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Cooperative Regulation of Cell Polarity and Growth by Drosophila Tumor Suppressors

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Science  07 Jul 2000:
Vol. 289, Issue 5476, pp. 113-116
DOI: 10.1126/science.289.5476.113

Abstract

Loss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. We provide evidence from Drosophila that a group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation. Scribble (Scrib) is a cell junction–localized protein required for polarization of embryonic and, as demonstrated here, imaginal disc and follicular epithelia. We show that the tumor suppressors lethal giant larvae(lgl) and discs-large (dlg) have identical effects on all three epithelia, and that scribalso acts as a tumor suppressor. 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. Our data indicate that the three tumor suppressors act together in a common pathway to regulate cell polarity and growth control.

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. We have used Drosophila genetics to study the morphogenesis of epithelia, in order to illuminate the relation between epithelial organization and oncogenesis.

The multi-PDZ (PSD-95, Dlg, ZO-1) domain protein Scrib is required for proper polarity and morphogenesis of embryonic epithelia (1). To test whether the requirement for Scrib is limited to the embryo, we examined the role of scrib in follicle cells, a monolayered epithelium of somatic cells that encases the germ line in the adult female ovary. Scrib was localized to lateral follicle cell membranes (Fig. 1A), and clones of cells (2) that lack scrib function became round and multilayered (Fig. 1C) 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.

Figure 1

Scrib is required for follicular epithelium morphogenesis. (A) Ovary costained for Scrib (green) and cortical actin (red) reveals expression in the lateral membranes of the follicle cell epithelium from stages 2b to 6. Scrib is also found in region 1 of the germarium and at low levels on germ cell membranes. (B) Wild-type ovary expressing green fluorescent protein (GFP) stained with rhodamine phalloidin displays the regular monolayered cuboidal epithelium. (C) Follicle cells homozygous for scrib (lacking GFP) (2) are rounded and often multilayered. Groups of cells carrying a WTscrib allele (arrow) maintain the WT epithelial monolayer.

In a screen to identify additional mutations causing epithelial defects in follicle cell clones (FCCs), we isolated a mutation (4w3) that produces a phenotype similar to that of scrib. Mapping and complementation analysis demonstrated that 4w3 is a strong allele of lgl, which encodes a cytoplasmic, myosin-associated protein with WD-40 repeats (3,4). FCCs of the null allele lgl4 showed loss of cell shape and monolayer organization as seen inlgl4w3 FCCs (Fig. 2B). Furthermore, this phenotype resembles that seen in FCCs for strong alleles of dlg (Fig. 2C) (5), which encodes a multi-PDZ domain protein (6). The phenotypes of scrib,lgl, and dlg FCCs are indistinguishable.

Figure 2

lgl and dlg phenocopyscrib in follicular and embryonic epithelia. lgland dlg mutations cause defects in follicle cell morphology (A to C), embryonic adherens junction formation at stage 10 (D to F), polarization of apical proteins at stage 11 (G to I), and cuticle deposition (J to L), phenotypes also exhibited by scrib mutations. Stains: (A to C) GFP, green; and phalloidin, red; (D to F) anti-Arm seen en face; (G to I) anti-Crb seen in cross section with apical surface oriented upward.

Because follicle cell epithelia require scrib,lgl, and dlg, we examined the function oflgl and dlg in the embryonic epidermis, wherescrib acts to restrict apical proteins and adherens junctions to their appropriate positions within the cell membrane (1). We stained embryos lacking both maternal and zygotic contributions of lgl and dlg[(7), hereafter referred to as lgl anddlg embryos] with antibodies to polarized proteins and cellular junction components (8). During mid-embryogenesis,lgl and dlg embryos showed defects in apicobasal polarity, revealed by aberrant distribution of the apical protein Crumbs (Crb) (9) (Fig. 2, H and I) and disruption of adherens junctions (Fig. 2, E and F). These defects are similar to those of scrib embryos; the terminal phenotypes ofscrib, lgl, and dlg embryos, as indicated by cuticle deposition, are also nearly identical (Fig. 2, K and L) (6, 10). Thus, lgl anddlg, like scrib, act to properly localize apical proteins and adherens junctions to organize epithelial architecture in embryos.

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, we compared the scribphenotype 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 (11) were profoundly disorganized and also massively overgrown. scribdiscs (Fig. 3, C and D) contained 4.7 times as many cells as wild-type (WT) discs (4.17 × 105 versus 8.82 × 104) (11) and consisted 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 (Fig. 3E) was absent inscrib discs (Fig. 3F). This loss of epithelial organization accompanied by overproliferation corresponds to the phenotype described for lgl and dlg zygotic mutant discs (12,13). Additional features of lgl and dlglarval phenotypes, such as overgrowth of brain tissue, were also present in scrib larvae (Fig. 3H). Together, these data indicate that scrib and the two previously characterizedDrosophila malignant neoplastic tumor suppressors,lgl and dlg (12), share a role in growth control as well as epithelial polarity.

Figure 3

scrib is an imaginal disc tumor suppressor. Confocal microscope sections of WT (A andB) and scrib (C and D) wing discs stained for phalloidin (green) and propidium iodide (red) to reveal cell shape and DNA, respectively. The well-organized, folded monolayer epithelium in WT is apparent; the much larger scrib disc shows no signs of organization. High-magnification images of WT (E) and scrib (F) discs stained as above demonstrate that the regular shape and apical polarization of actin seen in WT cells are totally absent in scrib (a↔b, apicobasal polarity). Wild-type (G) and scrib(H) larval brains stained with phalloidin. Thescrib brain lobes (outlined in dots) are much larger than in WT, and the axon-rich medulla (m) is disorganized.

The equivalent requirements for scrib, lgl, anddlg in epithelial development could result from independent activity of each gene in a separate pathway or from collaborative activity of the three genes in a single pathway. To address this issue, we tested for genetic interactions between the three mutations and found strong interactions of dlg and lgl withscrib. Most embryos zygotically mutant for scribhatch and survive into late larval stages (see above). However, embryos homozygous for scrib and additionally heterozygous fordlg died before hatching, with evident defects in dorsal closure (Fig. 4A). Dorsal closure phenotypes are characteristic of reduced activity of both dlg(10) and lgl (14). Additionally, embryos homozygous for both lgl andscrib displayed a cuticle phenotype nearly as severe as those of lgl or scrib null embryos (Fig. 4B). Heterozygosity for lgl also enhanced the imaginal disc phenotype of scrib hypomorphic larvae. These dose-sensitive interactions of dlg and lgl with scribsuggest that the three genes function in a common pathway.

Figure 4

dlg and lglgenetically interact with scrib. Zygoticscrib/scrib embryos hatch into larvae, but dlg/+; scrib/scrib embryos die with defects in dorsal closure (A, arrow). Zygotic lgl/lgl; scrib/scribembryos (B) die exhibiting phenotypes similar to that of complete absence of maternal and zygotic lgl orscrib gene product.

We further explored the relation between scrib,lgl, and dlg by comparing the subcellular localization of the gene products (8). Scrib and Dlg colocalized throughout development, in particular at the apical margin of the lateral membrane (ALM) of the embryonic epidermal epithelium (Fig. 5A). Colocalization at the ALM occurred after gastrulation and persisted in mature epithelia, where the ALM is the site of the septate junction (15). Lgl protein was not exclusively associated with the plasma membrane and was not polarized along it (4); however, it overlapped substantially with Dlg and Scrib at the ALM (Fig. 5B).

Figure 5

Scrib, Dlg, and Lgl are mutually dependent for proper subcellular localization. (A) Stage 16 WT embryonic epidermis costained for Dlg (green, left) and Scrib (red, center) reveals that the two proteins colocalize at the ALM (yellow, right). (B) Stage 16 WT embryo costained for Dlg (green, left) and Lgl (red, center). Lgl is found predominantly at the plasma membrane, including the ALM (yellow, right). In WT stage 11 embryos (C), Scrib is enriched at the ALM. However, inlgl embryos (D), Scrib is mislocalized throughout the basolateral membrane. scrib embryos show a similar basolateral misdistribution of Dlg (E), whereasdlg embryos (F) both mislocalize Scrib and display reduced levels of Scrib at the membrane. By stage 16 little Scrib is visible in dlg embryos (G); in contrast, stage 16 lgl embryos (H) retain ∼WT levels of misdistributed Dlg. En face sections of WT blastoderm (I) and stage 11 epidermis (J) demonstrate the localization of a significant proportion of Lgl (green) at cell membranes (marked with anti-Neurotactin in red). Similar sections of scrib embryos (K, blastoderm; L, stage 11) reveal that Lgl is almost exclusively cytoplasmic. In dlg embryos, Lgl is localized near to membranes at blastoderm (M) but becomes cytoplasmic by stage 11 (N).

We investigated epistatic relations betweenscrib, lgl, and dlg by determining the localization of each protein in embryos mutant for the other two genes. We first assayed the localization of Dlg and Scrib to the ALM. In all mutant blastoderms, Scrib and Dlg were associated with ingrowing cell membranes, as in WT. However, after gastrulation, when WT embryos display an enrichment of Scrib in the ALM (Fig. 5C), lglembryos showed Scrib (Fig. 5D) and Dlg (Fig. 5H) localized throughout the basolateral cell membrane, a misdistribution that persists into late embryogenesis. Like lgl embryos, scribembryos failed to polarize Dlg to the ALM (Fig. 5E), whiledlg embryos not only failed to polarize Scrib to the ALM (Fig. 5F), but also displayed a progressive loss of membrane-associated Scrib (Fig. 5G).

We next examined the distribution of Lgl, which normally has both a membrane-bound and a cytosolic component (4); reduction of activity in an lgl temperature-sensitive mutant correlates with loss of the membrane-bound pool (14). In WT embryos, Lgl was in close apposition to cell membranes (Fig. 5, I and J). However, in scribblastoderms and embryos, Lgl was distributed throughout the cytoplasm (Fig. 5, K and L). dlg blastoderms showed intermediate defects in Lgl distribution (Fig. 5M), but by mid-embryogenesis loss of membrane-localized Lgl was evident (Fig. 5N). 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 that scrib is required for the cortical association of Lgl; all three genes act to localize Scrib and Dlg to the ALM.

Our 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 (16), only scrib shares with dlg and lgl the concomitant loss of tissue organization that groups the three together as malignant neoplastic tumor suppressors (12). Previous analyses have described a role for dlg and lgl in imaginal disc polarity (17, 18); the demonstration in this work of genetic interactions withscrib 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 (19). Our 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.

The correlation between loss of membrane-associated Lgl inscrib 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 (20,21). 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 (22). In vertebrate epithelia, exocyst components are found at the tight junction (23), a structure analogous to the septate junction where Dlg and Scrib localize (15). InDrosophila 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.

In many epithelial-derived cancers, cytoarchitectural changes are hallmarks of oncogenic transformation. The disruption of epithelial architecture seen in scrib, dlg, andlgl 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 (24); indeed, a human Dlg homolog has been shown to bind APC and associate with β-catenin, the human homolog of Arm (29). 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 (25–28) is warranted. Further analysis of the mechanisms by which Scrib, Dlg, and Lgl keep Drosophilacell growth in check will likely enhance our understanding of mammalian oncogenesis as well.

  • * To whom correspondence should be addressed. E-mail: bilder{at}rascal.med.harvard.edu

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