discs large 1

DEVELOPMENTAL BIOLOGY

Embryonic

DLG is localized in an apical belt of the lateral cell membrane, at the position of the septate junction (Woods, 1991). Localization is to the cytoplasmic face of salivary gland cells (Woods, 1996).

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).

Larval

The distribution of proteins in the apico-lateral cell junctions in Drosophila imaginal discs was examined. The subcellular distribution of these proteins in normal and mutant proliferating cells was analyzed with marker antibodies and confocal microscopy. Antibodies to phosphotyrosine (PY), Armadillo (Arm) and Drosophila E-cadherin (DE-cad) as well as FITC phalloidin marking filamentous actin, labeled the site of the adherens junction, whereas antibodies to Discs large (Dlg), Fasciclin III (FasIII) and Coracle (Cor) labeled the more basal septate junction. The junctional proteins labeled by these antibodies underwent specific changes in distribution during the cell cycle. A loss-of-function dlg mutation, which causes neoplastic imaginal disc overgrowth, leads to loss of the septate junctions and the formation of what appear to be ectopic adherens junctions (Woods et al., 1996).

The current study was expanded to examine the effects of mutations in other genes that also cause imaginal disc overgrowth. Based on staining with PY and Dlg antibodies, the apico-lateral junctional complexes appear normal in tissue from the hyperplastic overgrowth mutants fat facets, discs overgrown, lethal (2) giant discs and warts. However, imaginal disc tissue from the neoplastic overgrowth mutants dlg and lethal (2) giant larvae show abnormal distribution of the junctional markers including a complete loss of apico-basal polarity in loss-of-function dlg mutations. These results support the idea that some of the proteins of apico-lateral junctions are required both for apico-basal cell polarity and for the signaling mechanisms controlling cell proliferation, whereas others are required more specifically in cell-cell signaling (Woods, 1997).

In third instar larval muscles, Dlg is concentrated primarily at type I neuromuscular junctions (NMJs). Closer examination reveals evenly distributed, weak immunoreactive spots at the muscle surface. In addition, a subcortical immunoreactive network, localized to the same range of optical sections as the muscle nuclei, was observed. Both types of extrasynaptic staining are specific to Dlg since they are not observed in mutant flies that express extremely low levels of a truncated form of Dlg (dlgX1-2 mutants) (Thomas, 2000).

To determine whether Dlg localization at extrasynaptic regions might represent intermediate trafficking steps, Dlg expression in muscles was analyzed during development. A highly sensitive anti-Dlg antibody (anti-DlgPDZ antibody) was used to determine the time course of Dlg expression at extrasynaptic regions. In stage 16 embryos, Dlg immunoreactivity is apparent in the ventral nerve cord, but virtually absent in both presynaptic termini and body wall muscles. At stage 17, after the initial formation of synaptic boutons, Dlg is detected at sites of contact between nerves and muscles. Double labeling with the neuron-specific anti-HRP antibody confirms that this immunoreactivity is presynaptic and that Dlg is still absent from the postsynaptic junctional region. At the same stage, however, extrasynaptic Dlg immunoreactivity becomes detectable, both as spots distributed throughout the muscle surface and as a subcortical network. Thus, expression of Dlg at distinct extrasynaptic sites clearly precedes its concentration at the postsynaptic membrane (Thomas, 2000).

A continuous shift of Dlg immunoreactivity from extrasynaptic to synaptic sites is observed during larval development. By the first instar stage, when Dlg begins to accumulate at the postsynaptic junctional membrane, extrasynaptic Dlg immunoreactivity at both the muscle membrane and the subcortical network is strong. By the second instar stage, synaptic Dlg localization is very strong, whereas extrasynaptic Dlg localization is significantly reduced. In third instar larvae, Dlg is almost exclusively localized at synapses and only a little Dlg can still be detected at the surface and subcortical region (Thomas, 2000).

The developmental analysis suggests a stepwise postsynaptic targeting of Dlg. To determine which domains of Dlg are required for distinct targeting steps, the subcellular distribution of FLAG-epitope-tagged Dlg deletion variants upon targeted expression in body wall muscles was studied. For this, advantage was taken of the GAL4-UAS expression system, using the GAL4 strain C57 as a muscle-specific activator. To evaluate the possible influence of endogenous Dlg on the localization of transgenic Dlg variants, expression of each construct was targeted in dlgX1-2 mutants (Thomas, 2000).

In all constructs, the last carboxy-terminal 40 amino acids of Dlg were replaced by the FLAG epitope. The importance of the carboxyl terminus for the synaptic localization of another MAGUK, PSD-95, is controversial. Several observations have demonstrated, however, that carboxy-terminal FLAG tagging has no detectable effect on the subcellular localization of Dlg. The hypomorphic allele dlg1P20 gives rise to a truncated protein lacking the same carboxy-terminal amino acids. The distribution of this gene product appears indistinguishable from the wild type. Upon targeted expression in muscles, Dlg-FLAG becomes enriched postsynaptically around type I boutons. Prominent FLAG immunoreactivity is also detected extrasynaptically at both the surface and the subcortical network. The extrasynaptic immunoreactivity in Dlg-FLAG-expressing flies is clearly stronger than in the wild type. However, a very similar increase in extrasynaptic immunoreactivity is observed upon expression of transgenic, non-tagged Dlg with an intact carboxyl terminus. This suggests that overexpression per se rather than the carboxy-terminal truncation is responsible for increased extrasynaptic localization of Dlg-FLAG (Thomas, 2000).

Invasive cell behavior during Drosophila imaginal disc eversion is mediated by the JNK signaling cascade

Drosophila imaginal discs are monolayered epithelial invaginations that grow during larval stages and evert at metamorphosis to assemble the adult exoskeleton. They consist of columnar cells, forming the imaginal epithelium, as well as squamous cells, which constitute the peripodial epithelium and stalk (PS). A new morphogenetic/cellular mechanism for disc eversion has been uncovered. Imaginal discs evert by apposing their peripodial side to the larval epidermis and through the invasion of the larval epidermis by PS cells, which undergo a pseudo-epithelial-mesenchymal transition (PEMT). As a consequence, the PS/larval bilayer is perforated and the imaginal epithelia protrude, a process reminiscent of other developmental events, such as epithelial perforation in chordates. When eversion is completed, PS cells localize to the leading front, heading disc expansion. The JNK pathway is necessary for PS/larval cells apposition, the PEMT, and the motile activity of leading front cells (Pastor-Pareja, 2004).

One hallmark of epithelial cells is their distinct apico-basal cell polarity. This polarity depends on a set of intercellular connections, which encircle epithelial cells at the border of the apical and basal-lateral membrane domains. The cells in insect epithelial tissues are interconnected by zonula adherens (ZAs), which function in both cellular adhesion and signaling. DE-cadherin is the major constituent of the ZAs in a complex with Armadillo (Arm, ß-catenin) and Dalpha-catenin. In addition, epithelia of flies and other invertebrates exhibit septate junctions, which are located basally to the ZAs. Septate junctions prevent diffusion through the pericellular space and are functionally equivalent to vertebrate tight junctions (Pastor-Pareja, 2004).

All imaginal disc cells at the third instar larval stage presented ZAs in an apical belt. During disc eversion, however, it was found that ZAs components delocalize from the free edges of the PS cells, remaining cytoplasmic at the edges of the perforations arising through the PS/larval bilayer and in those PS cells leading the spreading of the discs over the larval tissues. As a consequence, ZAs are lost in these cells. Moreover, septate junction components, such as Coracle and Disc Large are also found to be missing from the membranes of leading front cells (Pastor-Pareja, 2004).

The loss of apico/basal polarity and adhesion of the PS cells during disc eversion is reminiscent of an epithelial-mesenchymal transition (EMT), as described for mesoderm and neural crest cells in vertebrates, and for the acquisition of the invasive phenotype in carcinomas (Pastor-Pareja, 2004).

In summary, the evagination of imaginal disc can be divided into the following sequential steps: (1) an overall positional change of the imaginal discs leading to the confrontation and apposition of the PS and the larval epidermis; (2) a regulated modulation (PEMT) of PS cells, which involves the downregulation of their cell-cell adhesion systems and allows them to move into their local neighborhood and invade the larval epithelium; (3) the fenestration of the peripodial/larval bilayer and the formation of an unbound peripodial leading front, which will direct imaginal spreading by planar cell intercalation, and (4) a bulging of the imaginal tissue (Pastor-Pareja, 2004).

Once the hole is opened, the planar intercalation of PS cells ensures that, first in the hole and later in the leading front, all four dorsal, ventral, anterior, and posterior compartments of the wing disc are represented. This mechanism also guarantees the maintenance of a continuous epithelial barrier (Pastor-Pareja, 2004).

Oogenesis

A Fasciclin 2 morphogenetic switch organizes epithelial cell cluster polarity and motility: Fas2 polarization is then directed by PC Dlg and Lgl

Little is known about how intercellular communication is regulated in epithelial cell clusters to control delamination and migration. This problem has been investigated using Drosophila border cells as a model. Just preceding cell cluster delamination, expression of transmembrane immunoglobulin superfamily member, Fasciclin 2, is lost in outer border cells, but not in inner polar cells (PCs) of the cluster. Loss of Fasciclin 2 expression in outer border cells permits a switch in Fasciclin 2 polarity in the inner polar cells. This polarity switch, which is organized in collaboration with neoplastic tumor suppressors Discs large and Lethal-giant-larvae, directs cluster asymmetry essential for timing delamination from the epithelium. Fas2-mediated communication between polar and border cells maintains localization of Discs large and Lethal-giant-larvae in border cells to inhibit the rate of cluster migration. These findings are the first to show how a switch in cell adhesion molecule polarity regulates asymmetry and delamination of an epithelial cell cluster. The finding that Discs large and Lethal-giant-larvae inhibit the rate of normal cell cluster movement suggests that their loss in metastatic tumors may directly contribute to tumor motility. Furthermore, these results provide novel insight into the intimate link between epithelial polarity and acquisition of motile polarity that has important implications for development of invasive carcinomas (Szafranski, 2004).

How does developmentally programmed loss of Fas2 expression in BCs permit Fas2 polarization in PCs? The data indicate that this is a multistep process. Initially, Fas2 homophilic interactions between BCs and PCs are lost, and several experiments indicate that they are replaced by Fas2 heterophilic interactions with a putative BC receptor. These interactions are essential for maintaining Fas2 in PC membranes contacting BCs. Next, loss of Fas2 from BCs causes relocation of the majority of PC Fas2 to the interface between PCs, where it is maintained because of homophilic interactions with Fas2 from the adjacent PC. In support of this interpretation, misexpression of Fas2 in PCs appears to oversaturate Fas2 between PCs, causing its circumferential accumulation at the contact sites with BCs. It is concluded that the accumulation of Fas2 between PCs ensures that Fas2 is kept at sufficiently low levels at the sites of contact with BCs to allow its polarization to the leading half of PCs. Fas2 polarization is then directed by PC Dlg and Lgl, as evidenced by the observation that loss of function of either protein causes loss of Fas2 polarity. However, Fas2 can also polarize Dlg and Lgl; loss of Fas2 causes loss of Dlg and Lgl polarity, while ectopic Fas2 redirects Dlg and Lgl localization. Thus, Fas2 is in a positive feedback loop with Dlg and Lgl that ensures the build up of a PC signaling and adhesion complex at the leading half of the PCs. These data indicate that Fas2 is involved in intercellular interactions crucial for organizing polarity, an important criterion for a function specifically involved in regulation of motility in multicellular clusters (Szafranski, 2004).

Significantly, the results indicate that molecules used for polarizing epithelial cells are reorganized to polarize a motile cell cluster. The timing of the reorganization of epithelial polarity is crucial for timing delamination. Fas2 therefore plays a direct role in mediating intercellular interactions that modulate movement, a second property proposed for a function specifically involved in regulating cluster motility as opposed to single cells. It is concluded that the Fas2 morphogentic switch facilitates development of motile polarity essential for timely BC delamination. A similar switch mechanism may be important in other processes that crucially depend on timing of Fas2 activity, such as axon pathfinding, and learning and memory (Szafranski, 2004).

Fas2 polarity appears to compartmentalize PCs into distinct functional domains in order to control functionally distinct intercellular communication with leading versus trailing BCs. Leading BCs play a functionally distinct role by pioneering invasion between germ cells while simultaneously detaching from the epithelium. Trailing BCs are likely to play a less active role in invasion, but must mediate precisely timed detachment from the epithelium. Fas2 polarization is thus likely to be crucial for facilitating coordination of the distinct functional requirements of leading versus trailing BCs, by establishing distinct sets of intercellular contact and communication between the PCs and leading versus trailing BCs. In support of this hypothesis, previous studies have suggested that leading and trailing BCs are functionally distinct. In BC clusters comprising a mixture of wild type and slbo, jing, taiman or DE-cadherin mutant cells, wild-type BCs always lead invasion. Furthermore, additional structural evidence has been documented for cluster asymmetry. Amphiphysin, a vesicle trafficking protein that regulates Dlg and Lgl localization, is expressed at higher level in trailing BCs compared to leading BCs. Amph, Dlg and Lgl, are thus good candidates for proteins that differentially regulate cortical and cell surface activities needed to mediate distinct interactions of leading and trailing BCs with adjacent epithelial cells and germ cells during the delamination process (Szafranski, 2004).

Since only Dlg and Lgl are mislocalized in Fas2 clusters, but not Fas3, alpha-Spec or Crb, the data suggest that Fas2 directs localization of specific molecules within distinct regions of different cells of the cluster to control motility. A putative Fas2-binding BC receptor may be another molecule whose polarity is controlled by Fas2. Interaction with this putative receptor appears to facilitate organization of the global polarity of the cluster, since the orientation of delamination, mediated by the BCs, directly correlates with Fas2 polarity in PCs. These data thus suggest that Fas2 coordinates directional mass motion between cells that are potentially capable of motion in any direction, and that it helps to determine the locomotive-active regions of these cells, additional criteria for a function specifically involved in regulating cluster motility. Thus, because Fas2 is required for regulation of several activities that distinguish how single cells versus clusters move, the data provide the first molecular model for understanding the organization of epithelial cluster polarity during delamination and movement. One argument against this proposal might be that the PCs appear to be highly specialized. However, it is thought that this is likely to be of less significance, since PCs express epithelial polarity proteins in a pattern similar to adjacent follicle epithelial cells (Szafranski, 2004).

As has been shown for BC clusters, several vertebrate studies have shown that transmembrane proteins are differently expressed within different cell subpopulations in migrating clusters. Furthermore, the structure and functions of Fas2, Dlg and Lgl homologs are conserved across phylogeny. Thus, the involvement of Fas2, Dlg and Lgl in organizing cell cluster motility also may be conserved. It is concluded that although the precise mechanism of cluster movement may not be conserved in vertebrates, the information gleaned about how BCs regulate epithelial polarity to dynamically organize cluster polarity and movement will be generally useful for understanding how cell cluster motility is organized across phylogeny (Szafranski, 2004).

The role of Fas2 in regulating migration is discussed. Loss- and gain-of-function experiments demonstrate that PC Fas2 acts as a signal to inhibit the rate of BC migration. This work builds on previous studies demonstrating the importance of PCs in determining BC fate. However, this work is the first example of an intercellular signal that specifically organizes cluster movement, rather than determining cell fate. Fas2 clearly has a signaling function, since PCs do not contact the migration substrate. Thus, these data demonstrate for the first time the existence of intercellular communication between cells of a migratory cluster, that is specifically required to modulate migration (Szafranski, 2004).

PC Fas2 signaling inhibits the rate of cluster movement by maintaining Dlg and Lgl localization in BCs. The putative BC receptor with which Fas2 interacts may control Dlg and Lgl localization in BCs. Since Dlg is localized to the cortex of BCs, Dlg must inhibit the rate of migration through cortical activities in BCs. One cortical activity controlled by Dlg is the recruitment of Lgl to the membrane. Since lgl clusters have very similar migration phenotypes to dlg clusters, the data indicate that Lgl and Dlg cooperate to inhibit BC movement. The importance of Dlg and Lgl in regulating cell movement probably derives from the same scaffolding activities they use to organize and control membrane, cytoskeletal and signaling specialization during the polarization of epithelial and neuronal cells. It is proposed that Dlg and Lgl scaffolding organizes and integrates transmembrane signaling and adhesion proteins with signaling, trafficking and cytoskeletal effectors in the cortex of BCs to mediate contact-inhibition of cluster movement (Szafranski, 2004).

BCs resemble mutant dlg invasive tumor cells in that they lose epithelial polarity by accumulating Dlg and Lgl around their circumference, but in contrast to BCs, mutant dlg tumor cells migrate between germ cells without temporal or spatial control. The data demonstrate that Dlg and Lgl not only control polarity and delamination of epithelial clusters, but also actively inhibit movement. Thus, mutant dlg tumor invasion is likely to be caused by a combination of loss of epithelial polarity and over-activation of motility pathways. In this context the results appear to be paradoxical in that loss of epithelial polarity is generally considered to be crucial for facilitating acquisition of motility, but it is seen that loss of polarity in normal migrating clusters delays initiation of movement. The data resolve this paradox in that during normal development, molecules used for polarizing epithelial cells are reorganized to polarize a motile cell cluster. It therefore seems likely that in carcinomas, inappropriate loss of epithelial polarity simultaneously disrupts acquisition of motile polarity, but this phenomenon is not appreciated because ultimately the tumor cells migrate. Thus, it is postulated that overactivation of motility pathways, as is seen with loss of Dlg and Lgl in BCs, may be especially crucial for achieving carcinoma invasion. Consistent with this hypothesis, some dlg mutations that cause loss of epithelial polarity do not lead to tumor invasion, suggesting that acquisition of motility is a separate Dlg function (Szafranski, 2004).

Gene expression data for human cancers suggests that mutations that promote tumor formation, through loss of epithelial polarity and increased proliferation, may be the same mutations that subsequently cause tumor cell invasion. Based on the observation that Dlg is required to maintain polarity, inhibit proliferation and inhibit movement, it is proposed that tumor suppressors such as Dlg that regulate signaling and adhesion at epithelial junctions may unify human gene expression data by providing an ultrastructural target that controls contact inhibition of both proliferation and movement. Progressive deterioration of epithelial junctions may thus provide a common mechanism through which multiple tumor suppressor pathways impact the cascade from cell proliferation to tumor invasion, either through mutation or mislocalization of critical junctional proteins (Szafranski, 2004).

Effects of Mutation or Deletion

Mutations of the discs large-1 tumor suppressor gene causes a non-epithelial overgrowth or neoplastic transformation, resulting in tumor-like imaginal discs and enlarged larvae that never pupariate. dlg mutant wing discs develop convoluted monolayers of epithelial cells characterized by well-defined apical-basal polarity. These layered cells secrete large amounts of basement membrane material. Drosophila laminin and collagen are components of this matrix. Late in development clusters or 'rosettes' of separated cells form, lacking both cell-cell junctions and apical-basal polarity. In in vitro culture experiments, dlg wing discs do not respond to a pulse of exogenous ecdysone by secreting cuticle or losing basement membrane as do normal discs. These results are consistent with the hypothesis that cell-cell interaction and communication is required for termination of disc cell proliferation, which must occur prior to a cellular response to ecdysone (Abbott, 1991).

A weak mutation of dlg results in minor bristle defects, including missing and duplicate bristles. A stronger allele fails to hatch and dies during late embryogenesis with a failure of dorsal closure [Images] and terminal defects. Mutants in an intermediate allele die as pharate adults with severe bristle and eye defects. The antennae and legs of these animals have small overgrowth regions and the eyes show defects in the planar polarity of the ommatidial bristles: they are found at random around the ommatidial cluster (Woods, 1996).

DLG is expressed at one type of glutamatergic synapse of the neuromuscular junction and is associated with both presynaptic and postsynaptic membranes. Mutations in dlg alter the expression of dlg and cause striking changes in the structure of the subsynaptic reticulum, a postsynaptic specialization at these synapses. These results indicate that dlg is required for normal synaptic structure and offers insights regarding the role of dlg homologs at vertebrate synapses (Lahey, 1994).

During oogenesis Drosophila Discs large (Dlg) protein is involved in maintaining the structure of one population of follicular cells (the follicular epithelium or FE), and in the migratory activity of another population of follicular cells (border cells or BC). These two follicle cell populations migrate at stage 9 of oogenesis. Most follicle cells (the FE cells) surrounding nurse cells move to the oocyte as an epithelial sheet along the outside of the egg chamber, whereas the BCs move through the center of the egg chamber, in concert with the epithelium. Six to seven BCs break from the follicular epithelium and adopt a mesenchymal-like morphology, then migrate to the oocyte, as they contact anterior nurse cells, then posterior nurse cells, before reaching their destination. The interaction of BCs with posterior nurse cells is particularly dramatic. Nurse cells maintain an invariant quadrihedral-like architecture throughout most stages of oogenesis, but following initiation of BC movement, one nurse cell adjacent to the oocyte extends a cytoplasmic process that contacts the migrating cells. Nurse cell processes are never observed preceding BC migration, although BC fate has already been established. Nurse cell processes show a clear directionality. They are never observed to extend from anterior nurse cells to BCs that have moved close to the oocyte at the posterior of the egg chamber. These observations suggest that nurse cells adjacent to the oocyte play an active role in guiding the movement of BCs to the oocyte (Goode, 1997).

Drosophila Dlg is required to block cell invasion. Loss of dlg activity during oogenesis causes FE cells to change shape and invade in a pattern similar to BCs, yet dlg mutant cells have not adopted a border cell fate. Specifically these defectively migrating FEs do not express slow border cells or FasIII, both diagnostic indicators of the BC fate. dlg-invasive FE cells share an apolar morphology with BCs but other aspects of BC morphology, such as absence of lamellipodia-like structures, are not shared with BCs. Nevertheless, both oocyte and nurse cells attached to the oocyte extend processes that contact invasive FEs (Goode, 1997).

Both functional and morphological evidence indicates that cooperation between germ cell and follicle cell Dlg, probably mediated by Dlg PDZ domains, is crucial for regulating cell mixing, suggesting a novel developmental mechanism and mode of action for the Dlg family of molecules. Dlg is expressed in both germ and follicle cells from the time that the germ cell cyst becomes surrounded by follicle cells in the germarium. Dlg appears to be expressed at equivalent levels in both tissues throughout the growth phases, as the germ cell cyst expands in size and the number of follicular epithelial cells increases greater than 10-fold. Following cessation of follicle cell proliferation, levels of Dlg protein appear to dramatically decrease in germ cells, corresponding to the time just preceding and including BC migration to the oocyte. At the cellular level, Dlg is localized to sites of contact between follicle cells and to sites of contact between germ cells, but appears to be excluded at sites of contact between germ cells and follicle cells. Dlg is required in both germ cells and follicle cells. When dlg function is eliminated in both follicle cells and germ cells, follicle cells always invade along the BC pathway. Dlg also appears to be required to prohibit cell proliferation of follicular cells at the poles of the egg chambers. Mutations in SH3 or GuK domains of Dlg fail to confer premature cell mixing. Dlg is also required to prevent premature BC migration These findings suggest that Dlg does not simply inhibit individual cell behaviors during oogenesis, but rather acts in a developmental pathway essential for blocking cell proliferation and migration in a spatio-temporally defined manner. It is suggested that the PDZ domains of Dlg are required for prohibiting follicle cell invasion (Goode, 1997).

Loss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. Cells in epithelial sheets are characterized by columnar or cuboidal shape, strong cell-cell adhesion, and pronounced apicobasal polarity. However, tumors of epithelial origin lose these characteristics as they progress from benign growth to malignant carcinoma, and this loss is associated with poor clinical prognosis. Evidence is provided that a group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation. Scribbled (Scrib) is a cell junction-localized protein required for polarization of embryonic and imaginal disc and follicular epithelia. The tumor suppressor scrib was isolated in a screen for maternal effect mutations that disrupt aspects of epithelial morphogenesis such as cell adhesion, shape and polarity. scrib encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein. The structure of the embryonic cuticle was used to reflect the organization of the underlying epithelial epidermis that secretes it. The wild-type cuticle forms a smooth, continuous sheet, but embryos that are maternally and zygotically mutant for scrib produce a corrugated cuticular surface that is riddled with holes, hence the name scribbled (Bilder, 2000a).

Two tumor suppressors, lethal giant larvae (lgl) and discs-large (dlg), have the identical effects as scrib mutation on epithelial structure. Scrib and Dlg colocalize and overlap with Lgl in epithelia; activity of all three genes is required for cortical localization of Lgl and junctional localization of Scrib and Dlg. scrib, dlg, and lgl show strong genetic interactions. Thus, these three tumor suppressors act together in a common pathway to regulate cell polarity and growth control (Bilder, 2000b).

Because follicle cell epithelia require scrib, lgl, and dlg, the functions of lgl and dlg were examined in the embryonic epidermis, where scrib acts to restrict apical proteins and adherens junctions to their appropriate positions within the cell membrane (Bilder, 2000a). Embryos lacking both maternal and zygotic contributions of lgl and dlg, (hereafter referred to as lgl and dlg embryos) were stained with antibodies to polarized proteins and cellular junction components. During mid-embryogenesis, lgl and dlg embryos show defects in apicobasal polarity, revealed by aberrant distribution of the apical protein Crumbs (Crb) and disruption of adherens junctions. These defects are similar to those of scrib embryos; the terminal phenotypes of scrib, lgl, and dlg embryos, as indicated by cuticle deposition, are also nearly identical. Thus, lgl and dlg, like scrib, act to properly localize apical proteins and adherens junctions to organize epithelial architecture in embryos (Bilder, 2000b).

The similarity of mutant phenotypes in different epithelia suggests that the three proteins are components of the fundamental machinery that creates the distinctive architecture of epithelial cells and tissues. To test this assertion, the scrib phenotype was compared to that of lgl and dlg in a third major epithelium, the larval imaginal disc. Discs isolated from late third instar larvae zygotically mutant for scrib are profoundly disorganized and also massively overgrown. scrib discs contain 4.7 times as many cells as wild-type (WT) discs and consist of spherical masses of tightly packed cells, as opposed to the folded monolayer epithelium seen in WT larvae. The apical polarization of actin evident in WT discs is absent in scrib discs. This loss of epithelial organization accompanied by overproliferation corresponds to the phenotype described for lgl and dlg zygotic mutant discs. Additional features of lgl and dlg larval phenotypes, such as overgrowth of brain tissue, are also present in scrib larvae. Together, these data indicate that scrib and the two previously characterized Drosophila malignant neoplastic tumor suppressors, lgl and dlg, share a role in growth control as well as epithelial polarity. Epistatic relations between scrib, lgl, and dlg were investigated by determining the localization of each protein in embryos mutant for the other two genes. These experiments have shown 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 apical margin of the lateral membrane (ALM) of the embryonic epidermal epithelium (Bilder, 2000b).

These results provide strong evidence that Scrib, Dlg, and Lgl act in a common pathway to regulate cell architecture and cell proliferation control. Of the ~50 Drosophila genes in which mutation gives rise to overproliferation, only scrib shares with dlg and lgl the concomitant loss of tissue organization that groups the three together as malignant neoplastic tumor suppressors. Previous analyses have described a role for dlg and lgl in imaginal disc polarity; the demonstration in this work of genetic interactions with scrib and codependence for protein localization indicates a functional link between the three tumor suppressors. Furthermore, involvement of the tumor suppressors in embryonic epithelial polarity provides a well-studied context in which to understand their activities. These findings suggest that, in the WT gastrula, intrinsic, perhaps adhesion-based cues localize Dlg at the ALM; Dlg stabilizes Scrib at this position, and finally Scrib acts on the cortical cytoskeleton to bring Lgl to the membrane. The three proteins may then collaborate to maintain the proper distribution of polarized factors, including themselves (Bilder, 2000b).

The correlation between loss of membrane-associated Lgl in scrib and dlg mutants and defective cell polarity suggests models of action for this group of proteins. Whereas the PDZ domains of Scrib and Dlg are likely to bind to transmembrane proteins that organize the epithelial cell surface, the role of Lgl in polarity determination may derive from its function in targeted secretion of membrane proteins. Lgl homologs from humans and yeast can bind to plasma membrane t-SNARE proteins and promote the fusion of cargo-carrying vesicles with target membranes. In yeast undergoing polarized growth, the broadly distributed Lgl homologs function primarily at the bud tip, the site of the 'exocyst' complex required for vesicle trafficking and addition. In vertebrate epithelia, exocyst components are found at the tight junction, a structure analogous to the septate junction where Dlg and Scrib localize. In Drosophila epithelia, recruitment of Lgl into the proximity of membrane t-SNAREs requires proper localization of Scrib and Dlg, thus potentially linking the transmembrane proteins that establish polarity to the protein-targeting system that preserves it (Bilder, 2000b and references therein).

In many epithelial-derived cancers, cytoarchitectural changes are hallmarks of oncogenic transformation. The disruption of epithelial architecture seen in scrib, dlg, and lgl animals could affect growth control by several mechanisms. Many growth factor receptors are polarized to a specific membrane domain, and mislocalization of such proteins may affect signaling pathways that maintain cells in a differentiated, nonproliferative state. Additionally, the aberrant cell-cell junctions formed in scrib, dlg, and lgl mutants could compromise contact inhibition. Finally, disruption of cell-cell contacts may release junction-localized signaling components, such as Arm or APC, that have been implicated in regulating cell proliferation; indeed, a human Dlg homolog has been shown to bind APC and associate with beta-catenin, the human homolog of Arm. Because the modes of action of Scrib, Dlg, and Lgl are likely to be conserved between vertebrates and invertebrates, investigation into a tumorigenic role for the multiple human homologs of these genes is warranted. Further analysis of the mechanisms by which Scrib, Dlg, and Lgl keep Drosophila cell growth in check will likely enhance an understanding of mammalian oncogenesis as well (Bilder, 2000b).

The Drosophila tumor suppressor Scribbled (Scrib) is required for maintaining epithelial cell polarity. At the larval neuromuscular junction, Scrib 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).

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 dlg^XI-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).

A cell-adhesion molecule Fasciclin 2, which is required for synaptic growth, and Still life (Sif), an activator of Rac, were found to localize in the surrounding region of the active zone, defining the periactive zone in Drosophila neuromuscular synapses. betaPS integrin and Discs large, both involved in synaptic development, also decorate the zone. However, Shibire (Shi), the Drosophila dynamin that regulates endocytosis, is found in the distinct region. Mutant analyses show that sif genetically interacts with Fas2 in synaptic growth and that the proper localization of Sif requires Fas2, suggesting that they are components in related signaling pathways that locally function in the periactive zones. It is proposed that neurotransmission and synaptic growth are primarily regulated in segregated subcellular spaces, active zones and periactive zones, respectively (Sone, 2000).

To characterize the periactive zone, especially in identifying its functional significance, the distribution patterns of other molecules were examined with the aid of Pak staining. Monoclonal antibody, MAb1D4, against Fas2 labels the boutons in a complementary pattern with Pak staining. Fas2 staining surrounds the Pak-positive regions and forms concentric patterns as observed for Sif staining. The cross-section profile also shows similar patterns as Sif and Pak double staining. Sif and Fas2 are indeed co-localized in overlapping network-like patterns. Fas2 is involved in synaptic growth, stabilization and structural plasticity, possibly through its homophilic adhesion. These data suggest that Fas2 controls these synaptic events locally in the periactive zones. Thus, the periactive zone is characterized by the specific localization of two distinct types of molecules: a cell adhesion molecule (Fas2) that controls synaptic development and an intracellular molecule (Sif) that is a GEF to Rac (Sone, 2000).

In an attempt to understand the function of the periactive zone further, other molecular markers that stain the zone were sought. MAb6G11, the monoclonal antibody against betaPs integrin (Myospheroid) that is structurally similar to the vertebrate integrin beta1 subunit also shows staining that is complementary to Pak staining. Unlike Sif and Fas2, however, MAb6G11 staining is observed much more diffusely on the muscle surfaces surrounding the outside of the bouton, suggesting the staining in the postsynaptic specialization: the subsynaptic reticulum. In mutants of the mys gene the extent of the cell contact between nerve terminals and muscles is altered by either a primary or secondary effect of the mutation, and the growth of larval neuromuscular synapses is affected. These synaptic defects observed in the betaPs integrin mutants may represent its function in the periactive zones (Sone, 2000).

The polyclonal antibody against Dlg protein stains synaptic boutons in a way similar to MAb6G11. The Dlg staining also appears to be moderately diffused on the muscle surfaces surrounding the bouton. This pattern is complementary with the anti-Pak staining when the bouton is scanned at the surface level. In dlg mutants, the structural properties of synapses, including the formation of subsynaptic reticulum at the postsynapses and the number of active zones at the presynapses, are altered. Furthermore Dlg regulates the synaptic localization of Fas2 by binding directly to the cytoplasmic tail of Fas2. Therefore, one of the roles for Dlg in synaptic development is probably the localization of Fas2 to the periactive zone. These observations indicate that two additional molecules, betaPs integrin and Dlg, are present in synaptic areas including the periactive zones. They are both involved in the structural development of the neuromuscular synapses, and therefore appear to participate in the control of synaptic development in the periactive zones (Sone, 2000).

The mutant larvae could be distinguished from the wild type by the blind test for the Sif and Pak co-staining patterns. Similar results were obtained in the heterozygotes with Fas2^e76 and a Fas2 null allele, suggesting that the alteration of Sif localization is not due to a second-site mutation on the Fas2^e76 chromosome. To investigate the altered distribution of Sif further, the mutant boutons were examined under the electron microscope. A large number of Sif signals are occasionally present in the medial portions of the electron-dense regions in the Fas2^e76 boutons and these signals are still associated with the plasma membrane, as are the signals observed in the wild type. It is therefore concluded that the reduction of Fas2 in the periactive zones results in the improper localization of Sif along the plasma membrane. Previous study has shown that the synaptic localization of Fas2 requires Dlg. Therefore, the localization of Sif was examined in the dlg mutant background, but no apparent alteration was found in the network pattern. A considerable amount of Fas2 is still present in the periactive zones of dlg mutant boutons, while faint or no staining is detected in the Fas2^e76 boutons. This residual Fas2 seems to be sufficient to sustain the proper localization of Sif in the dlg mutants (Sone, 2000).

The periactive zone has been indicated as a region for the control of synaptic development. The periactive zone surrounds the active zone, which is the site for vesicle exocytosis or neurotransmission. This concentric organization suggests that the two zones specialize for the different cellular functions and constitute an elemental unit for the presynaptic structure. Investigation of how these zones are incorporated into the synaptic bouton during development will be of interest. The segregated distribution of the two zones suggests that the mechanisms controlling synaptic development and neurotransmission may be separable. This view is supported by the mutant analyses for Fas2 and Sif; both mutations affect structural properties of synapses without changing basic electrophysiological functions. In the NMJs of Fas2 mutants, the bouton number is decreased or increased depending on the alleles but the total synaptic strength is maintained at the normal level. Functional strength of the synapse is regulated only through the activity of a transcription factor, cAMP-response-element-binding protein (CREB), which functions independently of Fas2. Also in sif mutants, the basic electrophysiological properties of NMJs are normal. These observations clearly contrast with the mutant phenotypes for the proteins controlling vesicle exocytosis: Synaptotagmin, Cysteine string protein, n-Synaptobrevin and Syntaxin 1A. Mutants in genes coding for all these proteins show impaired EJPs. Taken together, these results indicate that synaptic development and neurotransmission are genetically separable phenomena and are regulated by independent pathways. It is proposed that these genetically separable phenomena are spatially segregated into the two zones on the presynaptic plasma membrane, although the possibility that the two zones interact with each other cannot be excluded (Sone, 2000).

In Drosophila, neuroblasts undergo typical asymmetric divisions to produce another neuroblast and a ganglion mother cell. At mitosis, neural fate determinants, including Prospero and Numb, localize to the basal cortex from which the ganglion mother cell buds off; Inscuteable and Bazooka, which regulate spindle orientation, localize apically. Lethal (2) giant larvae (Lgl) is essential for asymmetric cortical localization of all basal determinants in mitotic neuroblasts, and is therefore indispensable for neural fate decisions. Lgl, which itself is uniformly cortical, interacts with several types of Myosin to localize the determinants. Another tumor-suppressor protein, Lethal discs large (Dlg), participates in this process by regulating the localization of Lgl. The localization of the apical components is unaffected in lgl or dlg mutants. Thus, Lgl and Dlg act in a common process that differentially mediates cortical protein targeting in mitotic neuroblasts, and that creates intrinsic differences between daughter cells (Ohshiro, 2000).

In mitotic neuroblasts, the Prospero transcription factor and Numb, an antagonist of Notch signaling, associate with their respective adapter proteins, Miranda and Partner of Numb (Pon), and thereby localize to the basal cortex. In contrast, Inscuteable (Insc), Bazooka (Baz) and Partner of Inscuteable (Pins) form a ternary complex at the apical cortex independently of the basal determinants. However, the mechanisms that underlie the asymmetric protein sorting in neuroblasts are not known. To address this issue, chromosomal deficiencies have been sought that affect the subcellular distribution of Miranda. Such screening identified the lgl tumor-suppressor gene that encodes a protein containing WD40 repeats. In wild-type neuroblasts, Miranda, which localizes apically during interphase, accumulates at the basal cortex upon mitosis after a transient spread into the cytoplasm. In germline clone embryos lacking both maternal and zygotic lgl activity (lglGLC embryos), Miranda does not localize asymmetrically in mitotic neuroblasts, but rather is distributed uniformly throughout the cortex as well as in the cytoplasm, where it is concentrated along microtubule structures. Consequently, Miranda segregates into both the daughter neuroblast and the ganglion mother cell (GMC). Numb and Pon are also distributed uniformly at the cortex and in the cytoplasm (Ohshiro, 2000).

Whether other tumor-suppressor genes contribute to protein localization in neuroblasts was investigated. The tumor-suppressor gene dlg encodes a membrane-associated guanylate kinase homolog. Germline clone embryos lacking both maternal and zygotic dlg activity (dlgGLC embryos) exhibit defective localization of Miranda and Numb essentially identical to that of lglGLC embryos, suggesting that both tumor-suppressor proteins function in the same process in neuroblasts. To investigate the relationship between the roles of Dlg and Lgl, their subcellular localization in neuroblasts was compared. Both Lgl and Dlg are distributed mainly throughout the cortex, whereas the amount of Lgl in the cytoplasm appears to be greater in mitosis than in interphase. The cortical localization of Lgl appears to be important for its function -- whereas the mutant protein encoded by the temperature-sensitive allele lgl^ts3 is distributed normally at the permissive temperature (18°C), it fails to localize cortically at the restrictive temperature (29°C). The wild-type Lgl protein exhibits a similar, abnormal cytoplasmic distribution in dlgGLC embryos, whereas Dlg localization is not affected in lglGLC embryos. Thus, the cortical localization of Lgl requires dlg activity, suggesting that Dlg may function in localization of cell-fate determinants in neuroblasts by positioning Lgl at the cortex (Ohshiro, 2000).

There are two important processes associated with the asymmetric division: (1) the asymmetric localization of cell-fate determinants, which is achieved by specific adapter proteins that themselves localize asymmetrically to the cortex in neuroblasts; and (2) the orientation of the mitotic spindle and its coordination with the polarized localization of the determinants, which requires the apical Baz-Insc-Pins complex. This study has revealed another important process mediated by Dlg, Lgl and Myosins, which is responsible for the cortical anchoring of the determinant-adapter complexes. This process occurs upstream of the first and independently or parallel to the second of those two aspects of asymmetric division, as the localization of Lgl and Dlg is independent of apical or basal components. Both Lgl and Dlg contribute to the generation or maintenance of epithelial polarity, and zygotic mutants of the corresponding genes develop epithelial cell tumors as well as brain tumors at late larval stages. These previous observations with epithelial cells, together with the data on the roles of Lgl and Dlg in protein targeting in neuroblasts, suggest that aberrant sorting of intracellular proteins may be responsible for the tumor formation apparent in larval stages of lgl and dlg mutants (Ohshiro, 2000).

Drosophila neuroblasts are a model system for studying asymmetric cell division: they divide unequally to produce an apical neuroblast and a basal ganglion mother cell that differ in size, mitotic activity and developmental potential. During neuroblast mitosis, an apical protein complex orients the mitotic spindle and targets determinants of cell fate to the basal cortex, but the mechanisms of these two processes are unknown. The tumor-suppressor genes lethal (2) giant larvae (lgl) and discs large (dlg) regulate basal protein targeting, but not apical complex formation or spindle orientation, in both embryonic and larval neuroblasts. Dlg protein is apically enriched and is required for maintaining cortical localization of Lgl protein. Basal protein targeting requires microfilament and myosin function, yet the lgl phenotype is strongly suppressed by reducing levels of myosin II. It is concluded that Dlg and Lgl promote, and myosin II inhibits, actomyosin-dependent basal protein targeting in neuroblasts (Peng, 2000).

Embryonic Drosophila neuroblasts develop from an apical/basal polarized epithelium. Individual cells delaminate into the embryo, enlarge to form neuroblasts, and begin a series of asymmetric cell divisions; these divisions result in the production of a large mitotically active apical cell (neuroblast), and a smaller basal cell (ganglion mother cell, GMC) that differentiates into two neurons or glia. A growing number of proteins are known to be asymmetrically localized in mitotic neuroblasts: apically localized proteins include Bazooka (Baz), Inscuteable (Insc) and Partner of Inscuteable (Pins); basally targeted proteins include Miranda, Prospero, Partner of Numb (Pon) and Numb, which are important for GMC development. Miranda and Prospero are apically localized at late interphase before their mitosis-dependent transport to the basal cortex. The Baz/Insc/Pins apical complex is required for both apical/basal spindle orientation and basal protein targeting, but little is known about how this complex regulates either process (Peng, 2000).

To identify genes required for apical/basal protein targeting in neuroblasts, deficiency stocks were screened looking for defects in Prospero basal localization in neuroblasts. This screen identified the lgl gene, which encodes a WD-40 repeat protein with homologues in many species, including the closely related 'Lgl family' genes Lgl1/Lgl2 (human), Lgl1 (mouse), U51993 (Caenorhabditis elegans); the slightly more divergent 'Tomosyn family' genes Tomosyn (rat), KIAA1006 (human), C617762 (Drosophila ), and M01A10 (C. elegans); and recently duplicated genes similar to both families: sro7/sro77 (budding yeast). In Drosophila, lgl mutations affect protein targeting to epithelial apical junctions, epidermal cell-shape changes, and produce tumors of the brain and the imaginal disc. This spectrum of phenotypes has been noted for another tumor-suppressor gene, discs large. This study explores the role of Lgl and Dlg in regulating neuroblast cell polarity (Peng, 2000).

Apical and basal protein targeting are compared in neuroblasts from wild-type embryos and embryos that lack all maternal and zygotic Lgl or Dlg function (called lglGLC or dlgGLC embryos). Wild-type metaphase neuroblasts show apical Insc/Pins localization, and basal Miranda/Prospero/Pon crescents. In addition, Miranda and Prospero proteins can be observed around the apical centrosome and weakly on the mitotic spindle in wild-type neuroblasts. In contrast, all lglGLC and dlgGLC metaphase neuroblasts show cytoplasmic Pon and uniformly cortical and strongly spindle-associated Miranda/Prospero; the apical proteins Insc/Pins are normal or slightly expanded. Although lglGLC and dlgGLC embryos show striking defects in neuroblast basal protein localization, they also show an early loss of embryonic epithelial apical/basal polarity, which could indirectly cause the observed neuroblast defects (Peng, 2000).

To determine the neuroblast-specific function of Lgl and Dlg, Lgl- or Dlg-depleted neuroblasts were studied in embryos or larvae where epithelial development occurs normally. Initially, homozygous null lgl⁴ embryos were studied, in which maternal Lgl protein allows normal embryonic epithelial development (including Armadillo, Crumbs and Dlg localization). In stage 16-17 lgl⁴ embryos, mitotic neuroblasts show normal Baz/Insc/Pins apical crescents, and normal spindle orientation, but Miranda/Prospero are delocalized onto the spindle and around the cortex and Pon is cytoplasmic. This phenotype is less severe in early embryos but fully penetrant in older embryos, presumably due to progressive loss of maternal Lgl protein. Next, neuroblasts were assayed in lgl³³⁴⁴ or dlg^v55 homozygous larvae -- these larval neuroblasts are persistent embryonic neuroblasts that develop from a normal embryonic epithelium due to maternal Lgl and Dlg protein function. Wild-type larval metaphase neuroblasts have Insc/Pins crescents the opposite of Miranda/Prospero/Pon/Numb crescents, whereas homozygous lgl³³⁴⁴ or dlg^v55 larval metaphase neuroblasts show normal Insc/Pins crescents but Miranda/Prospero/Pon proteins are cytoplasmic, uniformly cortical, and weakly spindle-associated. It is concluded that Lgl and Dlg are required specifically in neuroblasts for basal protein targeting, without affecting apical protein localization or spindle orientation (Peng, 2000).

The lgl and dlg neuroblast basal localization phenotype is cell-cycle dependent. lgl and dlg mutant embryonic and larval neuroblasts show a fully penetrant loss of basal protein targeting at metaphase, but by late anaphase or telophase most neuroblasts show normal basal protein localization; 'telophase rescue' of basal protein localization also occurs in baz, insc and pon mutants. These results indicate that there are probably multiple mechanisms for basal protein localization (Peng, 2000).

Delocalization of the Prospero and Numb proteins produces defects in the nervous system and other tissues, so lgl mutant embryos were scored for cell fate defects. lglGLC embryos have severe morphological defects that preclude analysis, and lgl⁴ embryos can only be scored for late embryonic phenotypes, due to persistence of maternal Lgl protein. lgl⁴ embryos show a decrease in Even-skipped lateral (EL) neuron number at stage 17. A similar but stronger phenotype is seen in numb mutants, suggesting that the lgl phenotype may be due to delocalization of Numb during the GMC divisions that produce the EL neurons. The relatively mild lgl phenotype could be due to 'telophase rescue' of Numb protein in these GMCs, or to maternal Lgl protein (Peng, 2000).

In wild-type embryonic neuroblasts, Dlg protein is cortical with an apical crescent from late interphase to the end of mitosis that co-localizes with Baz/Insc/Pins, whereas Lgl protein is uniformly cortical and weakly cytoplasmic. Similarly, larval neuroblasts show apical Dlg and uniform cortical/cytoplasmic Lgl localization. lgl mutants show normal Dlg localization, but dlg mutants show loss of cortical Lgl protein. Thus, Dlg acts upstream of Lgl for its localization, but not necessarily its function (Peng, 2000).

Thus, in neuroblasts Lgl and Dlg regulate targeting of all known basal proteins without affecting apical protein localization or spindle orientation. In epithelia, Lgl and Dlg are necessary to restrict proteins to the apical membrane domain. Lgl could promote protein targeting to specific membrane domains in both neuroblasts (basal) and epithelia (apical), similar to the role of Lgl-related proteins in facilitating secretory vesicle fusion at specific membrane domains in yeast and mammals. If so, Lgl must act in neuroblasts via a secretory pathway that is independent of brefeldin A, because it has been shown that treatment with brefeldin A disrupts Golgi, inhibits Wingless secretion, but does not block basal protein targeting. Alternatively, Lgl may actively promote actomyosin-dependent localization of basal proteins and/or function to keep myosin II levels low so that they do not interfere with myosin-dependent basal localization. A general function of the Lgl protein family may be to increase the fidelity of protein targeting to specific domains of the plasma membrane (Peng, 2000).

Membrane-associated guanylate kinases (MAGUKs) assemble ion channels, cell-adhesion molecules and components of second messenger cascades into synapses, and are therefore potentially important for co-ordinating synaptic strength and structure. The targeting of the Drosophila MAGUK Discs-large (Dlg) to larval neuromuscular junctions has been examined. During development, Dlg is first found associated with the muscle subcortical compartment and plasma membrane, and later is recruited to the postsynaptic membrane. Using a transgenic approach, a study of how mutations in various domains of the Dlg protein affect Dlg targeting was undertaken. Deletion of the HOOK region -- the region between the Src homology 3 (SH3) domain and the guanylate-kinase-like (GUK) domain -- prevents association of Dlg with the subcortical network and renders the protein largely diffuse. Loss of the first two PDZ domains leads to the formation of large clusters throughout the plasma membrane, with scant targeting to the neuromuscular junction. Proper trafficking of Dlg missing the GUK domain depends on the presence of endogenous Dlg. It is concluded that postsynaptic targeting of Dlg requires a HOOK-dependent association with extrasynaptic compartments, and interactions mediated by the first two PDZ domains. The GUK domain routes Dlg between compartments, possibly by interacting with recently identified cytoskeletal-binding partners (Thomas, 2000).

The sequence preceding PDZ1 exhibits only weak or no homology between various MAGUKs. Several studies have implicated the amino terminus of both PSD-95 and SAP97 in junctional targeting. To determine whether the amino terminus of Dlg is of similar importance, transgenic Dlg missing the amino terminus (DeltaN), was expressed. This construct exhibits a synaptic localization indistinguishable from the control (Dlg-FLAG). This result is found both when DeltaN is expressed in the presence or absence (in dlgX1-2 mutants) of endogenous Dlg (Thomas, 2000).

In both the wild type and dlg mutant background, deletion of any single PDZ domain does not affect the synaptic localization of Dlg. Deletion of PDZ3 in combination with either PDZ1 or PDZ2 also does not affect localization. In contrast, deletion of both PDZ1 and PDZ2 (DeltaPDZ1+2) has dramatic effects on localization. This variant becomes localized at the surface of the muscle, with little synaptic localization. The immunoreactivity at the plasma membrane appears as large spots or clusters distributed throughout the muscle membrane. The DeltaPDZ1+2 variant can also be detected at the subcortical network, although the intensity of FLAG immunoreactivity is significantly weaker than in Dlg-FLAG-expressing muscles. Thus, in the absence of PDZ1 and 2, Dlg becomes transported to the muscle membrane, but fails to be directed to synaptic sites, and instead accumulates in ectopic clusters. This conclusion was reached by expressing DeltaPDZ1+2 in both the wild type and in dlgX1-2 mutants. To determine whether the distribution of endogenous Dlg is altered by the presence of these ectopic clusters, double labeling experiments were performed, in which DeltaPDZ1+2 and endogenous Dlg proteins were discriminated using anti-DlgGUK. The anti-DlgGUK antibody recognizes both endogenous and transgenic Dlg, but the anti-DlgPDZ antibody does not recognize DeltaPDZ1+2. Notably, endogenous Dlg does not become trapped in clusters containing the DeltaPDZ1+2 variant, but remains synaptically localized (Thomas, 2000).

Interestingly a transgenic protein comprising the amino terminus and all three PDZ domains (DeltaC1/2) fails to localize to the plasma membrane or synapses, and is instead found highly enriched in nuclei and cytoplasm. This result indicates that the PDZ1 and 2 domains are necessary but not sufficient for synaptic targeting (Thomas, 2000).

The involvement of the SH3 domain in synaptic targeting of MAGUKs has been controversial. To determine the role of the SH3 domain in Dlg targeting, tests were performed on both the allele dlgm30, in which the SH3 domain is affected by a point mutation, and flies expressing a Dlg variant in which the SH3 domain was deleted (DeltaSH3). In both dlgm30 and DeltaSH3 flies, Dlg is normally targeted to synaptic sites. In the case of the DeltaSH3 line, similar results were obtained in the presence and absence of endogenous Dlg (Thomas, 2000).

The HOOK region has been implicated in the association of Dlg with septate junctions in epithelial cells. This domain is among the least conserved regions of MAGUKs, but two sub-regions are moderately conserved among specific subsets of MAGUKs. A band 4.1-binding motif known as I3 is also found in SAP97, PSD-93 and p55 and may link these MAGUKs to the actin/spectrin cytoskeleton. Another short stretch (E-F region), presumed to form an alpha-helix at the amino terminus of the HOOK region, is found in all Dlg-like MAGUKs, and has been implicated in calmodulin-dependent dimerization of PSD-95 and SAP102 (Thomas, 2000 and references therein).

Deletion of the entire HOOK region (DeltaHOOK) dramatically affects the synaptic localization of Dlg both in the presence and absence of endogenous Dlg. In general, some synaptic localization is observed, although weaker than in Dlg-FLAG controls. In addition, the FLAG signal appears in the muscle nuclei and throughout the cytoplasm. Extrasynaptic localization at the plasma membrane is weak compared with Dlg-FLAG controls, and virtually no specific immunoreactivity is detected at the subcortical network. The relative intensity of the signals at synapses versus nuclei varies, even at the muscles within the same sample. Strikingly, very similar results are obtained when the amino-terminal helix within the HOOK region is disrupted by deleting only 13 amino acids (DeltaE-F). Deletion of the I3 region (DeltaI3) also results in weak synaptic localization of the transgenic protein when expressed in wild type. Unlike DeltaHOOK and DeltaE-F, however, deletion of I3 does not result in nuclear localization. Surprisingly, DeltaI3 exhibits strong synaptic localization when expressed in dlgX1-2 mutants (Thomas, 2000).

The importance of the HOOK region for targeting to both septate and synaptic junctions appears surprising, because it is not well conserved among MAGUKs. Nevertheless, a short stretch of amino acids at the amino terminus of the HOOK region of all Dlg-like MAGUKs is predicted to form an alpha-helix with a cluster of basic residues on one side. This helix has been reported to mediate a calmodulin-dependent dimerization of PSD-95 and SAP102. The finding that disruption of this helix mimics the effect of the entire DeltaHOOK deletion, confirms that it is of particular importance in vivo. It remains to be determined whether it functions in synaptic targeting by promoting dimerization of Dlg, by contributing to an intramolecular interaction, and/or by mediating heterophilic interactions (Thomas, 2000).

The role of the GUK domain in MAGUKs has remained obscure. In the case of fly epithelial cells, deletion of the GUK domain does not alter localization of Dlg to septate junctions. Various cytoskeletal and synapse-associated proteins have been reported to bind to the GUK domain of mammalian MAGUKs, suggesting that this domain may be important for synaptic localization. To ascertain the role of GUK in synaptic targeting, transgenic flies expressing a Dlg variant lacking the GUK domain (DeltaGUK) were examined. In the presence of endogenous Dlg, DeltaGUK becomes localized to synaptic sites, although this synaptic expression appeared consistently weaker than in Dlg-FLAG-expressing controls. Notably, however, synaptic localization of DeltaGUK is barely or not detectable upon expression in dlgX1-2 mutants. It is therefore concluded that endogenous Dlg mediates synaptic targeting of the DeltaGUK variant (Thomas, 2000).

None of these deletions resulted in an obvious accumulation solely at the subcortical network. This suggests that no additional domains are required to leave this compartment. A role for the GUK domain to direct subsequent transport is supported. Recent evidence suggests that targeting of PSD-95 and PSD-93 involves a microtubule-dependent transport of vesiculo-tubular structures. These MAGUKs can be linked to microtubules by binding of the PDZ3 and GUK domains to the microtubule-associated proteins CRIPT or MAP1A, respectively. Although these binding partners are suggestive for a role of these domains in MAGUK trafficking, no such function has previously been unraveled. Removal of PDZ3 has no obvious effect on synaptic localization of Dlg. Synaptic localization of the DeltaGUK protein is, however, diminished in wild-type flies and virtually abolished in dlgX1-2 mutants. This dependency on endogenous Dlg might be explained by dimerization. Alternatively, endogenous Dlg could promote vesiculo-tubular trafficking and thus allow the truncated version to hitchhike on the same vesicles. This explanation would also apply to the finding that the GUK domain is dispensable for targeting of PSD-95 in cultured neurons or slices, in which the endogenous MAGUK is expressed (Thomas, 2000).

Consistent with other studies of MAGUK targeting, it was found that the PDZ domains are neither sufficient to target Dlg to specific extrasynaptic sites nor to dock the protein at the synapse. Nonetheless, the PDZ1 and 2 domains are found to contribute to synaptic targeting. It is suggested that either the PDZ1 or PDZ2 domain is required for the final step in Dlg targeting, from plasma membrane to synapses, but is not necessary to direct Dlg to the plasma membrane. Thus, it may be assumed that the interaction with at least one PDZ binding protein is required to transport the protein to the synapse. A double mutant, in which the only known binding partners for the PDZ1 and 2 domains of Dlg, Shaker and FasII were abolished or dramatically reduced, has no obvious effect on synaptic targeting of Dlg. Interestingly, a non-synaptic PDZ-binding protein, Cypin, may regulate synaptic targeting of PSD-95 and SAP102 at extrasynaptic sites (Thomas, 2000 and references therein).

It remains to be determined whether the association of Dlg with intracellular membrane compartments serves solely to target Dlg itself. As discussed for GRIP1 and PSD-95, this step could also contribute to the sorting and co-transport of other synaptic molecules such as ion channels. The pre-formation of a junctional protein complex at intracellular compartments has been exemplified by the interaction of E-cadherin and beta-catenin at the endoplasmic reticulum. The clustering of glycine receptors by gephyrin provides a contrary example, however, since gephyrin traps receptor molecules only at developing synapses. Unfortunately, the limited sensitivity of available Shaker-specific antibodies has impaired attempts to distinguish between intracellular and synaptic assembly of the Dlg-Shaker complex. It appears obvious, however, that each targeting step represents an additional site at which the molecular composition of synaptic junctions could be regulated (Thomas, 2000).

Drosophila Bazooka and atypical protein kinase C are essential for epithelial polarity and adhesion. Wild-type bazooka function is required during cell invasion of epithelial follicle cells mutant for the tumor suppressor discs large. Clonal studies indicate that follicle cell Bazooka acts as a permissive factor during cell invasion, possibly by stabilizing adhesion between the invading somatic cells and their substratum, the germline cells. Genetic epistasis experiments demonstrate that bazooka acts downstream of discs large in tumor cell invasion. In contrast, during the migration of border cells, Bazooka function is dispensable for cell invasion and motility, but rather is required cell-autonomously in mediating cell adhesion within the migrating border cell cluster. Taken together, these studies reveal Bazooka functions distinctly in different types of invasive behaviors of epithelial follicle cells, potentially by regulating adhesion between follicle cells or between follicle cells and their germline substratum (Abdelilah-Seyfried, 2003).

Border cell migration during Drosophila oogenesis is one well-studied example of invasive and directed migration. Border cells are specified within the anterior follicular epithelium that surrounds the germ cells in each egg chamber. At late egg chamber stage 8, approximately eight border cells delaminate from the monolayer epithelium and, in a highly stereotyped fashion, invade the germ cell cluster within the developing egg chamber. First, they undergo directed cell migration between nurse cells towards the anterior margin of the oocyte and then turn dorsally, coming to rest at the dorsal anterior corner of the egg chamber next to the underlying oocyte nucleus. Border cell migration displays several features that are reminiscent of metastasis by cancer cells. Initially, border cells are polarized epithelial cells that lose some homophilic cell adhesion, undergo an epithelial-to-mesenchymal transition, acquire adhesion with the substratum, and undergo cell migration. However, not all epithelial characteristics are lost during migration. The migrating cells remain attached to each other and intercellular polarized junctions containing DE-cadherin (Shotgun), Armadillo (Arm) and Crumbs are present. DE-cadherin has been demonstrated to play an essential role in both migrating border cells, and their substratum, the germ cells. These studies suggest homophilic interactions between transmembrane receptors, such as DE-cadherin, may provide the necessary adhesion between invasive cells and their substratum (Abdelilah-Seyfried, 2003 and references therein).

The results suggest that baz is an essential component of dlg mutant follicle cell invasion into the germline. During border cell migration baz is dispensable for invasion and motility but appears to be required for correct cell adhesion within the migrating cluster. baz acts downstream of dlg in controlling follicle cell invasion. Taken together, these results suggest that loss of dlg initiates epithelial-to-mesenchymal transition and results in increased follicle cell motility. One role of wild-type baz may be to ensure the proper adherence between invading cells and their substratum (Abdelilah-Seyfried, 2003).

Two lines of evidence suggest mechanistic differences between tumor cell and border cell invasion. (1) While both dlg tumor cell and border cell invasion undergo a series of similar morphogenetic behaviors, the molecular mechanisms regulating each cellular repertoire appear, at least in part, to be distinct. Whereas tumor cell invasion is dependent on baz, border cell invasion and motility are not. Therefore, baz genetically discriminates between these processes. Conversely, border cell migration requires slbo function, whereas dlg mutant follicle cell invasion can occur with much lower levels of Slbo and FasIII proteins and therefore dlg mutant cells appear not to adopt a border cell fate. (2) A second line of evidence for mechanistic differences is that the patterns of cell invasion are distinct. The timing, direction and cohesion of border cells during their migration is highly stereotyped. In contrast, dlg tumor cells can invade at any stage in egg chamber development and in any orientation relative to the oocyte, possibly due to the position where follicle cell over-accumulation and multi-layering occur. Moreover, invasion also occurs in the absence of an oocyte, for example when the germline is dlg^m52;baz^EH171 double mutant (Abdelilah-Seyfried, 2003).

The data suggest that wild-type Baz is a permissive factor required for follicle cell invasion but that baz gene function is dispensable for border cell specification and invasion. Therefore, in the absence of baz, the specification of Slbo-positive cells and activation of the appropriate downstream targets that are required for the orchestration of border cell migration is normal. The activation of slbo and its target genes may largely mask the permissive role of baz in follicle cell migration, a requirement that is uncovered in the context of the slbo-independent type of follicle cell invasion caused by the loss of dlg. Moreover, in contrast to DE-cadherin, another gene with an essential function during border cell migration, Baz and DaPKC levels are not increased in border cells prior to and during border cell migration. The defects observed in baz mutant border cell migration are best explained by the lack of adhesion within the border cell cluster rather than by migratory defects (Abdelilah-Seyfried, 2003).

This study provides an example of a genetic interaction between the apical PAR complex and basolateral tumor suppressor genes. This interaction was assessed based on tumor cell invasion. baz is epistatic over (functions downstream of) dlg in regulating this process. One possible explanation for the mechanism by which Dlg, a basolateral protein absent from the sites of contact between follicle and germ cells, regulates motility is that it acts via another protein complex. Evidence is presented that the apical PAR complex may serve such a function. A model is suggested in which follicle cell invasion is a two-step process: first, the loss of dlg releases a repression of motility and, second, the apical PAR complex protein Baz serves as a permissive factor for invasion. Based on mosaic analysis, a model is proposed in which invasion might be mediated by two separate baz-dependent interactions between follicle and germline cells. During invasion of dlg mutant follicle cells, Baz functions as a permissive factor to promote follicle cell invasive behavior. This invasive behavior is blocked in the absence of follicle cell Baz, since dlg^m52 baz^EH171 or baz^EH171 mutant follicle cells lack invasive properties. Within the germline, Baz functions as both a permissive factor during invasion of dlg^m52 mutant follicle cells that express Baz, possibly by stabilizing adhesion between the invading somatic cells and the germline cells and, in the absence of follicle cell Baz, as a repressor of follicle cell invasion, possibly by regulating germ cell adhesion and preventing invasion of Baz-deficient follicle cells. The repression of Baz-deficient follicle cell invasion is neutralized in dlg^m52 baz^EH171 mutant germ cell clones possibly by a reduction of germ cell adhesion that may increase the ease with which dlg^m52 baz^EH171 mutant follicle cells can invade. These observations raise the question as to the molecular machinery and the adhesion molecules that mediate baz-dependent invasion and to the mechanisms that are in place in dlg^m52 baz^EH171 mutant follicle and germ cells in which invasion occurs. An alternative explanation to the loss of motility is that the removal of a second cell polarity system from follicle cells may cause such severe disturbances as to prevent cell invasion. However, dlg^m52 baz^EH171 double mutant follicle cells retain their capability to invade into dlg^m52 baz^EH171 double mutant germline proper, contradicting this explanation (Abdelilah-Seyfried, 2003).

The data presented in this study raise the possibility that DaPKC serves similar, essential functions during dlg tumor cell invasion. However, this hypothesis was not tested since it was genetically not possible to generate dlg DaPKC double mutant follicle and germline clones. During border cell migration, there is a different requirement for Baz and DaPKC. Whereas Baz appears to affect adhesion within the migratory border cell cluster, DaPKC function is dispensable for normal border cell invasion, migration, and adherence (Abdelilah-Seyfried, 2003).

In contrast to previous findings, the results indicate dlg predominantly functions cell-autonomously to prevent invasion of follicle cells. This finding is consistent with the data on lgl, which also functions cell-autonomously within the follicle cell layer to prevent heterogeneous cell mixing and invasion. Indeed, cases of cell-autonomous invasions of follicle cells into the germline have been documented; these support the notion that, despite quantitative differences between the studies, dlg functions cell-autonomously within the follicle cell layer. The FLP/FRT technique combined with GFP imaging used in the study allows for the unambiguous identification of mosaic tissues, clarifying issues of cell-autonomous gene function (Abdelilah-Seyfried, 2003).

The multiple PDZ domain protein Baz and its vertebrate homolog ASIP is a membrane scaffolding factor required for assembly and sub-membrane attachment of the apical PAR complex. The effects of the PAR complex on dlg mutant follicle cell invasion may be exerted via a separate but baz-dependent transmembrane adhesion complex, the nature of which is currently unknown. In contrast to its function during border cell migration, in humans, loss of E-cadherin correlates with and appears to promote the occurrence of invasive tumor formation. It has been suggested, therefore, that E-cadherins serve distinct functions in different cell types, either by promoting or inhibiting cell motility. Further studies are required to test whether the homologous proteins of Baz (ASIP) and DaPKC (atypical PKCs iota and zeta) serve conserved functions in mammalian cells and, in contrast to E-cadherin function, whether their loss prevents tumor cell invasion. Moreover, it is unclear whether baz function is restricted to the behavior of dlg mutant follicle cells or is essential in other forms of tumor cell invasions (Abdelilah-Seyfried, 2003).

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