of GUKh might interact directly or indirectly with the synaptic cytoskeleton. WH1 domains in other proteins bind F-actin, actin-associated proteins such as zyxin, vinculin, and profilin, or the spectrin-bound scaffolding protein Shank/ProSAP. Association of GUKh with cytoskeletal elements might also be mediated by those sequences that exhibit moderate similarity to the actin binding protein Kelch (Mathew, 2002).

While an association of GUKh with the actin-based synaptic cytoskeleton currently remains hypothetical, the C-terminal tETAL motif specifically binds to the second PDZ domain of Scrib. Anatomical and biochemical experiments suggest that in vivo, Dlg, Scrib, and GUKh may exist in the same complex at the NMJ. Alternatively, the three proteins could interact pairwise, forming separate heterodimers. Since GUKh was found to still localize normally at dlg mutant NMJs, it is proposed that Dlg and GUKh act in concert rather than in a hierarchical manner to recruit Scrib. As a possible mechanism, binding to the GUK domain of Dlg could cause sterical changes in GUKh, such that the tETAL motif becomes available for interaction with Scrib. A caveat to this study is that hypomorphic gukh mutants were used, and therefore, a requirement of GUKh in Dlg localization cannot be ruled out (Mathew, 2002).

While Dlg and Scrib are colocalized along the rims of synaptic boutons, which, as has been demonstrated for Dlg, comprise both the presynaptic membrane and the postsynaptic junctional region (SSR), GUKh intersects that region only in a narrow strip. Yet, in budding boutons, GUKh displays a complementary pattern to Dlg. These observations suggest that GUKh may not be continuously bound to Dlg but rather may be involved in transient interactions. The process of bouton budding is a dynamic process that is characterized by equally dynamic changes in both GUKh and Dlg distribution. The accumulation of GUKh at the core of budding boutons and the disappearance of Dlg at the border of buds suggest that both proteins serve different roles during this process. Interestingly, FasII, a molecule that mediates synapse stabilization but that also imposes an adhesive constraint on synaptic growth, faithfully resembles the changes in distribution of Dlg during budding, consistent with a role for Dlg in synaptic localization. The presence of GUKh at budding regions may represent a role for this protein in destabilizing regions of the synaptic bouton, thereby allowing for bud formation (Mathew, 2002).

In contrast to GUKh, Scrib is expressed throughout the SSR in exact colocalization with Dlg. Nonetheless, Scrib localization at distal regions of the SSR is also affected in gukh mutants. In fact, considering the hypomorphic character of the gukh alleles that were used in this study, the effect on Scrib localization appears remarkably strong. This observation might indicate that GUKh activity is required only temporarily and/or in a locally restricted fashion to prime a secondary mechanism by which Scrib becomes associated with the SSR, e.g., through a more direct interaction with Dlg. Interestingly, presynaptic expression of GUKh-C is largely sufficient to restore postsynaptic Scrib localization at gukh mutant NMJs. Together, these observations suggest a second, more indirect mechanism by which GUKh contributes to the recruitment of Scrib to the postsynaptic SSR; such a mechanism may involve trans-synaptic signaling (Mathew, 2002).

These studies provide evidence for one mechanism by which scaffolding proteins with different interaction domains may be linked to form a network of multiprotein complexes. GUKh, in physically linking Dlg and Scrib, can therefore bring together these complexes and their associated proteins. Since a single protein forms this link, it would be a straightforward point at which to also separate the complexes, along with their actions, to regulate different aspects of synapse formation. Examples would be during synapse stabilization and during synapse growth through bouton budding. Thus, this work provides a means by which macromolecular complexes can mediate and finely tune various structural changes at the highly dynamic structure of the synapse (Mathew, 2002).

Polarized cells contain numerous membrane domains, but it is unclear how the formation of these domains is coordinated to create a single integrated cell architecture. Genetic screens of Drosophila embryos have identified three complexes, each containing one of the PDZ domain proteins -- Stardust (Sdt), Bazooka (Baz) and Scribble (Scrib) -- that control epithelial polarity and formation of zonula adherens. These complexes can be ordered into a single regulatory hierarchy that is initiated by cell adhesion-dependent recruitment of the Baz complex to the zonula adherens. The Scrib complex represses apical identity along basolateral surfaces by antagonizing Baz-initiated apical polarity. The Sdt-containing Crb complex is recruited apically by the Baz complex to counter antagonistic Scrib activity. Thus, a finely tuned balance between Scrib and Crb complex activity sets the limits of the apical and basolateral membrane domains and positions cell junctions. These data suggest a model in which the maturation of epithelial cell polarity is driven by integration of the sequential activities of PDZ-based protein complexes (Bilder, 2003).

Domain mapping of Scrib reveals a multistep localization mechanism and domains necessary for establishing cortical polarity

The Drosophila tumor suppressor protein Scribble is required for epithelial polarity, neuroblast polarity, neuroblast spindle asymmetry and limiting cell proliferation. It is a member of the newly described LAP protein family, containing 16 leucine rich repeats (LRRs), four PDZ domains and an extensive carboxyl-terminal (CT) domain. LRR and PDZ domains mediate protein-protein interactions, but little is know about their function within LAP family proteins. This study has determined the role of the LRR, PDZ and CT domains for Scribble localization in neuroblasts and epithelia, and for Scribble function in neuroblasts. It was found that the LRR and PDZ domains are both required for proper targeting of Scribble to septate junctions in epithelia; that the LRR domain is necessary and sufficient for cortical localization in mitotic neuroblasts, and that the PDZ2 domain is required for efficient cortical and apical localization of Scribble in neuroblasts. In addition, it is shown that the LRR domain is sufficient to target Miranda protein to the neuroblast cortex, but that LRR+PDZ will exclude Miranda from the cortex. These results highlight the importance of both LRR and PDZ domains for the proper localization and function of Scribble in neuroblasts (Albertson, 2004).

The results demonstrate that the Scrib LRRs are absolutely required for Scrib cortical localization in neuroblasts and epithelial cells. How might the LRRs direct Scrib to the plasma membrane? Little is known about binding specificities of LRRs in LAP proteins, yet LRRs in other cytoplasmic, transmembrane and extracellular proteins have been extensively studied. One LRR subfamily has an LRR-containing extracellular domain that is responsible for high-affinity binding to ligands. Another LRR protein subfamily binds small cytoplasmic GTPases, such as Ras and Ran. Based on these reports, activated small G proteins (Cdc42, Rac1, Ran) are candidates for cortical targeting of the Scrib LRR domain, although the distribution of these proteins in neuroblasts has not been determined, nor have they been tested for physical interactions with Scrib LRRs (Albertson, 2004).

Scrib proteins lacking all four PDZ domains, or just PDZ2, are detected in the cytoplasm and uniformly around the neuroblast cortex, with only occasional weak apical enrichment. Scrib PDZ2 is likely to target Scrib to the neuroblast cortex, and more specifically to the apical neuroblast cortex, by associating with Gukh and Dlg proteins. Gukh binds to both Scrib PDZ2 and Dl; Scrib, Gukh and Dlg are all co-localized at the neuroblast cortex; dlg mutants have cytoplasmic Scrib protein. One simple model consistent with these data is that Dlg binds Gukh, which binds Scrib PDZ2, resulting in the observed localization of Scrib within neuroblasts. Nevertheless, the PDZ domains are not sufficient for proper Scrib localization: Scrib proteins that contain the PDZs but lack the LRRs (DeltaLRR or PDZ) are completely cytoplasmic. A two-step model is proposed for Scrib localization in which Scrib LRR-dependent cortical localization precedes and is a prerequisite for Scrib PDZ2-dependent apical enrichment. It is noted that proteins lacking all four PDZ domains still show weak apical enrichment in some neuroblasts, however, indicating a minor role for the LRR domain in apical Scrib targeting (Albertson, 2004).

The data suggest a mechanism in which Scrib neuroblast and epithelial localization involves distinct steps, moving from cytoplasm to cortex then apically. Other proteins targeted to specific cortical domains within neuroblasts show a similar cortical-to-asymmetric localization mechanism. The Insc protein contains a 158 aa region that directs Insc to the neuroblast cortex; the addition of a separate 100 aa domain confers apical localization. Similarly, the Pins protein contains four C-terminal GoLoco repeats that are sufficient for cortical localization, but addition of the first three of seven N-terminal tetratricopeptide repeats (TPRs) is required for asymmetric apical targeting. Stepwise targeting of Dlg has also been observed in epithelia and neuromuscular junctions. In epithelia, the Hook domain targets Dlg to the cell cortex while PDZ2 is required to restrict Dlg to the septate junction. At neuromuscular junctions, the Hook domain is necessary to localize Dlg to the plasma membrane and both PDZ1 and PDZ2 domains promote transport to the synapse. These observations suggest cortical localization may be a prerequisite for subsequent targeting to specific membrane domains and raise the possibility that similar transport mechanisms are shared among protein complexes in neuroblasts, epithelia and synapses (Albertson, 2004).

Despite the fact that Scrib has a plethora of protein-protein interaction motifs, only one binding partner for Drosophila Scrib has been identified, Gukh, which binds to Scrib PDZ2. Binding assays such as yeast two-hybrid assays and mass spectrometry will be essential to identify additional Drosophila Scrib binding partners, as has been shown for mammalian LAP proteins. Such discoveries will enable the determination of upstream and downstream players in Scrib-mediated pathways and will further an understanding of many cellular functions, including establishment of epithelial cell polarity, stem cell division, mitotic spindle biology, cell cycle progression and synapse formation and homeostasis (Albertson, 2004).

Scrib proteins lacking LRRs fail to be properly targeted to the lateral membrane and septate junctions of mature epithelial cells, whereas Scrib proteins lacking all four PDZ domains, or just PDZ2, still show some septate junction enrichment with an elevated level of cytoplasmic protein. Thus, the LRR domains are the primary determinant of cortical/septate junction targeting, and PDZ2 increases the efficiency or stability of this localization. Scrib localization in epithelia also requires Dlg, but the role of the Dlg and Scrib PDZ2-binding protein Gukh have not been examined in epithelia. The ability of each Scrib domain to establish or maintain epithelial polarity could not be tested in this system, however, because zygotic scrib mutant embryos develop normal epithelial polarity because of maternally derived Scrib protein (even though there is no detectable maternal Scrib protein in stage 15 or later embryos) (Albertson, 2004).

The proteins that interact with the Scrib LRRs to mediate cortical association in epithelia are unknown. More is known about the role of PDZ domains in junctional targeting of LAP proteins. The mammalian LAP proteins Erbin and Densin180 show PDZ-mediated interactions with p120-catenins, and Erbin-catenin p0071 colocalize to adherens junctions and desmosomes in cultured epithelial cells. Disruption of Erbin-p0071 interactions leads to aberrant cell morphology and disruption of cell-cell contacts. Similarly, Drosophila Scrib is required for proper morphology and formation of septate junctions in epithelia, and the PDZ domains are necessary for efficient Scrib localization to the epithelial cell septate junctions. Thus, Drosophila catenins are excellent candidates for recruiting Scrib to the septate junction (Albertson, 2004 and references therein).

In the absence of all Scrib function, Mira is predominantly localized to the cytoplasm and mitotic spindle of neuroblasts. Expression of just the Scrib LRR domain results in uniform cortical Scrib LRR distribution and the restoration of uniform cortical Mira localization. Conversely, all Scrib proteins that lack the LRR domain (PDZ, DeltaLRR and CT) fail to efficiently target Mira to the cortex. These results reveal a positive role for the Scrib LRR domain in targeting Mira to all regions of the neuroblast cortex. A similar 'uniform cortical Mira' phenotype is also observed in certain aPKC and lgl genetic backgrounds. Neuroblasts lacking aPKC show uniform cortical Mira and neuroblasts misexpressing a dephospho-Lgl protein also show uniform cortical Mira. In addition, loss of lgl leads to cytoplasmic Mira localization in neuroblasts. This has led to a model in which the apically-localized aPKC phosphorylates Lgl to inactivate it, thus restricting active dephospho-Lgl to the basal cortex, where it promotes cortical localization of Mira. The Scrib LRRs could act upstream of aPKC and Lgl, perhaps by blocking aPKC/Lgl interactions, and thus allowing activated Lgl to target Mira to the entire cortex. Alternatively, the Scrib LRRs could act downstream of aPKC and Lgl, perhaps by allowing both dephospho- and phospho-Lgl to target Mira to the cortex. In addition, loss of jaguar (myosin VI) leads to cytoplasmic localization of Mira, raising the possibility that the Scrib LRRs could stimulate myosin VI activity around the neuroblast cortex to promote uniform cortical Mira localization. The identification of Scrib LRR-binding proteins will help distinguish between these models (Albertson, 2004).

Addition of the PDZ domains back to the Scrib LRR protein dramatically alters the function of the protein. Whereas the Scrib LRR protein is uniformly cortical and promotes Mira cortical localization, Scrib LRR+PDZ proteins (FL, DeltaCT) are apically enriched and exclude Mira from the apical cortex. Thus, addition of the PDZ domains switches Scrib from promoting cortical Mira localization to excluding cortical Mira localization. The PDZ domains could carry out this function of excluding Mira from the apical cortex in at least three different ways. (1) The Scrib PDZ domains could promote aPKC-Lgl interactions, thereby leading to the phosphorylation and inactivation of apical Lgl; this would restrict active Lgl to the basal cortex, where it promotes cortical Mira localization. (2) The Scrib PDZ domains could promote myosin II (zipper) activity at the apical cortex; myosin II is a known inhibitor of Lgl, and thus this would restrict active Lgl to the basal cortex where it could promote cortical Mira localization. (3) The Scrib PDZ domains could provide directionality to the actin-myosin VI cytoskeleton, which could transport Mira specifically to the basal cortex. Identification of proteins that interact with the Scrib PDZ domains would help distinguish between these models (Albertson, 2004).

Little is known about how Dlg, Scrib and Lgl regulate cell size asymmetry and spindle asymmetry. LRR and PDZ domains of Scrib are necessary for this function. How might Scrib regulate cell size and spindle asymmetry? Two good candidate effectors are Ran GTPase and Pins. LRRs are known to physically interact with Ran, which promotes spindle assembly through several target proteins. For example, Ran stimulates the activity of NuMA (a microtubule motor accessory protein that promotes spindle assembly) by destabilizing inhibitory complexes associated with NuMA. LGN (a mammalian Pins homolog) is essential for mitotic spindle assembly and binds NuMA; release from LGN is an important event in the activation of mitotic NuMA. In Drosophila, Pins physically interacts with Dlg and is asymmetrically localized to the apical cortex of mitotic neuroblasts, where it promotes spindle asymmetry. These data suggest possible links between Dlg, Scrib, Ran and Pins and establishment of mitotic spindle asymmetry. Genetic and biochemical studies investigating interactions between Scrib, Ran and Pins may further understanding of spindle asymmetry establishment in Drosophila neuroblasts (Albertson, 2004).

Deletion of the CT domain has no effect on Scrib localization or its ability to rescue all tested scrib mutant phenotypes in neuroblasts; the CT domain alone is cytoplasmic and has no rescuing ability in any assay performed. It is concluded that the CT domain is not essential for any aspect of Scrib localization or function tested here (Albertson, 2004).

scrib transgenic lines that specifically lack the LAPSDa/b domains have not been assayed, however, some conclusions can be drawn based on existing Scrib domain analysis. The Scrib DeltaPDZ protein contains both LAPSDa/b domains, is membrane targeted but not enriched apically; it fails to promote basal Mira targeting, and it is defective for asymmetric mitotic spindle and cell size asymmetry. Thus, the LAPSD domains are insufficient for apical enrichment of Scrib and all of its tested functions in neuroblasts. The Scrib LRR protein lacking the LAPSDb domain is still membrane-associated, showing that the LAPSDb domain is not required for Scrib membrane targeting. Some evidence was found that the LAPSDb domain regulates nuclear import/export of the Scrib protein. The LRR protein contains just the LRRs and the LAPSDa domain and is targeted to the nucleus; this shows that there is a nuclear import signal or binding site for a nuclear protein within the LRR/LAPSDa domains, although a predicted nuclear localization signal is not detectable within these domains. In contrast, the DeltaPDZ protein contains the same LRR/LAPSDa domains plus the LAPSDb and CT domains, and it is excluded from the nucleus. This shows that the LAPSDb or CT domains can prevent nuclear import of the LRR/LAPSDa protein; it is highly likely that this function is provided by the LAPSDb domain, because deletion of the CT domain from an otherwise wild-type Scrib protein (i.e., DeltaCT) does not result in nuclear localization. These results are in contrast to the role of the LAPSDa/b domains in the related C. elegans Let-413 protein, where the LAPSDa/b domains are required for establishing epithelial polarity but not Let-413 protein localization. It will be interesting to determine whether the Scrib LAPSDa/b domains play a similar role in Scrib epithelial localization (Albertson, 2004).

The leading edge during dorsal closure as a model for epithelial plasticity: Pak is required for recruitment of the Scribble complex and septate junction formation

Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. DC and the follicular epithelium were used to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. It is proposed that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. It is concluded that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion (Bahri, 2010).

Some embryos lacking zygotic Pak function successfully bring the epidermal flanks together at the dorsal midline but fail to restore septate junctions and adherens junctions at the LE in the DME cells. Thus, Pak at the LE membrane of the dorsal-most epithelial cells (DME) is regulating establishment of apicobasal polarity during a mesenchymal-epithelial transition. It is suspected that Pak is acting through different routes in its regulation of adherens junction formation versus septate junction formation. This study has focused on Pak regulation of the Scrib complex in septate junction formation at the LE. The data indicate that Pak is a component of the Scrib complex at the lateral membrane. Although Pak might be associating with the Scrib complex throughout epithelia, it might only be required for recruitment of the Scrib complex in epithelia derived from a mesenchymal-like intermediate such as the follicular epithelium and the LE. With the exception of the LE, apicobasal polarity in the epidermis is determined much earlier in development with formation of the blastoderm by cellularization. The epidermis is therefore a primary epithelium that does not arise from a mesenchymal intermediate, and Pak function does not appear to be required for apicobasal polarity in primary epithelia (Bahri, 2010).

Localization of Pak at the lateral membrane in both the follicular epithelium and in the epidermis is integrin-dependent. Studies using organ culture of embryonic kidney mesenchyme and MDCK cells demonstrate a requirement for integrins in apicobasal polarity of epithelia derived from MET, and this study has shown that βPS-integrin is required for Scrib complex and septate junction protein recruitment at the LE and in the follicular epithelium. Furthermore, previous studies in the follicular epithelium and another Drosophila epithelium derived from MET, the midgut, have demonstrated a requirement for integrins in the maintenance of apicobasal polarity. It is proposed that, at the LE, the absence of the septate junction diffusion barrier allows the accumulation of integrin complexes along the lateral membrane. These lateral integrin complexes recruit Pak, around which the Scrib complex is assembled. Thus, the absence of septate junctions allows the recruitment of proteins needed for the assembly of septate junctions. The model suggests that there might be transient Pak-mediated links between integrin and the Scrib complex. Interestingly, Dlg and βPS-integrin have been shown to co-immunoprecipitate from fly head extracts, consistent with these proteins existing in a complex in the nervous system and/or in epithelia (Bahri, 2010).

The data and recent studies on the amnioserosa support the idea that septate junctions restrict the accumulation of lateral integrins. The amnioserosa is devoid of septate junction proteins such as FasIII, and this might be owing to absence in this tissue of the transcription factor Grainy head, which promotes expression of septate junction proteins. The wild-type amnioserosa has high levels of lateral βPS-integrin, but ectopic expression of Grainy head in the amnioserosa leads to an accumulation of septate junction proteins and an accompanying disruption of βPS-integrin localization. Similarly, at the completion of DC, septate junctions appear at the LE and this is accompanied by downregulation of LE lateral integrins. In pak14pak376A and cora14 embryos where LE septate junctions are deficient, lateral LE βPS-integrin persists (Bahri, 2010).

A recent study in mammalian cell culture indicates that Scrib recruits Pak to the LE (Nola, 2008), and this study has shown that Pak localization in the follicular epithelium is Scrib-dependent. This study of the LE at the end of DC demonstrates that the relationship between Cdc42/Rac signaling complexes and Scrib can act in the opposite direction: membrane-localized Pak recruits the Scrib complex. A bidirectional interaction between the Scrib complex and Cdc42/Rac signaling complexes, including Pak, might be a crucial regulator of events at the LE of closing epithelia during both wound healing and development in diverse systems. Scrib at the newly formed LE can lead to recruitment of the Cdc42/Rac signaling complex, allowing acquisition of mesenchymal characteristics and polarized cell migration. When the opposing epithelial flanks meet up, events can be reversed with Pak recruiting the Scrib complex to the lateral membrane, contributing to restoration of apicobasal polarity and cell adhesion at the LE during MET. The Scrib/Pak complex is believed to be a 'toggle switch', enabling the epithelial membrane to shift back and forth between a migratory state characterized by actin-based extensions and an apicobasal polarized state characterized by cell-cell adhesion (Bahri, 2010).

Aurora A triggers Lgl cortical release during symmetric division to control planar spindle orientation

Mitotic spindle orientation is essential to control cell-fate specification and epithelial architecture. The tumor suppressor Lgl localizes to the basolateral cortex of epithelial cells, where it acts together with Dlg and Scrib to organize apicobasal polarity. Dlg and Scrib also control planar spindle orientation but how the organization of polarity complexes is adjusted to control symmetric division is largely unknown. Lgl redistribution during epithelial mitosis is reminiscent of asymmetric cell division, where it is proposed that Aurora A promotes aPKC activation to control the localization of Lgl and cell-fate determinants. This study shows that the Dlg complex is remodeled during Drosophila follicular epithelium cell division, when Lgl is released to the cytoplasm. Aurora A controlled Lgl localization directly, triggering its cortical release at early prophase in both epithelial and S2 cells. This relied on double phosphorylation within the putative aPKC phosphorylation site, which was required and sufficient for Lgl cortical release during mitosis and could be achieved by a combination of aPKC and Aurora A activities. Cortical retention of Lgl disrupted planar spindle orientation, but only when Lgl mutants that could bind Dlg were expressed. Taken together, Lgl mitotic cortical release is not specifically linked to the asymmetric segregation of fate determinants, and the study proposes that Aurora A activation breaks the Dlg/Lgl interaction to allow planar spindle orientation during symmetric division via the Pins (LGN)/Dlg pathway (Carvalho, 2015).

Evolutionarily conserved polarity complexes establish distinct membrane domains and the polarized assembly of junctions along the apicobasal axis has been extensively characterized. One general feature is that it relies on mutual antagonism between apical atypical protein kinase C (aPKC) and Crumbs complexes and a basolateral complex formed by Scribble (Scrib), Lethal giant larvae (Lgl), and Discs large (Dlg). This study used the Drosophila follicular epithelium as an epithelial polarity model to address how polarity is coordinated during symmetric division. Dlg and Scrib have been shown to provide a lateral cue for planar spindle orientation. Accordingly, Scrib and Dlg remain at the cortex during follicle cell division. In contrast, Lgl is released from the lateral cortex to the cytoplasm during mitosis. This subcellular reallocation begins during early prophase, since Lgl starts to be excluded from the cortex prior to cell rounding, one of the earliest mitotic events, and is completely cytoplasmic before nuclear envelope breakdown (NEB). Thus, the Dlg complex is remodeled at mitosis onset in epithelia (Carvalho, 2015).

The subcellular localization of Lgl is controlled by aPKC-mediated phosphorylation of a conserved motif, which blocks Lgl interaction with the apical cortex. To address the mechanism of cortical release during mitosis, nonphosphorytable form Lgl3A-GFP was expressed in the follicular epithelium. Lgl3A-GFP remains at the cortex throughout mitosis indicating that Lgl dynamics during epithelial mitosis also rely on the aPKC phosphorylation motif. Although the apical aPKC complex depolarizes during follicle cell division, Lgl cortical release precedes aPKC depolarization. Using Par-6-GFP as a marker for the aPKC complex and the Lgl cytoplasmic accumulation as readout of its cortical release, it was found that maximum cytoplasmic accumulation of Lgl occurs when most Par-6 is still apically localized (~70% relative to interphase levels). Thus, Lgl cortical release is the first event of the depolarization that characterizes follicle cell division, indicating that Lgl reallocation does not require extension of aPKC along the lateral cortex (Carvalho, 2015).

Although the major pools of Lgl and aPKC are segregated during interphase, Lgl has a dynamic cytoplasmic pool that rapidly exchanges with the cortex. Thus, further activation of aPKC at mitosis onset would be expected to shift the equilibrium toward cytoplasmic localization. Lgl dynamic redistribution in epithelia is similar to the neuroblast, where activation of Aurora A (AurA) leads to Par-6 phosphorylation and subsequent aPKC activation. To test whether a similar mechanism induced Lgl cortical release during epithelial mitosis, Lgl subcellular localization was analyzed in aPKC mutants and in par-6 mutants unphosphorylatable by AurA. Lgl cytoplasmic accumulation is unaffected in par-6; par-6S34A mutant cells. Temperature-sensitive aPKCts/aPKCk06403 mutants display strong cytoplasmic accumulation of Lgl during prophase, with a minor delay relatively to the wild-type). Moreover, homozygous mutant clones for null (aPKCk06403) and kinase-defective (aPKCpsu141) alleles also display Lgl cortical release during mitosis. These results implicate that although aPKC activity may contribute for Lgl mitotic dynamics, the putative aPKC phosphorylation motif is under the control of a different kinase, which triggers Lgl cortical release in the absence of aPKC (Carvalho, 2015).

AurA is a good candidate to induce Lgl cortical release as it controls polarity during asymmetric division. Furthermore, Drosophila AurA is activated at the beginning of prophase, which coincides with the timing of Lgl cytoplasmic reallocation. To examine whether AurA controls Lgl dynamics in the follicular epithelium, homozygous mutant clones were generated for the kinase-defective allele aurA37. In contrast to wild-type cells, only low amounts of cytoplasmic Lgl were detected during prophase in aurA37 mutants, which display a pronounced delay in the cytoplasmic reallocation of Lgl during mitosis. This delayed cortical release of Lgl has been previously reported during asymmetric cell division in aurA37 mutants, possibly resulting from residual kinase activity. Thus, AurA is essential to trigger Lgl cortical exclusion at epithelial mitosis onset (Carvalho, 2015).

The idea that Lgl mitotic reallocation is directly controlled by a mitotic kinase implies that Lgl should display similar dynamics regardless of the polarized status of the cell. Consistently, Lgl-GFP is also released from the cortex before NEB in nonpolarized Drosophila S2 cells. Furthermore, Lgl3A-GFP is retained in the cortex during mitosis, revealing that Lgl cortical release is also phosphorylation dependent in S2 cells. Treatment with a specific AurA inhibitor (MLN8237), or with aurA RNAi, strongly impairs Lgl cortical release during prophase, as Lgl is present in the cortex at NEB. However, inhibition of AurA still allows later cortical exclusion, which could result from the activity of another kinase. Despite their distinct roles, AurA and Aurora B (AurB) phosphorylate common substrates in vitro. Therefore, whether AurB could act redundantly with AurA was analyzed. Inactivation of AurB with a specific inhibitor, Binucleine 2, enables normal Lgl cytoplasmic accumulation before NEB and still allows later cortical exclusion in cells treated simultaneously with the AurA inhibitor As AurB does not seem to participate on Lgl mitotic dynamics, RNAi directed against aPKC was used to examine whether it could act redundantly with AurA. aPKC depletion did not block Lgl cortical exclusion, but it was slightly delayed. However, simultaneous AurA inhibition and aPKC RNAi produced almost complete cortical retention of Lgl during mitosis. Thus, AurA induces Lgl release during early prophase, but aPKC retains its ability to phosphorylate Lgl during mitosis (Carvalho, 2015).

To address which serine(s) within the phosphorylation motif of Lgl control its dynamics during mitosis, individual and double mutants were enerated. As complete cortical release occurs before NEB, the ratio of cytoplasmic to cortical mean intensity of Lgl-GFP at NEB was quantified to compare each different mutant. All the single mutants displayed similar dynamics to LglWT, exiting to the cytoplasm prior to NEB. In contrast, all double mutants were cortically retained during mitosis, indicating that double phosphorylation is both sufficient and required to efficiently block Lgl cortical localization (Carvalho, 2015).

The ability to doubly phosphorylate Lgl would explain how AurA drives Lgl cortical release. Accordingly, the sequence surrounding S656 perfectly matches AurA phosphorylation consensus, whereas the S664 surrounding sequence shows an exception in the -3 position. In contrast, the sequence surrounding S660 does not resemble AurA phosphorylation consensus, and AurA does not directly phosphorylate S660 in vitro as detected by phosphospecific antibodies against S660. That S656 is directly phosphorylated by recombinant AurA was confirmed in vitro using a phosphospecific antibody for S656. Moreover, AurA inhibition or aurA RNAi results in a similar cortical retention at NEB to LglS656A,S664A, suggesting that AurA also controls S664 phosphorylation during mitosis, whereas aPKC would be the only kinase active on S660. Consistent with this, aPKC RNAi increases the cortical retention of LglS656A,S664A, mimicking the localization of Lgl3A. Furthermore, whereas S660A mutation does not significantly affect the cytoplasmic accumulation of Lgl in aPKC RNAi, S656A and S664A mutations disrupt Lgl cortical release in aPKC-depleted cells, leading to the degree of cortical retention of LglS656A,S660A and LglS660A,S664A, respectively. Altogether, these results support that AurA controls S656 and S664 and that these phosphorylations are partially redundant with aPKC phosphorylation to produce doubly phosphorylated Lgl, which is released from the cortex (Carvalho, 2015).

RNAi-mediated knockdown of Lgl in vertebrate HEK293 cells results in defective chromosome segregation. Furthermore, overexpressed Lgl-GFP shows a slight enrichment on the mitotic spindle suggesting that relocalization of Lgl could be important to control chromosome segregation. However, lgl mutant follicle cells assemble normal bipolar spindles, and although it was possible to detect minor defects on chromosome segregation, the mitotic timing (time between NEB and anaphase) is indistinguishable between lgl and wild-type cells. Additionally, loss of Lgl activity allows proper chromosome segregation in both Drosophila S2 cells and syncytial embryos. Thus, Lgl does not seem to have a general role in the control of faithful chromosome segregation in Drosophila (Carvalho, 2015).

Nevertheless, Lgl cortical release could per se play a mitotic function, as key mitotic events are controlled at the cortex. In fact, the orientation of cell division requires the precise connection between cortical attachment sites and astral microtubules, which relies on the plasma membrane associated protein Pins (vertebrate LGN). Pins uses its TPR repeat domain to bind Mud (vertebrate NUMA), which recruits the dynein complex to pull on astral microtubules, and its linker domain to interact with Dlg, which participates on the capture of microtubule plus ends. Notably, Pins/LGN localizes apically during interphase in Drosophila and vertebrate epithelia, being reallocated to the lateral cortex to orient cell division. Pins relocalization relies on aPKC in some epithelial tissues, but not in chick neuroepithelium and in the Drosophila follicular epithelium, where Dlg provides a polarity cue to restrict Pins to the lateral cortex. Dlg controls Pins localization during both asymmetric and symmetric division, and a recent study has shown that vertebrate Dlg1 recruits LGN to cortex via a direct interaction. However, Dlg uses the same phosphoserine binding region within its guanylate kinase (GUK) domain to interact with Pins/LGN and Lgl. Thus, maintenance of a cortical Dlg/Lgl complex during mitosis is expected to impair the ability of Dlg to bind Pins and control spindle orientation (Carvalho, 2015).

Interaction between the Dlg's GUK domain and Lgl requires phosphorylation of at least one serine within the aPKC phosphorylation site. Although the phosphorylation-dependent binding of Lgl to Dlg remains to be shown in Drosophila, crystallographic studies revealed that all residues directly involved in the interaction with p-Lgl are evolutionarily conserved from C. elegans to humans. Thus, whereas Lgl3A does not form a fully functional Dlg/Lgl polarity complex, double mutants should bind Dlg's GUK domain and are significantly retained at the cortex during mitosis due to the inability to be double phosphorylated. This led to an examination of their ability to support epithelial polarization during interphase and to interfere with mitotic spindle orientation. Rescue experiments were performed in mosaic egg chambers containing lgl27S3 null follicle cell clones. lgl mutant clones display multilayered cells with delocalization of aPKC. This phenotype is rescued by Lgl-GFP, but not by Lgl3A-GFP. More importantly, in contrast to LglS660A,S664A, which extends to the apical domain in wild-type cells and fails to rescue epithelial polarity in lgl mutant cells, LglS656A,S660A and LglS656A,S664A can rescue epithelial polarity, localizing with Dlg at the lateral cortex and below aPKC. Hence, aPKC-mediated phosphorylation of S660 or S664 is sufficient on its own to control epithelial polarity and to confine Lgl to the lateral cortex (Carvalho, 2015).

Whether exclusion of Lgl from the cortex and the consequent release from Dlg would be functionally relevant for oriented cell division was examined. Expression of Lgl-GFP or Lgl3A-GFP does not affect planar spindle orientation during follicle cell division. In contrast, Lgl double mutants display metaphasic cells in which the spindle axis, determined by centrosome position, is nearly perpendicular to the epithelial layer. Live imaging revealed that these spindle orientation defects were maintained throughout division as it was possible to follow daughter cells separating along oblique and perpendicular angles to the epithelia. Moreover, equivalent defects on planar spindle orientation were detected upon expression of LglS656A,S664A in the lgl or wild-type background, indicating that cortical retention of Lgl exerts a dominant effect. Interestingly, LglS656A,S660A and LglS656A,S664A induce higher randomization of angles, whereas LglS660A,S664A, which is less efficiently restricted to the lateral cortex, produces a milder phenotype. Altogether, these results indicate that retention of Lgl at the lateral cortex disrupts planar spindle orientation only if Lgl can interact with Dlg (Carvalho, 2015).

Despite the ability of LglS656A,S660A-GFP to rescue epithelial polarity in lgl mutants, strong overexpression of LglS656A,S660A-GFP, but not of other Lgl double mutants, can dominantly disrupt epithelial polarity during the proliferative stages of oogenesis. One interpretation is that LglS656A,S660A forms the most active lateral complex of the mutant transgenes, disrupting the balance between apical and lateral domains. Therefore whether the dominant effect of Lgl cortical retention on spindle orientation could solely result from Dlg mislocalization was assessed. Dlg is properly localized at the lateral cortex in LglS656A,S660A-expressing cells presenting misoriented spindles, but this position does not correlate with the orientation of the centrosomes. Thus, cortical retention of Lgl interferes with Dlg's ability to transmit its lateral cue to instruct spindle orientation, which may result from an impairment of the Dlg/Pins interaction (Carvalho, 2015).

In conclusion, these findings outline a mechanism that explains how the lateral domain is remodeled to accomplish oriented epithelial cell division, unveiling that AurA has a central role in controlling the subcellular distribution of Lgl. AurA regulates the activity of aPKC at mitotic entry during asymmetric division, and these results are consistent with the ability of aPKC to phosphorylate and collaborate in Lgl cortical release. However, in epithelia, aPKC accumulates in the apical side during interphase, where it induces apical exclusion of Lgl, in part by generating a phosphorylated form that binds Dlg. Consequently, aPKC has a reduced access to the cortical pool of Lgl at mitotic entry and would be unable to rapidly induce Lgl cortical exclusion. These data show that cell-cycle-dependent activation of AurA removes Lgl from the lateral cortex through AurA's ability to control Lgl phosphorylation on S656 and S664 independently of aPKC. Thus, AurA and aPKC exert the spatiotemporal control of Lgl distribution to achieve unique cell polarity roles in distinct cell types (Carvalho, 2015).

It is proposed that release of Lgl from the cortex allows Dlg interaction with Pins to promote planar cell division in Drosophila epithelia. Lgl cortical release requires double phosphorylation, indicating that whereas Lgl-Dlg association involves aPKC phosphorylation, multiple phosphorylations break this interaction, acting as an off switch on Lgl-Dlg binding. Triple phosphomimetic Lgl mutants display weak interactions with Dlg, suggesting that multiple phosphorylations could directly block Lgl-Dlg interaction. Alternatively, the negative charge of two phosphate groups may suffice to induce association between the N- and C-terminal domains of Lgl, impairing its ability to interact with the cytoskeleton and plasma membrane as previously proposed. This would reduce the local concentration of Lgl available to interact with Dlg, enabling the interaction of Dlg's GUK domain with the pool of Pins phosphorylated by AurA. Therefore, AurA converts the Lgl/Dlg polarity complex generated upon aPKC phosphorylation into the Pins/Dlg spindle orientation complex. This study, underlines the critical requirement of synchronizing the cell cycle with the reorganization of polarity complexes to achieve precise control of spindle orientation in epithelia (Carvalho, 2015).

Scribbled optimizes BMP signaling through its receptor internalization to the Rab5 endosome and promote robust epithelial morphogenesis

Epithelial cells are characterized by apical-basal polarity. Intrinsic factors underlying apical-basal polarity are crucial for tissue homeostasis and have often been identified to be tumor suppressors. Patterning and differentiation of epithelia are key processes of epithelial morphogenesis and are frequently regulated by highly conserved extrinsic factors. However, due to the complexity of morphogenesis, the mechanisms of precise interpretation of signal transduction as well as spatiotemporal control of extrinsic cues during dynamic morphogenesis remain poorly understood. Wing posterior crossvein (PCV) formation in Drosophila serves as a unique model to address how epithelial morphogenesis is regulated by secreted growth factors. Decapentaplegic (Dpp), a conserved bone morphogenetic protein (BMP)-type ligand, is directionally trafficked from longitudinal veins (LVs) into the PCV region for patterning and differentiation. These data reveal that the basolateral determinant Scribbled (Scrib) is required for PCV formation through optimizing BMP signaling. Scrib regulates BMP-type I receptor Thickveins (Tkv) localization at the basolateral region of PCV cells and subsequently facilitates Tkv internalization to Rab5 endosomes, where Tkv is active. BMP signaling also up-regulates scrib transcription in the pupal wing to form a positive feedback loop. These data reveal a unique mechanism in which intrinsic polarity genes and extrinsic cues are coupled to promote robust morphogenesis.

This study shows that the Scrib complex, a basolateral determinant, is a novel feedback component that optimizes BMP signaling in the PCV region of the Drosophila pupal wing (Gui, 2016).

During PCV development, limited amounts of Dpp ligands are provided by the Dpp trafficking mechanism. Furthermore, amounts of receptors appear to be limited since tkv transcription is down-regulated in the cells in which the BMP signal is positive, a mechanism that serves to facilitate ligand diffusion and sustain long-range signaling in the larval wing imaginal disc. To provide robust signal under conditions in which both ligands and receptors are limiting, additional molecular mechanisms are needed. Previous studies suggest that two molecules play such roles. Crossveinless-2 (Cv-2), which is highly expressed in the PCV region, serves to promote BMP signaling through facilitating receptor-ligand binding. Additionally, the RhoGAP protein Crossveinless-c (Cv-c) provides an optimal extracellular environment to maintain ligand trafficking from LVs into PCV through down-regulating integrin distribution at the basal side of epithelia. Importantly, both cv-2 and cv-c are transcriptionally regulated by BMP signaling to form a feedback or feed-forward loop for PCV formation (Gui, 2016).

Scrib, a third component, sustains BMP signaling in the PCV region in a different manner. First, to utilize Tkv efficiently, Scrib regulates Tkv localization at the basolateral region in the PCV cells, where ligand trafficking takes place. Regulation of receptor localization could be a means of spatiotemporal regulation of signaling molecules during the dynamic process of morphogenesis. Second, to optimize the signal transduction after receptor-ligand binding, Scrib facilitates Tkv localization in the Rab5 endosomes. Localization of internalized Tkv is abundant at Rab5 endosomes in the PCV region of wild-type, but not scrib RNAi cells. While the physical interaction between Scrib, Tkv and Rab5 in the pupal wing remains to be addressed, the data in S2 cells suggest that physical interactions between these proteins are key for preferential localization of Tkv at the Rab5 endosomes. Recently, Scrib has been implicated in regulating NMDA receptor localization through an internalization-recycling pathway to sustain neural activity. Therefore, Scrib may be involved in receptor trafficking in a context-specific manner, the molecular mechanisms of which, however, remain to be characterized. Third, BMP/Dpp signaling up-regulates scrib transcription in the pupal wing. Moreover, knockdown of scrib leads to loss of BMP signaling in PCV region but not loss of apical-basal polarity. These facts suggest that upregulation of Scrib is key for optimizing BMP signaling by forming a positive feedback loop (Gui, 2016).

Previous studies indicate that cell competition takes place between scrib clones and the surrounding wild-type tissues in the larval wing imaginal disc. Moreover, cell competition has been documented between loss-of-Dpp signal and the surrounding wild-type tissues. It is presumed that the mechanisms proposed in this study are independent of cell competition for the following reasons. First, scrib RNAi and AP-2μ RNAi data reveal that loss of BMP signal in the PCV region is produced without affecting cell polarity. Thus, cell competition is unlikely to occur in this context. Second, BMP signal is intact in scrib mutant clones of the wing imaginal disc, suggesting that cell competition caused by scrib clones is not a direct cause of loss of BMP signaling in scrib mutant cells (Gui, 2016).

Previous studies established that receptor trafficking plays crucial roles in signal transduction of conserved growth factors, including BMP signaling. Several co-factors have been identified as regulators of BMP receptor trafficking. Some of them down-regulate BMP signaling while others help maintain it. It is proposed that the Scrib-Rab5 system is a flexible module for receptor trafficking and can be utilized for optimizing a signal. During larval wing imaginal disc development, BMP ligands are trafficked through extracellular spaces to form a morphogen gradient. Although previous studies indicate that regulation of receptor trafficking impacts BMP signaling in wing imaginal discs, BMP signaling persists in scrib or dlg1 mutant cells in wing discs. Wing disc cells interpret signaling intensities of a morphogen gradient. In this developmental context, an optimizing mechanism might not be beneficial to the system. In contrast, cells in the PCV region use the system to ensure robust BMP signaling (Gui, 2016).

Taken together, these data reveal that a feedback loop through BMP and Scrib promotes Rab5 endosome-based BMP/Dpp signaling during PCV morphogenesis. Since the components (BMP signaling, the Scrib complex, and Rab5 endosomes) discussed in this work are highly conserved, similar mechanisms may be widely utilized throughout Animalia (Gui, 2016).



To determine the subcellular localization of Scrib, antibodies were generated against Scrib peptides. In fixed tissue, Scrib is present at low levels in precellular embryos, where it is found in the actin caps that overlay the still-dividing blastoderm nuclei. As cellularization proceeds during cycle 14, Scrib staining becomes associated with the cell membranes as they invaginate basally towards the centre of the embryo. During gastrulation, however, Scrib relocates specifically to a relatively apical position along the lateral cell membrane of ectodermal cells. The apicolateral epithelial staining seen at gastrulation continues into late embryogenesis, by which time it has consolidated into a narrow subapical region (Bilder, 2000a).

Because the subapical epithelial membrane is the site of cell-cell junctions, it was determined whether Scrib localizes specifically to a junction. In mature Drosophila epithelia, the adherens junction and the septate junction (analagous to the vertebrate tight junction) are adjacent structures, located at the margins of the apical and basolateral surfaces, respectively. Co-staining for Scrib and the adherens junction marker Armadillo (Arm), the homolog of vertebrate beta-catenin, reveals that Scrib is located immediately basal to the adherens junction, whereas co-staining for Scrib and the septate junction marker Coracle (Cor), a protein 4.1 family member, reveals coincident protein distributions. These results show that Scrib localizes to septate junctions. Although the septate junction does not appear ultrastructurally until late embryogenesis at stage 14, Scrib is enriched subjacent to the adherens junction after gastrulation at stage 8. By contrast, the initial expression of Cor at stage 12 is spread throughout the basolateral membrane, with apicolateral enrichment visible only from stage 14. These data indicate that Scrib is an early marker for the site of the future septate junction, at the apical boundary of the basolateral compartment (Bilder, 2000a).

The relation between scrib, lgl, and dlg were explored by comparing the subcellular localization of the gene products. Scrib and Dlg colocalize throughout development, in particular at the apical margin of the lateral membrane (ALM) of the embryonic epidermal epithelium. Scrib is localized to the epithelial septate junction, the analog of the vertebrate tight junction, at the boundary of the apical and basolateral cell surfaces. Colocalization at the ALM occurs after gastrulation and persists in mature epithelia, where the ALM is the site of the septate junction. Lgl protein is not exclusively associated with the plasma membrane and is not polarized along it; however, it overlaps substantially with Dlg and Scrib at the ALM (Bilder, 2000b).

The tumor suppressor gene scribbled (scrib) is required for epithelial polarity and growth control in Drosophila. The identification and embryonic expression pattern is reported of two Scrib protein isoforms resulting from alternative splicing during scrib transcription. Both proteins are first ubiquitously expressed during early embryogenesis. Then, during morphogenesis each Scrib protein displays a specific pattern of expression in the central and peripheral nervous systems, CNS and PNS, respectively. During germ band extension, the expression of the longer form Scrib1 occurs predominantly in the neuroblasts derived from the neuro-ectoderm and later becomes restricted to CNS neurons as well as to the pole cells in the gonads. By contrast, the shorter form Scrib2 is strongly expressed in the PNS and a subset of CNS neurons (Li, 2001).

A P-element mutagenesis led to the identification of a recessive mutation Pvartul causing late larval lethality and producing abnormal imaginal disc growth with a complex syndrome reminiscent of that observed in mutations of tumor suppressors. Both the larval brain hemispheres and a subset of the imaginal discs displayed massive overgrowth with a loss of cell polarity. This gene was consequently classified as a tumor suppressor (Li, 2001).

The putative gene was cloned by plasmid rescue and chromosomal walk. Sequencing and characterization of isolated cDNAs and ESTs showed that it encodes three size classes of transcripts with a length of 7.2, 6.0 and 4.6 kb, respectively, sharing identical 5' ends but divergent 3' ends. Northern blot analysis confirmed the occurrence of three size classes of transcripts. Sequence alignment of the three classes of cDNAs on the genomic sequence revealed the gene structure. The 7.2 and 6.0 kb transcripts differ by the length of their 3' UTR and contain an open reading frame (ORF) of 5218 nucleotides encoding a polypeptide of 1756 amino acids that is basically identical with Scribbled and is hereafter designated as Scrib1. The alternatively spliced 4.6 kb mRNA encodes a shorter protein (Scrib2) made of 1247 residues. Both Scrib1 and Scrib2 share identical sequence over the first 1143 residues with a set of 16 leucine-rich repeats and two PDZ domains and belong to the LAP protein family. In addition, Scrib1 contains two additional PDZ domains in its unique C-terminal segment of 613 residues. Scrib2 carries a shorter C-terminal segment of 104 residues displaying no sequence similarity with the C-terminal domain of Scrib1 (Li, 2001).

To compare the expression patterns of Scrib1 and Scrib2, antibodies were raised in rabbits against a C-terminal 20-mer peptide specific for each protein. Western blots of Drosophila proteins showed that anti-Scrib1 antibodies detected a prominent band with a molecular mass of ~250 kDa, whereas the anti-Scrib2 antibodies recognized a ~180 kDa protein band. The sizes of the detected proteins correspond to those of the polypeptides translated in vitro from Scrib1 and Scrib2 cDNAs. These results show that scrib encodes two distinct polypeptides (Li, 2001).

Developmental Western blot analysis reveals that Scrib1 is expressed at a relatively high level during the first half of embryogenesis (0-12 h) and then at a lower level (12-24 h). Scrib1 expression is nearly undetectable in larvae, pupae and adults. Similarly, Scrib-2 is more intensively expressed in 0-12 h embryos than in 12-24 h embryos and remains nearly undetectable at later stages of development (Li, 2001).

Immuno-histochemistry and confocal microscopy reveals that, from the onset of blastoderm formation up to germ band elongation, Scrib1 expression is ubiquitous. Then, Scrib1 expression becomes preferentially expressed in the neuroblasts and more particularly in the developing CNS. During late embryogenesis Scrib1 is intensively expressed in a limited number of cells, including the ventral nerve chord, and is expressed at a much lower level in other epithelial structures and muscle fibers. To confirm the pattern of Scrib1 expression in the developing CNS, embryos were double stained with anti-Scrib1 and either anti-Neurotactin antibodies that recognize neuroblasts, or BP102 revealing CNS neurons. During germ band elongation Scrib1 and Neurotactin expression patterns overlap in the neuroblasts derived from the neuroectoderm. In the CNS of late embryos (stage 16-17) the most intense Scrib1 staining coincides with that of BP102 and strongly decorates the longitudinal and transversal fibers of the connectives and commissures, respectively. In addition Scrib1 is also expressed in midline cells located at the level of the anterior and posterior commissures. Strong Scrib1 expression is also detected in the pole cells of the gonads after completion of the dorsal closure (Li, 2001).

During early embryogenesis the expression of Scrib2 resembles that of Scrib1, with the difference that Scrib2 is predominantly distributed in the basal domain of the forming blastoderm cells. In contrast, Scrib1 is first localized in the apical domain and then decorates the growing plasma membrane. During late embryogenesis the tissue distribution of Scrib2 differs markedly from that of Scrib1. This is particularly evident in the CNS where Scrib2 is predominantly localized in neuronal cell bodies and Scrib1 in the axons. In addition, Scrib2 is strongly expressed in both neuronal cell bodies and projections of the peripheral nervous system (PNS). Double staining with anti-Scrib2 antibodies and 22C10 shows an intense Scrib2 expression in all 22C10 decorated cells, confirming the specific expression of Scrib2 in the PNS sensory organs (Li, 2001).

Amphiphysin family members are implicated in synaptic vesicle endocytosis, actin localization and one isoform is an autoantigen in neurological autoimmune disorder; however, there has been no genetic analysis of Amphiphysin function in higher eukaryotes. Drosophila Amphiphysin is localized to actin-rich membrane domains in many cell types, including apical epithelial membranes, the intricately folded apical rhabdomere membranes of photoreceptor neurons and the postsynaptic density of glutamatergic neuromuscular junctions. Flies that lack all Amphiphysin function are viable, lack any observable endocytic defects, but have abnormal localization of the postsynaptic proteins Discs large, Lethal giant larvae and Scribbled, altered synaptic physiology, and behavioral defects. Misexpression of Amphiphysin outside its normal membrane domain in photoreceptor neurons results in striking morphological defects. The strong misexpression phenotype coupled with the mild mutant and the lack of phenotypes suggest that Amphiphysin acts redundantly with other proteins to organize specialized membrane domains within a diverse array of cell types. In other words, Drosophila Amphiphysin functions in membrane morphogenesis, but an additional role in endocytosis cannot yet be dismissed (Zelhof, 2001).

Asymmetric cell division is important in generating cell diversity from bacteria to mammals. Drosophila neuroblasts are a useful model system for investigating asymmetric cell division because they establish distinct apical-basal cortical domains, have an asymmetric mitotic spindle aligned along the apical-basal axis, and divide unequally to produce a large apical neuroblast and a small basal daughter cell (GMC). Discs large (Dlg), Scribble (Scrib) and Lethal giant larvae (Lgl) tumour suppressor proteins regulate multiple aspects of neuroblast asymmetric cell division. Dlg/Scrib/Lgl proteins show apical cortical enrichment at prophase/metaphase, and then have a uniform cortical distribution. Mutants have defects in basal protein targeting, a reduced apical cortical domain and reduced apical spindle size. Defects in apical cell and spindle pole size result in symmetric or inverted neuroblast cell divisions. Inverted divisions correlate with the appearance of abnormally small neuroblasts and large GMCs, showing that neuroblast/GMC identity is more tightly linked to cortical determinants than cell size. It is concluded that Dlg/Scrib/Lgl are important in regulating cortical polarity, cell size asymmetry and mitotic spindle asymmetry in Drosophila neuroblasts (Albertson, 2003).

Effects of Mutation or Deletion

Because cuticles of embryos mutant for scrib suggest a broad defect in epidermal organization, the morphology of scrib embryos was examined. scrib embryos proceed normally through precellular development, and the epithelial blastoderm forms as in wild type. After gastrulation, however, organization of the ectodermal epithelium is disrupted as cells lose their columnar shape and planar arrangement. These defects become progressively more severe as development proceeds. Instead of the wild-type tightly integrated columnar monolayer epithelium, the epidermis of late scrib embryos is frequently interrupted and consists of groups of irregular rounded cells that are separating from one another. Confocal microscope sections reveal that most of the epidermis is organized into multilayered strips or tubes of cells that have lost contact with the underlying tissue (Bilder, 2000a).

The septate junction localization of Scrib suggests that cell junctions might be defective in scrib mutants. The presence of cellular junctions was investigated using antibody probes specific for either the adherens or the septate junction. The initial assembly of adherens junctions in scrib cellular blastoderms is normal, but at stage 8, anti-Arm staining reveals a severe disruption of the forming circumferential adherens junction belts. The normally continuous 'honeycomb' pattern seen in tangential apical sections of the epidermis is severely fragmented. Tissue cross-sections show that, rather than being lost, Arm is misdistributed. Instead of its wild-type localization exclusively at the apical tip of contacting cell membranes, much Arm is found around the cell periphery on the inside of the epidermis, in the midst of opposing cell membranes. At these sites, Arm colocalizes with E-cadherin (Ecad), the transmembrane component of the adherens junction, indicating that adherens junctions form at ectopic basolateral membrane positions in scrib embryos (Bilder, 2000a).

The formation of adherens junctions at ectopic positions within scrib cells raise the issue of whether these cells have lost polarity. In the wild-type epidermis, apical proteins such as Stranded at Second (Sas) are localized to the non-contacting cell membrane at the surface of the embryo, whereas basolateral proteins such as Fasciclin III (Fas III) are found in a complementary domain along the contacting membranes of cells. In scrib epidermal cells at stage 11, Sas is distributed throughout the plasma membrane, in both contacting and non-contacting cell surfaces. By contrast, Fas III localization is much less affected; in particular, it is not significantly mislocalized into apical regions. This preferential loss of apical restriction was seen with a panel of both cytoplasmic and transmembrane proteins through mid-embryogenesis, after which late embryos repolarize some apical markers by a scrib-independent pathway. Thus, the polarity defects in scrib epithelial cells arise not from a total loss of cell polarity but from a specific misdistribution of apical proteins (Bilder, 2000a).

The equivalent requirements for scrib, lgl, and dlg in epithelial development could result from independent activity of each gene in a separate pathway or from collaborative activity of the three genes in a single pathway. To address this issue, genetic interactions between the three mutations were tested and strong interactions of dlg and lgl with scrib were found. Most embryos zygotically mutant for scrib hatch and survive into late larval stages. However, embryos homozygous for scrib and additionally heterozygous for dlg die before hatching, with evident defects in dorsal closure. Dorsal closure phenotypes are characteristic of reduced activity of both dlg and lgl. Additionally, embryos homozygous for both lgl and scrib display a cuticle phenotype nearly as severe as those of lgl or scrib null embryos. Heterozygosity for lgl also enhances the imaginal disc phenotype of scrib hypomorphic larvae. These dose-sensitive interactions of dlg and lgl with scrib suggest that the three genes function in a common pathway (Bilder, 2000b).

Epistatic relations between scrib, lgl, and dlg were investigated by determining the localization of each protein in embryos mutant for the other two genes. The localization of Dlg and Scrib to the ALM was examined. In all mutant blastoderms, Scrib and Dlg are associated with ingrowing cell membranes, as in WT. However, after gastrulation, when WT embryos display an enrichment of Scrib in the ALM, lgl embryos show Scrib and Dlg localized throughout the basolateral cell membrane, a misdistribution that persists into late embryogenesis. Like lgl embryos, scrib embryos fail to polarize Dlg to the ALM, while dlg embryos not only fail to polarize Scrib to the ALM, but also display a progressive loss of membrane-associated Scrib (Bilder, 2000b).

Also examined was the distribution of Lgl, which normally has both a membrane-bound and a cytosolic component; reduction of activity in an lgl temperature-sensitive mutant correlates with loss of the membrane-bound pool. In WT embryos, Lgl is in close apposition to cell membranes. However, in scrib blastoderms and embryos, Lgl is distributed throughout the cytoplasm. dlg blastoderms shows intermediate defects in Lgl distribution, but by mid-embryogenesis loss of membrane-localized Lgl is evident. The dissociation of Lgl from the membranes of dlg embryos parallels the loss of Scrib seen in these embryos. These data indicate that dlg is required for the stable association of Scrib with the cell membrane and scrib is required for the cortical association of Lgl; all three genes act to localize Scrib and Dlg to the ALM (Bilder, 2000b).

Regulation of synaptic plasticity and synaptic vesicle dynamics by the PDZ protein Scribble

At the larval neuromuscular junction, Scribbled colocalizes and indirectly interacts with another tumor suppressor and PDZ protein, Discs-Large (Dlg). Dlg is critical for development of normal synapse structure and function, as well as for normal synaptic Scrib localization. Scrib is also an important regulator of synaptic architecture and physiology. The most notable ultrastructural defect in scrib mutants is an increase in the number of synaptic vesicles in an area of the synaptic bouton thought to contain the reserve vesicle pool. Additionally, the number of active zones is reduced in scrib mutants. Functionally, the scrib synapse behaves relatively normally at low-frequency stimulation. However, several forms of plasticity at this synapse are drastically altered in the mutants. Specifically, scrib mutants exhibit loss of facilitation and post-tetanic potentiation, and faster synaptic depression. In addition, FM1-43 imaging of recycling synaptic vesicles shows that vesicle dynamics are impaired in scrib mutants. These results identify Scrib as an essential regulator of short-term synaptic plasticity. Taken together, these results are consistent with a model in which Scrib is required to sustain synaptic vesicle concentrations at their sites of release (Roche, 2002).

Mutations in dlg lead to prominent defects in both synapse structure and function. At the ultrastructural level these defects include an increase in bouton size and number of active zones, as well as a poorly developed subsynaptic reticulum (SSR), an elaborate folding of the postsynaptic membrane at the NMJ. Dlg is colocalized with Scrib, and mutations in dlg also result in severe mislocalization of synaptic Scrib. In contrast, although localization of Scrib to the NMJ is completely disrupted in scrib mutants, the localization of Dlg is not affected. Analysis of the NMJs in scrib mutants has shown that the general morphology is not affected. It is hypothesized that some of the defects in dlg mutants might be the consequence of Scrib mislocalization. This hypothesis was tested by serially sectioning type I synaptic boutons in several scrib mutant allelic combinations and examining their ultrastructure using electron microscopy. It was found that the synaptic structure in these mutants was drastically altered; however, these defects were quite distinct from those in dlg mutants. One of the most prominent defects was an abnormally high density of synaptic vesicles. In wild type, synaptic vesicles are organized into at least two pools: a pool in direct proximity to the T-shaped active zones [thought to represent the readily releasable pool (RRP)], and a pool localized in a broad area at the periphery of the entire synaptic bouton [representing the reserve vesicle pool (RP)]. Typically, the central region of the bouton is devoid of synaptic vesicles and contains endosomes and mitochondria, as well as other nonvesicular material. In contrast, boutons in the null allele scrib2 and scrib2/Df, but not those from the less severe allele scrib1, are filled with synaptic vesicles and lack an empty core. The number and area of mitochrondrial profiles, however, is unchanged in scrib mutant boutons. Overall, in these mutants there is a significant increase in synaptic vesicle density, as measured by determining the total number of vesicles at the central cross section of the boutons divided by the area of this cross section. In addition to this striking defect in vesicle distribution and density, many boutons contained morphologically abnormal vesicular material at the core (Roche, 2002).

To determine whether both the RRP and the RP are affected in scrib mutants, the number of vesicles was counted in an area 100, 150, and 200 nm around the active zone, which likely encompasses the RRP. The number of vesicles in these areas of scrib2/Df mutant boutons was not significantly different from wild type. Thus, it is the distribution and density of the RP that appear to be specifically affected in scrib mutants (Roche, 2002).

In addition to the defect in the RP, the average number of active zones in both scrib2 and scrib2/Df is slightly lower than wild type, although this difference was statistically different only at scrib2 homozygous boutons. Unlike dlg mutants, the SSR appeared normal, and neither the number of SSR layers nor the SSR density is significantly different from wild-type controls. This is in contrast to the observations in severe dlg mutants, in which the number of active zones is increased several fold and the SSR length is reduced (Roche, 2002).

Another phenotype in scrib mutants is the presence of an abnormally thick basal lamina. In ~30% of boutons examined, the muscle basal lamina appeared to be composed of several layers of normally sized basal lamina. However, at the majority of scrib mutant boutons, the basal lamina had a uniform electron density. These data suggest that scrib is involved in the normal development of several aspects of synapse structure (Roche, 2002).

Transgenic Scrib was expressed in motor neurons and muscles of scrib2 mutants, using the Gal4 drivers C380 and BG487. In these transgenic animals the density of synaptic vesicles was significantly decreased from the levels found in scrib mutants. In fact, the density of synaptic vesicles was significantly lower even than the densities in wild-type boutons. A similar effect was seen when analyzing the number of active zones, which was significantly higher than not only scrib mutants but also wild-type boutons. These data suggest that expression of Scrib in the mutant background not only rescues the scrib mutant defect, but causes a new phenotype in the opposing direction and likely results from an overexpression of Scrib compared with wild type. In contrast, the defect in the basal lamina was only partially rescued by transgenic Scrib expression. Thus, both the synaptic vesicle density and the number of active zones can be modified in either direction by manipulating the concentration of Scrib at the synapse (Roche, 2002).

To understand the physiological significance of the structural defects observed in scrib mutants, synaptic function was examined. Both evoked synaptic events [excitatory junctional currents (EJCs)] and spontaneous events [miniature EJCs (mEJCs)] were measured by two-electrode voltage-clamp experiments in muscle 6 of third instar larvae. Changes in the frequency of miniature synaptic currents result from alterations in presynaptic function, caused by alterations either in the probability of release (pR) or in the number of release sites. Conversely, changes in the amplitude of miniature events are generally considered to result from postsynaptic alterations, usually a change in receptor concentration or function. In scrib2/Df larvae, there was a significant decrease in the frequency of miniature synaptic events. Additionally, there was a slight but significant decrease in the amplitude of miniature synaptic events. Notably, presynaptic expression of transgenic Scrib using the Gal4 driver C380 in the scrib2 mutant background not only rescued the decrease in mEJC frequency but resulted in a significant increase in mEJC frequency compared with wild-type controls. This is in striking agreement with the ultrastructural studies, which show that expression of transgenic Scrib not only rescues the decrease in active zone number but results in a significant increase in the number of active zones compared with wild type. This result also is a strong indicator that, in this case, mEJC frequency is a reflection of the number of release sites (Roche, 2002).

Presynaptic expression of Scrib in the scrib2/Df background has no effect on mEJC amplitude, indicating that the reduced mEJC amplitude in scrib2/Df larvae arises from a postsynaptic mechanism (Roche, 2002).

Evoked release was measured by stimulating the segmental nerve innervating muscle 6 using a glass suction electrode. The nerve was stimulated with suprathreshold voltage at a frequency of 1 Hz, and the resulting EJCs were recorded. Surprisingly, there was no significant change in the amplitude of evoked EJCs when scrib2/Df was compared with wild type. There was a small but statistically insignificant increase in the quantal content of scrib2/Df larvae resulting from the small decrease in mEJC size. There was a slight divergence in wild-type and scrib mutant EJC amplitudes at low Ca2+ concentrations, with a significant difference only at 0.3 mM Ca2+. A double logarithmic plot of EJC amplitude in the linear portion of the Ca2+ concentration curve, an indicator of Ca2+ cooperativity, reveals a slight change in the slope of the linear fit, from 3.4 ± 0.1 for wild type to 2.8 ± 0.4 for scrib2/Df. Thus, vesicle release in scrib2/Df is slightly more sensitive to external Ca2+ levels than wild-type larvae. However, this change in sensitivity is very small in comparison to other Drosophila mutations that alter the Ca2+ sensitivity of vesicle release, perhaps a result of a mislocalization of release machinery with respect to cytoplasmic Ca2+ rather than a complete elimination of a critical component of the Ca2+ sensing process (Roche, 2002).

The similarity of evoked responses was surprising in view of the drastic changes in the ultrastructure and the decrease in spontaneous release frequency at synapses of the scrib mutant larvae. The response to high-frequency stimulation was tested to determine whether underlying defects may be uncovered during conditions in which the speed and accuracy of vesicle dynamics play a more crucial role. In wild type at 0.5 mM Ca2+, 10 Hz stimulation results in short-term facilitation of the synaptic current. This is thought to be caused by increased vesicular release resulting from residual Ca2+ remaining in the neuronal cytoplasm from previous depolarizations, acting on unknown targets to increase vesicle release. In contrast to wild type, facilitation was severely reduced or absent in scrib2/Df mutants, and unlike some other mutants that do not exhibit facilitation, the baseline transmission of scrib2/Df was equivalent to wild type at 0.5 mM Ca2+. Presynaptic expression of transgenic Scrib restored the response of the synapse to high-frequency stimulation (Roche, 2002).

To test whether the effect seen at 0.5 mM Ca2+ resulted from the specific inability to show facilitation or was a more global defect in synaptic vesicle replenishment at high stimulation frequencies, the synaptic response was tested at higher Ca2+ concentrations (1.0 mM), where no facilitation is seen in wild-type larvae. At 1.0 mM Ca2+, little change in EJC amplitude is seen after switching to 10 Hz stimulation frequencies, presumably because of a balance between the number of vesicles released and the resupply of the RRP. In contrast to the maintained EJC amplitude seen in wild-type larvae at 10 Hz, a significant depression is seen in scrib2/Df mutants, becoming apparent immediately after the first few stimuli. Thus, high-frequency stimulation causes scrib2/Df mutant synapses to exhibit both a lack of facilitation at low Ca2+ concentrations and faster synaptic depression at higher Ca2+ concentrations. These data suggest that scrib2/Df is defective not in the ability to exhibit facilitation but rather in the ability to resupply the RRP during high-frequency firing (Roche, 2002).

Another form of activity-dependent short-term plasticity exhibited at the Drosophila NMJ is post-tetanic potentiation (PTP). After a short train (30 sec) of high-frequency (10 Hz) stimuli, the EJC is potentiated for a period of 3-4 min relative to EJCs before the tetanus. The amplitude of wild-type EJCs is potentiated more than twofold after the train of high-frequency stimulation. scrib2/Df, in contrast, shows only a modest potentiation after the same stimulation protocol. Thus, multiple forms of activity-dependent plasticity are altered at the scrib synapse (Roche, 2002).

The alteration in short-term plasticity observed in response to high-frequency stimulation in scrib mutants, combined with the ultrastructural observations showing a remarkable increase in vesicle density, suggest that vesicle dynamics might be affected in the mutants. For example, if the rapid recruitment of vesicles to the RRP is altered, then synaptic transmission is expected to be normal at low stimulation frequencies, because at these frequencies the RP does not contribute to vesicle release. However, at high frequencies the RRP size must be maintained by rapid recruitment of vesicles. The vesicles are replenished to some degree by endocytosis of recently released vesicles. The increased density of vesicles in scrib mutants suggests that the defects in short-term plasticity are not likely a result of vesicle depletion, as is seen in the endocytotic mutant shibire, but more likely a result of the inability to rapidly recruit vesicles into the RRP. This hypothesis was tested by activity-dependent labeling of recycling vesicles using the styryl dye FM1-43 (Roche, 2002).

For these studies, preparations were dissected in 0.1 mM Ca2+ HL-3 saline and subsequently depolarized for 2 min in HL-3 saline containing 90 mM K+ in the presence of 5 µM FM1-43. After washing and fixing, samples were imaged by confocal microscopy. In wild type, strong FM1-43 fluorescence was observed at type I boutons. This labeling paradigm has been shown to label what has been termed the exo/endo cycling pool (ECP) in Drosophila, a pool that has been shown to contribute to both low- and high-frequency vesicle release (Roche, 2002).

This loading procedure resulted in fluorescence that was observed in a broad area at the periphery of synaptic boutons, whereas the central core of the boutons was devoid of fluorescence. This is in agreement with reports showing a similar distribution of synaptic vesicles. In scrib2/Df mutants a 54 ± 5% reduction in FM1-43 fluorescence was found compared with wild-type levels. In addition, the distribution of the FM1-43 label was affected in the mutants, which showed a significantly larger percentage of boutons with diffuse FM1-43 throughout the bouton surface. Thus, scrib2/Df mutants exhibit a decrease in activity-dependent endocytosis or altered distribution of endocytosed vesicles, or both. Because scrib2/Df larvae have very high synaptic vesicle densities in contrast to endocytotic mutants such as shibire and AP2, which show synaptic vesicle depletion, it is proposed that the defect in scrib mutants arises from a defect in exocytosis that secondarily causes effects on vesicle distribution, rather than a specific defect in endocytosis or endocytotic sorting. Indeed, the number of vesicles contained in the ECP is known to affect further loading of synaptic vesicles (Roche, 2002).

These data indicate that Scrib has a prominent role in synapse function and, taking into account its multiple PDZ domains, is likely involved in the precise localization of proteins necessary for vesicle dynamics. Facilitation is a Ca2+-dependent process that takes place in many synapses. This process is thought to result from residual Ca2+ accumulation, which acts on unidentified components of the exocytotic pathway. The precise mechanism by which high-frequency stimulation may lead to an increase in quantal content is currently unknown; however, several possibilities exist, including an increase in the pR of docked vesicles or an increase in the number of docked vesicles. There are several mutations in Drosophila that have effects on facilitation and PTP, including dunce (cAMP-specific phosphodiesterase II), rutabaga (Ca2+/calmodulin-dependent adenylyl cyclase), volado (alphaPS3-integrin), leonardo (a 14-3-3 protein family member), and latheo (an origin of replication complex protein). Of these mutants, scrib behaves most like rutabaga and latheo, in that both of these lack facilitation when baseline synaptic transmission is equivalent to wild type, and both have altered Ca2+ sensitivity of vesicle release, although the alterations in Ca2+ sensitivity are much more dramatic in both rutabaga and dunce mutants than in scrib mutants. It will be interesting to determine whether the localization of either of these proteins is altered in scrib mutants (Roche, 2002).

Three alternative models, or a combination of these, may contribute to the functional abnormalities observed in scrib mutants. One possibility is that the lack of facilitation observed at low Ca2+ in scrib mutants is caused by the inability of these synapses to recruit, transport, or convert vesicles into the RRP. This notion is supported by the FM1-43 studies, showing that endocytosis of vesicles in scrib mutants is greatly reduced, because endocytosis would be influenced by the amount of vesicles released as well as the number of vesicles already contained in the synapse. In other words, the cellular machinery underlying facilitation may be intact in scrib mutants, but the inability to recruit additional vesicles may mask the expression of facilitation. Similarly, during high-frequency stimulation at higher Ca2+, a decreased capability to recruit vesicles to the RRP would result in increased magnitude of depression in scrib mutants (Roche, 2002).

A second model is based on evidence indicating the existence of a feedback mechanism at the Drosophila NMJ that operates to maintain the excitability of the muscle within a narrow range. Because of this compensatory mechanism, the decrease in number of active zones in scrib mutants may lead to a maximal increase in pR, thus maintaining EJC amplitude at low frequencies. Stimulation of the terminal at frequencies that in wild type lead to facilitation, however, would be unable to further increase pR in scrib mutants, precluding further increases in the amplitude of the EJC. In other words, compensatory mechanisms in scrib mutants bring the mutant synapse to a facilitated state even at low-frequency stimulation, preventing a further increase in EJC amplitude at higher frequencies. The rapid depression observed in scrib mutants could be explained according to this model by the inability of the terminal to fulfill the increased demand for primed vesicles that is imposed by the increase in pR. In intact Drosophila larvae, motoneurons stimulate the muscles by firing trains of high-frequency action potentials. In scrib mutants, loss of high-frequency responsiveness might lead to the buildup of synaptic vesicles, perhaps explaining the enhanced density of synaptic vesicle in scrib mutants. Thus this model is consistent with the electrophysiological and ultrastructural observations in the mutants (Roche, 2002).

A third possible mechanism involves altered buffering of neuronal Ca2+ levels. Cytoplasmic Ca2+ buffering is important in regulating synaptic vesicle release, especially during high-frequency stimulation. A prevalent form of buffering Ca2+ at synapses is mitochondrial Ca2+ uptake, and this form of Ca2+ uptake has been shown to have effects on high-frequency synaptic firing patterns. The number of mitochondria in scrib boutons, however, was unchanged from wild type, making this possibility less likely. However, elimination or mislocalization of other critical Ca2+ buffering or sensing molecules may play a role in the defects seen in scrib synapses. Ultimately, the identification of Scrib binding partners may shed light on the precise mechanisms by which facilitation and sustained release are affected in scrib mutants (Roche, 2002).

Another defect in scrib mutant synapses was an abnormally thick basal lamina. The synaptic basal lamina has long been recognized as containing important elements for postsynaptic differentiation and for the clustering of neurotransmitter receptors. The effects of Scrib on the basal lamina may reflect the inability of Scrib to selectively recruit synaptic components to their correct destination (Roche, 2002).

PDZ proteins are characterized by multiple modular sequences involved in protein-protein interactions and for this reason are frequently called scaffolding proteins. These proteins bring together components necessary for certain cellular functions by binding to distinct partners through their multiple interaction domains. Scrib contains four PDZ domains and hence could form a multiprotein complex with at least four different proteins. Elucidation of the identity of these binding partners will be important in understanding the precise role that Scrib plays in synaptic vesicle dynamics. In epithelial cells it has been suggested that Scrib is involved in vesicle sorting, an important mechanism involved in segregating transmembrane proteins in these cells. Another PDZ domain-containing protein, EBP50, has also been shown to be involved in vesicular sorting, in particular, sorting of endocytotic vesicles involved in recycling ß adrenergic receptors. A similar mechanism, perhaps operating through a conserved cassette of proteins, may be altering vesicle dynamics at the synapse (Roche, 2002).

One might hypothesize, on the basis of the mislocalization of Scrib in dlg mutants, that by mutating dlg one would see, in addition to a dlg-specific phenotype, the scrib phenotype as well. Indeed this seems to be the case in other cell types in which mutation of either dlg or scrib causes similar phenotypes: formation of tumors and loss of cell polarity. However, at the Drosophila NMJ the effects of scrib mutation are quite different from those in several dlg mutants. These differences may stem from the fact that residual synaptic Scrib is still present in dlgXI-2 mutants, although at a lower level, and hence the remaining Scrib may be sufficient to override the synaptic scrib phenotype. Interestingly, levels of synaptic Scrib have an opposite influence on the regulation of the number of active zones than do levels of synaptic Dlg. Although a decrease in Dlg levels in severe dlg mutants causes an increase in active zone number, the same phenotype is observed by increasing Scrib levels. This observation is consistent with the notion that at synapses Scrib may negatively regulate Dlg function. This is in contrast to the observation in epithelial cells, where Dlg and Scrib appear to function in a similar manner during the determination of cell polarity and tumor suppression. This may reflect the ability of Dlg and Scrib to bind different protein partners with different functions in the two cell types. Indeed, partners such as Fasciclin II bind to Dlg at synapses but are absent in epithelial cells. Thus, the specific influence of scaffolding proteins in different cell types may be highly dependent on the availability of specific binding partners (Roche, 2002).

In conclusion, this study has introduced a relatively new function for a member of the PDZ protein family, regulation of synaptic vesicles. It will be important to understand the specific subset of proteins that interacts with Scrib, because this is likely the key to understanding how this protein is involved in synaptic vesicle regulation (Roche, 2002).

scribbled mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila

Cancer is a multistep process involving cooperation between oncogenic or tumor suppressor mutations and interactions between the tumor and surrounding normal tissue. This study is the first description of cooperative tumorigenesis in Drosophila, and uses a system that mimics the development of tumors in mammals. The MARCM system was used to generate mutant clones of the apical-basal cell polarity tumor suppressor gene, scribbled, in the context of normal tissue. scribbled mutant clones in the eye disc exhibit ectopic expression of cyclin E and ectopic cell cycles, but do not overgrow due to increased cell death mediated by the JNK pathway and the surrounding wild-type tissue. In contrast, when oncogenic Ras or Notch is expressed within the scribbled mutant clones, cell death is prevented and neoplastic tumors develop. This demonstrates that, in Drosophila, activated alleles of Ras and Notch can act as cooperating oncogenes in the development of epithelial tumors, and highlights the importance of epithelial polarity regulators in restraining oncogenes and preventing tumor formation (Brumby, 2003).

A clonal approach, more closely resembling the clonal nature of mammalian cancer, was used to analyze the effects of removing Scrib function on tumor formation. This analysis indicates that Drosophila scrib- tumors: (1) lose tissue architecture, including apical-basal cell polarity; (2) fail to differentiate properly; (3) exert non-cell-autonomous effects upon the surrounding wild-type tissue; (4) upregulate cyclin E and undergo excessive cell proliferation; (5) are restrained from overgrowing by the surrounding wild-type tissue via a JNK-dependent apoptotic response, and (6) show strong cooperation with oncogenic alleles of Ras and Notch to produce large amorphous tumors. These conclusions are summarized in a model for tumor development in Drosophila. It is suggested that the role of epithelial cell polarity regulators in restraining oncogenes is likely to be of general significance in mammalian tumorigenesis (Brumby, 2003).

The model suggests that the wild-type larval eye disc is a monolayered columnar epithelium, in which cell proliferation is tightly regulated. Cell architecture is maintained by the formation of adherens junctions, the apical localization of Scribbled, and adhesion to the basement membrane. Mutation of scrib results in loss of apical-basal polarity, leading to multilayering and rounding up of cells. scrib- tissue also shows impaired differentiation, and ectopic cyclin E expression (by an unknown mechanism) leads to ectopic cell proliferation. Unrestrained overgrowth and tumor formation of scrib- cells is held in check by compensatory JNK-mediated apoptosis, dependent upon the presence of surrounding wild-type cells. Secondary mutations are required to avoid this apoptotic fate. If JNK activity is blocked within scrib- cells, by expressing a dominant-negative form of JNK, apoptosis is prevented, resulting in tissue overgrowth and lethality. Even more aggressive overgrowth results from the addition of activating oncogenic alleles of Ras or Notch. In addition to promoting cell survival, these oncogenes must also promote tumor cell proliferation; however, it is proposed that other downstream effectors of these oncogenes are likely also to be important, since it was not possible to mimic the cooperative overgrowth effects of RasACT or NACT on scrib- tissue by simply blocking apoptosis and enhancing cell proliferation (Brumby, 2003).

scrib- clones ectopically express cyclin E and undergo ectopic S phases and mitoses. Since cyclin E is rate limiting for cell cycle progression in the developing eye, it is likely that upregulation of cyclin E in scrib- clones is critical for the ectopic cell proliferation. Indeed, alleles of scrib and lgl were originally isolated as dominant suppressors of a hypomorphic cyclin E allele, DmcycEJP, suggesting that these cell polarity genes normally play a critical role in limiting cyclin E expression. Currently being investigated is which signaling pathways are altered in scrib, dlg or lgl mutants that could be responsible for cyclin E upregulation. A recent study in human lung epithelial cells shows that disrupting cell polarity allows mixing of the heregulin-alpha ligand and the erbB2-4 receptor, which are normally physically separated, resulting in activation of the pathway and cell proliferation. Further studies are required to determine whether the ectopic expression of cyclin E observed in the absence of Scrib is simply a consequence of the tissue disorganization induced by disrupting cell polarity, or if Scrib has a direct role in limiting cell proliferation independent of cell polarity. Interestingly, the rounding up of cells in the absence of Scrib appears to be predominantly a cell-autonomous effect, yet clearly non-cell-autonomous defects are also apparent, including the upregulation of cyclin E. This would suggest that altered cell-cell interactions between wild-type and mutant cells can also alter signaling pathways within wild-type cells, and that the loss of apical-basal polarity and collapse of the columnar epithelium is not intrinsically responsible for the deregulated expression of cyclin E. A deeper understanding of the relationship between epithelial cell polarization and cell proliferation is clearly important for understanding the development of cancer, since a loss of cell polarity often accompanies tumor progression and metastasis (Brumby, 2003).

Overproliferation of scrib- clones in the eye disc is compensated for by JNK-mediated apoptosis. Blocking JNK pathway activity in scrib- eye clones greatly increases the proportion of clonal tissue, and results in lethality to the host. Since downregulating JNK pathway activity in otherwise wild-type clones of tissue does not induce increased cell proliferation, it is suggested that JNK pathway activity in scrib- clones induces apoptosis. This is consistent with previous reports on the pro-apoptotic effects of the JNK pathway in the Drosophila eye and the current observations. Recent studies in mammals would also suggest that activation of the JNK pathway can limit the growth of tumors in situ, possibly by increasing apoptosis (Brumby, 2003).

How JNK-mediated apoptosis is induced in scrib- clones is not known. While Scrib could play a direct role in repressing JNK pathway activity, it is also possible that the JNK pathway is activated indirectly, in response to other cellular defects. In the wing disc, removal of cells by JNK-mediated apoptosis is linked to discontinuities in a cell's response to morphogen gradients, most notably the antero-posterior patterning regulator, Dpp, in a process probably related to cell competition, with the purpose of eliminating aberrant or slow growing cells. Although this form of compensatory JNK-mediated apoptosis has not yet been demonstrated within the eye disc, the observation that the surrounding wild-type tissue context plays an important role in limiting the overgrowth of scrib- tissue argues against a simple cell-intrinsic apoptotic response of scrib- cells to a loss of cell polarity, and is more consistent with an integrative response mediated by both the tumor cells and the surrounding wild-type cells, as exemplified by cell competition. Whether this is dependent on a failure of scrib- cells to transduce Dpp signaling is not known; however, other interesting possibilities also warrant further investigation. Notable is the recent identification of a tumor necrosis factor-induced apoptotic signaling pathway involving the JNK pathway. It is also important to keep in mind the involvement of the JNK pathway in orchestrating cell shape changes during the morphogenetic movements of dorsal and thorax closure and wound healing. clones of scrib- tissue expressing BskDN (JNKDN) appear morphologically different from those expressing the apoptosis inhibitor p35; most notably, the clones are generally larger and less rounded than those expressing p35. This would imply either that p35 is not as effective as BskDN in preventing cell death, or that there are other effectors of the JNK pathway that are important in the inhibition of scrib- tumor overgrowth. The possibility that JNK activation could play a role in eliminating scrib- tissue from the epithelium in a process reminiscent of wound healing is currently being investigated (Brumby, 2003).

dlg and lgl mutant clones also show poor viability, suggesting that JNK-mediated apoptosis could be a common response to the loss of cell polarity and overproliferation induced by the absence of these tumor suppressors. Indeed, while other regulators of epithelial cell polarity, such as Crumbs and E-cadherin, apparently do not act as tumor suppressors in Drosophila, the effects of these mutations on cell proliferation when cell death is blocked warrant further examination. Interestingly, in mammalian systems, the polarized nature of epithelia is also important in protecting cells from an apoptotic response, and this acts as a brake on tumor development when polarity is disrupted (Brumby, 2003).

In Drosophila, activated Ras exerts its oncogenic effects through Raf and the MAPK pathway. Downstream targets of MAPK in the eye disc promote differentiation, cell survival and cell proliferation. This work also demonstrates that Ras can increase cyclin E protein levels in the eye disc. In combination with scrib-, the differentiation output of RasACT signaling appears to be attenuated, and the proliferative and anti-apoptotic responses prevail (Brumby, 2003).

Activated Notch also cooperates with scrib-, resulting in neoplastic overgrowth, and although no anti-apoptotic role for Notch signaling in the eye has been described previously, NACT exerts hyperproliferative effects in flies, and Notch signaling is required for proliferation of eye disc cells. Although it is not known if NACT induces the same critical downstream targets as RasACT to cause overgrowth of scrib- tissue, removing ras function in scrib- cells overexpressing NACT rescues the overgrowth phenotype, suggesting that the effects of NACT are at least partially dependent on Ras (Brumby, 2003).

Initially it seemed likely that the cooperative effects of RasACT or NACT on scrib- tissue could be explained by the ability of these oncogenes to promote cell proliferation while blocking apoptosis. However, the expression of neither cyclin E nor E2F1/DP, in combination with the apoptosis inhibitor p35 (or with the inhibitor of JNK pathway activity, BskDN), was capable of phenocopying the effect of RasACT or NACT in scrib- clones. It is therefore suggested that other downstream effectors, apart from anti-apoptotic and cell cycle regulators, must be important in mediating the oncogenic effects of RasACT or NACT. In fact, in Drosophila, Ras has also been shown to be a potent inducer of cellular growth, while cyclin E and E2F1 mainly promote cell cycle progression. Whether NACT also promotes cell growth in Drosophila has not been examined in detail. If growth promotion targets downstream of RasACT or NACT are critical in promoting the overgrowth of scrib- tumors, these are likely to be independent of the PI3 kinase pathway since ectopic PI3 kinase signaling in scrib- clones does not induce synergistic overgrowth, and RafACT is able to induce overgrowth as equally extensive as RasACT (Brumby, 2003).

Finally, it is noted that in mammalian systems, evidence exists for a role for Ras signaling in modulating cell junction complexes and enhancing epithelial to mesenchymal transitions, and in Drosophila also, constitutive RasACT signaling in clones alters cell affinities and changes the levels of E-cadherin and ß-catenin. Whether RasACT or NACT signaling destabilizes adherens junctions in Drosophila and this potentiates scrib- neoplastic overgrowth or whether alterations in the structure of the adherens junction resulting from the absence of Scrib alters a cells response to constitutive activation of these oncogenes are important future questions (Brumby, 2003).

This study has described a novel multi-hit model of tumorigenesis in Drosophila. Furthermore, although it has been suspected that disruptions to cell polarity could potentiate tumor progression and metastasis, this work demonstrates for the first time how the oncogenic effects of activated Ras and Notch are unleashed in the absence of epithelial polarity regulators. It is predicted that in mammals also, defects in apical-basal polarity could cooperate with oncogenes during neoplastic development. This approach in Drosophila can now be used to screen for novel oncogenes that, when specifically overexpressed in scrib- clones, are capable of inducing cooperative tumorigenesis, and can also be extended to identify cooperative interactions between other tumor suppressors and oncogenes within a whole animal context (Brumby, 2003).

Oncogenic mutations produce similar phenotypes in Drosophila tissues of diverse origins

An emerging interest in oncology is to tailor treatment to particular cancer genotypes, i.e. oncogenic mutations present in the tumor, and not the tissue of cancer incidence. Integral to such a practice is the idea that the same oncogenic mutation(s) produces similar outcomes in different tissues. To test this idea experimentally, tumors were studied driven by a combination of RasV12 and scrib1 mutations in Drosophila larvae. Tumors induced in tissues of neural ectodermal and mesodermal origins were found to behave similarly in every manner examined: cell cycle checkpoints, apoptosis, cellular morphology, increased aneuploidy and response to Taxol. It is concluded that oncogenic effects override tissue-specific differences, at least for the mutations, tissues, and phenotypes in this study (Stickel, 2014).

Scribble is essential for olfactory behavior in Drosophila melanogaster

The ability to discriminate and respond to chemical signals from the environment is an almost universal prerequisite for survival. Reported here is evidence that the scaffold protein Scribble is essential for odor-guided behavior in Drosophila. A P-element insert line has been identified with generalized sexually dimorphic smell impairment, smi97B. The transposon in this line is located between the predicted promoter region and the transcription initiation site of scrib. A deficiency in this region, Df(3R)Tl-X, and two scrib null alleles fail to complement the smell-impaired phenotype of smi97B. Wild-type behavior is restored by precise excision of the P element, scrib mRNA levels correspond with mutant and wild-type phenotypes, and introduction of a full-length scrib transgene in the smi97B mutant rescues the olfactory deficit. Expression of Scrib is widespread in olfactory organs and the central nervous system. Finally, alternative splicing of scrib generates transcripts that differ in the number of leucine-rich repeats and PDZ domains (Ganguly, 2003).

The smi97B mutation is one of the strongest smi mutations among a set of previously identified smi lines. The mutation is recessive: olfactory ability, quantified by avoidance responses to benzaldehyde, was reduced in smi97B homozygotes, compared to the P-element-free coisogenic host strain, Sam; ry506 (Sam), whereas smi97B/Sam heterozygotes display avoidance responses that are indistinguishable from wild type. Furthermore, the magnitude of the mutational effect of smi97B is sexually dimorphic; even at high odorant concentrations: males are hyposmic, while females are anosmic (Ganguly, 2003).

To determine whether smi97B flies experience smell impairment throughout their life cycle, whether larvae also display aberrant olfactory responsiveness was examined. Since larvae are attracted to most odorants, even those that elicit avoidance behavior in adults, the kinetics were compared of odor-guided responses of wild-type and smi97B third instar feeding larvae toward benzaldehyde and isoamylacetate. The effect of genotype in the two-way analysis of variance was significant for both odorants, but time and genotype x time interaction terms were not significant for either odorant. Larval motility in the absence of an odor cue was not significantly different between the two genotypes. Thus, the smi97B mutation causes olfactory deficits in both larvae and adults (Ganguly, 2003).

The genomic fragment flanking the 3' end of the P[lArB] element was sequenced and the insertion site was determined to be 1084 bp upstream of the open reading frame of scrib. scrib is a pleiotropic gene essential for localization of polarity determinants in developing epithelia, and synaptic maturation and modulation of short-term plasticity at the larval neuromuscular junction. smi97B is an allele of scrib (Ganguly, 2003).

The smi97B mutation was mapped to the region of the third chromosome including scrib. Df(3R)Tl-X (breakpoints 97B2;97D2), which uncovers scrib, failed to complement smi97B; hemizygotes generated by crossing Df(3R)Tl-X to smi97B displayed reduced avoidance responses compared to Df(3R)Tl-X/Sam controls. The smell-impaired phenotype of Df(3R)Tl-X/smi97B hemizygotes was also sex specific, with significantly more smell impairment in females than in males (Ganguly, 2003).

Next, to demonstrate that olfactory deficits in smi97B arise from the P[lArB] element and are not a linked mutation, precise excision of P[lArB] was shown to restore the wild-type phenotype; avoidance responses of precise excision alleles, like smi97B16A, were not significantly different from Sam. Further, mutations generated by imprecise excision of P[lArB] provided evidence for sex-specific regulation of scrib. The smi97B15A mutation contains a 3.6-kb P[lArB] fragment at the original insertion site that resulted in male-specific olfactory deficits: smi97B15A males were smell impaired compared to controls, while olfactory responses in females were not statistically different from those in wild type. In contrast, a 2.5-kb P[lArB] insertion at the same site in smi97B2A was correlated with mild hyposmia in females. In agreement with the sexually dimorphic phenotype, scrib transcripts were markedly reduced in smi97B15A males, but not in females, whereas transcriptional differences could not be resolved in smi97B2A, in line with the subtle female-specific phenotype of this imprecise revertant (Ganguly, 2003).

To further implicate scrib in olfactory behavior, complementation tests were conducted with previously identified scrib alleles. Two null alleles, scrib1 and scrib2, failed to complement the smell-impaired phenotype of smi97B. Avoidance responses of scrib1/smi97B and scrib2/smi97B heterozygotes were significantly lower than those of the scrib1/Sam and scrib2/Sam controls. No sexual dimorphism in smell impairment was observed, possibly due to the disparate genetic backgrounds of the scrib stocks and smi97B. scribS0421405 and scribj7B3 alleles, which contain P[lacW] insertions in the 5' untranslated region of scrib and the second intron, respectively, were tested. Avoidance responses of scribS0421405/smi97B females were significantly lower than those of scribS0421405/Sam females, while male responses were not significantly different from those of control males. Hence, scribS0421405 failed to complement the olfactory deficit caused by smi97B, but only in females. However, scribj7B3 fully complemented the smell-impaired phenotype of smi97B; avoidance responses of scribj7B3/smi97B flies were not significantly different from those of scribj7B3/Sam controls. Interallelic complementation is consistent with alternative splicing of scrib, which may involve the generation of sex-specific gene products involved in olfaction. Rescue of the smi97B phenotype was demonstrated by functional complementation with a wild-type scrib allele (Ganguly, 2003).

The sexually dimorphic olfactory phenotype of smi97B and evidence for interallelic complementation led to an examination of whether males and females express alternative splice variants of scrib. Three major RNA species were detected on Northern blots probed with a full-length scrib cDNA: a universal 5.9-kb transcript present in adults and to a lesser extent in larvae, a 7.1-kb transcript expressed predominantly in males and larvae of both sexes, and a 4.6-kb female-specific transcript. Transcript levels were correlated with sex-specific behavioral phenotypes, as evidenced by reductions in the 7.1-kb transcript in smi97B larvae and the 5.9- and 4.6-kb transcripts in mutant females compared to controls (Ganguly, 2003).

To investigate the existence of less prominent variants of scrib, a Drosophila head cDNA library was screened with a full-length scrib probe. Seven unique clones were identified and sequenced. The sizes of three inserts correspond with splice variants detected on Northern blots. A 7.1-kb clone encoding 16 LRRs, four PDZ domains, and a unique 3' untranslated exon corresponds to the transcript expressed in males and larvae; a 5.9-kb clone identical to the 7.1-kb fragment, but without the 3' untranslated region, corresponds to the transcript present in both sexes and larvae; and a 4.6-kb clone encoding 16 LRRs and PDZ domains I and II corresponds to the female-specific transcript. In addition, the 4.6-kb clone also contains a unique coding exon at the 3' end. The first 285 bases of this exon were shared by four additional clones, two of which were identical except for the number of LRRs they encode. In contrast to the 4.6-kb female-specific transcript that encodes only PDZ domains I and II, two clones were identified that encode only PDZ domains III and IV. Since LRRs and PDZ domains mediate protein-protein interactions, these differences suggest variability in the composition of protein assemblies recruited by the various Scrib isoforms (Ganguly, 2003).

Monospecific antibodies raised against a carboxyl-terminal peptide of Scrib did not visualize the expected 200-kD polypeptide encoded by the transgene. Instead they detected a 120-kD band in both sexes and an 80-kD female-specific immunoreactive band, likely due to high sensitivity of the protein in adult flies to proteolysis, which cleaves the expected 200-kD translation product in 120- and 80-kD immunoreactive polypeptides in females, whereas in males the latter fragment is proteolyzed further into smaller fragments (Ganguly, 2003).

Visualization of scrib expression in adult tissues with a riboprobe complementary to the scrib coding region revealed staining in the third antennal segment and maxillary palps and the major olfactory organs of Drosophila as well as in Johnston's organ in the second antennal segment, the primary auditory organ. Staining was also observed in cortical regions of the brain. Staining was not observed when hybridizations were performed with sense riboprobes. No differences in scrib expression were observed between males and females under these experimental conditions (Ganguly, 2003).

To localize Scrib in CNS projection areas, immunohistochemistry was performed. Staining in the brain was particularly intense in the antennal nerves and the ventrolateral and superior medial protocerebrum. Widespread deposition of Scrib was also detected in the antennae and maxillary palps (Ganguly, 2003).

Domains controlling cell polarity and proliferation in the Drosophila tumor suppressor Scribble

Cell polarity and cell proliferation can be coupled in animal tissues, but how they are coupled is not understood. In Drosophila imaginal discs, loss of the neoplastic tumor suppressor gene scribble, which encodes a multidomain scaffolding protein, disrupts epithelial organization and also causes unchecked proliferation. Using an allelic series of mutations along with rescuing transgenes, domain requirements for polarity, proliferation control, and other Scrib functions have been identified. The leucine-rich repeats (LRR) tether Scrib to the plasma membrane, are both necessary and sufficient to organize a polarized epithelial monolayer, and are required for all proliferation control. The PDZ domains, which recruit the LRR to the junctional complex, are dispensable for overall epithelial organization. PDZ domain absence leads to mild polarity defects accompanied by moderate overproliferation, but the PDZ domains alone are insufficient to provide any Scrib function in mutant discs. A model is suggested in which Scrib, via the activity of the LRR, governs proliferation primarily by regulating apicobasal polarity (Zeitler, 2004).

These results highlight the central role of the LRR in Scrib function. Animals with absent or mutant LRR have phenotypes identical to those entirely lacking Scrib, with dramatic effects on both epithelial polarity and growth control. Of the five evolutionarily conserved protein-protein interaction domains in Scrib, only expression of the LRR can provide polarizing and proliferation-controlling activity, with high levels sufficient to effect nearly full rescue. The LRR is also sufficient to mediate membrane localization, whereas a protein lacking the LRR remains in the cytoplasm. Interestingly, alteration of a conserved leucine in the 10th LRR disrupts all Scrib functions and displaces the protein into the cytoplasm. A related alteration in the 13th LRR of C. elegans (Let-413) also prevents membrane localization and function. However, it is clear that the LRR functions as more than a membrane attachment domain because the Scrib PDZs alone are incapable of rescuing any aspect of the mutant phenotype, even when provided with an exogenous membrane targeting signal (Zeitler, 2004).

How can the LRR, which is broadly localized in the absence of PDZ domains, convey information to specifically polarize the apicobasal axis? It has been suggested that a critical role of Scrib in epithelial polarity is the recruitment of Lgl to the lateral cell cortex. Lgl itself is not highly polarized in its distribution, and while it is displaced from the cortex in scrib null GLC embryos, immunofluorescense reveals that Lgl is indeed cortically localized in LRR-expressing scrib 4 GLC embryos, consistent with proper apicobasal polarization in these animals. Therefore, it appears that membrane-localized LRR can mediate interactions that effect cortical recruitment of Lgl, where Lgl can perform its still unknown activities in regulating protein trafficking (Zeitler, 2004).

A surprising result of these experiments is that epithelia can achieve apicobasal organization in the absence of Scrib PDZ domains. Requirement of the PDZ domains for epithelial polarization was expected because of the frequent occurrence of these motifs in proteins that regulate cell polarity. However, Let-413 PDZ domains are also not required to rescue polarity suggesting that LAP protein PDZ domains may be generally dispensable for organizing the apicobasal axis. This finding has implications for understanding the biological roles of LAP protein PDZ-binding partners. In the case of Scrib, it was found that high level expression of a protein lacking the PDZ domains can provide function similar to low levels of expression of full-length protein. The data thus suggest that under physiological conditions PDZ domain interactions contribute to Scrib function in polarity and proliferation control quantitatively rather than qualitatively (Zeitler, 2004).

The quantitative contribution of the PDZ domains may involve their role in polarizing Scrib along the plasma membrane. Analysis of tagged transgenes suggests that Scrib is localized by a two-part mechanism. In the first step, interactions mediated by the LRR bring the protein to the plasma membrane. In the second step, PDZ domain interactions enrich membrane-bound Scrib at the future site of the SJ. This mechanism mirrors the gradual polarization of Scrib during embryonic development, where Scrib is initially localized homogeneously basolaterally, but becomes focused apicolaterally as the final junctional complex forms. Scrib localization to the SJ thus parallels the maturation of the epithelium. Cell junctions are known to be sites of polarized vesicle trafficking, and proteins that control polarity of the entire epithelial cell membrane are localized to the small perijunctional region. Efficient regulation of cell polarity may require high Scrib levels at this location, with the PDZ domains serving to increase the local concentration of the active LRR (Zeitler, 2004).

The data provide direct evidence of a role for Scrib in SJ formation. Although Scrib and Dlg both localize to the SJ, an unambiguous demonstration that either protein is an SJ component has proven difficult. Physical interactions of SJ components with Scrib or Dlg have not yet been found, and whereas dlg and scrib null mutant discs have no SJs, the dramatic disorganization of these tissues raises the possibility that SJ loss might be secondary to the gross polarity disruption. In this work, scrib mutant discs and embryos were identified that polarize and form normal AJs but nevertheless contain severely disrupted SJs. Interestingly, SJ proteins are zygotically produced and first become concentrated at the apical region of the lateral membrane during embryonic stage 14, when the SJ ultrastructurally appears. By contrast, Scrib and Dlg, which are maternally provided, are enriched in this membrane region at stage 9, long before SJs develop. Scrib may therefore prepattern the site of the future SJ to mediate the subsequent coalescence of other SJ components (Zeitler, 2004).

Loss of scrib from imaginal epithelia causes two major defects: mispolarization, reflected in the ectopic distribution of apical and AJ proteins, and overproliferation, resulting in an enormous increase in cell numbers. A key question is whether these effects, which are seen in all three Drosophila neoplastic tumor suppressor gene mutants, are independent or if they are causally interrelated. The independent model posits the existence of Scrib domains that control proliferation and cell polarity without influencing the other function. By contrast, the interdependent model posits that Scrib acts primarily to regulate apicobasal polarity, and that loss of polarity itself disrupts proliferation control (Zeitler, 2004).

Previous views on control of polarity and proliferation by the neoplastic tumor suppressor genes have favored an independent model. These views are influenced by a study of Dlg functional domains, in which deletion of two PDZ domains caused overproliferation within a maintained epithelial monolayer. This finding led to the proposal that Dlg has separable functions in polarity and growth control, with the latter mediated by PDZ domain interactions. Using analogous methods in this study, it was found that deletion of Scrib PDZ domains similarly causes overproliferation in the absence of gross epithelial disorganization. However, the quantitative analysis reveals that PDZ domain deletion does not entirely disrupt, but only partially compromises, proliferation control. Moreover, this proliferation defect can also result from lower levels of full-length Scrib, and in fact can be rescued by expressing high levels of a single Scrib domain, the LRR. These analyses do not rule out the possibility that the PDZ domains, when covalently linked to the LRR, directly contribute to cell proliferation signaling. Nevertheless, any such contribution is likely minor because high levels of LRR alone restore null mutant discs to nearly WT size (Zeitler, 2004).

A formal demonstration of independent functions requires the identification not only of mutant proteins that rescue polarity without restoring proliferation control but also of those that rescue proliferation control without restoring polarity. Such proteins have not been found, using either random mutagenesis or rescue constructs engineered with a knowledge of conserved domains. Both polarity and proliferation control are simultaneously lost when the LRR is mutated, and expression of domains dispensable for polarity (DeltaLRR, myr-DeltaLRR) does not provide any proliferation control, even to a polarized disc. Although the possibility cannot be excluded that for instance specific LRR mutations could create a protein incapable of polarizing tissue but still allows the proper cessation of proliferation, the failure to identify such proteins encourages the consideration of alternative models (Zeitler, 2004).

Several of these data indicate that proliferation control by the Scrib LRR is intimately linked to its polarity-regulating activity. As with C. elegans Let-413, the LRR is both necessary and sufficient to polarize epithelial cells, including embryonic cells that do not overproliferate in scrib mutants. An LRR-specific missense mutation causes a phenotype equivalent to the protein-null condition. By contrast, in hypomorphic mutant animals, moderate disc polarity defects are accompanied by moderate proliferation defects, and in rescue experiments improvements in epithelial architecture accompany reductions in overproliferation. Because hyperproliferation itself is not sufficient to induce mispolarization, the data are consistent with a model in which the primary role of Scrib is to govern cell polarity, and overproliferation is a consequence of polarity disruption (Zeitler, 2004).

The finding that polarized but nevertheless hyperproliferative scrib 4 cells contain specific mislocalized proteins points to a possible mechanism for how polarity disruption could alter proliferation control. The survey revealed misdistributed SJ components, but the altered shapes of scrib 4 (as well as scrib 5 and j7b3) cells suggest that additional proteins may be aberrantly or inefficiently localized. Polarized proteins include growth factor receptors, which are often clustered near cell junctions, and mislocalization of these receptors can lead to altered activity. Although a growth-regulatory protein mislocalized in scrib 4 discs has not yet been identified, the role of the PDZs in Scrib localization to and assembly of the SJ suggests that such a partner might require junctional localization for efficient signaling. A test of this model, and a mechanistic understanding of Scrib function, awaits the identification of Scrib-binding partners (Zeitler, 2004).

The coupling of proliferation control to cell polarity demonstrated in the fly indicates that polarization loss may contribute to human oncogenesis, and not only in neoplastic tumors. It is generally thought that polarity defects are among the last steps during the development of carcinoma in situ, promoting primarily invasion and metastasis. The data suggest that loss of polarity-regulating proteins might also play a role at earlier steps in tumor development by disorganizing specific growth control pathways. Future work will identify, in both flies and mammals, the effectors of cancerous properties altered in depolarized tumors (Zeitler, 2004).

A genetic screen for dominant modifiers of a cyclin E hypomorphic mutation identifies novel regulators of S-phase entry in Drosophila

Cyclin E together with its kinase partner Cdk2 is a critical regulator of entry into S phase. To identify novel genes that regulate the G1- to S-phase transition within a whole animal use was made of a hypomorphic cyclin E mutation, DmcycEJP, which results in a rough eye phenotype. The X and third chromosome deficiencies were screened, candidate genes were tested, and a genetic screen of 55,000 EMS or X-ray-mutagenized flies was carried out for second or third chromosome mutations that dominantly modify the DmcycEJP rough eye phenotype. Focused was placed on the DmcycEJP suppressors, S(DmcycEJP), to identify novel negative regulators of S-phase entry. There are 18 suppressor gene groups with more than one allele and several genes that are represented by only a single allele. All S(DmcycEJP) tested suppress the DmcycEJP rough eye phenotype by increasing the number of S phases in the postmorphogenetic furrow S-phase band. By testing candidates several modifier genes were identifed from the mutagenic screen as well as from the deficiency screen. DmcycEJP suppressor genes fall into five general classes: (1) chromatin remodeling or transcription factors; (2) signaling pathways, and (3) cytoskeletal, (4) cell adhesion, and (5) cytoarchitectural tumor suppressors. The cytoarchitectural tumor suppressors include scribble, lethal-2-giant-larvae (lgl), and discs-large (dlg), loss of function of which leads to neoplastic tumors and disruption of apical-basal cell polarity. The genetic interactions of scribble with S(DmcycEJP) genes were further explored and it was shown that hypomorphic scribble mutants exhibit genetic interactions with lgl, scab (alpha PS3-integrin -- cell adhesion), phyllopod (signaling), dEB1 (microtubule-binding protein -- cytoskeletal), and moira (chromatin remodeling). These interactions of the cytoarchitectural suppressor gene, scribble, with cell adhesion, signaling, cytoskeletal, and chromatin remodeling genes, suggest that these genes may act in a common pathway to negatively regulate cyclin E or S-phase entry (Brumby, 2004).

This work has led to the identification of many genes that when mutated have the ability to dominantly modify the DmcycEJP adult rough eye phenotype and S-phase defect in third instar larval eye imaginal discs. In addition to genes already known to be regulators of Drosophila cyclin E or G1-S progression [such as E2F1; retinoblastoma (Rbf); ago (cdc4) encoding a protein involved in Cyclin E degradation; the EGF receptor pathway genes Egfr and Ras85D, which act to promote Cyclin E protein accumulation, and Hh signaling pathway genes, which act to promote cyclin E transcription], this screen led to the identification of many novel cyclin E interactors. This study mainly concentrated on the suppressors of DmcycEJP, although from the deficiency screen and specifically testing candidates, axin (an inhibitor of Wg signaling), rho1, and crumbs as enhancers of DmcycEJP, which therefore may act as novel positive regulators of G1-S progression, were identified. The suppressors of DmcycEJP identified include the following classes: (1) chromatin remodeling genes brm, mor, Trl, or the transcription factor Zn72D; (2) signaling pathway genes phyl, sina, trio, Abl, RpS6, wg and Wg pathway effectors dsh and arm; (3) genes encoding cytoskeletal proteins dEB1 (encoding a microtubule-binding protein) and expanded (encoding a FERM domain cytoskeletal protein and hyperplastic tumor suppressor); (4) genes encoding cell adhesion proteins scab (encoding an alpha-integrin), cadN (N-Cadherin), shg (E-Cadherin), and fat (encoding an atypical-cadherin and hyperplastic tumor suppressor); and (5) cytoarchitectural tumor suppressor genes scribble, lgl, and dlg, required for apical-basal cell polarity and cell proliferation inhibition. While some of these genes (brm, mor, expanded, fat, scribble, and lgl) have been previously shown or implicated to play a role in negatively regulating G1-S, a potential role for Trl, Znf72D, phyl, sina, trio, Abl, RpS6, wg, dsh, arm, dEB1, scab, cadN, and shg in inhibiting G1-S progression in Drosophila is novel. Further studies are required to determine whether Abl, RpS6, wg, dsh, arm, and shg do indeed suppress DmcycEJP by acting at the S-phase level and to understand the mechanism by which these genes act in G1-S regulation. The identification of novel classes of presumptive negative regulators of cyclin E or G1-S progression highlights the power of Drosophila whole-animal genetics as a tool for revealing new cell proliferation pathways (Brumby, 2004).

Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila

Apparent defects in cell polarity are often seen in human cancer. However, the underlying mechanisms of how cell polarity disruption contributes to tumor progression are unknown. Using a Drosophila genetic model for Ras-induced tumor progression, a molecular link has been shown between loss of cell polarity and tumor malignancy. Mutation of different apicobasal polarity genes activates c-Jun N-terminal kinase (JNK) signaling and downregulates the E-cadherin/β-catenin adhesion complex, both of which are necessary and sufficient to cause oncogenic RasV12-induced benign tumors in the developing eye to exhibit metastatic behavior. Furthermore, activated JNK and Ras signaling cooperate in promoting tumor growth cell autonomously, since JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras. The finding that such context-dependent alterations promote both tumor growth and metastatic behavior suggests that metastasis-promoting mutations may be selected for based primarily on their growth-promoting capabilities. Similar oncogenic cooperation mediated through these evolutionarily conserved signaling pathways could contribute to human cancer progression (Igaki, 2006).

Most human cancers originate from epithelial tissues. These epithelial tumors, except for those derived from squamous epithelial cells, normally exhibit pronounced apicobasal polarity. However, these tumors commonly show defects in cell polarity as they progress toward malignancy. Although the integrity of cell polarity is essential for normal development, how cell polarity disruption contributes to the signaling mechanisms essential for tumor progression and metastasis is unknown. To address this, a recently established Drosophila model of Ras-induced tumor progression triggered by loss of cell polarity has been used. This fly tumor model exhibits many aspects of metastatic behaviors observed in human malignant cancers, such as basement membrane degradation, loss of E-cadherin expression, migration, invasion, and metastatic spread to other organ sites (Pagliarini, 2003). In the developing eye tissues of these animals, loss of apicobasal polarity is induced by disruption of evolutionarily conserved cell polarity genes such as scribble (scrib), lethal giant larvae (lgl), or discs large (dlg), three polarity genes that function together in a common genetic pathway, as well as other cell polarity genes such as bazooka, stardust, or cdc42. Oncogenic Ras (RasV12), a common alteration in human cancers, causes noninvasive benign overgrowths in these eye tissues (Pagliarini, 2003). Loss of any one of the cell polarity genes somehow strongly cooperates with the effect of RasV12 to promote excess tumor growth and metastatic behavior. However, on their own, clones of scrib mutant cells are eliminated during development in a JNK-dependent manner; expression of RasV12 in these mutant cells prevents this cell death (Igaki, 2006).

To better quantify the metastatic behavior of tumors in different mutant animals, the analysis focused on invasion of the ventral nerve cord (VNC), a process in which tumor cells leave the eye-antennal discs and optic lobes (the areas where they were born) and migrate to and invade a different organ, the VNC. It was further confirmed that the genotypes associated with the invasion of the VNC in this study also resulted in the presence of secondary tumor foci at distant locations, although the number and size of these foci were highly variable (Pagliarini, 2003; Igaki, 2006).

In analyzing the global expression profiles of noninvasive and invasive tumors induced in Drosophila developing eye discs, it was observed that expression of the JNK phosphatase puckered (puc) was strongly upregulated in the invasive tumors. Upregulation of puc represents activation of the JNK pathway in Drosophila. Therefore an enhancer-trap allele, puc-LacZ, was used to monitor the activation of JNK signaling in invasive tumor cells. Strong ectopic JNK activation was present in invasive tumors, while only a slight expression of puc was seen in restricted regions of RasV12-induced noninvasive overgrowth. Intriguingly, more intense JNK activation was seen in tumor cells located in the marginal region of the eye-antennal disc and tumor cells invading the VNC. Analysis of clones of cells with a cell polarity mutation alone revealed that JNK signaling was activated by mutation of cell polarity genes. Notably, JNK signaling was not activated in a strictly cell-autonomous fashion. JNK activation in these cells was further confirmed by anti-phospho-JNK antibody staining that detects activated JNK (Igaki, 2006).

To examine the contribution of JNK activation to metastatic behavior, the JNK pathway was blocked by overexpressing a dominant-negative form of Drosophila JNK (BskDN). As previously reported (Pagliarini, 2003), clones of cells mutant for scrib, lgl, or dlg do not proliferate as well as wild-type clones, while combination of these mutations with RasV12 expression resulted in massive and metastatic tumors. Strikingly, inhibition of JNK activation by BskDN completely blocked the invasion of the VNC, as well as secondary tumor foci formation. Drosophila has two homologs of TRAF proteins (DTRAF1 and DTRAF2), which mediate signals from cell surface receptors to the JNK kinase cascade in mammalian systems. It was found that RNAi-mediated inactivation of DTRAF2, but not DTRAF1, in the tumors strongly suppressed their metastatic behavior. Inactivation of dTAK1, a Drosophila JNK kinase kinase (JNKKK), or Hep, a JNKK, also suppressed metastatic behavior. Drosophila has two known cell surface receptors that act as triggers for the JNK pathway, Wengen (TNF receptor) and PVR (PDGF/VEGF receptor). Intriguingly, it was found that RNAi-mediated inactivation of Wengen partially suppressed tumor invasion. Inactivation of PVR, in contrast, did not show any suppressive effect on metastatic behavior. It was also found that the metastatic behavior of RasV12-expressing tumors that were also mutated for one of three other cell polarity genes, bazooka, stardust, or cdc42, was also blocked by BskDN. These data indicate that loss of cell polarity contributes to metastatic behavior by activating the evolutionarily conserved JNK pathway (Igaki, 2006).

Next, whether JNK activation is sufficient to trigger metastatic behavior in RasV12-induced benign tumors was examined. Two genetic alterations can be used to activate JNK in Drosophila. First, JNK signaling can be activated by overexpression of Eiger, a Drosophila TNF ligand. While mammalian TNF superfamily proteins activate both the JNK and NFκB pathways, Eiger has been shown to specifically activate the JNK pathway through dTAK1 and Hep. Indeed, the eye phenotype caused by Eiger overexpression could be reversed by blocking JNK through Bsk-IR (Bsk-RNAi). Second, overexpression of a constitutively activated form of Hep (HepCA) can also activate JNK signaling. However, the eye phenotype caused by HepCA overexpression was only slightly suppressed by Bsk-IR, suggesting that HepCA overexpression may have additional effects other than JNK activation. Therefore Eiger overexpression was used to activate JNK in RasV12-induced benign tumors, and it was found that the RasV12+Eiger-expressing tumor cells did not result in the invasion of the VNC. This indicates that loss of cell polarity must induce an additional downstream effect(s) essential for metastatic behavior. A strong candidate for the missing event is downregulation of the E-cadherin/catenin adhesion complex, since this complex is frequently downregulated in malignant human cancer cells and is also downregulated by loss of cell polarity genes in Drosophila invasive tumors (Pagliarini, 2003). In addition, it has been recently reported that higher motility of mammalian scrib knockdown cells can be partially rescued by overexpression of E-cadherin-catenin fusion protein, suggesting a role of E-cadherin in preventing polarity-dependent invasion. Furthermore, overexpression of E-cadherin blocks metastatic behavior of RasV12/scrib−/− tumors (Pagliarini, 2003), indicating that loss of E-cadherin is essential for inducing tumor invasion in this model. It was found that loss of the Drosophila E-cadherin homolog shotgun (shg), combined with the expression of both RasV12 and Eiger, induced the invasion of the VNC. Intriguingly, loss of shg in RasV12-expressing clones also showed a weak invasive phenotype at lower penetrance. In agreement with the essential role of JNK in tumor invasion, clones of shg−/− cells weakly upregulated puc expression. It was further found that JNK activation in dlg−/− clones is not blocked by overexpression of E-cadherin, suggesting that mechanism(s) other than loss of E-cadherin also exist for inducing JNK activation downstream of cell polarity disruption. The metastatic behavior of RasV12+Eiger/shg−/− tumors was completely blocked by coexpression of BskDN, indicating a cell-autonomous requirement of JNK activation for this process. Furthermore, it was found that loss of the β-catenin homolog armadillo also induced metastatic behavior in RasV12-induced benign tumors. In contrast, overexpression of HepCA in RasV12/shg−/− cells resulted in neither enhanced tumor growth nor metastatic behavior. Together, these results suggest that, although the RasV12+Eiger/shg−/− does not completely phenocopy the effect of RasV12/scrib−/−, activation of JNK signaling and inactivation of the E-cadherin/catenin complex are the downstream components of cell polarity disruption that trigger metastatic behavior in RasV12-induced benign tumors (Igaki, 2006).

Aside from its evolutionarily conserved role in cell migration and invasion, JNK signaling is also a potent activator of cell death in Drosophila and mammals. Although RasV12-expressing tissues showed a weak and restricted activation of JNK at later stages of development, mutation of cell polarity genes in combination with RasV12 expression constitutively activated JNK signaling. Striking acceleration of tumor growth occurred during days 5 and 6, and these tumors outcompeted surrounding wild-type tissues, resulting in a loss of the unlabeled wild-type cells and a dramatic increase in the GFP-expressing mutant tissue. The activated JNK was correlated with this accelerated tumor growth, suggesting that JNK signaling may play a role in tumor growth. Indeed, in addition to blocking metastatic behavior, inactivation of JNK pathway components strongly suppressed the accelerated tumor growth caused by cell polarity disruption. These results reveal that JNK signaling activated by loss of cell polarity also stimulates tumor growth (Igaki, 2006).

Since JNK signaling is required for both tumor growth and invasion, it was next asked whether these two phenotypes are separable processes. To address this, different types of tumors caused by alterations in genes involved in cell proliferation, growth, and cell polarity were analyzed. Day 6 RasV12/scrib−/− tumors showed moderate tumor growth and VNC invasion phenotypes. Loss of the Akt gene, a component of insulin growth signaling, considerably reduced the tumor load of RasV12/scrib−/− animals but did not impair metastatic behavior. In contrast, overexpression of Akt, combined with mutations in both the scrib gene and the lats gene, a potent tumor suppressor, did not cause metastatic behavior despite accelerated tumor growth comparable to RasV12/scrib−/−. In addition, although RasV12/Tsc1−/− mutant cells resulted in extremely large tumors, these tumor cells never exhibited metastatic behavior. These data indicate that tumor growth and invasion are separable processes in this model system (Igaki, 2006).

It was found that JNK signaling is indeed activated in polarity-deficient cells, and acridine orange staining revealed that most of these cells die. Interestingly, ectopic cell death was mostly blocked within clones of polarity-deficient cells also expressing RasV12, despite strong JNK activation. In addition, coexpression of RasV12 and Eiger, a potent inducer of cell death, resulted in accelerated tumor growth, although neither RasV12 alone nor Eiger alone caused dramatic overgrowth. This massive overgrowth was completely blocked by coexpression of BskDN. Moreover, stimulation of JNK signaling by expressing Eiger dramatically enhanced tumor growth of RasV12/shg−/− tissues, although Eiger/shg−/− clones were very small, probably because of cell death of these mutant clones. The accelerated growth of the RasV12+Eiger/shg−/− tumors was again completely blocked by BskDN. Together, these data indicate that, in the context of oncogenic Ras, JNK activation is the primary mediator of tumor growth downstream of cell polarity disruption. These observations suggest that JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras, and that the dramatic tumor growth is caused by cooperation between oncogenic Ras and JNK signaling (Igaki, 2006).

This study provides a molecular link between loss of cell polarity and tumor malignancy, namely activation of JNK signaling and inactivation of the E-cadherin/catenin complex in the context of oncogenic Ras activation. Although RasV12 alone only induces noninvasive overgrowth, and loss of cell polarity alone results in JNK-mediated cell death, the combination of these two alterations promotes both tumor growth and invasion through oncogenic cooperation. Thus, the tumor-promoting alterations caused by loss of cell polarity do not function alone and rather act as oncogenic Ras modifiers or “oncomodifiers” (Igaki, 2006).

The JNK signaling is essential for a variety of biological processes such as morphogenesis, cell proliferation, migration, invasion, and cell death. Genetic studies in Drosophila have demonstrated that JNK signaling is essential for epithelial cell movements and invasive behavior during normal development. A genetic study in mice revealed that TNF-triggered JNK signaling stimulates epidermal proliferation. These studies suggest that JNK may play an important role in tumorigenesis, tumor growth, and metastasis. Indeed, a substantial body of evidence indicates that JNK activation and c-Jun phosphorylation play important roles in cancer development. In mammalian cell culture systems, Ras acts cooperatively with JNK or c-Jun to enhance cellular transformation. Furthermore, knockin mice expressing a mutant form of c-Jun (JunS63A,S73A) suppress development of skin tumors in response to Ras activation and also block development of intestinal epithelial cancers caused by APC mutation. Moreover, liver-specific inactivation of c-Jun impairs development of chemically induced hepatocellular carcinomas. Furthermore, JNK signaling is activated in many tumor types. On the contrary, however, it has been also shown that JNK functions as a negative regulator for tumor development in Ras/p53-transformed fibroblasts. Thus, the role of JNK signaling seems to be highly dependent on cellular context, and, this study provides the first evidence for a cell-autonomous oncogenic cooperation between JNK and Ras signaling that promotes tumor growth and malignancy (Igaki, 2006).

How is JNK signaling activated? Loss of cell polarity may directly influence activity of a JNK pathway component. Alternatively, cell polarity defects may activate a cell surface receptor that triggers JNK signaling. The genetic analysis of multiple JNK pathway components suggests that the pathway is activated through a cell surface receptor, Wengen. It would be interesting to further investigate whether mislocalization or disregulation of Wengen, which should be normally tightly regulated in polarized epithelial cells, results in stimulation of JNK pathway signaling (Igaki, 2006).

The discovery that metastasis-promoting alterations (i.e., JNK activation) also increase tumor growth may explain why tumor cells acquire such mutations; that is, they primarily provide a selective advantage in tumor growth. Given that cell polarity defects are frequently associated with human tumor malignancy, and that the pathways identified in Drosophila are evolutionarily conserved, similar molecular mechanisms could be involved in human tumor progression. It would be particularly interesting to study these processes in human tumors with high frequencies of Ras mutations. If such processes prove conserved, components of these pathways, especially JNK signaling, could serve as potential therapeutic targets against such cancers (Igaki, 2006).

Basolateral junctions utilize warts signaling to control epithelial-mesenchymal transition and proliferation crucial for migration and invasion of Drosophila ovarian epithelial cells

Fasciclin2 (Fas2) and Discs large (Dlg) localize to the basolateral junction (BLJ) of Drosophila follicle epithelial cells and inhibit their proliferation and invasion. To identify a BLJ signaling pathway a genome-wide screen was performed for mutants that enhance dlg tumorigenesis. Two genes were identified that encode known BLJ scaffolding proteins, lethal giant larvae (lgl) and scribble (scrib), and several not previously associated with BLJ function, including warts (wts) and roughened eye (roe/rotund), which encode a serine-threonine kinase and a transcription factor, respectively. Like scrib, wts and roe also enhance Fas2 and lgl tumorigenesis. Further, scrib, wts, and roe block border cell migration, and cause noninvasive tumors that resemble dlg partial loss of function, suggesting that the BLJ utilizes Wts signaling to repress EMT and proliferation, but not motility. Apicolateral junction proteins Fat (Ft), Expanded (Ex), and Merlin (Mer) either are not involved in these processes, or have highly spatio-temporally restricted roles, diminishing their significance as upstream inputs to Wts in follicle cells. This is further indicated in that Wts targets, CyclinE and DIAP1, are elevated in Fas2, dlg, lgl, wts, and roe cells, but not Fat, ex, or mer cells. Thus, the BLJ appears to regulate epithelial polarity and dynamics not only as a localized scaffold, but also by communicating signals to the nucleus. Wts may be regulated by distinct junction inputs depending on developmental context (Zhao, 2008).

The purpose of this work was to gain greater insight into how the BLJ suppresses epithelial tumorigenesis and invasion by identifying and understanding the function of new genes important for BLJ function. To do so, a genomewide screen was completed for enhancers of dlg, which encodes a scaffolding protein that is a crucial organizer of the BLJ and is a potent repressor of follicle epithelial cell tumorigenesis and invasion. Deficiencies that cumulatively span ∼80% of the autosomes, or 64% of the Drosophila genome were systematically screened. A relatively small number of enhancers, ∼1 per 1000 genes screened, were detected indicating that the screen selected for loci specifically required for dlg function. Thus, the novel dlg enhancer genes that were identified, wts, roe, ebi, as well as at least two genes yet to be identified, are likely to be key collaborators with dlg in suppressing epithelial invasion. The specificity of the interactions between dlg and these enhancers is further indicated in that more than one allele of each gene showed an interaction, in several dlg backgrounds, and the strengths of enhancement were similar to deficiencies defining each locus. wts, roe, and ebi also enhanced Fas2 and lgl, indicating that they are not just important for dlg function, but for the function of the BLJ as a whole. In addition, overexpression of all enhancers except ebi suppressed dlg and Fas2 tumorigenesis, further confirming that the identified genes function in a BLJ network (Zhao, 2008).

BLJ pathway components in the nucleus and their putative relationship to Notch: ebi encodes an F-box protein with WD repeats that promotes protein degradation of specific targets. The failure of ebi overexpression to suppress Fas2 or dlg, and the relatively mild ebi phenotypes (midoogenesis small-nucleus and epithelial-organization defects, but no defects in germinal vesicle localization), suggest that ebi may function in only one of the three branches of BLJ signaling or in a parallel pathway to the BLJ. In the eye, ebi is important for promoting differentiation and inhibiting proliferation, which appear to be separable functions. Thus ebi could enhance Fas2 and dlg tumorigenesis by functioning within the proliferation-repressing branch of the BLJ, or the importance of ebi for differentiation suggests that it could function in the EMT branch of the BLJ or both. In contrast, ebi promotes protein degradation in response to Notch (N) and Drosophila EGF receptor (EgfR) signals, suggesting that it may act in a parallel pathway. Both Ebi and its mammalian homolog, TBL1, function in a corepressor complex through association with nuclear hormone transcriptional corepressor SMRTER/SMRT (Zhao, 2008).

Interestingly, although most N appears to be localized on the apical surface of follicle cells, some N is also localized in BLJs. Thus, it is possible that N localized to the BLJ may signal directly to Ebi. Consistent with this possibility, it was found that all of the genes in the BLJ network share some midoogenesis defects with N, including the small nucleus phenotype, epithelial stratification defects, and mislocalization of the germinal vesicle. The epithelial defects are also reminiscent of N-pathway mutants brainiac and egghead, which are required in the germ line for regulating N that is localized on the apical surface of the follicle cells abutting the germ line. Thus one possibility is that N signaling activity is regulated by its localization to apical vs. basolateral junctions in response to several signaling pathways acting during midoogenesis (Zhao, 2008).

The other modest dlg enhancer that was identified, roe, encodes a Krüppel-family zinc-finger protein that appears to be a transcription factor. Roe is also implicated in Notch signaling and thus may function with Ebi in N-dependent processes as proposed above. However, in contrast to ebi, roe loss caused follicle cell tumors, suggesting that roe may function more directly in a BLJ pathway than ebi. Consistent with a direct role for Roe in BLJ signaling, it was found that roe overexpression suppressed Fas2 and dlg tumorigenesis. Further, as for Fas2, dlg, and wts, roe represses CycE and DIAP1 expression (Zhao, 2008).

Warts was of special interested because of the many similarities observed in the quality and strength of wts and scrib phenotypes, suggesting that they are components in a BLJ signaling pathway, rather than a parallel pathway that cross talks with BLJ signaling. wts encodes a serine/threonine kinase that is an ortholog of human tumor suppressors Lats1 and Lats2, both of which have been linked to highly aggressive breast cancers. The prevailing model for Wts signaling in Drosophila is based on signaling in eye and wing tissue. Wts appears to relay signals from apicolateral junction proteins Ft, Ex, and Mer in wing and eye tissues. However, the results from almost every assay, including early tumor formation, border cell migration, BrdU, PH3, CycE, and DIAP1 expression, indicated little functional overlap between Ft, ex, mer, or mer; ex and wts, thus diminishing the importance of apicolateral Ft-Ex-Mer for Wts activation in follicle cells. The exceptions were that during midoogenesis, Mer is required for border cell migration and Ex is required for the endocycle switch, while both are required for maintenance of epithelial integrity and positioning of the germinal vesicle. However, the involvement of Ex and Mer in these processes are fundamentally distinct from how they act in Wts-dependent processes in other tissues. (1) Ft is not involved; (2) no indication was observed of Ex-Mer synergism; (3) ex, mer, and mer; ex phenotypes are relatively mild when compared to wts. It is concluded that the model for Wts activation in which apicolateral junction proteins Ft, Ex, and Mer play the predominant role cannot be universally applicable in all cell types. Rather, the relative importance of Ex and Mer for Wts regulation appears to depend on developmental context (Zhao, 2008).

Consistent with this proposal, strong functional interdependence and phenotypic similarities were found between Fas2, dlg, lgl, scrib, and wts, thus indicating that the BLJ, not the apicolateral junction, plays the predominant role in Wts regulation during oogenesis. Although genetic evidence alone cannot completely rule out that Wts may act in a parallel pathway to the BLJ and impinge on a set of downstream targets that overlap with those targeted by the BLJ, the following observations favor a model in which the BLJ is more directly involved in Wts regulation (it is noted that these are not mutually exclusive alternatives): (1) over 50 tumor suppressor genes have been identified in Drosophila, but lgl, scrib, and wts were the only strong dlg enhancers identified in this genomewide screen; (2) wts showed strong genetic interactions with Fas2, dlg, and lgl, similar to or stronger than scrib, which encodes a known BLJ protein; (3) wts has early tumor phenotypes similar to dlg partial loss of function and to scrib; (4) wts has the same border cell migration phenotype as scrib; (5) wts has similar small nucleus, epithelial stratification, and germinal vesicle defects as Fas2, dlg, lgl, and scrib; (6) like lgl and scrib, wts overexpression suppressed Fas2 and dlg tumorigenesis; (7) Fas2, dlg, and wts have similar proliferation defects, and (8) Fas2, dlg, and wts similarly repress CycE and DIAP1 expression, which is especially crucial, because CycE and DIAP1 are downstream targets of Wts signaling, and ex and mer had no impact on their expression, contrary to results in other tissues. Thus, the data strongly indicate that the BLJ signals through Wts, and may impinge on Roe in the nucleus, thus suggesting the first BLJ signaling pathway in animal cells. This implies that the BLJ not only acts as a localized scaffold, but also signals to the nucleus to control gene expression, both of which cooperate to regulate epithelial polarity and dynamics (Zhao, 2008).

How can these results in follicle cells, which suggest that Wts acts predominantly downstream of the BLJ, be reconciled with findings in eye tissue, which indicate that Wts acts downstream of the apicolateral junction? Interestingly, the genetic data in the eye suggest that Ft, Ex, and Mer cannot account for all of the signals that activate Wts, because wts overgrowth and tissue disorganization phenotypes are more severe than ft or mer; ex. On the basis of these findings in follicle cells, it is possible that Wts activation in the eye requires additional input from the BLJ. This possibility may have been overlooked thus far because dlg does not appear to have an overgrowth phenotype in the eye. dlg may be essential for additional functions in the eye that are epistatic to its tumor suppressor function, thus preventing loss of cells from the epithelium that could mask an overgrowth phenotype. Consistent with this, when activated Rasv12 is combined with dlg loss, dramatic tumors develop that are larger and more invasive than those produced by Rasv12 alone (Zhao, 2008).

In contrast, Dlg may have a diminished role in Wts signaling in the eye, much as the evidence indicates a diminished role for Ex and Mer in Wts signaling in the ovary. According to this model, Wts receives predominant input from distinct lateral junctions depending on tissue context. One distinction is that ovarian follicle cells are derived from a mesodermal lineage, while the eye and wing tissues are from ectodermal lineages. Further, many genes that disrupt apical-basal polarity and epithelial morphology have only subtle phenotypes in the eye by comparison to the ovary or embryo. Finally, the follicular epithelium requires input from junctions on all three follicle cell surfaces, lateral, apical, and basal, whereas most epithelia require only two, lateral and apical or basal. Thus, ovarian and imaginal tissues are likely to organize signaling pathways acting downstream of epithelial junctions in similar, yet fundamentally different ways to meet the unique organizational requirements of their cell-tissue morphologies. Some or all of these differences may contribute to the suggested specificity observed in Wts signaling downstream of BLJs in follicle cells. In general, these findings raise the possibility for future investigation that depending on the cell-tissue morphologies of a given organ, one lateral junction may play a predominant organizational role, and Wts signaling may act as a universal signaling adapter for mediating contact inhibition from that junction (Zhao, 2008).

An especially interesting aspect of Mer and Ex function that was uncovered in follicle cells is that it appears to be restricted to predominantly postmitotic, differentiated cells, in contrast to the role of Mer and Ex in other tissues. Further, given the absence of an involvement of Ft and lack of Mer-Ex synergism it is concluded that if Mer and Ex would be involved in Wts activation in follicle cells, they would have to function via a fundamentally distinct mechanism than in other tissues. It is proposed that during early oogenesis, the BLJ alone may provide the predominant input to Wts. Then, during midoogenesis, Ex and Mer may become involved in novel interactions with Dlg or other components of the BLJ to activate Wts in spatiotemporally distinct populations of differentiating cells to help achieve their unique developmental functions (Zhao, 2008).

How do wts, scrib, and roe promote motility? It is proposed that Scrib, Wts, and Roe are all crucially involved in EMT. In EMT, cells (1) loose apical-basal polarity and become mesenchymal-like, and (2) adopt a polarity conducive to movement. scrib, wts, and roe cells clearly lose epithelial polarity and become mesenchymal-like as indicated by their rounded morphology and lateralized phenotype. However, scrib, wts, and roe tumors do not invade, and scrib, wts, and roe border cells do not move, suggesting that the second aspect of EMT, adoption of a polarity conducive to movement, is defective. Consistent with this, mammalian Scrib is required for migration and epithelial wound healing of cultured human breast epithelial cells, and is also required in vivo for wound healing in mice. Human Scrib directs migration by organizing several polarities crucial for migration, including the orientation of the microtubule and Golgi networks and the localization of Cdc42 and Rac1 to the cell's leading edge. Thus Scrib has a conserved function in directed cell migration by organizing a polarity conducive to movement. In mammalian PC12 cells Scrib is in complex with Rac1. Fly Rac1 is essential for border cell migration and invasion of Fas2 and dlg tumors, suggesting that an essential role of Scrib in Rac1 function may be of crucial importance for movement. The apparent conserved role of BLJ proteins in organizing EMT, and both promoting and repressing movement, reemphasizes the suggestion that BLJ proteins do more than merely maintain apical-basal polarity, but rather repress a cellular transformation from epithelial polarity to a mesenchymal, lateralized signature conducive to movement (Zhao, 2008).

How is the function of scrib, wts, and roe in promoting border cell movement consistent with the requirement of Fas2, dlg, and lgl in repressing border cell movement? Further, how do scrib and wts act as enhancers of dlg tumor invasion even though scrib and wts tumors are noninvasive? For border cell movement, Fas2 and dlg mutations not only accelerate movement, but also delay border cell delamination. The delay in border cell delamination suggests that the BLJ normally promotes motility, but this promoting function can be bypassed when the repression of motility branch of the BLJ pathway is simultaneously lost. Cumulative data indicate that scrib, wts, and roe act predominantly within the EMT and proliferation branches of the BLJ pathway, and not the repression of motility branch. It is suggested that without simultaneous loss of the repression of motility branch of the BLJ pathway, scrib and wts border cells cannot bypass the essential requirement for the second step of EMT, thus border cell motility is blocked (Zhao, 2008).

This interpretation is also consistent with the seemingly paradoxical function of scrib and wts as enhancers of dlg tumor invasion, even though Scrib and Wts promote rather than repress border cell movement. The noninvasive scrib and wts tumor phenotypes indicate that they are crucial for repressing the first step of EMT, loss of epithelial polarity and adoption of a lateralized, mesenchymal-like phenotype. It has been suggested that scrib and wts enhance dlg invasive tumorigenesis by increasing the rate at which dlg mutant follicle cells undergo EMT and further facilitate invasion by depressing proliferation control and increasing the number of follicle cells available for movement. Thus, even though scrib and wts are required to promote movement, it is suggested that in dlg; scrib/+ or dlg; wts/+ tumors this requirement can be bypassed because the branch of the BLJ pathway that represses motility is simultaneously disrupted (Zhao, 2008).

The noninvasive tumor phenotypes of scrib and wts are very similar to the phenotypes of dlg mutants that specifically disrupt Dlg SH3 and GuK domains. Thus Scrib and Wts may act specifically downstream of the Dlg SH3 and GuK domains. Consistent with this, Scrib appears to associate with the Dlg GuK domain in neuronal synapses via the linker protein GuK-holder. Further, whereas Fas2, dlg, and lgl cause faster border cell migration, border cell migration is very similar to wild type in the dlg SH3/GuK-specific mutants, suggesting that Dlg SH3/GuK predominantly represses the first step of EMT and proliferation but not motility. On the basis of this specificity, it is suggest that one reason that lgl may be a stronger dlg enhancer than scrib and wts is that lgl represses motility in addition to EMT and proliferation. For example, the de novo tumor formation observed when one copy of lgl, scrib, or wts is removed in dlghf/dlgsw ovaries suggests that a threshold level of BLJ activity essential for maintenance of polarity has been lost. However, the lgl interaction may be much stronger than scrib and wts because lgl additionally represses motility (Zhao, 2008).

Increased expression of CycE and DIAP1, known Wts targets, was observed in Fas2, dlg, lgl, scrib, wts, and roe cells. Thus the importance of CycE for proliferation control, and DIAP1 for control of EMT and motility, suggests that part of the mechanism by which Fas2-Dlg represses tumorigenesis is through activating Wts signaling. DIAP1 is in a complex with Rac1 and Profilin and enables border cell motility apparently by promoting actin turnover. Further, in the embryo, DIAP1 loss leads to Dlg cleavage and cellular rounding and dispersal. Too much DIAP1 also appears to be deleterious to movement, because targeted overexpression of DIAP1 specifically in border cells slows their migration (data not shown). Thus maintaining the proper balance of DIAP1 is critical for directed movement, and it may be part of the mechanism by which Scrib and Wts influence border cell movement, suggesting that interaction with Dlg and Rac1 may be another level at which Scrib regulates EMT and movement, consistent with the possibility that it functions downstream of Scrib and Wts in follicle cells to repress both EMT and proliferation (Zhao, 2008).

In contrast to the strong enhancement of dlg by scrib, Fas2 was only weakly enhanced by scrib. Given the complexity of coordinating EMT, proliferation, and motility within an epithelial field, perhaps the simplest model is that multiple Dlg complexes reside within the BLJ, each with a distinct set of ligands that control one or more morphogenetic activities (Zhao, 2008).

Another interesting difference in the enhancement of dlg and Fas2 by lgl, scrib, wts, and roe was that they all enhanced both dlg tumorigenesis and invasion, but only enhanced Fas2 tumorigenesis, without invasion. An important difference between these experiments may be that in Fas2null follicle cells, Dlg is missing Fas2 as a ligand, whereas in dlghf/dlgsw, dlghf/dlgip20, and dlghf/dlglv55 follicle cells, Fas2 is localized at sites of contact between follicle cells in both the native epithelium and in streams of invading cells, suggesting that Fas2 continues to act as a Dlg ligand in these cells. This is probably an important difference because Fas2-Dlg binding is expected to control the conformation of Dlg. Dlg conformations in turn may specify Dlg intra- and intermolecular interactions that determine the relative balance of EMT, proliferation, and invasion factors that associate with the BLJ scaffold. For example, in neuronal cells intramolecular interactions between Dlg SH3 and GuK domains regulate the strength of intermolecular binding of GuK-holder, which binds Scrib. The SH3-GuK intramolecular interaction is further modulated by intramolecular interactions with PDZ3, which are regulated by intermolecular interactions with neurolignin, a transmembrane ligand for PDZ3 (Zhao, 2008).

On the basis of this molecular model, it is proposed that in the absence of Fas2, Dlg has a distinct conformation that tilts the balance toward EMT and proliferation over invasion, when Lgl, Scrib, Wts, or Roe are reduced. This study has shown that lgl, scrib, wts, and roe are expected to act predominantly downstream of Dlg SH3 and GuK domains to repress EMT and proliferation. Thus, removal of one copy of lgl, scrib, wts, or roe in Fas2 cells may tip the ratio of factors controlling EMT, motility, and proliferation toward derepression of EMT and proliferation, masking the Fas2 requirement for invasion. One possibility is that lgl, scrib, wts, or roe are especially important for expression of a protein in the apicolateral junction, such as Par-3/Bazooka, which is essential for dlg invasion. Consistent with this, Ex upregulation is seen in both dlg and wts clones. Further, lgl enhancement at the lglts permissive temperature showed essentially the opposite trend from Fas2. Rather than enhance tumorigenesis over invasion, removal of one copy of Fas2, dlg, scrib, wts, or roe in lgl egg chambers favored invasion. Thus, it is suggested that tumor invasiveness associated with particular combinations of mutated BLJ proteins may be masked or unmasked on the basis of the balance of activities that are disrupted, rather than disruption of particular activities per se (Zhao, 2008).

In summary, this study has identified the first signaling pathway that acts downstream of the BLJ that specifically controls EMT and proliferation, and important clues have been gained as to how this signaling may be organized. Like the Drosophila follicular epithelium, the human ovarian surface epithelium, which is thought to be the site of origin of most ovarian cancers, is derived from a mesodermal lineage. The data suggest that the BLJ plays an especially crucial role in the follicle cells compared to ectodermal lineages in repressing epithelial invasion and that the follicular epithelium appears to organize signaling from epithelial junctions in distinct ways compared to other epithelia. Given the conservation in the lineage of the fly and human epithelia, and the sensitivity of this screen for detecting molecules important for invasive carcinogenesis, it is proposed that the fly egg chamber may serve as a prototype for identifying early molecular events that are crucial for invasion of human ovarian cancer and possibly other malignancies that remain undetected before they start to invade (Zhao, 2008).

Intrinsic tumor suppression and epithelial maintenance by endocytic activation of Eiger/TNF signaling in Drosophila

Oncogenic alterations in epithelial tissues often trigger apoptosis, suggesting an evolutionary mechanism by which organisms eliminate aberrant cells from epithelia. In Drosophila imaginal epithelia, clones of cells mutant for tumor suppressors, such as scrib or dlg, lose their polarity and are eliminated by cell death. This study shows that Eiger, the Drosophila tumor necrosis factor (TNF), behaves like a tumor suppressor that eliminates oncogenic cells from epithelia through a local endocytic JNK-activation mechanism. In the absence of Eiger, these polarity-deficient clones are no longer eliminated; instead, they grow aggressively into tumors. In scrib clones endocytosis is elevated, which translocates Eiger to endocytic vesicles and leads to activation of apoptotic JNK signaling. Furthermore, blocking endocytosis prevents both JNK activation and cell elimination. These data indicate that TNF signaling and the endocytic machinery could be components of an evolutionarily conserved fail-safe mechanism by which animals protect against neoplastic development (Igaki, 2009).

Clones of cells mutant for Drosophila tumor suppressor genes, such as scrib or dlg, are eliminated from imaginal discs, suggesting an evolutionarily conserved fail-safe mechanism that eliminates oncogenic cells from epithelia. This study reports that this elimination of mutant cells is accomplished by endocytic activation of Eiger/TNF signaling. Eiger is a conserved member of the TNF superfamily in Drosophila, but its physiological function has been elusive. Although ectopic overexpression of Eiger can trigger apoptosis, flies deficient for eiger develop normally and exhibit no morphological or cell death defect. This study shows that Eiger is required for the elimination of oncogenic mutant cells from imaginal epithelia. This not only provides an explanation for previous unexplained observations, but also argues that Eiger is a putative intrinsic tumor suppressor in a fashion similar to mammalian p53 or ATM, which causes no phenotype when mutated, but protects animals as tumor suppressors when their somatic cells are damaged (Igaki, 2009).

The intrinsic tumor suppression found in scrib mutant clones was also observed in dlg mutant clones, suggesting that this is a mechanism triggered by loss of epithelial basolateral determinants. Intriguingly, it was found that mutant clones of salvador, the hippo pathway tumor suppressor, are not susceptible to similar effect of Eiger. These data suggest that the Eiger-JNK pathway behaves as an intrinsic tumor suppressor that eliminates cells with disrupted cell polarity (Igaki, 2009).

It is intriguing that Eiger's tumor suppressor-like function is dependent on endocytosis. The data show that Eiger is translocated to endosomes through endocytosis and activates JNK signaling in these vesicles. Moreover, blocking endocytosis abolishes both JNK activation and Eiger-dependent cell elimination. Endocytic activation of signal transduction has been observed for EGF and β2-adrenergic receptor signaling in mammalian cells. After endocytosis, these ligand/receptor complexes localize to endosomes, where they meet adaptor or scaffold proteins that recruit downstream signaling components. Therefore, the endocytic activation of Eiger/TNF-JNK signaling might also be achieved by the recruitment of its downstream signaling complex to the endosomes, possibly through a scaffold protein that resides in endosomes. Recent studies in Drosophila have shown that components of the endocytic pathway -- vps25, erupted, and avalanche -- function as tumor suppressors (Lu, 2005; Moberg, 2005; Thompson, 2005; Vaccari, 2005). Furthermore, mutations in endocytosis proteins have been reported in human cancers. Thus, deregulation of endocytosis may contribute to tumorigenesis. This study provides new mechanistic insights into the role of endocytosis in tumorigenesis (Igaki, 2009).

Mammalian TNF superfamily consists of at least 19 members. While many have been shown to play important roles in immune responses, hematopoiesis, and morphogenesis, the physiological functions for other members have yet to be determined. Mechanisms that eliminate damaged or oncogenic cells from epithelial tissues are essential for multicellular organisms, especially for long-lived mammals like humans. The tumor suppressor role of Eiger might have evolved for host defense or elimination of dying/damaged cells, such as cancerous cells, very early in animal evolution. Given that components of the Eiger tumor suppressor-like machinery (such as Eiger, endocytic pathway components, and JNK pathway components) are conserved from flies to humans, it is also possible that Eiger and its mammalian counterparts are components of an evolutionarily conserved fail-safe by which animals maintain their epithelial integrity to protect against neoplastic development (Igaki, 2009).

Interaction between RasV12 and scribbled clones induces tumour growth and invasion

Human tumours have a large degree of cellular and genetic heterogeneity. Complex cell interactions in the tumour and its microenvironment are thought to have an important role in tumorigenesis and cancer progression. Furthermore, cooperation between oncogenic genetic lesions is required for tumour development; however, it is not known how cell interactions contribute to oncogenic cooperation. The genetic techniques available in the fruitfly Drosophila melanogaster allow analysis of the behaviour of cells with distinct mutations, making this the ideal model organism with which to study cell interactions and oncogenic cooperation. In Drosophila eye-antennal discs, cooperation between the oncogenic protein RasV12 and loss-of-function mutations in the conserved tumour suppressor scribbled (scrib) gives rise to metastatic tumours that display many characteristics observed in human cancers. This study shows that clones of cells bearing different mutations can cooperate to promote tumour growth and invasion in Drosophila. The RasV12 and scrib- mutations can also cause tumours when they affect different adjacent epithelial cells. This interaction between RasV12 and scrib- clones involves JNK signalling propagation and JNK-induced upregulation of JAK/STAT-activating cytokines, a compensatory growth mechanism for tissue homeostasis. The development of RasV12 tumours can also be triggered by tissue damage, a stress condition that activates JNK signalling. Given the conservation of the pathways examined in this study, similar cooperative mechanisms could have a role in the development of human cancers (Wu, 2010).

Clones of mutant cells marked with green fluorescent protein (GFP) can be generated in the eye-antennal imaginal discs of Drosophila larvae by mitotic recombination. Clones expressing RasV12, an oncogenic form of the Drosophila Ras85D protein, moderately overgrow. Clones mutant for scrib lose apico-basal polarity and die. In contrast, scrib clones simultaneously expressing RasV12 grow into large metastatic tumours. To understand better the cooperation between these two mutations, animals were produced in which cell division after a mitotic recombination event creates two daughter cells: one expressing RasV12 and the other mutant for scrib. Discs containing adjacent RasV12 (GFP-positive) and scrib- clones developed into large tumours, capable of invading the ventral nerve cord. This shows that RasV12 and scrib also cooperate for tumour induction when they occur in different cells. These tumours are referred to as RasV12//scrib- tumours, to denote interclonal oncogenic cooperation and distinguish them from RasV12scrib- tumours, in which cooperation occurs in the same cells intraclonally (Wu, 2010).

This study has used Drosophila to investigate how oncogenic cooperation between different cells can promote tumour growth and invasion. These experiments, addressed to understanding interclonal cooperation in RasV12//scrib- tumours, uncovered a two-tier mechanism by which scrib- cells promote neoplastic development of RasV12 cells: (1) propagation of stress-induced JNK activity from scrib- cells to RasV12 cells; and (2) expression of the JAK/STAT-activating Unpaired cytokines downstream of JNK. These findings, therefore, highlight the importance of cell interactions in oncogenic cooperation and tumour development. It was also shown that stress-induced JNK signalling and epigenetic factors such as tissue damage can contribute to tumour development in flies. Notably, tissue damage caused by conditions such as chronic inflammation has been linked to tumorigenesis in humans. Furthermore, expression of the Unpaired cytokines promotes tumour growth as well as an antitumoural immune response, which parallels the situation in mice and humans. Future research into phenomena such as compensatory growth and interclonal cooperation in Drosophila will provide valuable insights into the biology of cancer (Wu, 2010).

Tumor suppression by cell competition through regulation of the Hippo pathway

Homeostatic mechanisms can eliminate abnormal cells to prevent diseases such as cancer. However, the underlying mechanisms of this surveillance are poorly understood. This study investigated how clones of cells mutant for the neoplastic tumor suppressor gene scribble (scrib) are eliminated from Drosophila imaginal discs. When all cells in imaginal discs are mutant for scrib, they hyperactivate the Hippo pathway effector Yorkie (Yki), which drives growth of the discs into large neoplastic masses. Strikingly, when discs also contain normal cells, the scrib- cells do not overproliferate and eventually undergo apoptosis through JNK-dependent mechanisms. However, induction of apoptosis does not explain how scrib- cells are prevented from overproliferating. This study reports that cell competition between scrib- and wild-type cells prevents hyperproliferation by suppressing Yki activity in scrib- cells. Suppressing Yki activation is critical for scrib- clone elimination by cell competition, and experimental elevation of Yki activity in scrib- cells is sufficient to fuel their neoplastic growth. Thus, cell competition acts as a tumor-suppressing mechanism by regulating the Hippo pathway in scrib- cells (Chen, 2012).

This study shows that tumorigenic scrib- cells are removed from Drosophila imaginal discs by a cell-cell signaling event that suppresses elevated Yki activity in scrib- cells. Previous reports implicated JNK as a mediator of cell competition of scrib- clones, where it induces apoptosis and suppresses proliferation. However, it was not known how JNK prevents scrib- clones from hyperproliferating. This study now provides evidence that JNK prevents scrib- clones from hyperproliferating by regulating the activity of the Hippo pathway effector Yki. First, scrib- clones that do not face cell competition up-regulate Yki activity, which drives their hyperproliferation. Second, when scrib- clones do face cell competition, then JNK signaling prevents the upregulation of Yki activity. Third, experimental up-regulation of Yki activity is sufficient to rescue scrib- clones from being eliminated by cell competition. Fourth, experimental suppression of Yki activity in scrib- clones not subjected to cell competition is sufficient to suppress their hyperproliferation. Therefore, cell competition suppresses up-regulation of Yki activity in scrib- cells, and this suppression is important for the elimination of scrib- clones by cell competition. Previous reports showed that Hippo pathway reporters can be up-regulated in scrib- and lgl− mutant discs and clones and that Yki is required for the overgrowth of scrib-+BskDN cells not subjected to cell competition. However, these studies did not analyze the effects of cell competition on Yki activity in scrib- cells. This analysis now shows that scrib- cells facing cell competition do not up-regulate Yki activity and thereby identifies a mechanism that is critical for the elimination of scrib- cells. Although it was reported that scrib- and lgl clones can upregulate ex-lacZ expression and Yki activity (Chen, 2012).

However, upon quantification it was found that the majority of scrib- clones have normal or reduced levels of ex-lacZ expression, and only a small percentage of scrib- clones have elevated levels of ex-lacZ expression. Clones with elevated ex-lacZ expression were observed mainly in the hinge region of wing discs, which may provide an environment of reduced cell competition. Thus, outcompeted scrib- clones do not have elevated levels of Yki activity. In contrast, when scrib- clones are rescued from cell competition, they show highly elevated levels of ex-lacZ expression. Similarly, discs that are entirely mutant for scrib, thereby creating an environment that does not have competing normal cells, show hyperactivation of Yki. Cell competition thus prevents the hyperactivation of Yki in scrib- clones and turns a potential high-Yki 'supercompeting' scrib- cell into a cell of lower fitness and less resistance to apoptosis. Importantly, scrib- wts- and scrib-+Yki clones show greatly increased growth and survival compared with scrib- clones. These results show that elevated levels of Yki are sufficient to protect scrib- cells from being outcompeted. Thus, if Yki activity already was high in scrib- cells facing cell competition, those cells would not be outcompeted, and overexpression of Yki or loss of wts would not cause such dramatic effects on the survival and growth of scrib- clones. Apparently, Yki levels in scrib- cells facing cell competition are not high enough for these cells to evade cell competition. Thus, the amount of Yki activity in scrib- cells is a critical determinant of whether scrib- clones are eliminated or form tumorous tissue, and the suppression of Yki activity in scrib- clones is important for the elimination of scrib- clones by cell competition (Chen, 2012).

These studies show that JNK activity is required in scrib- cells for the suppression of Yki activity by cell competition. In contrast, JNK signaling can induce Yki activity during regeneration and compensatory proliferation in imaginal discs. Therefore, the effects of JNK signaling on Yki activity in scrib- cells are different from those in normal cells: JNK signaling activates Yki in normal cells promoted to regenerate but suppresses Yki in scrib- cells induced to be eliminated. Interestingly, both these effects are observed in discs with scrib- clones. In scrib- cells, JNK activity suppresses the hyperactivation of Yki, but in neighboring cells that are stimulated to proliferate and compensate for the loss of scrib- cells, the activities of both JNK and Yki are elevated. However, non-cell-autonomous effects on Yki reporters were still observed in egr−/− animals and in discs that ubiquitously inhibited JNK signaling by BskDN. Therefore, JNK-independent signals contribute to the non-cell-autonomous induction of Yki activity around scrib- clones. The regulation of Yki by JNK signaling thus is complex and context dependent and may involve several mechanisms (Chen, 2012).

The observation that wts- scrib- clones overgrow indicates that JNK and Wts function in parallel to regulate Yki or that JNK regulates the Hippo pathway upstream of Wts. JNK can phosphorylate and activate Yap1 to regulate apoptosis in mammalian cells. Notably, the JNK phosphorylation sites of Yap1 are different from the Lats phosphorylation sites, supporting a model in which JNK functions in parallel with Wts to regulate Yki activity. However, it is not known whether the same sites also act to suppress the activity of Yki in other contexts (Chen, 2012).

Although several models have been proposed to explain how cell-cell interactions between scrib- and normal cells lead to the elimination of scrib- clones from epithelia, it was not clear what properties normal cells must possess to perform this tumorsuppressive role. The data demonstrate that for scrib- cells to be eliminated they must be juxtaposed with cells that have higher levels of competitive fitness, not just proper cellular architecture. Overexpression of the Myc or RasV12 oncogenes in scrib- clones increases their fitness. As a result, in scrib- clones cell competition does not suppress Yki activity, which protects these clones from being eliminated. Interestingly, Myc expression also synergizes with loss of scrib to form tumors in mammals, and the data offer a model to explain this phenomenon. In addition to the cell-autonomous hyperproliferation, scrib- cells that are not removed from imaginal discs have profound non-cell-autonomous effects on the Hippo pathway. This non-cell-autonomous Hippo pathway-regulating signal may serve normally as a regenerative growth signal that facilitates the replacement of eliminated or dying cells, such as outcompeted scrib- cells. If scrib- clones are not eliminated efficiently, however, this signal may persist longer than required to restore the tissue, thereby causing overgrowth and deformation of neighboring tissue. Thus, continued residence of tumorigenic cells can stimulate growth beyond that needed for compensation, essentially hijacking the proliferation and regeneration programs of their normal neighbors. Therefore, the non-cell-autonomous activation of Yki by scrib- cells may have important implications for tumor-stromal interactions in human cancers (Chen, 2012).

In summary, it is concluded that cell competition is crucial in suppressing the tumorigenic capacity of scrib- cells and does so by regulating their Yki activity. Loss of this regulation results in overproliferation of both tumorigenic cells and neighboring wild-type cells. Efficient elimination of scrib- clones by cell competition prevents Yki-fueled overgrowth of mutant cells and prevents them from disrupting proliferation control of their normal neighbors. Thus, this study identified a tumor-suppression mechanism that depends on signaling between normal and tumorigenic cells. These data identify evasion of cell competition as a critical step toward malignancy and illustrate a role for wild-type tissue in preventing the formation of cancers (Chen, 2012).

Drosophila Src regulates anisotropic apical surface growth to control epithelial tube size.

Networks of epithelial and endothelial tubes are essential for the function of organs such as the lung, kidney and vascular system. The sizes and shapes of these tubes are highly regulated to match their individual functions. Defects in tube size can cause debilitating diseases such as polycystic kidney disease and ischaemia. It is therefore critical to understand how tube dimensions are regulated. This study identified the tyrosine kinase Src as an instructive regulator of epithelial-tube length in the Drosophila tracheal system. Loss-of-function Src42 mutations shorten tracheal tubes, whereas Src42 overexpression elongates them. Surprisingly, Src42 acts distinctly from known tube-size pathways and regulates both the amount of apical surface growth and, with the conserved formin dDaam, the direction of growth. Quantitative three-dimensional image analysis reveals that Src42- and dDaam-mutant tracheal cells expand more in the circumferential than the axial dimension, resulting in tubes that are shorter in length-but larger in diameter-than wild-type tubes. Thus, Src42 and dDaam control tube dimensions by regulating the direction of anisotropic growth, a mechanism that has not previously been described (Nelson, 2012).

To define the molecular mechanisms by which Src42 acts, loss-of-function mutations were tested in candidate Src interactors for a short-tracheal phenotype. dDaam (also known as DAAM, Dishevelled associated activator of morphogenesis), a conserved Diaphanous-related formin that has been shown to bind vertebrate Src, showed a mild, but significant, short-tracheal phenotype near the end of embryogenesis (stage 16). Shortening was more apparent after hatching, raising the possibility that the weaker embryonic phenotype was due to maternal dDaam (Matusek, 2006; Matusek, 2008); failure of dDaam maternal/zygotic embryos to cellularize (Matusek, 2008) precludes a direct test of this possibility). Similarly to Src42, dDaam acts autonomously in the tracheal system, because tracheal expression of Flag-tagged dDaam fully rescued the shortened dorsal trunk of dDaam mutants. Remarkably, despite only causing modest reductions in dorsal-trunk length at embryonic stage 16, zygotic dDaam mutations were still able to completely suppress the overelongation of septate-junction, aECM, apico-basal and PCP mutants. Further, whereas Src42; scrib mutants had gross embryonic defects, dDaam; scrib mutants underwent largely normal development and had trachea with the dDaam short-tracheal phenotype. Thus, dDaam seems to act downstream of or in parallel to all characterized control pathways for tracheal-tube size (Nelson, 2012).

Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules

Adherens junctions (AJs) and cell polarity complexes are key players in the establishment and maintenance of apical-basal cell polarity. Loss of AJs or basolateral polarity components promotes tumor formation and metastasis. Recent studies in vertebrate models show that loss of AJs or loss of the basolateral component Scribble (Scrib) cause deregulation of the Hippo tumor suppressor pathway and hyperactivation of its downstream effectors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ), homologs of Drosophila Yorkie. However, whether AJs and Scrib act through the same or independent mechanisms to regulate Hippo pathway activity is not known. This study dissects how disruption of AJs or loss of basolateral components affect the activity of the Drosophila YAP homolog Yorkie (Yki) during imaginal disc development. Surprisingly, disruption of AJs and loss of basolateral proteins produced very different effects on Yki activity. Yki activity was cell-autonomously decreased but non-cell-autonomously elevated in tissues where the AJ components E-cadherin (E-cad) or α catenin (α-cat) were knocked down. In contrast, scrib knockdown caused a predominantly cell-autonomous activation of Yki. Moreover, disruption of AJs or basolateral proteins had different effects on cell polarity and tissue size. Simultaneous knockdown of α-cat and scrib induced both cell-autonomous and non-cell-autonomous Yki activity. In mammalian cells, knockdown of E-cad or α-cat caused nuclear accumulation and activation of YAP without overt effects on Scrib localization and vice versa. Therefore, these results indicate the existence of multiple, genetically separable inputs from AJs and cell polarity complexes into Yki/YAP regulation. (Yang, 2014).

This report addresses the effects of AJs and basolateral cell polarity determinants on the activity of the Hippo pathway in Drosophila imaginal discs. Knockdown of AJs and basolateral components both induced ectopic activation of Yki. However, knockdown of AJs and basolateral proteins had strikingly different effects on Yki. Disruption of the basolateral module induced mainly a cell-autonomous increase in Yki activity, whereas knockdown of AJs caused non-autonomous induction of Yki reporters. Therefore, these data identify and genetically uncouple multiple different molecular pathways from AJs and the basolateral module that regulate Yki activity (Yang, 2014).

These studies further show that knockdown of AJs induces cell-autonomous reduction of Yki activity and causes cell death and decreased size of Drosophila imaginal discs. Likewise, E-cad and :alpha;-cat mutant clones do not survive in imaginal discs. This effect may be mediated by LIM domain proteins of the Zyxin and Ajuba subfamilies, which regulate Hippo signaling by directly inhibiting Wts/Lats kinases and by interacting with Salvador (Sav), an adaptor protein that binds to the Hpo/MST kinases. A recent report shows that α-Cat recruits Ajuba and indirectly Wts to AJs and loss of Ajuba leads to activation of Wts and hence phosphorylation and inhibition of Yki and diminished tissue size. Thus, α-cat mutant cells may inactivate Yki because they lose Ajuba function (Yang, 2014).

In contrast, in mammalian systems, several in vivo and in vitro studies have shown the opposite effect on Hippo signaling upon AJ disruption; knockdown of E-cad or α-cat caused an increase in cell proliferation and nuclear accumulation of YAP, and conditional knockout of α-cat in mouse skin cells caused tumor formation and elevated nuclear YAP staining. This suggests that AJ components have a tumor suppressor function in mammals. The observation that Scrib is mislocalized upon disruption of AJs in several different mammalian cell lines suggested that YAP activation could be due to the concomitant disruption of the basolateral module. However, the finding that acute disruption of AJs can cause YAP activation without disrupting Scrib localization and vice versa indicates that AJs and the basolateral module also act independently on the Hippo pathway in mammalian cells. In mammalian cells, α-Cat forms a complex with YAP and 14-3-3 proteins, thereby sequestering phosphorylated YAP at the plasma membrane. However, α-Cat may function as a tumor suppressor only in epidermal stem cells, as conditional deletion of α-cat in differentiated cells only caused a mild phenotype with no overgrowth and tumor formation. Therefore, it is possible that the negative regulation of YAP by α-Cat is cell type-specific, although further testing is required to fully address this issue (Yang, 2014).

The non-cell-autonomous effect of AJ knockdown on the Hippo pathway is an intriguing phenomenon. Several groups reported non-autonomous effects on the Hippo pathway in Drosophila in other mutant conditions. Disrupting the expression gradients of the atypical Cadherin Dachsous or that of its regulator Four-jointed, clones of cells mutant for the tumor suppressor genes vps25 or hyperplastic discs (hyd) , clones of cells overexpressing Src64, or overexpression of the proapoptotic gene reaper or the JNK signaling ligand eiger all cause non-autonomous activation of Yki. This non-autonomous activation of Yki may be part of a regenerative response that stimulates cell proliferation in cells neighboring tissue defects. The signals that activate Yki in these situations are not known, nor is it known whether these mutant conditions activate the same or different signaling mechanisms. The non-autonomous activation of Yki around cells with AJ knockdown may be mediated by changes in mechanical forces. AJs are important for maintaining tension between cells across epithelia, and disruption of AJs leads to an imbalance of apical tension. Mechanical forces are known to regulate the Hippo pathway, and YAP/TAZ act as mediators of mechanical cues from the cellular microenvironment such as matrix stiffness. In particular, the Zyxin and Ajuba family LIM domain proteins can act as sensors of mechanical forces and may be involved in the non-autonomous activation of Yki. The effects on Hippo signaling of solely changing Zyxin and Ajuba may not be as strong as those described here, and these proteins may thus cooperate with other molecular conduits to regulate the activity of the Hippo pathway in response to changes in AJ strength. Unraveling these mechanisms will provide important new insights into understanding how cells interact with neighboring cells to regulate proliferation, apoptosis, and the Hippo pathway (Yang, 2014).

It is currently unknown whether AJs also exert non-autonomous effects on the Hippo pathway in mammalian tissues. Amphiregulin, an EGF ligand, is a downstream target of YAP and can induce non-cell-autonomous cell proliferation through EGFR signaling. However, it is not known whether YAP itself is activated non-cell-autonomously to contribute to the hyper-proliferation phenotypes observed upon disruption of AJs in vivo and in vitro. It will be interesting to determine whether AJs and other cell-cell signaling mechanisms also have non-cell-autonomous effects on the activity of YAP in mammalian tissues, for example during regeneration (Yang, 2014).

Finally, the apical proteins aPKC and Crb modulate the activity of the Hippo pathway, and many Hippo pathway components are apically localized, which is important for their activity. The data presented in this study add to these findings. Disruption of AJs causes reduced Yki activity, despite the fact that Crb and Mer are mislocalized. Thus, AJs and cell polarity components regulate Yki activity through multiple, genetically separable inputs. It will be interesting to decipher all of the different underlying molecular mechanisms of how AJs and basolateral proteins regulate the Hippo pathway and how these mechanisms evolved in Drosophila and in mammals (Yang, 2014).

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).

The transcriptional response to tumorigenic polarity loss in Drosophila

Loss of polarity correlates with progression of epithelial cancers, but how plasma membrane misorganization drives oncogenic transcriptional events remains unclear. The polarity regulators of the Drosophila Scribble (Scrib) module are potent tumor suppressors and provide a model for mechanistic investigation. RNA profiling of Scrib mutant tumors revealed multiple signatures of neoplasia, including altered metabolism and dedifferentiation. Prominent among these was upregulation of cytokine-like Unpaired (Upd) ligands, which drive tumor overgrowth. This study identified a polarity-responsive enhancer in upd3, which was activated in a coincident manner by both JNK-dependent Fos and aPKC-mediated Yki transcription. This enhancer, and Scrib mutant overgrowth in general, were also sensitive to activity of the Polycomb Group (PcG), suggesting that PcG attenuation upon polarity loss potentiated select targets for activation by JNK and Yki. These results link epithelial organization to signaling and epigenetic regulators that control tissue repair programs, and provide insight into why epithelial polarity is tumor-suppressive (Bunker, 2015).

Interplay among Drosophila transcription factors Ets21c, Fos and Ftz-F1 drives JNK-mediated tumor malignancy

This study defines TF network that triggers an abnormal gene expression program promoting malignancy of clonal tumors, generated in Drosophila imaginal disc epithelium by gain of oncogenic Ras (RasV12) and loss of the tumor suppressor Scribble (scrib1). Malignant transformation of the rasV12scrib1 tumors requires TFs of distinct families, namely the bZIP protein Fos, the ETS-domain factor Ets21c and the nuclear receptor Ftz-F1, all acting downstream of Jun-N-terminal kinase (JNK). Depleting any of the three TFs improves viability of tumor-bearing larvae, and this positive effect can be enhanced further by their combined removal. Although both Fos and Ftz-F1 synergistically contribute to rasV12scrib1 tumor invasiveness, only Fos is required for JNK-induced differentiation defects and Matrix metalloprotease (MMP1) upregulation. In contrast, the Fos-dimerizing partner Jun is dispensable for JNK to exert its effects in rasV12scrib1 tumors. Interestingly, Ets21c and Ftz-F1 are transcriptionally induced in these tumors in a JNK- and Fos-dependent manner, thereby demonstrating a hierarchy within the tripartite TF network, with Fos acting as the most upstream JNK effector. Of the three TFs, only Ets21c can efficiently substitute for loss of polarity and cooperate with Ras(V12) in inducing malignant clones that, like rasV12scrib1 tumors, invade other tissues and overexpress MMP1 and the Drosophila insulin-like peptide 8 (Dilp8). While rasV12ets21c tumors require JNK for invasiveness, the JNK activity is dispensable for their growth. In conclusion, this study delineates both unique and overlapping functions of distinct TFs that cooperatively promote aberrant expression of target genes, leading to malignant tumor phenotypes. (Kulshammer, 2015).

Genome-wide transcriptome profiling in the Drosophila epithelial tumor model has generated a comprehensive view of gene expression changes induced by defined oncogenic lesions that cause tumors of an increasing degree of malignancy. These data allowed discovery of how a network of collaborating transcription factors confers malignancy to RasV12scrib1 tumors (Kulshammer, 2015).

This study revealed that the response of transformed RasV12scrib1 epithelial cells is more complex in comparison to those with activated RasV12 alone with respect to both the scope and the magnitude of expression of deregulated genes. Aberrant expression of more than half of the genes in RasV12scrib1 tumors requires JNK activity, highlighting the significance of JNK signaling in malignancy. Importantly, the tumor-associated, JNK-dependent transcripts cluster with biological functions and processes that tightly match the phenotypes of previously described tumor stages. Furthermore, the RasV12scrib1 transcriptome showed significant overlap (27% upregulated and 15% downregulated genes) with microarray data derived from mosaic EAD in which tumors were induced by overexpressing the BTB-zinc finger TF Abrupt (Ab) in scrib1 mutant clones as well as with a transcriptome of scrib1 mutant wing discs. It is proposed that 429 misregulated transcripts (e.g. cher, dilp8, ets21c, ftz-f1, mmp1, upd), shared among all the three data sets irrespective of epithelial type (EAD versus wing disc) or cooperating lesion (RasV12 or Ab), represent a 'polarity response transcriptional signature' that characterizes the response of epithelia to tumorigenic polarity loss. Genome-wide profiling and comparative transcriptome analyses thus provide a foundation to identify novel candidates that drive and/or contribute to tumor development and malignancy while unraveling their connection to loss of polarity and JNK signaling (Kulshammer, 2015).

In agreement with a notion of combinatorial control of gene expression by an interplay among multiple TFs, this study identified overrepresentation of cis-acting DNA elements for STAT, GATA, bHLH, ETS, BTB, bZIP factors and NRs in genes deregulated in RasV12scrib1 mosaic EAD, implying that transcriptome anomalies result from a cross-talk among TFs of different families. Many of the aberrantly expressed genes contained binding motifs for AP-1, Ets21c and Ftz-F1, indicating that these three TFs may regulate a common set of targets and thus cooperatively promote tumorigenesis. This is consistent with the occurrence of composite AP-1-NRRE (nuclear receptor response elements), ETS-NRRE and ETS-AP-1 DNA elements in the regulatory regions of numerous human cancer-related genes, such as genes for cytokines, MMPs (e.g., stromelysin, collagenase) and MMP inhibitors (e.g., TIMP) (Kulshammer, 2015).

Interestingly, Drosophila ets21c and ftz-f1 gene loci themselves contain AP-1 motifs and qualify as polarity response transcriptional signature transcripts. Indeed, this study has detected JNK- and Fos-dependent upregulation of ets21c and ftz-f1 mRNAs in RasV12scrib1 tumors. While JNK-mediated control of ftz-f1 transcription has not been reported previously, upregulation of ets21c in the current tumor model is consistent with JNK requirement for infection-induced expression of ets21c mRNA in Drosophila S2 cells and in vivo. Based on these data, it is proposed that Ftz-F1 and Ets21c are JNK-Fos-inducible TFs that together with AP-1 underlie combinatorial transcriptional regulation and orchestrate responses to cooperating oncogenes. Such an interplay between AP-1 and Ets21c is further supported by a recent discovery of physical interactions between Drosophila Ets21c and the AP-1 components Jun and Fos (Rhee, 2014). Whether regulatory interactions among AP-1, Ets21c and Ftz-F1 require their direct physical contact and/or the presence of composite DNA binding motifs of a particular arrangement to control the tumor-specific transcriptional program remains to be determined (Kulshammer, 2015).

Importantly, some of the corresponding DNA elements, namely AP-1 and STAT binding sites, have recently been found to be enriched in regions of chromatin that become increasingly accessible in RasV12scrib1 mosaic EAD relative to control. This demonstrates that comparative transcriptomics and open chromatin profiling using ATAC-seq and FAIRE-seq are suitable complementary approaches for mining the key regulatory TFs responsible for controlling complex in vivo processes, such as tumorigenesis (Kulshammer, 2015).

The prototypical form of AP-1 is a dimer comprising Jun and Fos proteins. In mammals, the Jun proteins occur as homo- or heterodimers, whereas the Fos proteins must interact with Jun in order to bind the AP-1 sites. In contrast to its mammalian orthologs, the Drosophila Fos protein has been shown to form a homodimer capable of binding to and activating transcription from an AP-1 element, at least in vitro (Kulshammer, 2015).

The role of individual AP-1 proteins in neoplastic transformation and their involvement in pathogenesis of human tumors remain somewhat elusive. While c-Jun, c-Fos and FosB efficiently transform mammalian cells in vitro, only c-Fos overexpression causes osteosarcoma formation, whereas c-Jun is required for development of chemically induced skin and liver tumors in mice. In contrast, JunB acts as a context-dependent tumor suppressor. Thus, cellular and genetic context as well as AP-1 dimer composition play essential roles in dictating the final outcome of AP-1 activity in tumors (Kulshammer, 2015).

This study shows that, similar to blocking JNK with its dominant-negative form, Bsk, removal of Fos inhibits ets21c and ftz-f1 upregulation, suppresses invasiveness, improves epithelial organization and differentiation within RasV12scrib1 tumors and allows larvae to pupate. Strikingly, depletion of Jun had no such tumor-suppressing effects. It is therefore concluded that in the malignant RasV12scrib1 tumors, Fos acts independently of Jun, either as a homodimer or in complex with another, yet unknown partner. A Jun-independent role for Fos is further supported by additional genetic evidence. Fos, but not Jun, is involved in patterning of the Drosophila endoderm Ÿand is required for expression of specific targets, e.g., misshapen (msn) and dopa decarboxylase (ddc), during wound healing. Future studies should establish whether the JNK-responsive genes containing AP-1 motifs, identified in this study, are indeed regulated by Fos without its 'canonical' partner (Kulshammer, 2015).

The current data identify Fos as a key mediator of JNK-induced MMP1 expression and differentiation defects in RasV12scrib1 tumors. Only Fos inhibition caused clear suppression of MMP1 levels and restoration of neurogenesis within clonal EAD tissue, thus mimicking effects of JNK inhibition. Improved differentiation and reduced invasiveness are, however, not sufficient for survival of animals to adulthood, because interfering with Fos function in RasV12scrib1 clones always resulted in pupal lethality (Kulshammer, 2015).

The systems approach of this paper, followed by genetic experiments, identified Ets21c and Ftz-F1 as being essential for RasV12scrib1-driven tumorigenesis. It was further shown that mutual cooperation of both of these TFs with Fos is required to unleash the full malignancy of RasV12scrib1 tumors (Kulshammer, 2015).

TFs of the ETS-domain family are key regulators of development and homeostasis in all metazoans, whereas their aberrant activity has been linked with cancer. ets21c encodes the single ortholog of human Friend leukemia insertion1 (FLI1) and ETS-related gene (ERG) that are commonly overexpressed or translocated in various tumor types. While FLI1 is considered pivotal to development of Ewing's sarcoma, ERG has been linked to leukemia and prostate cancer. As for Ftz-F1 orthologs, the human liver receptor homolog-1 (LRH-1) has been associated with colonic, gastric, breast and pancreatic cancer, whereas steroidogenic factor 1 (SF-1) has been implicated in prostate and testicular cancers and in adrenocortical carcinoma. However, the molecular mechanisms underlying oncogenic activities of either the ERG/FLI1 or the SF-1/LRH-1 proteins are not well understood (Kulshammer, 2015).

This study shows that removal of Ftz-F1 markedly suppressed invasiveness of RasV12scrib1 tumors, restoring the ability of tumor-bearing larvae to pupate. Additionally, and in contrast to Fos, Ftz-F1 inhibition also partly reduced tumor growth in the third-instar EAD and allowed emergence of adults with enlarged, rough eyes composed predominantly of non-clonal tissue. The reduced clonal growth coincided with downregulation of the well-established Yki target, expanded, implicating Ftz-F1 as a potential novel growth regulator acting on the Hpo/Yki pathway. It is further speculated that reduced viability of RasV12scrib1ftz-f1RNAi clones and induction of non-autonomous compensatory proliferation by apoptotic cells during the pupal stage could explain the enlargement of the adult eyes. The precise mechanism underlying compromised growth and invasiveness of RasV12scrib1ftz-f1RNAi tumors and improved survival of the host remains to be identified (Kulshammer, 2015).

In contrast, effects of Ets21cLONG knockdown in RasV12scrib1 tumors appeared moderate relative to the clear improvement conferred by either Fos or Ftz-F1 elimination. ets21cLONG RNAi neither reduced tumor mass nor suppressed invasiveness, and pupation was rescued only partly. However, unlike ftz-f1RNAi, ets21cLONG RNAi significantly reduced expression of dilp8 mRNA. Based on abundance of Ets21c binding motifs in the regulatory regions of tumor-associated genes and the normalized expression of >20% of those genes upon removal of Ets21c, it is further suggested that Ets21c acts in RasV12scrib1 tumors to fine-tune the tumor gene-expression signature (Kulshammer, 2015).

Dilp8 is known to be secreted by damaged, wounded or tumor-like tissues to delay the larval-to-pupal transition. This study has corroborated the role of JNK in stimulating dilp8 expression in RasV12scrib1 tumor tissue, and has further implicated Ets21c and Fos as novel regulators of dilp8 downstream of JNK. However, the data also show that elevated dilp8 transcription per se is not sufficient to delay metamorphosis. Unlike the permanent larvae bearing RasV12scrib1 tumors, those with RasV12scrib1ftz-f1RNAi tumors pupated despite the excessive dilp8 mRNA. Likewise, pupation was not blocked by high dilp8 levels in larvae bearing EAD clones overexpressing Abrupt. As Dilp8 secretion appears critical for its function, it is proposed that loss of Ftz-F1 might interfere with Dilp8 translation, post-translational processing or secretion (Kulshammer, 2015).

Consistent with the individual TFs having unique as well as overlapping functions in specifying properties of RasV12scrib1 tumors, knocking down pairwise combinations of the TFs had synergistic effects on tumor suppression compared with removal of single TF. This evidence supports the view that malignancy is driven by a network of cooperating TFs, and elimination of several tumor hallmarks dictated by this network is key to animal survival. An interplay between AP-1, ETS-domain TFs and NRs is vital for development. For example, the ETS-factor Pointed has been shown to cooperate with Jun to promote R7 photoreceptor formation in the Drosophila adult eye. In mosquitoes, synergistic activity of another ETS-factor, E74B, with the ecdysone receptor (EcR/USP) promotes vitellogenesis. It is thus proposed that tumors become malignant by hijacking the developmental mechanism of combinatorial control of gene activity by distinct TFs (Kulshammer, 2015).

Despite the minor impact of ets21cLONG knockdown on suppressing RasV12scrib1 tumors, Ets21cLONG is the only one of the tested TFs that was capable of substituting for loss of scrib in inducing malignant clonal overgrowth when overexpressed with oncogenic RasV12 in EAD. While invasiveness of such RasV12ets21cLONG tumors required JNK activity, JNK signaling appeared dispensable for tumor growth. Importantly, the overgrowth of RasV12ets21cLONG tumors was primarily independent of a prolonged larval stage, because dramatic tumor mass expansion was detected already on day 6 AEL. How cooperativity between Ets21cLONG and RasV12 ensures sufficient JNK activity and the nature of the downstream effectors driving tumor overgrowth remain to be determined. In contrast, co-expression of either Ftz-F1 or Fos with RasV12 resulted in a non-invasive, RasV12-like hyperplastic phenotype (Kulshammer, 2015).

Why does Ets21cLONG exert its oncogenic potential while Fos and Ftz-F1 do not? Simple overexpression of a TF may not be sufficient, because many TFs require activation by a post-translational modification (e.g., phosphorylation), interaction with a partner protein and/or binding of a specific ligand. Full activation of Fos in response to a range of stimuli is achieved through hyperphosphorylation by mitogen-activated protein kinases (MAPKs), including ERK and JNK. Indeed, overexpression of a FosN-Ala mutated form that cannot be phosphorylated by JNK was sufficient to phenocopy fos deficiency, indicating that Fos must be phosphorylated by JNK in order to exert its oncogenic function. Consistent with the current data, overexpression of FosN-Ala partly restored polarity of lgl mutant EAD cells. It is therefore conclude that the tumorigenic effect of Fos requires a certain level of JNK activation, which is lacking in EAD co-expressing Fos with RasV12. Nevertheless, the absence of an unknown Fos-interacting partner cannot be excluded (Kulshammer, 2015).

Interestingly, MAPK-mediated phosphorylation also greatly enhances the ability of SF-1 and ETS proteins to activate transcription. Two potential MAPK sites can be identified in the hinge region of Ftz-F1, although their functional significance is unknown. Whether Ets21c or Ftz-F1 requires phosphorylation and how this would impact their activity in the tumor context remains to be determined. Genetic experiments demonstrate that at least the overgrowth of RasV12ets21cLONG tumors does not require Ets21c phosphorylation by JNK (Kulshammer, 2015).

In addition, previous crystallography studies revealed the presence of phosphoinositides in the ligand binding pocket of LHR-1 and SF-1 and showed their requirement for the NR transcriptional activity. Although developmental functions of Drosophila Ftz-F1 seem to be ligand independent, it is still possible that Ftz-F1 activity in the tumor context is regulated by a specific ligand. An effect of Ftz-F1 SUMOylation cannot be ruled out (Kulshammer, 2015).

In summary, this work demonstrates that malignant transformation mediated by RasV12 and scrib loss depends on MAPK signaling and at least three TFs of different families, Fos, Ftz-F1 and Ets21c. While their coordinated action ensures precise transcriptional control during development, their aberrant transcriptional (Ets21c, Ftz-F1) and/or post-translational (Fos, Ftz-F1, Ets21c) regulation downstream of the cooperating oncogenes contributes to a full transformation state. The data implicate Fos as a primary nuclear effector of ectopic JNK activity downstream of disturbed polarity that controls ets21c and ftz-f1 expression. Through combinatorial interactions on overlapping sets of target genes and acting on unique promoters, Fos, Ftz-F1 and Ets21c dictate aberrant behavior of RasV12scrib1 tumors. Although originally described in Drosophila, detrimental effects of cooperation between loss of Scrib and oncogenic Ras has recently been demonstrated in mammalian tumor models of prostate and lung cancer. This study and further functional characterization of complex TF interactions in the accessible Drosophila model are therefore apt to provide important insight into processes that govern cancer development and progression in mammals (Kulshammer, 2015).


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scribbled: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 April 2017

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