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).
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).
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).
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).
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).
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).
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).
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).
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).
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