BIOLOGICAL OVERVIEW

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).

To place Scrib within the known pathway for Drosophila epithelial polarity determination, the effect of scrib mutations on Crumbs (Crb) was examined. Crb is an apically localized transmembrane protein that is necessary and sufficient to confer apical character on plasma membrane. In scrib embryos, Crb shows unrestricted localization in both apical and basolateral regions. Whether scrib mutants are identical to a gain-of-function crb phenotype was examined by comparing them with embryos in which GAL4-driven Crb is present throughout the cell membrane. Ectopic Crb that is produced in this manner is sufficient to phenocopy several aspects of scrib embryos, including mislocalization of apical proteins and the cuticle pattern. These data indicate that a major function of Scrib in epithelial polarity is to exclude Crb from the basolateral domain. Since ectopic Crb does not cause the epithelial morphology and multilayering defects seen in scrib embryos, Scrib may be required for the localization of additional epithelial determinants as well (Bilder, 2000a).

Analysis of the morphological and polarization phenotypes exhibited by scrib embryos shows that Scrib is a critical component of epithelial architecture in the Drosophila ectoderm, and suggests that its function is closely linked to that of Crb. Scrib is not required for the early localization of basal Discs lost (now redefined as Drosophila Patj) or apical Armadillo during blastoderm formation, and scrib mutants do not exhibit the defective cellularization or precipitous loss of cell adhesion seen when Discs lost or Armadillo, respectively, are depleted in the embryo. The increasingly severe cell shape, polarity and epithelial organization defects of scrib embryos are first manifested after gastrulation, coincident with the onset of defects in crb and stardust embryos. Loss of Crb results in loss of apical proteins from the plasma membrane and a failure to consolidate early adherens junction material into an apical band of zonula adherins (ZAs), while in scrib embryos early adherens junctions become misdistributed basolaterally. Together with the similarities between scrib loss-of-function and crb gain-of-function phenotypes, these data place Scrib and Crb in a pathway required for the progression from the initially differentiated blastoderm membrane domains into a fully polarized epithelium with mature junctions (Bilder, 2000a).

These results show that the junctional protein Scrib specifically restricts apical membrane determinants to the apical cell surface. This restriction allows the proper segregation of apical and basolateral domain components, and the appropriate placement of the adherens junction, resulting in full epithelial polarization. How does Scrib, a putative scaffolding protein whose localizaton bounds the apical domain, dictate the proper confinement of apical proteins? Two models suggest themselves. Scrib could assemble a diffusion barrier that physically separates apical and basolateral compartments, similar to the 'fence' function proposed for the vertebrate tight junction. To date, such a barrier has been shown to exist only for lipid diffusion in the outer leaflet of the plasma membrane. If scrib mutations disrupt such a mechanical barrier, then secondary retention systems must serve to maintain basolateral protein restriction from the apical cell surface. An alternative is that Scrib has a role in the polarized targeting of transport vesicles carrying apical proteins. The junctional complex, and in particular the tight junction, has been proposed to be a key sorting site for a subset of Golgi-derived vesicles. In this model, Scrib might interact with the 'exocyst', a secretory targeting apparatus localized to the tight junction and involved in polarized segregation of transmembrane proteins. PDZ domain proteins have been implicated at several different sites of the protein trafficking pathway, and occasional punctate intracellular staining of Scrib is reminiscent of vesicles. Distinction between these models will rely on the identification of binding partners for Scrib (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).

To test whether the requirement for Scrib is limited to the embryo, the role of scrib in follicle cells, a monolayered epithelium of somatic cells that encases the germ line in the adult female ovary, was examined. 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 that groups the three together as malignant neoplastic tumor suppressors. Previous analyses have described a role for dlg and lgl in imaginal disc polarity; the demonstration in this work of genetic interactions with scrib and codependence for protein localization indicates a functional link between the three tumor suppressors. Furthermore, involvement of the tumor suppressors in embryonic epithelial polarity provides a well-studied context in which to understand their activities. These findings suggest that, in the WT gastrula, intrinsic, perhaps adhesion-based cues localize Dlg at the ALM; Dlg stabilizes Scrib at this position, and finally Scrib acts on the cortical cytoskeleton to bring Lgl to the membrane. The three proteins may then collaborate to maintain the proper distribution of polarized factors, including themselves (Bilder, 2000b).

The correlation between loss of membrane-associated Lgl in scrib and dlg mutants and defective cell polarity suggests models of action for this group of proteins. Whereas the PDZ domains of Scrib and Dlg are likely to bind to transmembrane proteins that organize the epithelial cell surface, the role of Lgl in polarity determination may derive from its function in targeted secretion of membrane proteins. Lgl homologs from humans and yeast can bind to plasma membrane t-SNARE proteins and promote the fusion of cargo-carrying vesicles with target membranes (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).

PROTEIN STRUCTURE

Amino Acids - 1731

Structural Domains

Sequence analysis of Scribbled reveals the presence of two known protein-protein interaction motifs. At the amino terminus of Scrib is a set of 16 leucine-rich repeats (LRRs). This region is very similar to the Ras-binding LRRs of the proteins Sur-8 from Caenorhabditis elegans and adenylate cyclase from yeast. Distributed throughout the remainder of the protein are four PDZ domains. The PDZ domains of Scrib are highly similar to those of PDZ3 from PSD-95, a human homolog of fly Discs-large. Comparison of Scrib residues with those of PSD-95 indicates that all four Scrib PDZ domains are Type 1A; these domains predicted to bind to the consensus S/TXV at the carboxy terminus of proteins. Scrib has at least one human homolog, encoded by the KIAA0147 cDNA, which is called here hscrib1. No obvious signal sequences or transmembrane domains are predicted in either Scrib or hScrib1 by standard algorithms, suggesting that Scrib is a cytosolic protein. Densin, a protein with 16 LRRs and one PDZ domain, has been suggested to have transmembrane topology. The possibility that Scrib is an atypical transmembrane protein cannot be excluded (Bilder, 2000a).

date revised: 20 August 2000

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