Analysis of the spatio-temporal expression of the l(2)gl transcripts and proteins has shown that transcripts and proteins are produced ubiquitously during early embryogenesis, at a time when expression of the gene is required for preventing tumorigenesis. In the second half of embryogenesis, l(2)gl expression becomes restricted to tissues that do not show any phenotypic alteration in mutant animals. The l(2)gl protein exhibits two distinct intracellular localizations. It is preferentially found free in the cytoplasm but can become associated with the inner face of the plasma membrane where it is restricted to domains facing contiguous cells. In particular, the l(2)gl protein is absent from the basal and apical domains of the plasma membrane (Strand, 1991).
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).
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).
Epithelial tissues are composed of polarized cells with distinct apical and basolateral membrane domains. In the Drosophila ovarian follicle cell epithelium, apical membranes are specified by Crumbs (Crb), Stardust (Sdt), and the aPKC-Par6-cdc42 complex. Basolateral membranes are specified by Lethal giant larvae (Lgl), Discs large (Dlg), and Scribble (Scrib). Apical and basolateral determinants are known to act in a mutually antagonistic fashion, but it remains unclear how this interaction generates polarity. A computer model of apicobasal polarity was build that suggests that the combination of positive feedback among apical determinants plus mutual antagonism between apical and basal determinants is essential for polarization. In agreement with this model, in vivo experiments define a positive feedback loop in which Crb self-recruits via Crb-Crb extracellular domain interactions, recruitment of Sdt-aPKC-Par6-cdc42, aPKC phosphorylation of Crb, and recruitment of Expanded (Ex) and Kibra (Kib) to prevent endocytic removal of Crb from the plasma membrane. Lgl antagonizes the operation of this feedback loop, explaining why apical determinants do not normally spread into the basolateral domain. Once Crb is removed from the plasma membrane, it undergoes recycling via Rab11 endosomes. The results provide a dynamic model for understanding how epithelial polarity is maintained in Drosophila follicle cells (Fletcher, 2012).
These above results define an apical positive feedback loop that centers on endocytic regulation of Crb. If such a positive feedback loop exists, it must be antagonized by the basolateral determinants to prevent spreading of apical determinants into the basolateral domain. In the computer model, ectopic spreading of apical determinants caused by simulated inhibition of endocytosis (strongly reducing the rate at which apical determinants are removed from the plasma membrane) can be counteracted simply raising the number of basolateral determinants by 5-fold. In follicle cells, inhibiting endocytosis with RNAi against the AP2/clathrin component AP50 leads to ectopic spreading of apical determinants into the basolateral domain, as in the model. Overexpression of Lgl-GFP was sufficient to restore normal polarity even in the presence of AP50 RNAi, again similar to the simulations. Furthermore, expression of Lgl-GFP also rescued the spreading of apical determinants caused by Rab5 RNAi or overexpression of Crb. These results suggest that Lgl may be a rate-limiting basolateral determinant and that it acts to inhibit positive feedback among apical determinants and thereby promote endocytic removal of Crb from the basolateral membrane (Fletcher, 2012).
Once Crb has been endocytosed by the AP2/clathrin machinery, it could be either degraded in the lysosome or recycled. Recent evidence indicates that Crb avoids the lysosome due to the action of the retromer machinery. The recycling endosome protein Rab11 is essential for Crb to remain at the plasma membrane in embryos. By costaining for Crb and Rab11 in follicle cells, it was possible to detect many endosomes that are positive for both proteins. Furthermore, when Rab11 is knocked down by RNAi in follicle cells, a loss of Crb from the plasma membrane was detected and an accumulation in enlarged endosomes. In contrast, RNAi of Rab5 causes accumulation of Crb at the plasma membrane. Accordingly, the Rab11 RNAi phenotype -- unlike that of Rab5 -- cannot be suppressed by coexpression of Lgl-GFP. These results confirm that Crb undergoes Rab11-mediated recycling to maintain its polarized plasma membrane localization (Fletcher, 2012).
One difference between the computer model and in vivo data is that inactivation of apical determinants in the model leads to complete loss of apical determinants from the membrane. However, in follicle cells, mutation of crb does not cause complete loss of apical aPKC from the plasma membrane. This residual aPKC is due to the Bazooka protein (Baz/Par3), which-like Crb-is able to bind to aPKC-Par6 and normally localizes to adherens junctions but can also occupy the apical membrane in the absence of Crb. Whether the Baz system operates by the same positive feedback principle as the Crb system remains to be explored (Fletcher, 2012).
These findings indicate that polarization of Crb in the follicle cell epithelium depends on the combination of two principles: positive feedback and mutual antagonism. The apical domain forms where Crb can recruit additional Crb molecules via Crb-Crb interactions, recruitment of Sdt and aPKC-Par6-cdc42, aPKC phosphorylation of the Crb FERM-binding domain, and recruitment of the FERM-domain protein Ex and its binding partner Kib. Although direct binding between these factors in follicle cells was not shown, work in other model systems indicates that they do bind directly. Disruption of any element of this feedback loop results in endocytosis of Crb from the plasma membrane. In contrast, ectopic activation of various components of this feedback loop-by overexpression of Crb, cdc42V12, or aPKCdeltaN-stabilizes Crb and the other apical determinants at the plasma membrane. The basolateral domain forms where Crb is endocytosed from the plasma membrane because Lgl (which can bind to aPKC-Par6 and inhibit aPKC kinase activity), presumably prevents Crb from engaging in a productive interaction with the other apical determinants, thereby disrupting Crb self-recruitment (Fletcher, 2012).
In conclusion, the model explains how epithelial polarity is a property of a complex system that can emerge spontaneously from the nature of the interactions between apical and basolateral polarity determinants. The principle of combined positive feedback and mutual antagonism outlined in this study in Drosophila follicle cells may prove to be widely used in the generation of polarity in many different cellular contexts (Fletcher, 2012).
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).
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 other 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).
The equivalent requirements for scribbled, lgl, and dlg 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 show 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).
To test whether the requirement for Scrib is limited to the embryo, an examination was made of the role of scrib in follicle cells, a monolayered epithelium of somatic cells that encases the germ line in the adult female ovary. Scrib is localized to lateral follicle cell membranes, and clones of cells that lack scrib function become round and multilayered with polarity defects, similar to the phenotype of embryonic epithelia lacking scrib function. The epithelial defects of scrib mutant follicle cells are cell autonomous. These data indicate that scrib is required within cells from multiple tissues for proper epithelial structure (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, which 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 (Fujita, 1998; Lehman, 1999). 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 (TerBush, 1996). 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).
Recessive mutations at the l(2)gl locus of Drosophila cause a complex syndrome, which has as its most striking features the development of malignant neuroblastomas in the larval brain and tumors of the imaginal discs. A chromosomal segment containing the l(2)gl gene has been cloned. Within this segment a transcription unit has been localized that is structurally changed in all l(2)gl alleles examined. The developmental profile of expression of the two RNAs (6 and 4.5 kb) made by this transcription unit coincides with the two major terminal phases of cell proliferation in the developing fly, namely, early embryogenesis and late third instar larvae. Tumors are produced when both normal l(2)gl alleles are inactivated by deletion or insertional mutation. The normal function of the l(2)gl presumably controls the normal cell proliferation of the optic centers of the brain and the imaginal discs, as well as their post-mitotic differentiation (Mechler, 1985).
Homozygous mutations of the recessive oncogene lethal 2 giant larvae of Drosophila cause lethal neoplasms of the imaginal discs and the brain hemisphere. A 13-kb DNA segment spanning the l(2)gl+ locus has been inserted into P element vectors and used for P-mediated transformation. The P-l(2)gl+ transposons have been introduced into the germ line of heterozygous l(2)gl-/+ flies and were shown by backcrossing to fully rescue the homozygous l(2)gl deficient animals, which otherwise would have died of brain and imaginal disc neoplasms. Further genetic backcrossing with l(2)gl deficiencies characterized by deletions of increased sizes involving the left end of chromosome 2 indicated that a relatively large region of developmentally regulated DNA sequence adjacent to the l(2)gl gene is apparently not essential for the viability and fertility of the fly. These experiments indicate that all the genetic information specified by the l(2)gl+ gene is contained within this 13-kb DNA segment and demonstrate that the development of neuroblastomas and imaginal disc tumors results from the absence of l(2)gl function. When this function is restored, tumor development is completely suppressed (Opper, 1987).
Cell proliferation in Drosophila imaginal discs appears to be regulated by a disc-intrinsic mechanism involving local cell interactions that also control the formation of patterns of differentiation. This growth-control mechanism breaks down in animals homozygous for the mutation lethal (2) giant discs; such mutants remain as larvae for up to 9 days longer than normal. During this time cell proliferation continues in the imaginal discs as well as in the imaginal rings for the salivary glands, foregut, and hindgut, so that these tissues become greatly overgrown. When wild-type wing discs from mid-third instar larvae were removed and cultured for up to 28 days in wild-type female adult hosts, they grew and terminated growth at a cell number close to that which would be attained in situ by the time of pupariation. In contrast, wing discs from l(2)gd homozygotes grew rapidly and continuously when cultivated in wild-type hosts, reached an enormous size, and acquired abnormal folding patterns. Overgrowth of mutant imaginal rings also continued during culture of these tissues in wild-type hosts. It is concluded that overgrowth in this mutant is due to an autonomous defect in the imaginal primordia, which requires an extended larval period for its expression in situ (Bryant, 1985).
Homozygosity for lethal(2)giant larvae induces neoplasia of the imaginal discs. To explore the developmental capacities of lgl mutant cells, their growth and differentiation has been investigated in genetic mosaics. Adult wings mosaic for lgl display abnormal growth and differentiation of the lgl mutant and neighboring wild-type cells, suggesting aberrant cell-cell interactions during development. lgl mutant clones also straddle the anteroposterior boundary of the wing imaginal disc, apparently due to failure of the cells of the anterior and the posterior compartment to segregate at the boundary. To further test if anteroposterior compartmentalization takes place in the neoplastic imaginal discs of lgl mutant larvae, the expression of an engrailed (en)-specific lacZ reporter gene was studied during progressive stages of their tumorous growth. en is activated in the posterior compartments of the neoplastic imaginal discs. However, during later stages of tumorous overgrowth, the en-expressing and nonexpressing cells appear to show extensive intermixing. These observations suggest that neoplastic transformation of imaginal discs involves loss of their normal cell-cell interactions and signaling (Agrawal, 1995).
The lethal(2)giant larvae gene encodes a widely expressed cytoskeletal protein that acts in numerous biological processes during embryogenesis and oogenesis, including cell proliferation, and morphogenetic movements. Having identified the nucleotide change occurring in the l(2)glts3 sequence, the identical change was produced by site-directed mutagenesis leading to the substitution of a serine by a phenylalanine at position 311 of p127l(2)gl and the modified l(2)glF311 gene was introduced into l(2)gl flies. The transgene can fully rescue the development of l(2)gl flies raised at 22°C but causes drastic effects on their development at 29°C confirming the temperature sensitivity of the phenylalanine substitution at position 311. Fertility of females, albeit not of males, is strongly affected. Temperature-shift experiments and microscopic examination of ovaries showed that the mutation blocks egg chamber development at the onset of vitellogenesis (stages 8-9) with growth arrest of the oocyte, incomplete follicle cell migration over the oocyte associated with abnormal organization of the follicular epithelium, and apoptosis of the germline cells, as measured by TUNEL assays. By comparison to wildtype, it was found that p127F311 is already reduced in amount at 22°C and delocalizes from the cytoskeletal matrix, albeit without affecting the apical localization of myosin II, a major partner of p127. At 29°C, the level of p127F311 is even more reduced and the distribution of myosin-II becomes markedly altered at the apices of the follicle cells. These data indicate that during oogenesis p127 plays a critical function at the onset of vitellogenesis and regulates growth of the oocyte, follicle cell migration over the oocyte and their organization in a palisadic epithelium, as well as viability of the germline cells (De Lorenzo, 1999).
During Drosophila metamorphosis, larval tissues, such as the salivary glands, are histolysed whereas imaginal tissues differentiate into adult structures, to form at eclosion a fly-shaped adult. Inactivation of the lethal(2)giant larvae gene encoding the cytoskeletal associated p127 protein, causes malignant transformation of brain neuroblasts and imaginal disc cells with developmental arrest at the larval-pupal transition phase. At this stage, p127 is expressed in wild-type salivary glands that become fully histolysed 12-13 h after pupariation. By contrast to wild-type, administration of 20-hydroxyecdsone to l(2)gl-deficient salivary glands is unable to induce histolysis, although it releases stored glue granules and gives rise to a nearly normal pupariation chromosome puffing, indicating that p127 is required for salivary gland apoptosis. To unravel the l(2)gl function in this tissue, transgenic lines expressing reduced (approximately 0.1) or increased levels of p127 (3.0) were used. The timing of salivary gland histolysis displays an l(2)gl-dose response. Reduced p127 expression delays histolysis whereas overexpression accelerates this process without affecting the duration of third larval instar, prepupal and pupal development. Similar l(2)gl-dependence is noticed in the timing of expression of the cell death genes reaper, head involution defective and grim, supporting the idea that p127 plays a critical role in the implementation of ecdysone-triggered apoptosis. These experiments show also that the timing of salivary gland apoptosis can be manipulated without affecting normal development and provide ways to investigate the nature of the components specifically involved in the apoptotic pathway of the salivary glands (Farkas, 2000).
The abnormal wing discs (awd) gene of Drosophila is homologous to the nm23 gene of mammals, a gene whose expression is altered in metastatic tumors. Both awd and nm23 encode nucleoside diphosphate kinases (NDP kinases). The accumulation of AWD/NDP kinase has been examined during normal development by assaying enzyme activity in extracts. There is a nearly constant level of activity throughout larval and pupal development. The tissue-specific transcription of the awd gene was examined by RNA in situ hybridization and by reporter gene expression. In imaginal discs and brains there is no detectable awd gene expression until the beginning of the third larval instar, despite the constant level of enzyme activity measured in extracts of larvae and pupae. The most intense awd gene expression in imaginal discs and brains occurs after the end of larval development. awd gene expression was examined in neoplastic brain tumors caused by mutations in the lethal (2) giant larvae gene. In l(2)gl mutant brains, as in normal brains, awd gene expression begins during the third larval instar. No tumors form in brains from l(2)gl-;awd- double mutant larva, so awd gene expression is required for tumor formation and/or proliferation. There is more accumulation of AWD/NDP kinase in l(2)gl- mutant brains than there is in normal brains. Using an awd reporter gene, it has been shown that this is a consequence of an increased proportion of awd gene-expressing cells in mutant brains. Using the same awd reporter gene as a marker of donor cells, the invasiveness of l(2)gl-induced neuroblastomas has been confirmed (Timmons, 1993).
Loss of function mutations in the lethal(2)giant larvae gene causes neoplastic brain tumors in Drosophila. A lacZ reporter gene has been introduced into l(2)gl mutant cells and beta-galactosidase expression was used as a marker to monitor the growth of such tumors following transplantation into wild-type adult hosts. Whereas normal larval brains do not grow when transplanted, mutant brains can develop into enormous tumors that fill the entire abdominal cavity. To investigate whether these tumors are similar to mammalian tumors at the biochemical level, the accumulation of a specific protein was examined -- one which is differentially expressed in mammalian metastatic tumors and is likely to be involved in the invasive and/or metastatic mechanism. Increased accumulation of a 72 kilodalton (kDa) type IV collagenase has been observed in several metastatic human tumors. Using antibodies directed against this human 72 kDa type IV collagenase, it has been shown for the first time that Drosophila has a cross-reacting 49 kDa protein with gelatinase activity. In brains dissected from l(2)gl mutant larvae, the accumulation of this 49 kDa gelatinase of Drosophila is increased compared to the level in brains dissected from wild-type larvae. In tumors derived from mutant brains, all of the cells express this protein. Moreover, the tumor cells that invade host organs express this protein. These data suggest that the metastasis of Drosophila tumor cells is similar to the metastasis of some human tumors at the biochemical level as well as at the cellular level (Woodhouse, 1994).
The Drosophila tumor suppressor gene lethal(2) giant larvae (lgl) encodes a cytoskeletal protein required for the change in shape and polarity acquisition of epithelial cells, and also for asymmetric division of neuroblasts. lgl also participates in the release of Decapentaplegic (Dpp), a member of the transforming growth factor ß (TGFß) family that functions in various developmental processes. During embryogenesis, lgl is required for the dpp-dependent transcriptional activation of zipper (zip), which encodes the non-muscle myosin heavy chain (NMHC), in the dorsalmost ectodermal cells -- the leading edge cells. The embryonic expression of known targets of the dpp signaling pathway, such as labial or tinman is abolished or strongly reduced in lgl mutants. lgl mutant cuticles exhibit phenotypes resembling those observed in mutated partners of the dpp signaling pathway. In addition, lgl is required downstream of dpp and upstream of its receptor Thickveins (Tkv) for the dorsoventral patterning of the ectoderm. During larval development, the expression of spalt, a dpp target, is abolished in mutant wing discs, while it is restored by a constitutively activated form of Tkv (TkvQ253D). Taking into account that the activation of dpp expression is unaffected in the mutant, this suggests that lgl function is not required downstream of the Dpp receptor. Finally, the function of lgl responsible for the activation of Spalt expression appears to be required only in the cells that produce Dpp, and lgl mutant somatic clones behave non autonomously. The activity of lgl is therefore positioned in the cells that produce Dpp, and not in those that respond to the Dpp signal. These results are consistent with the same role for lgl in exocytosis and secretion as that proposed for its yeast ortholog, sro7/77 -- lgl might function in parallel or independently of its well-documented role in the control of epithelial cell polarity (Arquier, 2001).
Secretion relies on intracellular vesicular trafficking and on the polarized exocytosis machinery. Recent studies have demonstrated that Lgl function is essential for the establishment of the polarities of epithelial cells. An important issue is therefore to understand whether the role of Lgl in Dpp secretion is direct or simply a consequence of the loss of epithelial cell polarity. Analysis of the temporal requirement for Lgl function argues in favor of Lgl being necessary for the establishment of cell polarity, rather than for its maintenance. Moreover, alteration in Dpp signaling can be observed in lgl mutants in epithelial cells that are correctly polarized and this supports a direct function for Lgl in Dpp secretion (Arquier, 2001).
The epidermis is not affected in homozygous lgl4-null mutant larvae that no longer contain the maternal Lgl protein responsible for a normal embryonic development. lgl4 larvae develop a cuticle that possesses the hallmarks of a wild-type cuticle by all the criteria used, thus indicating that the apical secretion of cuticle components has not been altered. Markers for epithelial cell polarity are localized in the correct position in stage 16 embryos when Lgl is no longer detected. Likewise, lglts3 embryos in which the Lgl protein has lost its cortical location have maintained their typical epithelial cell polarity and their capacity to secrete normal cuticle components. In neuroblasts, Lgl seems to exert its action early during mitosis to recruit basal determinants to the cortex but it does not contribute to their maintenance in this latter location. The polarity of epithelial wing disc cells is preserved until the middle of the third instar larval stage, long after the maternal Lgl contribution has ceased (Arquier, 2001).
It seems reasonable to assume that there is a unique exocytosis pathway mediated by lgl to ensure both cell polarity control and secretion. Dlg and scrib might participate in this same pathway: indeed, they strongly interact genetically with lgl and share with this gene a large panel of identical mutant phenotypes. Lgl, however, does not strictly colocalize with Dlg and Scrib in either epithelial cells. In addition, the Dlg cortical localization does not require lgl function. One could therefore anticipate an lgl action, within a separate and distinct pathway, in parallel to that of dlg and scrib. Further experiments are needed to address this issue (Arquier, 2001).
In yeast, sro7/77-mediated polarized exocytosis relies on a complex regulation and interaction with the actomyosin cytoskeleton. Sro7/77 displays a strong genetic interaction with myo1 (encoding a Type II myosin homolog of NMHC) and with myo2 (encoding an unconventional Type V myosin). In addition, Myo1P can physically interact with Sro7P, in a manner resembling that prevailing between Lgl and Zipper/NMHC. These observations support the idea that Lgl serves as a functional link between the actomyosin cytoskeleton polarity and a specific polarized exocytosis pathway, although the precise function exerted by Lgl in such a process has yet to be deciphered. In yeast, as in flies, myo1 (zipper) and sro7/77 (lgl) display a negative genetic interaction. Loss-of-function alleles of lgl suppress the dorsal closure phenotype in homozygous zip mutants. Conversely, overexpression of lgl enhances the dorsal closure phenotype (Arquier, 2001).
In many developmental processes, polyploid cells are generated by a variation of the normal cell cycle called the endocycle in which cells increase their genomic content without dividing. How the transition from the normal mitotic cycle to endocycle is regulated is poorly understood. The transition from mitotic cycle to endocycle in the Drosophila follicle cell epithelium is regulated by the Notch pathway. Loss of Notch function in follicle cells or its ligand Delta function, in the underlying germline, disrupts the normal transition of the follicle cells from mitotic cycle to endocycle: mitotic cycling continues, leading to overproliferation of these cells. The regulation is at the transcriptional level, since Su(H), a downstream transcription factor in the pathway, is also required cell autonomously in follicle cells for proper transitioning to the endocycle. One target of Notch and Su(H) is likely to be the G2/M cell cycle regulator String, a phosphatase that activates Cdc2 by dephosphorylation. String is normally repressed in the follicle cells just before the endocycle transition, but is expressed when Notch is inactivated. Analysis of the activity of String enhancer elements in follicle cells reveals the presence of an element that promotes expression of String until just before the onset of polyploidy in wild-type follicle cells but well beyond this stage in Notch mutant follicle cells. This suggests that it may be the target of the endocycle promoting activity of the Notch pathway. A second element that is insensitive to Notch regulation promotes String expression earlier in follicle cell development, which explains why Notch, while active at both stages, represses String only at the mitotic cycle-endocycle transition (Deng, 2001).
In Drosophila, mutations in three neoplastic genes, discs large (dlg), lethal(2)giant larvae (lgl) and scribble (scrib), cause loss of apical-basal polarity accompanied by hyperproliferation. It is not understood how loss of apical-basal polarity results in hyperproliferation in these mutants. One attractive hypothesis, however, is that overproliferation is caused by mislocalization of critical plasma membrane proteins that are involved in cell cycle control. The Notch protein is indeed mislocalized throughout lgl mutant cells; this defect could reduce responsiveness to Delta from the germline and thus lead to overproliferation. In addition, many lgl mutant follicle cells existed in multilayered structures where they are less likely to receive the Delta signal because of lack of direct contact with the germline. By contrast, in Notch mutant follicle cells, the cell cycle defect is observed before any obvious polarity defect; normal Armadillo localization is observed in Notch but not in lgl clones. Additional factors probably also contributed to the overproliferation in lgl mutant clones because significantly more proliferation is observed in lgl than in Notch clones (Deng, 2001).
The tumor suppressor genes lethal giant larvae (lgl) and discs large (dlg) act together to maintain the apical basal polarity of epithelial cells in the Drosophila embryo. Neuroblasts that delaminate from the embryonic epithelium require lgl to promote formation of a basal Numb and Prospero crescent, which will be asymmetrically segregated to the basal daughter cell upon division to specify cell fate. Sensory organ precursors (SOPs) also segregate Numb asymmetrically at cell division. Numb functions to inhibit Notch signaling and to specify the fates of progenies of the SOP that constitute the cellular components of the adult sensory organ. In contrast to the embryonic neuroblast, lgl is not required for asymmetric localization of Numb in the dividing SOP. Nevertheless, mosaic analysis reveals that lgl is required for cell fate specification within the SOP lineage; SOPs lacking Lgl fail to specify internal neurons and glia. Epistasis studies suggest that Lgl acts to inhibit Notch signaling by functioning downstream or in parallel with Numb. These findings uncover a previously unknown function of Lgl in the inhibition of Notch and reveal different modes of action by which Lgl can influence cell fate in the neuroblast and SOP lineages (Justice, 2003).
The discovery that lgl function is required to specify cell fate within the SOP lineage, but does not affect asymmetric segregation of Numb, suggests that Lgl function is distinct from Dlg function in the SOP. Lgl function is most likely required after polarization of the SOP and somehow contributes to the selective inhibition of Notch activity that specifies the fate of the pIIb cell. How might Lgl fulfill this function? Lgl is a WD repeat-containing protein conserved in eukaryotes ranging from yeast to man. Similar to many other WD repeat-containing proteins, Lgl likely interacts with multiple partners in a dynamic manner. It binds type II myosins and t-SNAREs on the plasma membrane and is known to be involved in exocytosis in yeast and Drosophila by presumably targeting vesicles to the plasma membrane and thereby inserting membrane proteins at specific zones along the apical-basal axis of epithelial cells and releasing extracellular signaling molecules such as Dpp. The requirement for Lgl function, however, is not restricted to membrane proteins and secreted proteins that require vesicular transport. For example, formation of the basal crescent in neuroblasts involves cytoplasmic and cortical movements of globular proteins, such as Numb, Pon, Prospero, and Miranda, that attach to the cytoplasmic side of the membrane via lipid modifications or association with membrane proteins. One plausible scenario for the role of Lgl in mediating basal Numb crescent formation in neuroblasts is that Lgl and motor proteins form a complex that mediates basal transport of determinants. Such Lgl-containing adaptor complexes in the SOP must differ from those in embryonic neuroblasts under this scenario, given that anterior Numb crescent formation in the SOP is independent of Lgl (Justice, 2003).
A scenario is imagined in which Lgl is required to deliver components of the machinery required for Numb-mediated inhibition of Notch cannot be excluded. Alternatively, Lgl could directly participate in such a mechanism and could perhaps target endocytic vesicles containing Numb and Notch to the lysosome for degradation. A direct role for Lgl in the Notch pathway is supported by studies suggesting that vesicle trafficking of Notch and Delta plays a critical role during Notch pathway signaling. Lgl might bring Notch inhibitors to the plasma membrane or traffic endocytic vesicles in an inhibitory mechanism with Numb and alpha-Adaptin that specifies cell fates in the SOP lineage (Justice, 2003).
Cell fate diversity can be achieved through the asymmetric segregation of cell fate determinants. In the Drosophila embryo, neuroblasts divide asymmetrically and in a stem cell fashion. The determinants Prospero and Numb localize in a basal crescent and are partitioned from neuroblasts to their daughters (GMCs). Nonmuscle myosin II (Zipper) regulates asymmetric cell division by an unexpected mechanism, excluding determinants from the apical cortex. Myosin II is activated by Rho kinase and restricted to the apical cortex by the tumor suppressor Lethal (2) giant larvae. During prophase and metaphase, myosin II prevents determinants from localizing apically. At anaphase and telophase, myosin II moves to the cleavage furrow and appears to push rather than carry determinants into the GMC. Therefore, the movement of myosin II to the contractile ring not only initiates cytokinesis but also completes the partitioning of cell fate determinants from the neuroblast to its daughter (Barros, 2003).
Class II myosins are barbed end-directed motors that form bipolar filaments. The filaments bind actin and initiate contraction when the two ends of the bipolar filament pull in opposite directions. Myosin's mode of action makes it unlikely that myosin II could transport cargo from one side of the cell to the other, except perhaps by progressive contraction along the cortex. The lack of colocalization of myosin II and Miranda in neuroblasts further implies that myosin II does not transport Miranda directly. The data suggest, first, that myosin II is required to maintain an intact cortical actin cytoskeleton and, second, that active myosin modifies the actin cytoskeleton at the apical cortex to exclude Miranda binding. The C. elegans myosin II may act in a similar fashion, as it appears to limit PAR-3 to the anterior of the zygote (Barros, 2003).
Several alternative approaches were taken to inactivate myosin II in neuroblasts. First, germline clones of Sqh were analyzed. In severe sqh1GLC embryos, levels of the regulatory light chain are greatly reduced from early development, and the heavy chain is found only in inactive aggregates. The actin cytoskeleton is disrupted, and neither Lgl nor Miranda localize to the cortex. Miranda concentrates instead at the spindle microtubules. Therefore, active myosin is necessary from early development to organize the actin cytoskeleton, which is in turn required for Lgl and Miranda to localize to the cortex (Barros, 2003).
Myosin II is activated by phosphorylation of its regulatory light chain by Rho kinase. Myosin was inactivated at the time of neuroblast cell division by inhibition of Rho kinase. Myosin II no longer localizes at the apical neuroblast cortex but instead spreads into the cytoplasm, and basal protein localization is disrupted. Although F-actin and Lgl remain uniformly at the cortex, cell fate determinants are now found around the entire cell cortex, demonstrating that apical cortical myosin is required to confine determinants to the basal half of the cell. Inhibition of Rho kinase also blocks cytokinesis, although the defect in basal protein localization is unlikely to be the consequence of mitotic arrest or a block in cytokinesis. First, basal protein localization is not disrupted in neuroblasts arrested in mitosis by colcemid treatment. Second, mitosis occurs without cytokinesis in pebble mutants, but the resultant polyploid neuroblasts still localize Numb and Prospero asymmetrically. Finally, the loss of asymmetry resulting from Rho kinase inhibition can be rescued by expression of a constitutively active form of the myosin II regulatory light chain (SqhE20E21). It is concluded that myosin II is required to restrict cell fate determinants to the basal cortex (Barros, 2003).
Myosin II localizes to the apical cortex of metaphase neuroblasts. Why is myosin localization/activity asymmetric? Lgl binds myosin II heavy chain directly and inhibits myosin filament formation. This binding is regulated by phosphorylation of Lgl; this phosphorylation inhibits its interaction with myosin II in vitro. If Lgl negatively regulates myosin activity and localization, then myosin should be uniformly distributed in an lgl mutant. Indeed, in lgl1GLC mutants myosin II no longer concentrates apically but is found uniformly around the cortex. Most Miranda protein is released from the cortex and binds microtubules, again suggesting that myosin excludes Miranda from the cortex (Barros, 2003).
Myosin II localizes to the entire cortex in lgl mutants and thereby prevents Miranda binding basally. In Drosophila neuroblasts in which Lgl levels are reduced (zygotic lgl1 mutants), Miranda is released from the cortex. Miranda localization can be rescued by simultaneously reducing the level of myosin II (zip1 zygotic mutants). Reducing the level of active myosin may restore the balance between the levels of Lgl and myosin, enabling the remaining myosin to concentrate apically (Barros, 2003).
How does myosin II restrict neuroblast proteins to the basal side of the cell cortex? Myosin II and Miranda occupy primarily opposite sides at the neuroblast cortex: myosin II is concentrated at the apical cortex while Miranda localizes as a basal crescent. As myosin II shifts to the cleavage furrow, Miranda is segregated into the forming GMC. The apical F-actin compartment may be modified by myosin II to exclude binding of basal proteins like Miranda. Active myosin II requires Rho kinase activity and depends on inactivation of Lgl at the apical cortex by aPKC (Betschinger, 2003). Ectopic expression of a nonphosphorylatable form of Lgl, in which the conserved aPKC-dependent phosphorylation sites are mutated from Serines to Alanines (Lgl-3A), results in mislocalization of Miranda around the neuroblast cortex (Betschinger, 2003). The data support a spatially regulated interaction between myosin II and Lgl. Myosin is apically localized in wild-type neuroblasts, corresponding to the domain in which Lgl is inactivated by aPKC. In lgl mutants, myosin is no longer restricted apically but localizes around the entire cell cortex. Conversely, when nonphosphorylatable Lgl is expressed in neuroblasts, myosin is inhibited throughout the cell and drops off the cortex. It is proposed that myosin II is activated and can form filaments at the apical cortex, where phosphorylated Lgl is inactive and unable to bind myosin II. Myosin may then modify the actin cytoskeleton to prevent the binding of Miranda. At the basal cortex, in the absence of aPKC, Lgl is active and can bind and inhibit myosin. Myosin cannot form filaments, which are required for it to bind to the actin cortex. As a result, Miranda can bind to the basal cortex (Barros, 2003).
At anaphase, myosin II moves to the equator and appears to 'push' cell fate determinants into the daughter cell. This movement is regulated in an Lgl-independent fashion and occurs whether myosin is restricted to the apical cortex or is uniformly cortical (as in lgl mutants). Cortical myosin is essential, however, to efficiently segregate determinants into the GMC at telophase (telophase rescue). In neuroblasts expressing Lgl-3A, myosin II is cytoplasmic, and determinants are not partitioned to the daughter cell. Nonetheless, at telophase, myosin seems to be recruited from the cytoplasm, since it still accumulates to the cleavage furrow. Thus three separate steps of myosin regulation in neuroblasts can be defined. First, myosin forms an apical crescent. This is positively regulated by Rho kinase and negatively regulated by Lgl. Second, cortical myosin moves to the equator. This movement occurs independently of Lgl. Third, cortical and cytoplasmic myosin accumulates at the cleavage furrow, a step that is also Lgl independent. Rho Kinase activation seems to be important for all three steps of myosin II regulation. When Rho kinase is inhibited, myosin falls into the cytoplasm, and there is no cleavage furrow formation (Barros, 2003).
In conclusion, these results demonstrate that myosin II acts downstream of Lgl and the apical protein complex to regulate the segregation of cell fate determinants. Myosin II does not negatively regulate basal protein targeting, as has previously been suggested nor does it transport determinants directly. Instead, it is proposed that myosin II acts in a novel fashion, excluding determinants from the apical cortex and 'pushing' them into the GMC at anaphase and telophase. Myosin II might modify the actin cytoskeleton to prevent determinants binding, although the actual structure formed and the physical change in the actin cytoskeleton remains to be determined (Barros, 2003).
Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype; reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes (Rolls, 2003).
One of the more unexpected findings is that aPKC mutant neuroblasts show normal Baz/Par3 apical localization. The Baz/Par3-Par6-aPKC complex has been suggested to form a functional unit that is interdependent for localization in C. elegans, mammals, and Drosophila. Baz/Par3 shows normal apical localization in aPKC mutant neuroblasts, showing that normal Baz/Par3 localization can occur without being part of the Par3-Par6-aPKC complex. In addition, neuroblasts lacking apical aPKC and Par6 still form a molecularly defined apical cortical domain containing Baz/Par3, Insc, Pins, and Dlg. These results lead to the proposal of a hierarchy for apical protein localization in neuroblasts: Baz/Par3-Insc-Pins-Dlg --> aPKC --> Par6-Lgl. This hierarchy is consistent with recent biochemical analyses in which a protein complex was isolated containing Par6-aPKC-Lgl, but not Baz/Par3. It is suggested that aPKC may be required to anchor the Par6-aPKC-Lgl complex at the apical cortex of the neuroblast (Rolls, 2003).
Both aPKC and Lgl are required for Miranda basal localization in neuroblasts, and all available data support a model in which Lgl is required for targeting Miranda to the neuroblast cortex, whereas aPKC blocks Lgl function on the apical side of the neuroblast: (1) lgl mutants have little or no Miranda at the cortex; (2) aPKC mutants show uniform cortical Miranda localization; (3) a weak lgl phenotype can be suppressed by reducing aPKC levels, showing that aPKC activity antagonizes Lgl activity; (4) aPKC and Lgl physically interact; (5) an overexpressed nonphosphorylatable Lgl protein is uniformly cortical and able to induce uniform cortical Miranda localization, whereas phospho-Lgl is preferentially released from the cell cortex. This has led to a model in which Lgl acts as an anchor for Miranda at the basal cortex, but is absent from the apical cortex due to aPKC-mediated phosphorylation. Although this simple model is attractive, it is noted that Lgl has never been observed colocalized with Miranda in a basal cortical crescent, and a role for cytoplasmic Lgl in Miranda localization has not been definitively ruled out (Rolls, 2003).
The Baz/Par3-Par6-aPKC complex has a well-characterized role in regulating neuroblast spindle orientation. Spindle orientation can be measured relative to extrinsic landmarks around the neuroblast (e.g., perpendicular to the overlying ectoderm) or relative to intrinsic cues within each neuroblast (e.g., perpendicular to the Baz/Par3-Par6-aPKC apical crescent). Mutations in baz or par6 genes randomize embryonic neuroblast spindle orientation relative to the overlying ectoderm, but it is not clear whether these phenotypes are due to disruption of the ectodermal layer or to a cell-autonomous defect in the neuroblast. The function of aPKC in embryonic neuroblast spindle orientation cannot be assayed due to high levels of maternal aPKC protein present in aPKC zygotic mutant embryos. However, aPKC is not required for intrinsic spindle orientation in larval neuroblasts; the mitotic spindle is always perpendicular to the Baz/Par3-Insc-Pins apical crescent (Rolls, 2003).
The Baz/Par3 and Par6 proteins are required to establish epithelial polarity in Drosophila, and aPKC is also shown to be required for normal apical/basal epithelial cell polarity. The similar phenotype in baz, par6, and aPKC mutants may indicate that these proteins function together as a complex to provide a single function in epithelia, despite the evidence that they have independent functions in neuroblasts. One primary function may be the inhibition of Lgl activity because it is found that the lgl epithelial polarity defects can be strongly suppressed by reducing aPKC levels. Lgl-Dlg-Scrib activity can also antagonize Baz/Par3-Par6-aPKC activity, and it is tempting to speculate that aPKC inactivates Lgl by phosphorylation, whereas Lgl can inactivate aPKC by sequestering it into an Lgl-Par6-aPKC complex and out of the Baz/Par3-Par6-aPKC complex (Rolls, 2003).
The role of aPKC in cell proliferation has not been previously investigated in Drosophila. There are three lines of evidence showing that aPKC promotes cell proliferation in neuroblasts and epithelia. The number of cells in aPKC mutant mushroom body neuroblast clones is significantly lower than the number in wild-type clones. There appears to be a normal number of early-born gamma neurons in these clones, followed by only a few later-born alpha neurons. The normal number of early-born gamma neurons suggests that loss of aPKC does not lead to cell death in this population; moreover, no decrease is seen in the number of neuroblasts per brain lobe in aPKC mutant larvae. It is concluded that cell death is not contributing to the reduction in neurons observed in the clones, but rather, that the neuroblast stops dividing near the time the neuroblast switches over to generating alpha and ß neurons. The neuroblast may become arrested at some point in the cell cycle, or it may undergo a terminal division to generate a pair of GMCs (perhaps due to both daughter cells inheriting Miranda and Prospero GMC determinants). A second indication that aPKC promotes cell proliferation is that far fewer epithelial cells are observed in aPKC mutant eye imaginal discs compared with the wild type, even with an additional day of growth as second instar larvae. Finally, a 50% reduction in aPKC levels (aPKC/+) can strongly suppress the epithelial and brain overproliferation phenotypes of lgl mutants. Together, these data show that aPKC positively regulates cell proliferation in epithelia and neuroblasts. Interestingly, reduction in the function of the mammalian atypical PKCzeta (using overexpression of a dominant-negative kinase) can suppress Rac1/cdc42-induced overproliferation. Thus, aPKC may have an evolutionarily conserved role in promoting cell proliferation, as well as in the establishment of cell polarity (Rolls, 2003).
Although aPKC and Lgl act antagonistically to regulate many aspects of epithelial and neuroblast cell polarity and cell proliferation, they may share a common positive function in regulating neuroblast apical cell size. lgl zygotic mutants have some embryonic telophase neuroblasts with an abnormally small apical cortical domain, apical spindle pole, and neuroblast size. These defects are not suppressed by reducing aPKC levels, and in fact may be enhanced. Thus, it is proposed that Lgl and aPKC both act positively to promote large apical cell and spindle pole size. It has been reported that Baz/Par3-Par6-aPKC and Pins-Gαi act in parallel pathways to promote large apical cell size and apical spindle size; Lg could be acting as part of the Pins-Galphai pathway, or as a third input promoting apical cell and spindle size (Rolls, 2003).
How a cell chooses to proliferate or to differentiate is an important issue in
stem cell and cancer biology. Drosophila neuroblasts undergo self-renewal
with every cell division, producing another neuroblast and a differentiating
daughter cell, but the mechanisms controlling the self-renewal/differentiation
decision are poorly understood. This study tested whether cell polarity genes,
known to regulate embryonic neuroblast asymmetric cell division,
also regulate neuroblast self-renewal. Clonal analysis
in larval brains shows that pins mutant neuroblasts rapidly fail to
self-renew, whereas lethal giant larvae (lgl) mutant neuroblasts
generate multiple neuroblasts. Notably, lgl pins double mutant
neuroblasts all divide symmetrically to self-renew, filling the brain with
neuroblasts at the expense of neurons. The lgl pins neuroblasts show
ectopic cortical localization of atypical protein kinase C (aPKC), and a
decrease in aPKC expression reduces neuroblast numbers, suggesting that aPKC
promotes neuroblast self-renewal. In support of this hypothesis,
neuroblast-specific overexpression of membrane-targeted aPKC, but not a
kinase-dead version, induces ectopic neuroblast self-renewal. It is concluded that
cortical aPKC kinase activity is a potent inducer of neuroblast
self-renewal (Lee, 2005).
Drosophila neuroblasts are an
excellent model system in which to investigate the molecular control of
self-renewal versus differentiation. Larval neuroblasts repeatedly divide
asymmetrically to self-renew a neuroblast and to produce a smaller daughter
cell, called a ganglion mother cell (GMC), that typically makes two postmitotic
neurons; this process enables a single neuroblast to generate many hundreds of
neurons. Self-renewal is defined as the capacity of a
neuroblast to maintain all attributes of its cell type (molecular markers and
proliferation potential). In this regard, a neuroblast is very similar to a
germline stem cell: both maintain their stem cell identity while generating
differentiating progeny. About 100 neuroblasts per
brain lobe are formed during embryogenesis, where they proliferate briefly
before entering quiescence. Brain neuroblasts
re-enter the cell cycle between 10 and 72 h after larval hatching
(ALH), and then a stable population of ~100 mitotic, self-renewing neuroblasts is
maintained. This invariant neuroblast
number was used to screen for mutants altering self-renewal versus differentiation:
mutants in which a neuroblast makes two neuroblast progeny (ectopic
self-renewal) will have >100 neuroblasts, whereas mutants in which a
neuroblast makes two GMC progeny (failure in self-renewal) will have <100
neuroblasts. This assay was used to test known cell polarity mutants
for a role in neuroblast self-renewal (Lee, 2005).
Two classes of cell
polarity regulators were assayed for an effect on larval neuroblast self-renewal.
lgl and discs large (dlg) zygotic mutants were examined, because
these mutants form brain tumours and promote basal
protein targeting in embryonic and larval neuroblasts. Lgl
and Dlg have several protein interaction motifs and are localized around the
neuroblast cortex. In addition, pins
and Galphai zygotic mutants were examined; these genes regulate cell
polarity in embryonic neuroblasts, but have not
been well characterized in larval neuroblasts. Pins and Galphai are
colocalized with Inscuteable and the evolutionarily conserved
Bazooka-Par6- aPKC proteins at the apical cortex of mitotic
neuroblasts, and all of these proteins are partitioned into the neuroblast
during cytokinesis (Lee, 2005).
In wild-type larvae, a population of ~100 neuroblasts
could be identified by the markers Worniu,
Deadpan and Miranda, and by labelling with a pulse of 5-bromodeoxyuridine
(BrdU); by contrast, the thousands of differentiating GMCs and neurons rapidly
downregulate neuroblast markers and express nuclear Prospero and/or Elav.
A clear increase in neuroblast number is observed in
lgl and dlg mutants; there are supernumerary neuroblasts at all
stages examined; all extra neuroblasts expressed Deadpan and Miranda and
are proliferative on the basis of their ability to incorporate BrdU. Galphai
zygotic mutants have a complex phenotype that will be described
in a later publication; however, pins zygotic mutants show a marked decrease in
neuroblast number. Notably, this phenotype is not due to a subset of
neuroblasts remaining quiescent, because neuroblast numbers peak and then
decline over time, and it is not due to neuroblast
cell death. The relatively late onset of the pins phenotype
is probably due to the gradual depletion of maternal pins gene product
in these larvae (Lee, 2005).
To determine whether the pins and lgl larval brain phenotypes are
due to defects in neuroblast self-renewal, positively marked genetic
clones were induced in single neuroblasts to trace their
progeny. Clone induction
parameters were adjusted to ensure that each clone was derived from a single neuroblast (1.2
clones per lobe). In wild-type brains, neuroblast clones always
contained a single Worniu+ Miranda+
nuclear-Prospero- neuroblast and numerous smaller Worniu-
Miranda- nuclear-Prospero+ progeny,
confirming that wild-type neuroblasts always
divide to self-renew and to generate a smaller differentiating GMC.
By contrast, lgl mutant brains had an average of
2.3 neuroblasts per clone, with up to six neuroblasts per clone, showing that
lgl mutant neuroblasts can divide symmetrically to yield two neuroblasts.
The opposite phenotype was seen in pins
mutant brains: 72.8% of the clones had no neuroblast and the remainder had a
single neuroblast.
The neuroblasts did not die in the pins mutants as evidenced by the following: the cell death marker
caspase-3 was not upregulated, neuroblast-specific expression of the p35 cell death inhibitor
did not rescue the missing neuroblasts, and one
clone was observed in which the largest cell coexpressed neuroblast and GMC markers,
consistent with an intermediate stage in neuroblast-to-GMC differentiation. It is concluded
that wild-type neuroblasts exclusively
generate neuroblast/GMC siblings; lgl mutant neuroblasts occasionally
undergo ectopic self-renewal to generate neuroblast/neuroblast siblings; and
pins mutant neuroblasts occasionally fail to self-renew, resulting in
GMC/GMC siblings and termination of the lineage (Lee, 2005).
Next to be examined was whether lgl pins double mutants had fewer neuroblasts (like
pins mutants) or extra neuroblasts (like lgl mutants).
Unexpectedly, a phenotype was detected in which the larval brain was full of
cells expressing the neuroblast markers Worniu, Miranda and Deadpan and lacking
expression of the neuronal marker Elav. Additional markers that distinguish neuroblasts and
GMCs were examined to determine whether these cells were neuroblasts or a hybrid
neuroblast/GMC identity. Both wild-type neuroblasts and lgl
pins cells actively transcribed the worniu, deadpan,
miranda and prospero genes, maintained proliferation, did not
express the Elav neuronal differentiation marker, and did not extend axons.
The only potential GMC attribute found in lgl pins
neuroblasts was nuclear Prospero protein but, because wild-type
neuroblasts and GMCs both contain Prospero protein, which can accumulate in
neuroblast nuclei if not properly localized, this protein is not a definitive
marker for the GMC cell type. Thus, lgl pins brains contain large numbers
of ectopic, proliferating, self-renewing neuroblasts. Combining these
lgl, pins and lgl pins mutant data leads to the conclusion
that Lgl inhibits self-renewal, whereas Pins has dual functions in promoting and
inhibiting self-renewal (Lee, 2005).
To understand how Lgl and Pins
regulate neuroblast self-renewal at the cellular level, cortical
polarity marker localization was examined in mitotic larval neuroblasts. In wild-type larval
neuroblasts, the Par complex (Bazooka-Par6-aPKC) and
Pins-Galphai proteins forms an apical crescent at metaphase
and are partitioned into the self-renewing neuroblast at telophase, whereas the
Miranda and Prospero proteins form a basal crescent at metaphase and are
partitioned into the differentiating GMC at telophase. In lgl
pins double mutants, in which all neuroblasts divide symmetrically to
generate self-renewing neuroblast/neuroblast siblings, most mitotic neuroblasts
show uniform cortical aPKC, cytoplasmic Bazooka and Par6, and uniform cortical
Miranda at metaphase and telophase.
Thus, only aPKC maintained its correct subcellular localization and correlated
with neuroblast self-renewal (Lee, 2005).
aPKC localization was examined in lgl and pins single mutants, in which symmetric
divisions occurred at lower frequency. In lgl mutants, aPKC showed weak
ectopic cortical localization in about half the metaphase neuroblasts, whereas
Miranda was delocalized from the cortex; by telophase, however, both proteins
appeared to be localized normally. Ectopic cortical
aPKC was also observed in dlg mutant larval neuroblasts.
A role for Lgl in restricting aPKC to the apical cortex of neuroblasts has not
been reported but
would be consistent with the observation that basolateral Lgl restricts aPKC to
the apical surface of Drosophila and vertebrate epithelia and
Xenopus blastomeres. In pins mutants, aPKC and cytoplasmic Miranda
showed weak uniform cortical distribution in metaphase neuroblasts, but were properly
localized in most telophase neuroblasts
Thus, both Lgl and Pins are required to restrict aPKC to the apical cortex in
metaphase neuroblasts (Lee, 2005).
Whether aPKC is required for neuroblast self-renewal was examined.
aPKC mutant clones in larval
mushroom body neuroblasts showed premature lineage termination,
consistent with aPKC being required for neuroblast
self-renewal. In addition, aPKC null mutants died as second instar larvae
with reduced neuroblast numbers. Because this was a
relatively mild phenotype and there was no detectable aPKC protein at this
stage, it is likely that there are additional pathways for stimulating
neuroblast self-renewal. Next, whether aPKC is required for ectopic
neuroblast self-renewal in the lgl mutants was tested. lgl aPKC double
mutants had normal numbers of neuroblasts, showing
that aPKC is required for the ectopic neuroblast self-renewal seen in lgl
mutants. aPKC mutants also suppressed ectopic neuroblast self-renewal in
several independently isolated lgl mutations, further supporting a role
for aPKC in self-renewal. In addition, it was found that aPKC is fully epistatic to
lgl in regulating Miranda localization. Thus, aPKC is required for the ectopic
neuroblast self-renewal and Miranda delocalization phenotypes seen in lgl
mutants (Lee, 2005).
These data are most consistent with a model in which
Lgl negatively regulates aPKC, and aPKC directly promotes self-renewal. This
model is based on the observations that Lgl restricts aPKC localization to the
apical cortex of neuroblasts and that a reduction in aPKC blocks the lgl
self-renewal phenotype. To test this model,
worniu-Gal4 line was used to drive neuroblast-specific expression of
constitutively active aPKC or Lgl proteins, and an increase or
decrease in neuroblast numbers was assayed. Neuroblast-specific expression of aPKC targeted
to the plasma membrane with a CAAX prenylation motif
(UAS-aPKCCAAXWT)
resulted in ectopic cortical aPKC localization, loss of
cortical Miranda, and a large increase in the number
of neuroblasts. These effects were not observed after overexpression of wild-type
aPKC or a membrane-targeted kinase-dead aPKC
(UAS-aPKCCAAXKD).
Expression of a constitutively active aPKC
(UAS-aPKCDeltaN) that was predominantly
cytoplasmic gave only a slight increase in
neuroblast number, showing that cortical
localization of aPKC is essential to generate ectopic neuroblasts. By contrast,
neuroblast-specific expression of a constitutively active Lgl protein (Lgl3A)
resulted in the expected uniform cortical localization of Miranda, but no change in
neuroblast numbers. Combined overexpression of both
Lgl3A and aPKCCAAXWT, however, resulted in strong suppression of the
aPKCCAAXWT ectopic neuroblast phenotype,
even though Lgl3A alone had no effect on neuroblast
numbers, consistent with Lgl inhibiting aPKC function either directly or
through its downstream effectors. Thus, neuroblast-specific overexpression of
aPKC can expand the neuroblast population (most probably by promoting symmetric
neuroblast/neuroblast cell divisions) without eliminating the ability of these
neuroblasts to undergo asymmetric neuroblast/GMC divisions to generate
differentiating progeny. It is concluded that aPKC is sufficient to promote
neuroblast self-renewal, Lgl can inhibit aPKC function, and membrane-targeting
and kinase activity are essential for aPKC function (Lee, 2005).
This study has established
Drosophila larval neuroblasts as a model system for studying self-renewal
versus differentiation. A simple model is proposed in which Pins anchors
aPKC apically and Lgl inhibits aPKC localization basally, thereby restricting
aPKC to the apical cortex where it promotes neuroblast self-renewal.
In addition, aPKC can phosphorylate and directly
inhibit Lgl function, which together with the current
data provides evidence for mutual inhibition between Lgl and aPKC in
neuroblasts, similar to the mutual inhibition seen between these two proteins in
epithelia. Mutual inhibition between aPKC and Lgl would result in stabilization of apical aPKC localization and more reliable partitioning of aPKC into the neuroblast during mitosis. In pins mutants, aPKC is delocalized and nonfunctional owing to Lgl activity, thereby reducing self-renewal; in lgl mutants, aPKC shows weak ectopic cortical localization that increases self-renewal, and in lgl pins double mutants, aPKC is both delocalized and fully active: thus all neuroblasts undergo symmetric self-renewal. Although the targets of aPKC involved in self-renewal are unknown, aPKC may directly phosphorylate and inactivate GMC determinants, and/or phosphorylate and activate neuroblast-specific proteins. Notably, lgl1 mutant mice have neural progenitor hypertrophy and knockdown of a pins mammalian homologue (AGS3) leads to depletion of neural progenitors: phenotypes that are very similar to those described in this study. In the future, it will be important to determine the role of aPKC in mammalian neural progenitor self-renewal and to identify the aPKC-regulated phosphoproteins that regulate neuroblast self-renewal in Drosophila (Lee, 2005).
Fasciclin2 (Fas2) and Discs large (Dlg) localize to the basolateral junction (BLJ) of Drosophila follicle epithelial cells and inhibit their proliferation and invasion. To identify a BLJ signaling pathway a genome-wide screen was performed for mutants that enhance dlg tumorigenesis. Two genes were identified that encode known BLJ scaffolding proteins, lethal giant larvae (lgl) and scribble (scrib), and several not previously associated with BLJ function, including warts (wts) and roughened eye (roe/rotund), which encode a serine-threonine kinase and a transcription factor, respectively. Like scrib, wts and roe also enhance Fas2 and lgl tumorigenesis. Further, scrib, wts, and roe block border cell migration, and cause noninvasive tumors that resemble dlg partial loss of function, suggesting that the BLJ utilizes Wts signaling to repress EMT and proliferation, but not motility. Apicolateral junction proteins Fat (Ft), Expanded (Ex), and Merlin (Mer) either are not involved in these processes, or have highly spatio-temporally restricted roles, diminishing their significance as upstream inputs to Wts in follicle cells. This is further indicated in that Wts targets, CyclinE and DIAP1, are elevated in Fas2, dlg, lgl, wts, and roe cells, but not Fat, ex, or mer cells. Thus, the BLJ appears to regulate epithelial polarity and dynamics not only as a localized scaffold, but also by communicating signals to the nucleus. Wts may be regulated by distinct junction inputs depending on developmental context (Zhao, 2008).
The purpose of this work was to gain greater insight into how the BLJ suppresses epithelial tumorigenesis and invasion by identifying and understanding the function of new genes important for BLJ function. To do so, a genomewide screen was completed for enhancers of dlg, which encodes a scaffolding protein that is a crucial organizer of the BLJ and is a potent repressor of follicle epithelial cell tumorigenesis and invasion. Deficiencies that cumulatively span ∼80% of the autosomes, or 64% of the Drosophila genome were systematically screened. A relatively small number of enhancers, ∼1 per 1000 genes screened, were detected indicating that the screen selected for loci specifically required for dlg function. Thus, the novel dlg enhancer genes that were identified, wts, roe, ebi, as well as at least two genes yet to be identified, are likely to be key collaborators with dlg in suppressing epithelial invasion. The specificity of the interactions between dlg and these enhancers is further indicated in that more than one allele of each gene showed an interaction, in several dlg backgrounds, and the strengths of enhancement were similar to deficiencies defining each locus. wts, roe, and ebi also enhanced Fas2 and lgl, indicating that they are not just important for dlg function, but for the function of the BLJ as a whole. In addition, overexpression of all enhancers except ebi suppressed dlg and Fas2 tumorigenesis, further confirming that the identified genes function in a BLJ network (Zhao, 2008).
BLJ pathway components in the nucleus and their putative relationship to Notch: ebi encodes an F-box protein with WD repeats that promotes protein degradation of specific targets. The failure of ebi overexpression to suppress Fas2 or dlg, and the relatively mild ebi phenotypes (midoogenesis small-nucleus and epithelial-organization defects, but no defects in germinal vesicle localization), suggest that ebi may function in only one of the three branches of BLJ signaling or in a parallel pathway to the BLJ. In the eye, ebi is important for promoting differentiation and inhibiting proliferation, which appear to be separable functions. Thus ebi could enhance Fas2 and dlg tumorigenesis by functioning within the proliferation-repressing branch of the BLJ, or the importance of ebi for differentiation suggests that it could function in the EMT branch of the BLJ or both. In contrast, ebi promotes protein degradation in response to Notch (N) and Drosophila EGF receptor (EgfR) signals, suggesting that it may act in a parallel pathway. Both Ebi and its mammalian homolog, TBL1, function in a corepressor complex through association with nuclear hormone transcriptional corepressor SMRTER/SMRT (Zhao, 2008).
Interestingly, although most N appears to be localized on the apical surface of follicle cells, some N is also localized in BLJs. Thus, it is possible that N localized to the BLJ may signal directly to Ebi. Consistent with this possibility, it was found that all of the genes in the BLJ network share some midoogenesis defects with N, including the small nucleus phenotype, epithelial stratification defects, and mislocalization of the germinal vesicle. The epithelial defects are also reminiscent of N-pathway mutants brainiac and egghead, which are required in the germ line for regulating N that is localized on the apical surface of the follicle cells abutting the germ line. Thus one possibility is that N signaling activity is regulated by its localization to apical vs. basolateral junctions in response to several signaling pathways acting during midoogenesis (Zhao, 2008).
The other modest dlg enhancer that was identified, roe, encodes a Krüppel-family zinc-finger protein that appears to be a transcription factor. Roe is also implicated in Notch signaling and thus may function with Ebi in N-dependent processes as proposed above. However, in contrast to ebi, roe loss caused follicle cell tumors, suggesting that roe may function more directly in a BLJ pathway than ebi. Consistent with a direct role for Roe in BLJ signaling, it was found that roe overexpression suppressed Fas2 and dlg tumorigenesis. Further, as for Fas2, dlg, and wts, roe represses CycE and DIAP1 expression (Zhao, 2008).
Warts was of special interested because of the many similarities observed in the quality and strength of wts and scrib phenotypes, suggesting that they are components in a BLJ signaling pathway, rather than a parallel pathway that cross talks with BLJ signaling. wts encodes a serine/threonine kinase that is an ortholog of human tumor suppressors Lats1 and Lats2, both of which have been linked to highly aggressive breast cancers. The prevailing model for Wts signaling in Drosophila is based on signaling in eye and wing tissue. Wts appears to relay signals from apicolateral junction proteins Ft, Ex, and Mer in wing and eye tissues. However, the results from almost every assay, including early tumor formation, border cell migration, BrdU, PH3, CycE, and DIAP1 expression, indicated little functional overlap between Ft, ex, mer, or mer; ex and wts, thus diminishing the importance of apicolateral Ft-Ex-Mer for Wts activation in follicle cells. The exceptions were that during midoogenesis, Mer is required for border cell migration and Ex is required for the endocycle switch, while both are required for maintenance of epithelial integrity and positioning of the germinal vesicle. However, the involvement of Ex and Mer in these processes are fundamentally distinct from how they act in Wts-dependent processes in other tissues. (1) Ft is not involved; (2) no indication was observed of Ex-Mer synergism; (3) ex, mer, and mer; ex phenotypes are relatively mild when compared to wts. It is concluded that the model for Wts activation in which apicolateral junction proteins Ft, Ex, and Mer play the predominant role cannot be universally applicable in all cell types. Rather, the relative importance of Ex and Mer for Wts regulation appears to depend on developmental context (Zhao, 2008).
Consistent with this proposal, strong functional interdependence and phenotypic similarities were found between Fas2, dlg, lgl, scrib, and wts, thus indicating that the BLJ, not the apicolateral junction, plays the predominant role in Wts regulation during oogenesis. Although genetic evidence alone cannot completely rule out that Wts may act in a parallel pathway to the BLJ and impinge on a set of downstream targets that overlap with those targeted by the BLJ, the following observations favor a model in which the BLJ is more directly involved in Wts regulation (it is noted that these are not mutually exclusive alternatives): (1) over 50 tumor suppressor genes have been identified in Drosophila, but lgl, scrib, and wts were the only strong dlg enhancers identified in this genomewide screen; (2) wts showed strong genetic interactions with Fas2, dlg, and lgl, similar to or stronger than scrib, which encodes a known BLJ protein; (3) wts has early tumor phenotypes similar to dlg partial loss of function and to scrib; (4) wts has the same border cell migration phenotype as scrib; (5) wts has similar small nucleus, epithelial stratification, and germinal vesicle defects as Fas2, dlg, lgl, and scrib; (6) like lgl and scrib, wts overexpression suppressed Fas2 and dlg tumorigenesis; (7) Fas2, dlg, and wts have similar proliferation defects, and (8) Fas2, dlg, and wts similarly repress CycE and DIAP1 expression, which is especially crucial, because CycE and DIAP1 are downstream targets of Wts signaling, and ex and mer had no impact on their expression, contrary to results in other tissues. Thus, the data strongly indicate that the BLJ signals through Wts, and may impinge on Roe in the nucleus, thus suggesting the first BLJ signaling pathway in animal cells. This implies that the BLJ not only acts as a localized scaffold, but also signals to the nucleus to control gene expression, both of which cooperate to regulate epithelial polarity and dynamics (Zhao, 2008).
How can these results in follicle cells, which suggest that Wts acts predominantly downstream of the BLJ, be reconciled with findings in eye tissue, which indicate that Wts acts downstream of the apicolateral junction? Interestingly, the genetic data in the eye suggest that Ft, Ex, and Mer cannot account for all of the signals that activate Wts, because wts overgrowth and tissue disorganization phenotypes are more severe than ft or mer; ex. On the basis of these findings in follicle cells, it is possible that Wts activation in the eye requires additional input from the BLJ. This possibility may have been overlooked thus far because dlg does not appear to have an overgrowth phenotype in the eye. dlg may be essential for additional functions in the eye that are epistatic to its tumor suppressor function, thus preventing loss of cells from the epithelium that could mask an overgrowth phenotype. Consistent with this, when activated Rasv12 is combined with dlg loss, dramatic tumors develop that are larger and more invasive than those produced by Rasv12 alone (Zhao, 2008).
In contrast, Dlg may have a diminished role in Wts signaling in the eye, much as the evidence indicates a diminished role for Ex and Mer in Wts signaling in the ovary. According to this model, Wts receives predominant input from distinct lateral junctions depending on tissue context. One distinction is that ovarian follicle cells are derived from a mesodermal lineage, while the eye and wing tissues are from ectodermal lineages. Further, many genes that disrupt apical-basal polarity and epithelial morphology have only subtle phenotypes in the eye by comparison to the ovary or embryo. Finally, the follicular epithelium requires input from junctions on all three follicle cell surfaces, lateral, apical, and basal, whereas most epithelia require only two, lateral and apical or basal. Thus, ovarian and imaginal tissues are likely to organize signaling pathways acting downstream of epithelial junctions in similar, yet fundamentally different ways to meet the unique organizational requirements of their cell-tissue morphologies. Some or all of these differences may contribute to the suggested specificity observed in Wts signaling downstream of BLJs in follicle cells. In general, these findings raise the possibility for future investigation that depending on the cell-tissue morphologies of a given organ, one lateral junction may play a predominant organizational role, and Wts signaling may act as a universal signaling adapter for mediating contact inhibition from that junction (Zhao, 2008).
An especially interesting aspect of Mer and Ex function that was uncovered in follicle cells is that it appears to be restricted to predominantly postmitotic, differentiated cells, in contrast to the role of Mer and Ex in other tissues. Further, given the absence of an involvement of Ft and lack of Mer-Ex synergism it is concluded that if Mer and Ex would be involved in Wts activation in follicle cells, they would have to function via a fundamentally distinct mechanism than in other tissues. It is proposed that during early oogenesis, the BLJ alone may provide the predominant input to Wts. Then, during midoogenesis, Ex and Mer may become involved in novel interactions with Dlg or other components of the BLJ to activate Wts in spatiotemporally distinct populations of differentiating cells to help achieve their unique developmental functions (Zhao, 2008).
How do wts, scrib, and roe promote motility? It is proposed that Scrib, Wts, and Roe are all crucially involved in EMT. In EMT, cells (1) loose apical-basal polarity and become mesenchymal-like, and (2) adopt a polarity conducive to movement. scrib, wts, and roe cells clearly lose epithelial polarity and become mesenchymal-like as indicated by their rounded morphology and lateralized phenotype. However, scrib, wts, and roe tumors do not invade, and scrib, wts, and roe border cells do not move, suggesting that the second aspect of EMT, adoption of a polarity conducive to movement, is defective. Consistent with this, mammalian Scrib is required for migration and epithelial wound healing of cultured human breast epithelial cells, and is also required in vivo for wound healing in mice. Human Scrib directs migration by organizing several polarities crucial for migration, including the orientation of the microtubule and Golgi networks and the localization of Cdc42 and Rac1 to the cell's leading edge. Thus Scrib has a conserved function in directed cell migration by organizing a polarity conducive to movement. In mammalian PC12 cells Scrib is in complex with Rac1. Fly Rac1 is essential for border cell migration and invasion of Fas2 and dlg tumors, suggesting that an essential role of Scrib in Rac1 function may be of crucial importance for movement. The apparent conserved role of BLJ proteins in organizing EMT, and both promoting and repressing movement, reemphasizes the suggestion that BLJ proteins do more than merely maintain apical-basal polarity, but rather repress a cellular transformation from epithelial polarity to a mesenchymal, lateralized signature conducive to movement (Zhao, 2008).
How is the function of scrib, wts, and roe in promoting border cell movement consistent with the requirement of Fas2, dlg, and lgl in repressing border cell movement? Further, how do scrib and wts act as enhancers of dlg tumor invasion even though scrib and wts tumors are noninvasive? For border cell movement, Fas2 and dlg mutations not only accelerate movement, but also delay border cell delamination. The delay in border cell delamination suggests that the BLJ normally promotes motility, but this promoting function can be bypassed when the repression of motility branch of the BLJ pathway is simultaneously lost. Cumulative data indicate that scrib, wts, and roe act predominantly within the EMT and proliferation branches of the BLJ pathway, and not the repression of motility branch. It is suggested that without simultaneous loss of the repression of motility branch of the BLJ pathway, scrib and wts border cells cannot bypass the essential requirement for the second step of EMT, thus border cell motility is blocked (Zhao, 2008).
This interpretation is also consistent with the seemingly paradoxical function of scrib and wts as enhancers of dlg tumor invasion, even though Scrib and Wts promote rather than repress border cell movement. The noninvasive scrib and wts tumor phenotypes indicate that they are crucial for repressing the first step of EMT, loss of epithelial polarity and adoption of a lateralized, mesenchymal-like phenotype. It has been suggested that scrib and wts enhance dlg invasive tumorigenesis by increasing the rate at which dlg mutant follicle cells undergo EMT and further facilitate invasion by depressing proliferation control and increasing the number of follicle cells available for movement. Thus, even though scrib and wts are required to promote movement, it is suggested that in dlg; scrib/+ or dlg; wts/+ tumors this requirement can be bypassed because the branch of the BLJ pathway that represses motility is simultaneously disrupted (Zhao, 2008).
The noninvasive tumor phenotypes of scrib and wts are very similar to the phenotypes of dlg mutants that specifically disrupt Dlg SH3 and GuK domains. Thus Scrib and Wts may act specifically downstream of the Dlg SH3 and GuK domains. Consistent with this, Scrib appears to associate with the Dlg GuK domain in neuronal synapses via the linker protein GuK-holder. Further, whereas Fas2, dlg, and lgl cause faster border cell migration, border cell migration is very similar to wild type in the dlg SH3/GuK-specific mutants, suggesting that Dlg SH3/GuK predominantly represses the first step of EMT and proliferation but not motility. On the basis of this specificity, it is suggest that one reason that lgl may be a stronger dlg enhancer than scrib and wts is that lgl represses motility in addition to EMT and proliferation. For example, the de novo tumor formation observed when one copy of lgl, scrib, or wts is removed in dlghf/dlgsw ovaries suggests that a threshold level of BLJ activity essential for maintenance of polarity has been lost. However, the lgl interaction may be much stronger than scrib and wts because lgl additionally represses motility (Zhao, 2008).
Increased expression of CycE and DIAP1, known Wts targets, was observed in Fas2, dlg, lgl, scrib, wts, and roe cells. Thus the importance of CycE for proliferation control, and DIAP1 for control of EMT and motility, suggests that part of the mechanism by which Fas2-Dlg represses tumorigenesis is through activating Wts signaling. DIAP1 is in a complex with Rac1 and Profilin and enables border cell motility apparently by promoting actin turnover. Further, in the embryo, DIAP1 loss leads to Dlg cleavage and cellular rounding and dispersal. Too much DIAP1 also appears to be deleterious to movement, because targeted overexpression of DIAP1 specifically in border cells slows their migration (data not shown). Thus maintaining the proper balance of DIAP1 is critical for directed movement, and it may be part of the mechanism by which Scrib and Wts influence border cell movement, suggesting that interaction with Dlg and Rac1 may be another level at which Scrib regulates EMT and movement, consistent with the possibility that it functions downstream of Scrib and Wts in follicle cells to repress both EMT and proliferation (Zhao, 2008).
In contrast to the strong enhancement of dlg by scrib, Fas2 was only weakly enhanced by scrib. Given the complexity of coordinating EMT, proliferation, and motility within an epithelial field, perhaps the simplest model is that multiple Dlg complexes reside within the BLJ, each with a distinct set of ligands that control one or more morphogenetic activities (Zhao, 2008).
Another interesting difference in the enhancement of dlg and Fas2 by lgl, scrib, wts, and roe was that they all enhanced both dlg tumorigenesis and invasion, but only enhanced Fas2 tumorigenesis, without invasion. An important difference between these experiments may be that in Fas2null follicle cells, Dlg is missing Fas2 as a ligand, whereas in dlghf/dlgsw, dlghf/dlgip20, and dlghf/dlglv55 follicle cells, Fas2 is localized at sites of contact between follicle cells in both the native epithelium and in streams of invading cells, suggesting that Fas2 continues to act as a Dlg ligand in these cells. This is probably an important difference because Fas2-Dlg binding is expected to control the conformation of Dlg. Dlg conformations in turn may specify Dlg intra- and intermolecular interactions that determine the relative balance of EMT, proliferation, and invasion factors that associate with the BLJ scaffold. For example, in neuronal cells intramolecular interactions between Dlg SH3 and GuK domains regulate the strength of intermolecular binding of GuK-holder, which binds Scrib. The SH3-GuK intramolecular interaction is further modulated by intramolecular interactions with PDZ3, which are regulated by intermolecular interactions with neurolignin, a transmembrane ligand for PDZ3 (Zhao, 2008).
On the basis of this molecular model, it is proposed that in the absence of Fas2, Dlg has a distinct conformation that tilts the balance toward EMT and proliferation over invasion, when Lgl, Scrib, Wts, or Roe are reduced. This study has shown that lgl, scrib, wts, and roe are expected to act predominantly downstream of Dlg SH3 and GuK domains to repress EMT and proliferation. Thus, removal of one copy of lgl, scrib, wts, or roe in Fas2 cells may tip the ratio of factors controlling EMT, motility, and proliferation toward derepression of EMT and proliferation, masking the Fas2 requirement for invasion. One possibility is that lgl, scrib, wts, or roe are especially important for expression of a protein in the apicolateral junction, such as Par-3/Bazooka, which is essential for dlg invasion. Consistent with this, Ex upregulation is seen in both dlg and wts clones. Further, lgl enhancement at the lglts permissive temperature showed essentially the opposite trend from Fas2. Rather than enhance tumorigenesis over invasion, removal of one copy of Fas2, dlg, scrib, wts, or roe in lgl egg chambers favored invasion. Thus, it is suggested that tumor invasiveness associated with particular combinations of mutated BLJ proteins may be masked or unmasked on the basis of the balance of activities that are disrupted, rather than disruption of particular activities per se (Zhao, 2008).
In summary, this study has identified the first signaling pathway that acts downstream of the BLJ that specifically controls EMT and proliferation, and important clues have been gained as to how this signaling may be organized. Like the Drosophila follicular epithelium, the human ovarian surface epithelium, which is thought to be the site of origin of most ovarian cancers, is derived from a mesodermal lineage. The data suggest that the BLJ plays an especially crucial role in the follicle cells compared to ectodermal lineages in repressing epithelial invasion and that the follicular epithelium appears to organize signaling from epithelial junctions in distinct ways compared to other epithelia. Given the conservation in the lineage of the fly and human epithelia, and the sensitivity of this screen for detecting molecules important for invasive carcinogenesis, it is proposed that the fly egg chamber may serve as a prototype for identifying early molecular events that are crucial for invasion of human ovarian cancer and possibly other malignancies that remain undetected before they start to invade (Zhao, 2008).
Drosophila neural stem cells or neuroblasts undergo typical asymmetric cell division. An evolutionally conserved protein complex, comprising atypical protein kinase C (aPKC), Bazooka (Par-3) and Par-6, organizes cell polarity to direct these asymmetric divisions. Aurora-A (AurA) is a key molecule that links the divisions to the cell cycle. Upon its activation in metaphase, AurA phosphorylates Par-6 and activates aPKC signaling, triggering
the asymmetric organization of neuroblasts. Little is known, however, about how such a positive regulatory cue is counteracted to coordinate aPKC signaling with other cellular processes. During a mutational screen using the Drosophila compound eye, microtubule star (mts), which encodes a catalytic subunit of protein phosphatase 2A (PP2A), was identified as a negative regulator
for aPKC signaling. Impairment of mts function causes defects in neuroblast divisions, as observed in lethal (2) giant larvae (lgl) mutants. mts genetically interacts with
par-6 and lgl in a cooperative manner in asymmetric neuroblast division. Furthermore, Mts tightly associates with Par-6 and dephosphorylates AurA-phosphorylated Par-6. This genetic and biochemical evidence indicates that PP2A suppresses aPKC signaling by promoting Par-6 dephosphorylation in neuroblasts, which uncovers a novel balancing mechanism for aPKC signaling in the regulation of asymmetric cell division (Ogawa, 2009).
Polarity is a fundamental characteristic of cells and underlies a variety of cellular processes involved in the development and homeostasis of living organisms. In epithelial cells, which consist of the apical and basolateral membrane domains, cell polarity creates distinct subcellular compartments to arrange the cells into a well-ordered structure. In asymmetric cell division, cell polarity is coupled with mitosis. Cell polarity creates two subcellular domains with distinct characteristics in the mitotic mother cell and coordinates the mitotic spindle with the polarity axis to allow the two daughter cells to be distinct. Because these cell polarity events are tightly linked to other elementary processes such as the cell cycle and mitotic events, cell polarity is finely controlled to coordinate with those cellular processes (Ogawa, 2009).
Drosophila neural-stem-like cells, or neuroblasts, undergo typical asymmetric divisions, providing an excellent model for the study
of how cell polarity is controlled. Neuroblasts repeatedly divide into a large, self-renewing daughter (the neuroblast itself) and a smaller, differentiating daughter [the ganglion mother cell (GMC)]. Cell fate determinants, such as Prospero, Brain tumor (Brat) and Numb, are segregated to the GMC. The localization of these determinants and the coordination with mitotic spindle orientation are controlled by the apically localized protein complexes -- the aPKC-Par complex and the Pins complex -- which are mutually linked by Inscuteable (Insc). The aPKC-Par complex consists of atypical protein kinase C (aPKC), Bazooka (Baz) and Par-6 and is primarily involved in organizing cell polarity and the asymmetric distribution of the cell fate determinants along the axis of polarity. The Pins complex, which consists of Partner of Inscuteable (Pins), Locomotion defects (Loco) and Gαi, determines the orientation of the mitotic spindle relative to the cell polarity axis (Ogawa, 2009).
aPKC is a key enzyme involved in establishment of neuroblast polarity and definition of the apical cortex. A tumor suppressor protein, Lethal (2) giant larvae (Lgl), is thought to antagonize
aPKC as an inhibitory substrate. Although aPKC binds to non-phosphorylated
Lgl, Lgl that is phosphorylated by aPKC dissociates from it and is released from the cell cortex. In the absence of aPKC, the entire cortex becomes basal, and Miranda, an adaptor protein for Prospero and Brat, distributes uniformly throughout the cortex. However, loss of Lgl results in uniform activation of aPKC in the cortex just as if the entire cortex were apical. Consequently, Miranda misdistributes into the cytoplasm and concentrates on mitotic spindles (Ogawa, 2009).
The apical complex and the basal determinants dynamically change their localization as the cell cycle progresses. The apical complex accumulates at the apical cortex during late interphase,
retains its apical localization during metaphase, and then initiates expansion through the cortex in anaphase. Miranda and its cargos are temporally found in the apical cortex in late interphase and, after spreading into the cytoplasm at the onset of mitosis, form the basal crescent that is complementary to the localization of the apical complex during metaphase. At late anaphase onwards, they are restricted to the GMC compartment, which is separated by the contractile ring from the neuroblast compartment (Ogawa, 2009).
It was recently shown that the mitotic kinase Aurora-A (AurA) has an important role in linking the cell cycle to the asymmetric cell division of neuroblasts and sensory organ precursors (SOPs)
by phosphorylating Par-6. When AurA is inactive, aPKC binds to unphosphorylated Par-6 and Lgl and remains inactive. Phosphorylation of Par-6 by AurA blocks the interaction of Par-6 with aPKC, which in turn leads to activation of aPKC. Activated aPKC then phosphorylates Lgl to replace it with Baz. The Par complex that has recruited Baz is able to phosphorylate Numb, leading to an exclusion of Numb from the apical domain. Because AurA becomes active in the mitotic phase, it is able to synchronize aPKC activation with the entry into mitosis. Given the role of AurA as a positive regulator of aPKC signaling, it is also likely that dephosphorylation negatively regulates this signaling pathway (Ogawa, 2009).
The serine/threonine phosphatases are grouped into four major classes based on their sensitivity to inhibitors and requirement for divalent cations: protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), protein phosphatase 2B (PP2B) and protein phosphatase
2C (PP2C). PP2A holoenzyme functions as a heterotrimeric complex comprising a catalytic C subunit [Microtubule star (Mts)
in Drosophila], a scaffolding A subunit (PP2A-29B in Drosophila) and a regulatory B-subunit [Twins (Tws), Widerborst (Wdb) and PP2A-B' in Drosophila]. The A-subunit can serve as a linker between the C- and B-subunits, and the B-subunit can influence the enzymatic activity and substrate specificity of the holoenzyme. In a genetic screen using the Drosophila compound eye, mts was identified as an enhancer of the aPKC-induced eye phenotype. The genetic and biochemical evidence indicating that Mts suppresses aPKC activity by enhancing dephosphorylation of Par-6 in neuroblasts uncovered an antagonistic role of PP2A in the aPKC signaling pathway (Ogawa, 2009).
The Drosophila compound eye is composed of repetitive ommatidia
that contain epithelial retinal cells. Because of the crystal-like arrangement of ommatidia in the compound eye, it is sensitive to defects in epithelial polarity and therefore ideal for use in mutational screens for components involved in epithelial polarity. A modifier screen was undertaken under a sensitized background to look for mutations that affected epithelial polarity. When the membrane-tethered aPKC (aPKCCAAX) is expressed by GMR-GAL4, it becomes expressed in all differentiated retinal cells, the apicobasal polarity of the retinal cells is severely impaired, and the compound eye becomes small and rough. Kinase activity of aPKCCAAX is essential for inducing this eye phenotype, because the kinase-dead version, in which Lys293 is mutated to Trp (K293W), does not alter the eye morphology. Using this system, mutants were screened that modify the aPKCCAAX-induced eye phenotype among lethal mutants available from stock centers, and mts was identified as a phenotypic enhancer. When the GMR-GAL4/UAS-aPKCCAAX fly was crossed to the mts02496 or mtsXE-2258 mutant fly, a smaller and rougher eye was observed, suggesting that Mts acts as an antagonist for the aPKC signaling pathway (Ogawa, 2009).
The mts gene is expressed ubiquitously during embryogenesis and its protein product localizes to the cytoplasm in neuroblasts as well as in epithelial cells. Because a large amount of mts mRNA is maternally supplied, zygotic mts mutant embryos do not show significant defects with regard to cell polarity, and germline clones do not produce an egg. Therefore loss-of-function phenotypes were examined in neuroblasts by overexpressing a dominant-negative mutant of Mts (dnMts) (Hannus, 2002), which lacks the N-terminal region of the phosphatase domain. In wild-type neuroblasts, the protein complex containing aPKC, Par-6 and Baz localizes to the apical cortex and directs Miranda to the basal cortex at metaphase. In the dnMts-expressing embryos, the apical complex
localizes to the apical cortex, and its distribution is broader than normal. By contrast, localization of Miranda is severely affected, and it is distributed
less asymmetrically along the cell cortex and into the cytoplasm, where it is concentrated on the mitotic spindles. This phenotype resembles that of lgl, raising the possibility that Mts functions in the same pathway as Lgl (Ogawa, 2009).
This study shows that PP2A functions as a negative regulator of the aPKC signaling pathway in Drosophila neuroblasts. Although several studies have suggested that PP2A negatively regulates aPKC signaling in mammalian culture cells, the critical target(s) of PP2A is unknown in these studies. The substrates of aPKC, which include Lgl and aPKC itself, can also be substrates for PP2A. However, none of them has been molecularly identified as a target to be dephosphorylated by PP2A. This study has identified Par-6 as a direct
target of PP2A, which has its catalytic subunit encoded by the mts gene. Par-6 is known to be phosphorylated by AurA to trigger aPKC activation when neuroblasts and SOPs enter mitosis. Biochemical and genetic evidence reveals that Mts dephosphorylates Par-6 to suppress the aPKC pathway, suggesting an antagonistic role for Mts against AurA in the regulation of cell polarity that is governed by aPKC signaling (Ogawa, 2009).
Co-immunoprecipitation assays of overexpressed Par-6, aPKC or Lgl with Mts in S2 cells indicated that all these molecules can form a complex either directly or indirectly. Among these, the association of Mts with aPKC and Lgl is relatively weaker than the association with Par-6, although a previous study suggested
that PP2A associates with aPKC to suppress its kinase activity in mammalian cultured cells (Nunbhakdi-Craig, 2002). In the current study results indicate that, in S2 cells, Par-6 most efficiently forms a complex with Mts. Consistently, the in vitro dephosphorylation assay showed that PP2A effectively dephosphorylates AurA-phosphorylated Par-6 but not the auto-phosphorylated PKCzeta or the PKCzeta-phosphorylated Lgl. It is inferred from these results that Par-6 is a direct target of Mts. Substrate specificity of PP2A is greatly influenced by the B-subunit incorporated into the holoenzyme. Thus, differences in the affinity with Mts among the three tested
molecules in co-immunoprecipitation assays might, therefore, partly reflect the B-subunit(s) that is expressed in S2 cells endogenously. The Drosophila genome contains three genes for the B-subunit: tws, wdb and PP2A-B', all of which are ubiquitously expressed during embryogenesis, as is mts (Ogawa, 2009).
At present, it is not clear which of these three is used for targeting Par-6. Among them,
tws mutants often show bristle duplications that are due to defective cell fate decisions of the SOP, as
lgl4/lglts3 flies show. Since this lgl4/lglts3 phenotype is enhanced by
mts, mts is also likely to be involved in the same pathway. Furthermore, a recent study demonstrated that Tws, together with Mts, is included in the aPKC complex to regulate the asymmetric cell division of larval neuroblasts (Chabu, 2009). These results suggest that Mts uses Tws to target Par-6 in the asymmetric cell divisions of SOPs as well as of neuroblasts (Ogawa, 2009).
Par-6 is an essential cofactor for aPKC activity, and it is known to keep aPKC inactive in the absence of AurA-dependent phosphorylation of Par-6 in neuroblasts. The complete deprivation
of Par-6 results in the uniform distribution of Miranda into the cell cortex, which is reminiscent of the aPKC mutant phenotype. Thus, Par-6 is required to produce functional aPKC, and its kinase activity
becomes active only when Par-6 is phosphorylated by AurA. par-6
heterozygotes (par-6δ226/+) do not show any defect in Miranda localization during the embryonic stage, which indicates that one copy of
par-6 in addition to the maternal supply is sufficient to support normal aPKC function. When mts is further inactivated under this condition (par-6δ226/+, mtsXE-2258/mts02496), some neuroblasts exhibit an lgl-like phenotype in Miranda localization, which is indicative of aPKC hyperactivation and unlike the par-6 loss-of-function mutant. This result suggests that Par-6, because
of its hyper-phosphorylation, becomes unable to restrict aPKC activity within the normal range. Thus, a probable normal function of Mts is to promote the inhibitory function of Par-6 on aPKC without affecting its function as an essential subunit of the aPKC complex (Ogawa, 2009).
Whereas AurA seems to be active only during the mitotic phase in cell-cycling cells, mitotically inactive or interphase epithelial cells exhibit concrete apicobasal polarity. How do those cells activate aPKC signaling even though AurA is inactive? This apparent paradox raises several possibilities. Par-6 phosphorylation is required for aPKC activation in epithelial cells but might be mediated by kinase(s) other than AurA. Alternatively, aPKC might be activated by mechanisms other than the phosphorylation of Par-6. Indeed, it has been reported that the active form of Cdc42 binds to the CRIB domain of Par-6 to relieve its inhibitory effect on aPKC, leading to the activation of aPKC (Ogawa, 2009).
Although obvious defects are not detected in the epithelial cells of zygotic
mts mutant embryos, Oogenesis clones of mts show dramatic defects in their epithelial polarity. This follicle cell phenotype is different from that caused by hyperactivation of aPKC, as observed in the lgl mutant, suggesting that the action of Mts is mechanistically different in the maintenance of follicle cell polarity from that observed in neuroblasts. In photoreceptor cells, Mts operates antagonistically against Par-1 kinase, which restricts Baz to the adherens junctions. It is also known that Par-1 phosphorylates Baz directly to inhibit its incorporation into the apical aPKC complex, thereby restricting Baz to the apical domain in follicle cells. These data raise the possibility that Mts antagonizes Par-1 in Baz localization in follicle cells
by inhibiting Par-1-dependent Baz phosphorylation. If this is the case, Mts positively regulates the aPKC pathway in follicle cells and photoreceptor cells, unlike the situation in neuroblasts. To date, there is no report for Par-1 function in Drosophila neuroblasts. Further study is necessary to test whether a similar antagonism between Mts and Par-1 has a role in the regulation of neuroblast polarity (Ogawa, 2009).
AurA-mediated Par-6 phosphorylation is a key step in initiating the asymmetric segregation of the cell fate determinants in the neuroblast cell cycle. Once Par-6 is phosphorylated, aPKC will be continuously activated during the mitotic phase. The apical domain would overwhelm the entire cortex unless an antagonistic reaction occurred. PP2A will be able to balance AurA in Par-6 phosphorylation during mitosis. Thus, PP2A, together with the antagonistic ligand Lgl, might have a role in maintaining aPKC activity at an appropriate level to create both apical and basal domains in the cortex during mitosis. Although both Mts and Lgl negatively regulate aPKC signaling, Mts operates on aPKC activity by directly regulating the cell-signaling cascade, whereas Lgl does so through the direct physical association as a substrate. Therefore, they are different in their mechanisms of action (Ogawa, 2009).
When neuroblasts complete cell cleavage, the basal membrane is largely segregated into the GMC, and the entire cell cortex of neuroblasts appears to become apical. It is therefore necessary
to repolarize in order to make the apical and basal domains in the cell cortex for the onset of the next cell cycle. To do so, Par-6 phosphorylation must be removed before entering the next cell cycle, to reset the configuration of the apical complex. A model is proposed in which Mts actively dephosphorylates
Par-6 to reset the membrane polarity after the completion of each division cycle. In this context, it will be important to examine whether Mts function depends on the cell-cycle stage in neuroblasts (Ogawa, 2009).
In eukaryotes, serine/threonine phosphatases are categorized into four major groups: PP1, PP2A, PP2B and PP2C. Recent studies have shown that PP1α affects Par-3 activity through the regulation of a phosphorylation-dependent interaction of Par-3 with 14-3-3
or PKCzeta. This study also identified a
Pp1-87B mutation as an enhancer of the aPKCCAAX-induced eye phenotype in the genetic screen and found defects in localization of Miranda
as well as in epithelial cell polarity in the Pp1-87B mutant. Furthermore, it has been reported that protein phosphatase 4 (PP4), which is a PP2A family member, regulates Miranda localization in
Drosophila neuroblasts, although the direct substrate of PP4 is not yet clear. Thus, other classes of phosphatases in addition to PP2A are involved in the regulation of cell polarity in various
cellular contexts. Further delineation of phosphatase functions and the crosstalk between phosphatases should help lead to an understanding of the global control of cellular processes regulated by cell polarity (Ogawa, 2009).
Most cell types in an organism show some degree of polarization, which relies on a surprisingly limited number of proteins. The underlying molecular mechanisms depend, however, on the cellular context. Mutual inhibitions between members of the Par genes are proposed to be sufficient to polarize the C. elegans one-cell zygote and the Drosophila oocyte during mid-oogenesis. By contrast, the Par genes interact with cellular junctions and associated complexes to polarize epithelial cells. The Par genes are also required at an early step of Drosophila oogenesis for the maintenance of the oocyte fate and its early polarization. This study shows that the Par genes are not sufficient to polarize the oocyte early and that the activity of the tumor-suppressor gene lethal giant larvae (lgl) is required for the posterior translocation of oocyte-specific proteins, including germline determinants. Lgl localizes asymmetrically within the oocyte and is excluded from the posterior pole. Phosphorylation of Par-1, Par-3 (Bazooka) and Lgl is crucial to regulate their activity and localization in vivo. Adherens junctions locate around the ring canals, which link the oocyte to the other cells of the germline cyst. However, null mutations in the DE-cadherin gene, which encodes the main component of the zonula adherens, do not affect the early polarization of the oocyte. It is concluded that, despite sharing many similarities with other model systems at the genetic and cellular levels, the polarization of the early oocyte relies on a specific subset of polarity proteins (Fichelson, 2010).
One general strategy to establish polarity within a cell is to create non-overlapping membrane domains along one specific axis. In most cell types, four complexes are involved in the formation of these domains: (1) Par-3-Par-6-aPKC, (2) Crb-Sdt-dPatj, (3) Scrib-Lgl-Dlg and (4) Par-1. However, the activities and interactions of these complexes depend on the cellular context. In single-cell systems that lack intercellular junctions, such as the C. elegans embryo and vertebrate hippocampal neurons, mutual inhibitions between the Par-3-Par-6-aPKC complex and Par-1 (PAR-2) seem sufficient to establish polarity. By contrast, in the follicular epithelium, the Crb-Sdt-dPatj complex acts redundantly with Par-3-Par-6-aPKC to define the apical side, whereas the Scrib-Lgl-Dlg complex cooperates with Par-1 on the lateral cortex. Consistent with this redundancy, expression of non-phosphorylatable forms of either Par-3 or Par-1 is not able to disrupt the apical-basal polarity of the follicle cells, although they both localize ectopically. This study shows that the early polarization of the oocyte is an intermediate case. The results suggest that the Crb-Sdt-dPatj complex does not act redundantly with the Par-3-Par-6-aPKC complex as it is not required, whereas Lgl could function with Par-1. Consistent with this hypothesis, this study showed that the expression of a non-phosphorylatable form of Par-1 is able to disrupt the early polarization of the oocyte, whereas a non-phosphorylatable form of Par-3 is not able to counter the activities of both Lgl and Par-1. In addition, it was found that Par-1 localizes on the fusome independently of Lgl further suggesting that both could act in parallel pathways (Fichelson, 2010).
The results show that phosphorylation plays a crucial role to regulate the activity and localization of Par-1, Par-3 and Lgl during early oogenesis, but to different extents in each case. Par-1 phosphorylation might not be crucial for its localization within the germarium, since it was found that the non-phosphorylatable and wild-type forms of Par-1 had a similar localization, although Par-1-AEM (apical-lateral exclusion motif (AEM), in which a conserved threonine is replaced by an alanine) appears a bit more cytoplasmic. Its overexpression, however, induces very strong and penetrant polarity defects in the germline. These results contrast with the follicular epithelium, where Par-1-AEM-GFP localizes ectopically to the apical membrane but does not affect the polarization of those cells. Thus, the main function of Par-1 phosphorylation in the germarium might be to downregulate its kinase activity. In addition, it was shown that the microtubule cytoskeleton is a probable target of Par-1 activity, since a strong reduction in microtubules was observed in oocytes expressing Par-1-AEM (Fichelson, 2010).
The localization of endogenous Par-3, wild-type Par-3-GFP and non-phosphorylatable Par-3 (Baz-S151A,S1085A-GFP) also appear identical. However, Baz-S151A,S1085A was unable to localize properly in the absence of the endogenous Par-3. This failure is probably due to the inability of this mutant form of Par-3 to homodimerize. Phosphorylation thus plays an important role for Par-3 localization. This non-phosphorylatable form of Par-3 is, however, still active, as it is able to rescue par-3-null homozygous clones in the follicular epithelium and is also sufficient to induce polarity defects in the oocyte when expressed at later stages of oogenesis. In this latter case, Baz-S151A,S1085A was expressed in the presence of the endogenous Par-3 and was able to reach the cortex of the oocyte and localize ectopically to the posterior pole. The results suggest that this ectopic Par-3 is probably made of heterodimers of endogenous and non-phosphorylatable forms of Par-3. The data further show clear differences with the follicular epithelium, where expression of the non-phosphorylatable form of Par-3 in par-3 mutant clones not only rescues the absence of endogenous protein, but also localizes properly at the apical side only. This could be a consequence of redundant mechanisms in the follicle cells (Fichelson, 2010).
It was found that Lgl localization is strikingly asymmetric in the early oocyte as it is completely absent from the posterior cortex from stage 1 to stage 5-6 of oogenesis. By contrast, it becomes specifically enriched at the posterior cortex from stage 7 onward. This is the first time that such an asymmetry is described within the germline so early during oogenesis for any protein. It was further demonstrated that this asymmetric localization depends on Lgl phosphorylation, as Lgl-3A localizes around the entire oocyte cortex. Phosphorylation thus plays an important role for Lgl localization. Surprisingly, the ectopic Lgl-3A localization is not sufficient to disrupt the early polarization of the oocyte. By contrast, the same Lgl-3A construct induces much stronger polarity defects than wild-type Lgl when overexpressed in embryonic neuroblasts or in the oocyte at later stages of oogenesis. One possible explanation is that at least one of the mutated Serine in Lgl-3A is also required for Lgl activity during the early stages of oogenesis. Another possibility is that an unknown redundant pathway is able to counteract Lgl activity in the germarium (Fichelson, 2010).
One question remaining from this work is the relationship between the localization of Par-1, Par-3 and Lgl, and their function. It is difficult to relate Par-1 localization on the fusome in region 1 of the germarium and the polarization defects induced by its absence in region 3. Furthermore, this study shows that Par-3 localizes around the ring canals with DE-Cadherin and Armadillo on genuine AJs, which are structures playing key roles in the polarization of many epithelia. However, the absence of Par-3 on these junctions in DE-Cadherin mutant clones does not perturb the polarization of the oocyte. The relevant localization of Par-3 for its polarizing activity in the germarium thus remains unknown. Finally, although Lgl localization is clearly asymmetric, excessive or ectopic localization only affect oogenesis after the oocyte becomes polarized (Fichelson, 2010).
Several arguments strongly point to the microtubule cytoskeleton and associated proteins as key targets of the polarity complexes in the germarium: (1) short treatments of microtubules depolymerizing drugs allow the restriction of cytoplasmic proteins into the oocyte in region 2 of the germarium but disrupt their localization to the posterior of the oocyte in region 3; (2) the Orb protein localizes into the oocyte in hypomorphic combinations of dhc64C, which encodes the heavy chain of the minus-end-directed molecular motor Dynein, but fails to translocate to the posterior pole of the oocyte; (3) In strong allelic combinations of Bicaudal D, a binding partner of the dynein-dynactin complex, Orb and centrosomes also fail to migrate to the posterior of the oocyte. Furthermore, in all three cases, the oocyte becomes polyploid later on and reverts to the nurse cell fate. This study shows that overexpression of Par-1-AEM strongly reduces the level of microtubules and induces identical phenotypes. This confirms that the earliest step of oocyte polarization depends on microtubules and is consistent with the well-established function of Par-1 mammalian homologues, the MARKs, in destabilizing microtubules. It is, however, more difficult to explain why the oocyte nucleus becomes polyploid and why the oocyte loses its identity. Although the Par proteins could have a separate function within the nucleus, depolymerizing the microtubules leads to an identical phenotype, which rather suggests that polyploidization of the oocyte nucleus is a direct consequence of the absence of microtubules. A failure to polarize is, however, not sufficient to induce polyploidization of the oocyte nucleus. Indeed, several mutants were found to retain Orb at the anterior of the oocyte but did not produce egg chambers with 16 nurse cells. The link between the cytoplasmic polarization of the oocyte and its nuclear identity thus remains unclear and is an exciting line for future investigations (Fichelson, 2010).
Cancer stem cells (CSCs) are postulated to be a small subset of tumor cells with tumor-initiating ability that shares features with normal tissue-specific stem cells. The origin of CSCs and the mechanisms underlying their genesis are poorly understood, and it is uncertain whether it is possible to obliterate CSCs without inadvertently damaging normal stem cells. This study shows that a functional reduction of eukaryotic translation initiation factor 4E (eIF4E) in Drosophila specifically eliminates CSC-like cells in the brain and ovary without having discernable effects on normal stem cells. Brain CSC-like cells can arise from dedifferentiation of transit-amplifying progenitors upon Notch hyperactivation. eIF4E is up-regulated in these dedifferentiating progenitors, where it forms a feedback regulatory loop with the growth regulator dMyc to promote cell growth, particularly nucleolar growth, and subsequent ectopic neural stem cell (NSC) formation. Cell growth regulation is also a critical component of the mechanism by which Notch signaling regulates the self-renewal of normal NSCs. These findings highlight the importance of Notch-regulated cell growth in stem cell maintenance and reveal a stronger dependence on eIF4E function and cell growth by CSCs, which might be exploited therapeutically (Song, 2011).
The differential cell growth rates observed between ectopic NBs and normal or primary NBs and the correlation between cell growth defects and NB fate loss prompted a test of whether slowing down cell growth might selectively affect the formation of ectopic NBs. Attenuation of TOR signaling, a primary mechanism of cell growth regulation, through NB-specific overexpression of TSC1/2, a strong allele of eIF4E antagonist 4EBP [4EBP(LL)s], or a dominant-negative form of TOR (TOR.TED) all partially suppressed ectopic NB formation in α-adaptin (ada) mutants without affecting normal or primary NBs. Interestingly, RNAi-mediated knockdown of eIF4E, a stimulator of oncogenic transformation and a downstream effector of TOR signaling, showed a better suppression than manipulating other TOR pathway components, suggesting that eIF4E might play a more important role in ectopic NB formation. Strikingly, the brain tumor phenotypes caused by overactivation of N signaling - as in lethal giant larvae
(lgl) mutant, aPKCCAAX overexpression, or N overexpression conditions - were also fully suppressed by eIF4E knockdown. Furthermore, the brain tumor phenotypes of brat mutants were also completely rescued by eIF4E RNAi (Song, 2011).
In contrast, normal NB formation or maintenance was not affected by eIF4E knockdown. NBs with eIF4E knockdown remained highly proliferative, as evidenced by the mitotic figures, and displayed relatively normal apical basal cell polarity. There are several other eIF4E-like genes in the fly genome (Hernandez, 2005), which may play partially redundant roles in normal NB maintenance. eIF4E knockdown appeared to specifically block ectopic NB formation caused by the dedifferentiation of IPs in type II NB lineages, since it did not affect ectopic type I NB formation in cnn or polo mutants that are presumably caused by symmetric divisions of type I NBs. In addition, cell fate transformation induced by N overactivation in the SOP lineage was not affected by eIF4E RNAi, supporting the idea that eIF4E is particularly required for type II NB homeostasis. Supporting the specificity of the observed eIF4E RNAi effect, another eIF4E RNAi transgene (eIF4E-RNAi-s) also prevented ectopic NB formation. Moreover, a strong loss-of-function mutation of eIF4E also selectively eliminated ectopic NBs induced by N overactivation without affecting normal NBs, reinforcing the hypothesis that ectopic NBs exhibit higher dependence on eIF4E (Song, 2011).
To further support the notion that the ectopic NBs are particularly vulnerable to eIF4E depletion, a conditional expression experiment was carried out in which eIF4E-RNAi-s was turned on in brat mutants using the 1407ts system, after ectopic NBs had been generated. Whereas the brain tumor phenotype exacerbated over time in the brat mutants, 1407-GAL4-driven eIF4E-RNAi-s expression in brat mutants effectively eliminated ectopic NBs, leaving normal NBs largely unaffected (Song, 2011).
In normal type II NB lineage, eIF4E protein was enriched in the NBs. Ectopic NBs induced by N overactivation in ada mutants also expressed eIF4E at high levels, whereas spdo mutant NBs exhibited reduced eIF4E expression. Thus, eIF4E up-regulation correlates with N-induced ectopic NB formation in a dedifferentiation process that likely involves elevated cell growth (Song, 2011).
Given the coincidence of nucleolar size change with ectopic NB formation, the involvement of the growth regulator dMyc was tested. dMyc protein levels were up-regulated in normal or N overactivation-induced ectopic NBs, but were down-regulated in spdo mutant NBs. Furthermore, dMyc transcription, as detected with a dMyc-lacZ transcriptional fusion reporter, was also up-regulated in both normal and ectopic NBs in ada mutants. A previous study in Drosophila S2 cells identified dMyc as a putative N target. In vivo chromatin immunoprecipitation (ChIP) experiments were carried out to assess whether dmyc transcription is directly regulated by N signaling in NBs. Using chromatin isolated from wild-type larval brains and a ChIP-quality antibody against the N coactivator Suppressor of Hairless [Su(H)], specific binding was demonstrated of Su(H) to its putative binding sites within the second intron of dmyc (dmyc-A). No binding to an internal negative control region proximal to the first exon of dmyc (dmyc-B) or to the promoter region of the rp49 gene was detected. N signaling thus directly activates dMyc transcription in the NBs. Similar to eIF4E RNAi, knockdown of dMyc strongly suppressed ectopic NB formation induced by Brat or Ada inactivation or N overactivation. Intriguingly, the strong tumor suppression effect of eIF4E knockdown was partially abolished by dMyc overexpression. Furthermore, dMyc function, as reflected by its promotion of nucleolar growth in IPs, was attenuated by eIF4E RNAi, although eIF4E RNAi alone had no obvious effect. Different from the reported eIF4E regulation of Myc expression in mammalian cells (Lin, 2008), dMyc promoter activity or protein levels remained unaltered under eIF4E RNAi conditions, suggesting that eIF4E may modulate dMyc activity without altering its expression. One possibility is that eIF4E may enter the nucleus to interact with Myc and promote its transcriptional activity. To test this hypothesis, HEK293T cells were transfected with Flag-tagged human eIF4E alone or in combination with HA-tagged dMyc. Indeed, both Drosophila dMyc and endogenous human c-Myc specifically coimmunoprecipitated with human eIF4E from nuclear extracts, indicating a conserved interaction between eIF4E and Myc within the nuclei of proliferating cells. Consistent with these biochemical data, dMyc transcriptional activity within NBs, which could be monitored with an eIF4E-lacZ reporter, was drastically reduced upon eIF4E knockdown (Song, 2011).
In contrast, eIF4E transcription, as detected with an eIF4E-lacZ transcriptional fusion reporter, as well as eIF4E protein levels detected by immunostaining were up-regulated upon dMyc overexpression and down-regulated by dMyc RNAi. It is unlikely that the changes in eIF4E-lacZ activity were due to global increases or decreases in β-galactosidase (β-gal) translation caused by altered dMyc levels, since lacZ expression from a dMyc-lacZ reporter was unaffected under similar conditions. Furthermore, like dMyc protein, eIF4E-lacZ reporter expression was up-regulated in normal NBs or ectopic NBs in ada mutants, further supporting the notion that dMyc may up-regulate eIF4E transcription. Moreover, ChIP experiments using chromatins isolated from wild-type larval brains and a ChIP-quality antibody against dMyc demonstrated specific binding of dMyc to an eIF4E promoter region harboring a cluster of adjacent noncanonical E boxes, supporting a direct regulation of eIF4E transcription by dMyc. dMyc and eIF4E thus appeared to form a regulatory feedback loop that promoted NB growth and renewal. Consistent with this model, while knocking down either dMyc or eIF4E had no noticeable effect on type II NB maintenance and only a mild effect on NB nucleolar size in the case of dMyc RNAi, their simultaneous knockdown led to a significant reduction in nucleolar size, premature neuronal differentiation, and loss of NBs (Song, 2011).
If the dMyc-eIF4E axis of cell growth control is a crucial downstream effector of N signaling in regulating NB maintenance, its up-regulation might be able to rescue the type II NB depletion phenotype resulting from reduced N signaling. Indeed, the loss of NBs associated with reduced Notch signaling was preventable when cell growth was boosted by dMyc overexpression. Thus, while N-IR directed by 1407-GAL4 led to complete elimination of type II NBs, the coexpression of dMyc, but not CD8-GFP or Rheb, an upstream component of the TOR pathway, resulted in the preservation of approximately half of type II NBs with apparently normal cell sizes, cell fate marker expression, and lineage composition. A similar effect was observed when dMyc was coexpressed with N-IR using the conditional 1407ts system, with transgene expression induced at the larval stage. While both dMyc and Rheb promote cell growth, they do so through distinct mechanisms, with the former increasing nucleolar size and the latter expanding cytoplasmic volume. These results thus provide compelling evidence that control of cell growth, particularly nucleolar growth, is a critical component in the maintenance of NB identity by N signaling (Song, 2011).
The differential responses of normal and tumor-initiating stem cells to functional reduction of eIF4E prompted a test of whether chemicals that specifically inhibit eIF4E function might have therapeutic potential in preventing CSC-induced tumorigenesis. Indeed, the brain tumor phenotypes induced by N overactivation or ada loss of function were effectively suppressed by feeding animals with fly food containing Ribavirin, an eIF4E inhibitor that interferes with eIF4E binding to mRNA 5' caps and promotes the relocalization of eIF4E from the nucleus to the cytoplasm (Kentsis, 2004; Assouline, 2009) (Song, 2011).
The CSC hypothesis was initially developed based on studies in mammalian systems. Various studies have supported the notion that CSCs share many functional features with normal stem cells, such as signaling molecules, pathways, and mechanisms governing their self-renewal versus differentiation choice. However, the cellular origin of CSCs and the molecular and cellular mechanisms underlying their development or genesis remain poorly understood. It has been proposed that CSCs could arise from (1) an expansion of normal stem cell niches, (2) normal stem cells adapting to different niches, (3) normal stem cells becoming niche-independent, or (4) differentiated progenitor cells gaining stem cell properties. This study has showen that in the Drosophila larval brain, CSCs can arise from the dedifferentiation of transit-amplifying progenitor cells back to a stem cell-like state. Importantly, eIF4E was identified as a critical factor involved in this dedifferentiation process. More significantly, it was shown that reduction of eIF4E function can effectively prevent the formation of CSCs without affecting the development or maintenance of normal stem cells. This particular dependence on eIF4E function by CSCs appears to be a general theme, as reduction of eIF4E function also effectively prevented the formation of CSCs, but not normal GSCs, in the fly ovary. These findings may have important implications for stem cell biology and cancer biology, in terms of both mechanistic understanding and therapeutic intervention (Song, 2011).
This study also offers mechanistic insights into the cellular processes leading to the dedifferentiation of progenitors back to stem cells. In Drosophila type II NB clones with overactivated N signaling, ribosome biogenesis within ectopic NBs appears to be faster than in normal NBs, as shown by the fact that the ratio of nucleolar to cellular volume of the ectopic NBs is approximately fivefold higher than that of normal NBs. The faster growth rate is accompanied by the up-regulation of dMyc and eIF4E and appears to be essential for transit-amplifying progenitors to undergo complete dedifferentiation back to a stem cell-like state. When the function of cell growth-promoting factors such as eIF4E is attenuated, the faster cell growth of ectopic NBs can no longer be sustained and the dedifferentiation process stalls. As a result, brain tumor formation caused by uncontrolled production of ectopic NBs is suppressed. In contrast, normal NBs, which presumably have relatively lower requirements for cell growth and hence eIF4E function, maintain their stem cell fate and development under similar conditions. Therefore, a potential key to a successful elimination of CSC-induced tumors would be to find the right level of functional reduction in eIF4E, which causes minimal effects on normal stem cells but effectively obliterates CSCs. An ongoing clinical trial with Ribavirin in treating acute myeloid leukemia (AML) (Assouline, 2009), a well-characterized CSC-based cancer, demonstrated exciting proof of principle that such a strategy is feasible. The current version of Ribavirin, however, has certain limitations, such as its poor specificity and the high dosage (micromolar range) required for effective treatment. Thus, more specific and effective eIF4E inhibitors are urgently needed. The drug treatment experiments with Ribavirin validated Drosophila NBs as an excellent CSC model for searching further improved drugs. More importantly, the nuclear interaction between eIF4E and Myc unraveled by this biochemical analysis not only provides a new mechanistic explanation for the synergistic effects of eIF4E and Myc in tumorigenesis (Ruggero, 2004; Wendel, 2007), but also sheds new light on how to rationally optimize drug design and therapy for treating CSC-based cancer (Song, 2011).
The results offer new information on how N signaling helps specify and maintain NSC fate. N signaling regulates stem cell behavior in various tissues of diverse species. However, it remains unclear how differential N signaling determines distinct cell fate within the stem cell hierarchy. This study demonstrates that N signaling maintains Drosophila NSC fate at least in part through promoting cell growth. The following evidence supports that cell growth, but not cell fate, change is the early and primary effect of N signaling inhibition in type II NBs: (1) Pros expression is not immediately turned on in spdo mutant NBs with reduced cell sizes. Instead, it gradually increases during the course of spdo mutant NB divisions. (2) Up-regulation of Pros is not the cause of stem cell fate loss in spdo mutant NBs, as shown by spdo pros double-mutant analysis. (3) Cell growth defects precede the up-regulation of Ase expression in aph-1 mutant NBs. (4) Promotion of cell growth, and particularly nucleolar growth, by dMyc is sufficient to prevent NB loss caused by N inhibition. At the molecular level, N signaling appears to regulate the transcription of dMyc, which in turn up-regulates the transcription of eIF4E. Such a transcriptional cascade and feedback regulation of dMyc activity by eIF4E may help to sustain and amplify the activity of the Notch-dMyc-eIF4E molecular circuitry. Hence, differential N signaling within the lineage can lead to different cell growth rates, which partially determine differential cell fates. Consistent with this notion, knockdown of both eIF4E and dMyc results in defects of NB cell growth and loss of stem cell fate (Song, 2011).
While many signaling pathways and molecules have been implicated in the maintenance of stem cell identity, the question of how a stem cell loses its 'stemness' at the cellular level remains poorly understood. A stem cell may lose its stem cell fate by undergoing a symmetric division to yield two daughter cells that are both committed to differentiation or through cell death. Earlier studies provided intriguing hints that cell growth and translational regulation could influence stem cell maintenance in the Drosophila ovary. This study usded detailed clonal analyses of NSCs over multiple time points to provide direct evidence that a NSC with impaired N signaling will gradually lose its identity due to a gradual slowing down of cell growth and loss of cell mass. Remarkably, such loss of stem cell fate can be prevented when cell growth is restored by dMyc, but not Rheb, overexpression, demonstrating the functional significance of regulated cell growth, particularly nucleolar growth, in stem cell maintenance. More importantly, this information offers clues on how to specifically eliminate tumor-initiating stem cells. These studies suggest that a stem cell, normal or malignant, has to reach a certain growth rate in order to acquire and maintain its stemness, presumably because when the stem cell grows below such a threshold, its proliferative capacity becomes too low, whereas the concentration of differentiation-promoting factors becomes too high to be compatible with the maintenance of stem cell fate. Consistent with this notion are the strong correlation between the expression of ribosomal proteins and cellular proliferation (van Riggelen, 2010) as well as the correlation between the reduction of NB sizes and the up-regulation of differentiation-promoting factor Pros or Ase in different developmental contexts (Song, 2011).
The results also provide new insights into how the evolutionarily conserved tripartite motif and Ncl-1, HT2A, and Lin-41 (TRIM-NHL) domain proteins regulate stem cell homeostasis. The TRIM-NHL protein family, to which Brat and Mei-P26 belong, include evolutionarily conserved stem cell regulators that prevent ectopic stem cell self-renewal by inhibiting Myc. However, the downstream effectors of the TRIM-NHL proteins remain largely unknown. This study identified eIF4E as such a factor. NB-specific knockdown of eIF4E completely suppresses the drastic brain tumor phenotype caused by loss of Brat. Interestingly, eIF4E knockdown is even more effective than dMyc knockdown in this regard. N signaling and Brat have been proposed to act in parallel in regulating Drosophila type II NB homeostasis. However, at the molecular level, how deregulation of these two rather distinct pathways causes similar brain tumor phenotypes remain largely unknown. The current results suggest that these two pathways eventually converge on the dMyc-eIF4E regulatory loop to promote cell growth and stem cell fate. N overactivation and loss of Brat both result in up-regulation of eIF4E and dMyc in transit-amplifying progenitors, accelerating their growth rates and helping them acquire stem cell fate. Consistent with a general role of eIF4E and dMyc in stem cell regulation, it was shown that partial reduction of eIF4E or dMyc function in the Drosophila ovary effectively rescues the ovarian tumor phenotype due to the loss of Mei-P26. The vertebrate member of the TRIM-NHL family, TRIM32, is shown to suppress the stem cell fate of mouse neural progenitor cells, partially through degrading Myc. Whether eIF4E acts as a downstream effector of TRIM32 in balancing stem cell self-renewal versus differentiation in mammalian tissues awaits future investigation (Song, 2011).
Photoreceptor morphogenesis in Drosophila requires remodelling of apico-basal polarity and adherens junctions (AJs), and includes cell shape changes, as well as differentiation and expansion of the apical membrane. The evolutionarily conserved transmembrane protein Crumbs (Crb) organises an apical membrane-associated protein complex that controls photoreceptor morphogenesis. Expression of the small cytoplasmic domain of Crb in crb mutant photoreceptor cells (PRCs) rescues the crb mutant phenotype to the same extent as the full-length protein. This study shows that overexpression of the membrane-tethered cytoplasmic domain of Crb in otherwise wild-type photoreceptor cells has major effects on polarity and morphogenesis. Whereas early expression causes severe abnormalities in apico-basal polarity and ommatidial integrity, expression at later stages affects the shape and positioning of AJs. This result supports the importance of Crb for junctional remodelling during morphogenetic changes. The most pronounced phenotype observed upon early expression is the formation of ectopic apical membrane domains, which often develop into a complete second apical pole, including ectopic AJs. Induction of this phenotype requires members of the Par protein network. These data point to a close integration of the Crb complex and Par proteins during photoreceptor morphogenesis and underscore the role of Crb as an apical determinant (Muschalik, 2011).
Strikingly, CrbFLAGintra can only affect photoreceptor cell (PRC) shape and adhesion when expressed during late larval and early pupal development. During this period, PRCs undergo substantial morphogenetic changes to adopt their final shape. It is noteworthy that the epithelial cells of the imaginal disc are already well polarised, with an elaborated ZA encircling the apices of the cells. Therefore, the transition from a larval epithelial cell into the highly modified PRC does not require establishment of polarity, but rather mechanisms that control remodelling of polarity and AJs. This study shows that early expression of CrbFLAGintra interferes with this process. Similar conclusions were drawn from studies in the Malpighian tubules, where proper Crb levels are essential for maintenance of polarity and epithelial integrity only during the process of tube elongation, which depends on major cell rearrangements. Once most of the morphogenetic changes and remodelling of the ZA have been completed, PRCs are less susceptible to elevated CrbFLAGintra levels. This is reflected by the observation that cells in which the intracellular domain of Crb is expressed during late pupal development and in the adult, exhibit a normal polarised shape, although junctional and polarity proteins are severely mislocalised in these cells. Two explanations might account for this difference. First, the apical and basolateral membrane domain, as well as the ZA, might be more stable at later stages, so that ectopic apical and junctional components recruited by CrbFLAGintra are unable to affect apico-basal polarity and AJs. Second, some of the downstream factors required for ectopic apical pole formation might no longer be available at later stages. In fact, Baz is removed from the ZA at ~60% of pupal development and becomes enriched in the rhabdomere, similar to aPKC. Furthermore, Par-6 can be found at the basolateral membrane in adult PRCs. Although the polarised shape is unaffected, PRCs overexpressing CrbFLAGintra during later stages display defects in ZA positioning and show an increase in stalk membrane length, the development of which is regulated by crb (Muschalik, 2011).
Loss-of-function studies show that crb is not required for the development of an apical pole, yet, as shown in this study, overexpression of its cytoplasmic tail is sufficient to induce formation of ectopic apical membranes. This raises the question of how ectopic apical poles develop under these conditions. The results, from localisation studies and genetic interactions, indicate that, once initiated, development of an ectopic apical membrane domain relies on the same events and requires identical components to those required for formation of the original apical domain. It is suggested that CrbFLAGintra assembles a new Crb-dependent membrane-associated protein platform at the basolateral membrane domain, enabling the recruitment of effector proteins essential to develop apical features. One of these is βH-spec, which might stabilise the CrbFLAGintra complex by linking it to the underlying spectrin-based membrane skeleton. In fact, removal of one copy of kst strongly suppresses the overexpression phenotype and F-actin accumulates at CrbFLAGintra-positive membranes. In addition, the actin-based cytoskeleton is likely to be directly involved in the formation of ectopic rhabdomeres, as rhabdomeres are composed of microvilli and the terminal web, both of which are actin-rich structures (Muschalik, 2011).
In addition to βH-spec, Par-6 and aPKC are also recruited into the CrbFLAGintra complex and both are required to mediate the CrbFLAGintra-induced overexpression phenotype, as demonstrated by genetic interactions. Furthermore, by using different hypomorphic alleles of aPKC, the function of aPKC in this process could be shown to depend on its ability to bind Par-6 and the presence of an intact kinase domain. In the embryonic epidermis, aPKC ensures apical identity by phosphorylation of the tumour suppressor Lgl, thereby excluding it from the apical domain and restricting its activity to the basolateral side of the cells. Lgl, in contrast, prevents Baz from promoting apical membrane characteristics basolaterally. It is proposed that, upon overexpression of CrbFLAGintra, Lgl is removed from CrbFLAGintra-positive sites through phosphorylation by aPKC, which weakens basolateral membrane identity. The observation that other basolateral markers are absent from ectopic rhabdomeres and diminished at membranes surrounding ectopic rhabdomeres supports this assumption. Furthermore, removal of Lgl from the basolateral membrane upon overexpression of CrbFLAGintra would be consistent with the finding that the lgl loss-of-function phenotype of PRCs mimics the CrbFLAGintra overexpression phenotype. This is similar to the situation in Drosophila embryonic epithelia, and suggests that there is a conserved mechanism for both cell types. Moreover, it might explain why lowering the dose of lgl does not cause an enhancement of the overexpression phenotype. By contrast, an enhancement was found with yrt, which negatively regulates Crb activity, demonstrating that the experimental approach is suitable for the identification of enhancers. Besides Lgl, aPKC also phosphorylates Baz, as shown in the Drosophila follicle epithelium, the embryonic epidermis and PRCs. Phosphorylation of Baz is required to exclude it from the apical membrane, thereby restricting AJs to more basal positions. Apical exclusion of Baz also requires Crb, which prevents binding of Baz to Par-6. It is suggested that the following scenario occurs upon CrbFLAGintra overexpression. First, removal of Lgl from the basolateral membrane enables Baz to spread basolaterally. However, under these conditions, Baz becomes immediately excluded from CrbFLAGintra-positives sites by the same mechanisms occurring at the original apical domain. Delocalisation of Baz, in turn, affects AJs and alters the adhesive properties of the cells, as Baz localisation defines the position of the ZA. The model is consistent with observations from genetic interactions, which have shown that simultaneous expression of CrbFLAGintra and a non-phosphorylatable version of Baz (GFP-Baz-S980A) strongly suppressed the CrbFLAGintra overexpression phenotype. This suppression could be the result of Baz S980A either binding to aPKC-Par-6, or to Sdt, therefore preventing aPKC-Par-6 or Sdt from binding to CrbFLAGintra. Alterations in PRC adhesion might also explain the disruption of the basal lamina and the elimination of PRCs. As no obvious decrease in cell number was noticed at 45-55% of pupal development, elimination is likely to occur during late pupal development (Muschalik, 2011).
Formation of distinct membrane domains also requires polarised protein trafficking. The ectopic localisation of Rh1 and Spam (Eyes shut) upon overexpression of CrbFLAGintra during late larval and pupal development suggests that the apical secretory machinery becomes reorganised under these conditions. In Drosophila PRCs, delivery of various apical proteins, including Rh1, depends on the small GTPase Rab11 and the exocyst component Sec6. A redistribution of these proteins upon overexpression of CrbFLAGintra in developing PRCs might account for the delivery of apical transport vesicles to CrbFLAGintra-positive membranes, which facilitates the formation of a second apical pole. In case of cells with reversed apico-basal polarity the majority of apical vesicles might be targeted to the ectopic apical pole so that the original apical membrane domain receives only minor amounts of apical proteins, with it eventually adopting basolateral membrane identity (Muschalik, 2011).
Another crucial component in polarised vesicle delivery and targeting are phosphoinositides. In developing Drosophila PRCs, PtdIns(3,4,5)P3 is enriched at the apical membrane, whereas PtdIns(4,5)P2 predominantly localises at the ZA. Studies in MDCK (Madin-Darby canine kidney) cells have shown that ectopic localisation of either of the above two phosphoinositides is sufficient to cause a switch from one membrane identity to the other. Strikingly, Baz recruits the lipid phosphatase PTEN (phosphatase and tensin homolog) to the AJs of PRCs and embryonic epidermal cells, and Baz is delocalised upon CrbFLAGintra expression in pupal PRCs. Mutations in, or overexpression of, PTEN cause severe morphogenetic defects, including loss of PRCs and absence or splitting of rhabdomeres, phenotypes that are also observed upon overexpression of CrbFLAGintra. Given these data, it is tempting to speculate that ectopic CrbFLAGintra and its associated proteins cause a modification in the lipid composition of the basolateral membrane domain, thereby remodelling the polarity of PRCs (Muschalik, 2011).
Asymmetric cell division of Drosophila neural stem cells or neuroblasts is an important process which gives rise to two different daughter cells, one of which is the stem cell itself and the other, a committed or differentiated daughter cell. During neuroblast asymmetric division, atypical Protein Kinase C (aPKC) activity is tightly regulated; aberrant levels of activity could result in tumorigenesis in third instar larval brain. This study identified clueless (clu), a genetic interactor of parkin (park), as a novel regulator of aPKC activity. It preferentially binds to the aPKC/Bazooka/Partition Defective 6 complex and stabilizes aPKC levels. In clu mutants, Miranda (Mira) and Numb are mislocalized in small percentages of dividing neuroblasts. Adult mutants are short-lived with severe locomotion defects. Clu promotes tumorigenesis caused by loss of function of lethal(2) giant larvae (lgl) in the larval brain. Removal of clu in lgl mutants rescues Mira and Numb mislocalization and restores the enlarged brain size. Western blot analyses indicate that the rescue is due to the down-regulation of aPKC levels in the lgl clu double mutant. Interestingly, the phenotype of the park mutant, which causes Parkinson's Disease-like symptoms in adult flies, is reminiscent of that of clu in neuroblast asymmetric division. This study provides the first clue for the potential missing pathological link between temporally separated neurogenesis and neurodegeneration events; the minor defects during early neurogenesis could be a susceptible factor contributing to neurodegenerative diseases at later stages of life (Goh, 2013).
In this study, Clu, a protein expressed in L3 brain NBs, was found to be involved in regulating aPKC levels. In the absence of Clu, both Mira and Numb were delocalized in small percentages of mitotic NBs. Clu preferably binds to aPKC and Baz but not Lgl. In addition, this study also showed that Clu promotes a tumorigenesis phenotype in the lgl mutant. In the absence of Lgl, the function of Clu to maintain aPKC levels was sensitized. Drastic rescue was seen when clu was removed from the lgl mutant. The Western blot analyses indicated that in the lgl clu double mutant, both aPKC and p-aPKC levels were reduced, which was responsible for the rescue of lgl tumorigenesis phenotype. These data are most consistent with a model in which Clu is a member of aPKC/Baz/Par6 complex and presumably functions to maintain its stability (Goh, 2013).
If Clu is indeed involved in the maintenance of the aPKC/Baz/Par6 active complex, why does deletion of clu alone fail to generate any drastic phenotype? Deletion of clu only caused a low percentage of Mira and Numb mislocalization. The L3 brain of clu did not form tumors and the mutant could even survive all the way to adulthood, albeit it could only survive for 3-7 days. The key to that question may lie in its genetic interactor, lgl (Goh, 2013).
In the Western Blot analyses of whole larval brain lysate and knockdown cell line lysate of clu, a decrease was observed in Lgl as compared with the control. This may suggest that in clu mutant, there might also be a lower level of the inactive aPKC/Lgl/Par6 inactive complex. It is possible that the lack of Clu not only exposes the aPKC/Baz/Par6 complex to perturbations such as dephosphorylation or degradation, it also lowers Lgl levels, which in turn results in less aPKC/Lgl/Par6 complex. As a consequence, more aPKC can be released and forms a complex with Baz to replenish the decreasing pool of active complex. Thus, Lgl is able to fulfill its role as a molecular buffer in the clu mutant, shifting the equilibrium toward the more active aPKC/Baz/Par6 complex and compensating Clu function (Goh, 2013).
The primary aim of asymmetric cell division in NBs is to localize cell fate determinants such as Pros, Brat and Numb to the basal cortex so that after cytokinesis, they would only be inherited by the GMC. Numb and Mira have been found to be phosphorylated by aPKC and this phosphorylation resulted in apical exclusion of the proteins. The optimal levels of phosphorylation of Numb depend heavily on the equilibrium of two complexes, the active aPKC/Baz/Par6 complex, and the inactive aPKC/Lgl/Par6. Mira phosphorylation may depend on a similar aPKC complex. Studies so far had pointed to Lgl as the major buffering molecule to maintain this equilibrium, together with other phosphatases like PP4 and PP2A modulating the equilibrium or activity of the two complexes. There should be, however, additional molecules that help maintain this equilibrium in the Drosophila NBs (Goh, 2013).
Clu exhibited preferential binding capacity to the aPKC/Baz/Par6 complex rather than aPKC/Lgl/Par6, suggesting its participation in the maintenance or regulation of the equilibrium between the two complexes. Under extreme conditions, such as in the lgl mutant, lack of Lgl not only shifts the equilibrium entirely to the aPKC/Baz/Par6 complex, resulting in hyper-phosphorylation and subsequent delocalization of Numb and Mira, but also sensitizes Clu function on stabilizing aPKC/Baz/Par6. When clu is further deleted in the lgl mutant, the aPKC/Baz/Par6 complex could be destabilized, hence resulting in a significant decrease in the levels of both aPKC and p-aPKC, which in turn rescues Numb and Mira localization phenotypes in dividing NBs and restores L3 brain sizes. Western Blot analyses of whole larval brain lysate and knockdown BG3C2 cell lines supported this hypothesis (Goh, 2013).
Both clu and park have been shown previously to cause mitochondrial defects in adults. An initial suspicion was that ATP levels in clu NBs might contribute to the defects of neuroblast asymmetric cell division, as well as the rescue phenotype in the lgl mutant. A recent paper indicated that localization of mitochondria did not affect levels of ATP or cause any oxidative damage in the 3L brain, although they observed abnormal clustering in the clu mutant (Sen, 2013). Measurements of ATP levels in clu, lgl and lgl clu mutant brains also did not indicate any differences among them or the wild type brains. Thus, it is concluded that the clu phenotype observed in this study is very likely to be independent of mitochondrial activity (Goh, 2013).
Parkinson's disease is a neurodegenerative disease of the central nervous system which occurs in aged individuals. In patients with Parkinson's disease, there is massive loss of dopaminergic neurons which results in impairment in movement. The Drosophila ortholog of park encodes an E3 ligase, and they were found to have defective flight muscles and loss of dopaminergic neurons in park null mutants (Goh, 2013).
This has directly linked genes involved in neurodegenerative disease, clu and park, with lgl, a gene that is involved in NB asymmetric division during early development. Since these three genes are expressed in the same cells and are involved in the same process during neurogenesis, it is speculated that early defects in neurogenesis, although weak, may become a susceptible factor contributing to neurodegenerative diseases such as Parkinson disease at a much late stage of life. More in-depth studies on the links among genes involved in two developmentally separated events, asymmetric division during early neurogenesis and neurodegeneration due to aging, need to be done before the final conclusion can be reached (Goh, 2013).
Loss of the cell polarity gene could cooperate with oncogenic Ras to drive tumor growth and invasion, which critically depends on the c-Jun N-terminal Kinase (JNK) signaling pathway in Drosophila. By performing a genetic screen, this study identified Src42A, the ortholog of mammalian Src, as a key modulator of both RasV12/lgl -/-triggered tumor invasion and loss of cell polarity gene-induced cell migration. The genetic study further demonstrated that the Bendless (Ben)/dUev1a ubiquitin E2 complex is an essential regulator of Src42A-induced, JNK-mediated cell migration. Furthermore, this study showed that ectopic Ben/dUev1a expression induced invasive cell migration along with increased MMP1 production in wing disc epithelia. Moreover, Ben/dUev1a could cooperate with RasV12 to promote tumor overgrowth and invasion. In addition, it was found that the Ben/dUev1a complex is required for ectopic Src42A-triggered cell death and endogenous Src42A-dependent thorax closure. These data not only provide a mechanistic insight into the role of Src in development and disease but also propose a potential oncogenic function for Ubc13 and Uev1a, the mammalian homologs of Ben and dUev1a (Ma, 2013).
Neural development requires N-glycosylation regulation of intercellular signaling, but the requirements in synaptogenesis have not been well tested. All complex and hybrid N-glycosylation requires MGAT1 (UDP-GlcNAc:alpha-3-D-mannoside-beta1,2-N-acetylglucosaminyl-transferase I) function, and Mgat1 nulls are the most compromised N-glycosylation condition that survive long enough to permit synaptogenesis studies. At the Drosophila neuromuscular junction (NMJ), Mgat1 mutants display selective loss of lectin-defined carbohydrates in the extracellular synaptomatrix, and an accompanying accumulation of the secreted endogenous Mind the gap (MTG) lectin, a key synaptogenesis regulator. Null Mgat1 mutants exhibit strongly overelaborated synaptic structural development, consistent with inhibitory roles for complex/hybrid N-glycans in morphological synaptogenesis, and strengthened functional synapse differentiation, consistent with synaptogenic MTG functions. Synapse molecular composition is surprisingly selectively altered, with decreases in presynaptic active zone Bruchpilot (BRP) and postsynaptic Glutamate receptor subtype B (GLURIIB), but no detectable change in a wide range of other synaptic components. Synaptogenesis is driven by bidirectional trans-synaptic signals that traverse the glycan-rich synaptomatrix, and Mgat1 mutation disrupts both anterograde and retrograde signals, consistent with MTG regulation of trans-synaptic signaling. Downstream of intercellular signaling, pre- and postsynaptic scaffolds are recruited to drive synaptogenesis, and Mgat1 mutants exhibit loss of both classic Discs large 1 (DLG1) and newly defined Lethal (2) giant larvae [L(2)gl] scaffolds. It is concluded that MGAT1-dependent N-glycosylation shapes the synaptomatrix carbohydrate environment and endogenous lectin localization within this domain, to modulate retention of trans-synaptic signaling ligands driving synaptic scaffold recruitment during synaptogenesis (Parkinson, 2013).
This study began with the hypothesis that disruption of synaptomatrix N-glycosylation would alter trans-synaptic signaling underlying NMJ synaptogenesis (Dani, 2012). MGAT1 loss transforms the synaptomatrix glycan environment. Complete absence of the HRP epitope, α1-3-fucosylated N-glycans, is expected to require MGAT1 activity: key HRP epitope synaptic proteins include fasciclins, Neurotactin and Neuroglian, among others. This study shows that HRP epitope modification of the key synaptogenic regulator Fasciclin 2 is not required for stabilization or localization, suggesting a role in protein function. However, complete loss of Vicia villosa (VVA) lectin reactivity synaptomatrix labeling is surprising because the epitope is a terminal β-GalNAc. This result suggests that the N-glycan LacdiNAc is enriched at the NMJ, and that the terminal GalNAc expected on O-glycans/glycosphingolipids may be present on N-glycans in this synaptic context. Importantly, VVA labels Dystroglycan and loss of Dystroglycan glycosylation blocks extracellular ligand binding and complex formation in Drosophila, and causes muscular dystrophies in humans. This study shows that VVA-recognized Dystroglycan glycosylation is not required for protein stabilization or synaptic localization, but did not test functionality or complex formation, which probably requires MGAT1-dependent modification. Conversely, the secreted endogenous lectin MTG is highly elevated in Mgat1 null synaptomatrix, probably owing to attempted compensation for complex and hybrid N-glycan losses that serve as MTG binding sites. MTG binds GlcNAc in a calcium-dependent manner and pulls down a number of HRP-epitope proteins by immunoprecipitation (Rushton, 2012), although the specific proteins have not been identified. It will be of interest to perform immunoprecipitation on Mgat1 samples to identify changes in HRP bands. Importantly, MTG is crucial for synaptomatrix glycan patterning and functional synaptic development. MTG regulates VVA synaptomatrix labeling, suggesting a mechanistic link between the VVA and MTG changes in Mgat1 mutants. The MTG elevation observed in Mgat1 nulls provides a plausible causative mechanism for strengthened functional differentiation (Parkinson, 2013).
Consistent with recent glycosylation gene screen findings (Dani, 2012), Mgat1 nulls exhibit increased synaptic growth and structural overelaboration. Therefore, complex and hybrid N-glycans overall provide a brake on synaptic morphogenesis, although individual N-glycans may provide positive regulation. Likely players include MGAT1-dependent HRP-epitope proteins (e.g., fasciclins, Neurotactin, Neuroglian), and position-specific (PS) integrin receptors and their ligands, all of which are heavily glycosylated and have well-characterized roles regulating synaptic architecture. An alternative hypothesis is that Mgat1 phenotypes may result from the presence of high-mannose glycans on sites normally carrying complex/hybrid structures, suggesting possible gain of function rather than loss of function of specific N-glycan classes. NMJ branch and bouton number play roles in determining functional strength, although active zones and GluRs are also regulated independently. Thus, the increased functional strength could be caused by increased structure at Mgat1 null NMJs. However, muscle-targeted UAS-Mgat1 rescues otherwise Mgat1 null function, but has no effect on structural defects, demonstrating that these two roles are separable. Presynaptic Mgat1 RNAi also causes strong functional defects, showing there is additionally a presynaptic requirement in functional differentiation. Neuron-targeted Mgat1 causes lethality, indicating that MGAT1 levels must be tightly regulated, but preventing independent assessment of Mgat1 presynaptic rescue of synaptogenesis defects (Parkinson, 2013).
Presynaptic glutamate release and postsynaptic glutamate receptor responses drive synapse function. Using lipophilic dye to visualize SV cycling, this study found Mgat1 null mutants endogenously cycle less than controls, but have greater cycling capacity upon depolarizing stimulation. The endogenous cycling defect is consistent with the sluggish locomotion of Mgat1 mutants, whereas the elevated stimulation-evoked cycling is consistent with electrophysiological measures of neurotransmission. Similarly, mutation of dPOMT1, which glycosylates VVA-labeled Dystroglycan, decreases SV release probability (Wairkar, 2008), although dPOMT1 adds mannose not GalNAc. Null Mgat1 mutants display no change in SV cycle components (e.g. Synaptobrevin, Synaptotagmin, Synaptogyrin, etc.), but exhibit reduced expression of the key active zone component Bruchpilot. Other examples of presynaptic glycosylation requirements include the Drosophila Fuseless (FUSL) glycan transporter, which is critical for Cacophony (CAC) voltage-gated calcium channel recruitment to active zones, and the mammalian GalNAc transferase (GALGT2), whose overexpression causes decreased active zone assembly. Postsynaptically, Mgat1 nulls show specific loss of GLURIIB-containing receptors. Similarly, dPOMT1 mutants exhibit specific GLURIIB loss (Wairkar, 2008), although dystroglycan nulls display GLURIIA loss. Selective GLURIIB loss in Mgat1 nulls may drive increased neurotransmission owing to channel kinetics differences in GLURIIA versus GLURIIB receptors (Parkinson, 2013).
Bidirectional trans-synaptic signaling regulates NMJ structure, function and pre/postsynaptic composition. This intercellular signaling requires ligand passage through, and containment within, the heavily glycosylated synaptomatrix, which is strongly compromised in Mgat1 mutants. In testing three well-characterized signaling pathways, this study found that Wingless (Wg) accumulates, whereas both GBB and JEB are reduced in the Mgat1 null synaptomatrix. WG has two N-glycosylation sites, but these do not regulate ligand expression, suggesting WG build-up occurs owing to lost synaptomatrix N-glycosylation. Importantly, WG overexpression increases NMJ bouton formation similarly to the phenotype of Mgat1 nulls, suggesting a possible causal mechanism. GBB is predicted to be N-glycosylated at four sites, but putative glycosylation roles have not yet been tested. Importantly, GBB loss impairs presynaptic active zone development similarly to Mgat1 nulls, suggesting a separable causal mechanism. JEB is not predicted to be N-glycosylated, indicating that JEB loss is caused by lost synaptomatrix N-glycosylation. Importantly, it has been shown that loss of JEB signaling increases functional synaptic differentiation similarly to Mgat1 nulls (Rohrbough, 2013). In addition, jeb mutants exhibit strongly suppressed NMJ endogenous activity, similarly to the reduced endogenous SV cycling in Mgat1 nulls. Moreover, the MTG lectin negatively regulates JEB accumulation in NMJ synaptomatrix, consistent with elevated MTG causing JEB downregulation in Mgat1 nulls (Parkinson, 2013).
Trans-synaptic signaling drives recruitment of scaffolds that, in turn, recruit pre- and postsynaptic molecular components. Specifically, DLG1 and L(2)GL scaffolds regulate the distribution and density of both active zone components (e.g. BRP) and postsynaptic GluRs, and both of these scaffolds are reduced at Mgat1 null NMJs. Importantly, dlg1 mutants display selective loss of GLURIIB, with GLURIIA unchanged, similar to Mgat1 nulls, suggesting a causal mechanism. Moreover, l(2)gl mutants display both a selective GLURIIB impairment as well as reduction of BRP aggregation in active zones, similarly to Mgat1 nulls, suggesting a separable involvement for this synaptic scaffold. DLG1 and L(2)GL are known to interact in other developmental contexts, indicating a likely interaction at the developing synapse. Although synaptic ultrastructure has not been examined in l(2)gl mutants, dlg1 mutants exhibit impaired NMJ development, including a deformed SSR. These synaptogenesis requirements predict similar ultrastructural defects in Mgat1 mutants, albeit presumably due to the combined loss of both DLG1 and L(2)GL scaffolds. Future work will focus on electron microscopy analyses to probe N-glycosylation mechanisms of synaptic development (Parkinson, 2013).
The Drosophila melanogaster junctional neoplastic tumor suppressor, Lethal-2-giant larvae (Lgl), is a regulator of apicobasal cell polarity and tissue growth. Previous studies have shown in the developing Drosophila eye epithelium that, without affecting cell polarity, depletion of Lgl results in ectopic cell proliferation and blockage of developmental cell death due to deregulation of the Hippo signaling pathway. This study shows that Notch signaling is increased in lgl-depleted eye tissue, independently of Lgl's function in apicobasal cell polarity. The upregulation of Notch signaling is ligand dependent and correlates with accumulation of cleaved Notch. Concomitant with higher cleaved Notch levels in lgl- tissue, early endosomes (Avalanche [Avl+]), recycling endosomes (Rab11+), early multivesicular bodies (Hrs+), and acidified vesicles, but not late endosomal markers (Car+ and Rab7+), accumulate. Colocalization studies revealed that Lgl associates with early to late endosomes and lysosomes. Upregulation of Notch signaling in lgl- tissue requires dynamin- and Rab5-mediated endocytosis and vesicle acidification but is independent of Hrs/Stam or Rab11 activity. Furthermore, Lgl regulates Notch signaling independently of the aPKC-Par6-Baz apical polarity complex. Altogether, these data show that Lgl regulates endocytosis to restrict vesicle acidification and prevent ectopic ligand-dependent Notch signaling. This Lgl function is independent of the aPKC-Par6-Baz polarity complex and uncovers a novel attenuation mechanism of ligand-activated Notch signaling during Drosophila eye development (Parsons, 2014).
This study demonstrates a novel function for the cell polarity regulator lgl in regulation of endocytosis and Notch signaling. In the developing Drosophila eye epithelium that (1) Notch targets, E(spl)m8, CycA, and Rst, are upregulated in lgl- tissue; (2) Notch upregulation contributes to the lgl- mosaic adult eye phenotype; (3) Notch upregulation in lgl- clones is ligand dependent and requires endocytosis; (4) Lgl colocalizes with intracellular Notch and endocytic markers; (5) lgl- tissue accumulates Avl+ EEs, Hrs+MVBs, Rab11+ REs, endocytic compartments, and acidified vesicles, but not LE markers, Rab7, and Car; (6) Notch upregulation in lgl- clones is independent of the ESCRT-0 complex (Hrs/Stam) or Rab11, but it requires Rab5 function and acidification of endosomes; and (7) Notch upregulation in lgl- clones is independent of aPKC-Baz-Par6. Altogether, these data reveal a novel role for Lgl in attenuating ligand-activated Notch signaling via restricting the acidification of endocytic compartments, independently of its role in regulating the aPKC-Baz-Par6 cell polarity complex (Parsons, 2014).
The data have revealed that increased vesicle acidification in lgl- tissue is responsible for elevated Notch signaling. Because S3 cleavage (by γ-secretase) of Notch extracellular domain (Next) to form the intracellular domain (Nicd) depends on acidification of endosome, this suggests that increased vesicle acidification in lgl- tissue leads to aberrant γ-secretase activity and cleavage of Next to Nicd, resulting in upregulation of Notch signaling (Parsons, 2014).
The precise endocytic compartment in which the Notch receptor undergoes γ-secretase-mediated S3 proteolytic processing is controversial. In Drosophila epithelial tissues, V-ATPase function is implicated in Notch activation in the EEs or the MVBs; however, whether this is ligand dependent or independent is unclear. In contrast, ligand-independent generation of Nicd in the LE and/or lysosome compartment has been described. Because increased Notch signaling in lgl- tissue depends on Rab5 EE activity, but Rab7 and Car LE compartments were not perturbed in lgl- tissue, a model is favored in which Lgl restricts vesicle acidification and activation of Notch signaling in the EE and/or early MVB compartments (Parsons, 2014).
It is speculated that Lgl regulates Notch signaling by two possible mechanisms: (1) via direct regulation of vesicle acidification or (2) via regulation of endosomal maturation, which indirectly affects vesicle acidification. In the first model, Lgl might inhibit V-ATPase activity by regulating levels and/or subunit composition or the association and/or dissociation of the V-ATPase complex. In the second model, Lgl might regulate endosomal maturation, and alterations in this process subsequently lead to accumulation of acidic vesicles and ectopic Notch activation. The data showing that lgl- tissue accumulates EEs (Avl+), REs (Rab11+), and early MVBs (Hrs+), but not LEs (Rab7+ or Car+), suggest that Lgl regulates a specific step in endosome maturation after early MVB formation. Further studies are required to determine whether Lgl controls vesicle acidification by affecting the V-ATPase or endosome maturation to regulate Notch signaling (Parsons, 2014).
Previous work has revealed that alterations in components of endocytic compartments, such as Rab5 overexpression or mutation of tsg101/ept or vps25, disrupt epithelial cell polarity and upregulate Notch signaling. However, in these cases, it is unclear whether perturbation of endocytosis alters Notch signaling via cell polarity disruption or whether changes in endocytic compartments directly impact on Notch. This study shows that without cell polarity loss, lgl- tissue displays altered endocytic compartments and upregulates Notch signaling, indicating that changes in endocytosis alone are sufficient to upregulate Notch pathway activity. Moreover, this study shows that reducing aPKC-Baz-Par6 complex activity does not rescue Notch pathway upregulation in lgl- tissue, revealing that Lgl's roles in regulating cell polarity and endocytosis are separable. Interestingly, the Crb polarity protein also has separable roles in the regulation of cell polarity and Notch signaling and endocytosis, via different mechanisms to Lgl (Parsons, 2014).
The discovery that Lgl depletion increases Notch activation without cell polarity loss has implications for tumorigenesis. In the developing eye epithelium, increased Notch signaling results in upregulation of the cell cycle regulator, CycA, and the cell survival regulator, Rst, which in lgl- tissue, is expected to contribute to increased cell proliferation and survival, concomitant with impaired Hippo signaling. Because elevated Notch signaling is associated with various human cancers, the finding that Lgl regulates Notch signaling warrants investigation of whether elevated Notch signaling in human cancer is associated with Lgl depletion. Notably, Lgl1 knockout in the mouse brain induces hyperproliferation and decreased differentiation, associated with increased Notch signaling, and mutation of zebrafish Lgl disrupts retinal neurogenesis, dependent on increased Notch signaling. Moreover, the finding that Lgl plays a novel role in regulating endosomal acidification and the striking suppressive effect of chloroquine on the adult lgl- mosaic phenotype reveal the importance of acidification in tumor growth, perhaps by also modulating Hippo signaling. The data, together with evidence that many cancers show higher acidity due to increased V-ATPase activity, which contributes to tumorigenesis, posit the question of whether Lgl dysfunction might contribute to acidification defects in human cancer (Parsons, 2014).
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date revised: 21 December 2021
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