BIOLOGICAL OVERVIEW

stardust (sdt) encodes multiple PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. The polarized architecture of these epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical domain comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, whose cytoplasmic domain recruits the PDZ-protein Discs Lost (now redefined as Drosophila Patj) into the complex. stardust provides an essential component of the SAC. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Thus, Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of the Crumbs cytoplasmic tail. Nevertheless, Stardust and Crumbs expression patterns in sensory organs differ. The polarity of neuroblasts requires the function of Bazooka, but not of Stardust or Crumbs. In conclusion, the Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity (Bachmann, 2001; Hong, 2001).

The sdt mutant phenotype is characterized by the loss of epithelial integrity and cell shape. sdt mutant embryos fail to concentrate the scattered spot adherens junctions into a continuous ZA. As a consequence, cells lose contact and the epithelium becomes multilayered. The assembly of the ZA requires a functional SAC. Formation of the SAC depends on an intact cytoplasmic tail of Crumbs, which recruits the PDZ protein Discs Lost (Dlt) into the complex. In sdt mutant embryos, the SAC components Crumbs (Crb) and Dlt fail to localize apically from stage 8 onwards. Because Dlt is correctly associated with the ingrowing plasma membrane during cellularization in sdt embryos, it is suggested that sdt is, similarly to crb, essential for maintenance, but not for initiation of Dlt localization. The transmembrane protein Stranded at Second (Sas: Schonbaum, 1992) is lost from the apical pole, whereas the basolateral marker Neurotactin, initially retained at the lateral membranes, is delocalized in later stages. Lack of sdt also affects localization of the PDZ protein Bazooka (Baz) in epithelial cells. Baz forms, together with the PDZ protein DmPar-6 and Atypical protein kinase C (DaPKC), another apical protein complex. This complex is essential for the polarity of epithelial cells and neuroblasts, the progenitors of the central nervous system. In the CNS Baz controls spindle orientation and localization of determinants. Lack of sdt function does not affect Baz localization in neuroblasts, showing that sdt, unlike baz, is not required for the apicobasal polarity of neuroblasts (Bachmann, 2001).

sdt encodes several isoforms, which are constituents of the SAC. The presence of Sdt proteins containing either a MAGUK or a GUK domain has no precedent among other MAGUK proteins. Recent results have shown that the GUK domain of the rat MAGUK proteins SAP97 and PSD-95/SAP-90 and of hCASK and hDlgcan form intra- as well as inter-molecular interactions with the SH3 domain of the same or other MAGUK proteins, respectively. It has also been demonstrated that intramolecular interactions between the SH3 and the GUK domain of SAP97 modulate binding of its GUK domain to its partner GKAP. Several issues are yet to be solved: how Crb, Dlt and Sdt participate in the organization of the SAC; in which temporal sequence they are recruited; and whether Dlt and Sdt compete for the Crb binding site. Sdt and Dlt behave differently on overexpression of Crb, in that Dlt is redistributed around the plasma membrane, whereas Sdt is depleted from the apical membrane (Bachmann, 2001).

The different Sdt isoforms now make it possible to isolate additional components of the SAC. The mouse homolog Pals1 has been isolated by virtue of its interaction with mouse Lin-7, another PDZ protein. Homologs of Lin-7 and its C. elegans binding partners Lin-2 and Lin-10 have been identified in Drosophila and it is of interest to see whether they form part of the SAC. The analysis of all the interactions necessary to establish a functional SAC, the control of its assembly and its role in the establishment of the ZA will allow further unravelling of the mechanisms involved in the control of apicobasal polarity of epithelial cells (Bachmann, 2001).

Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts

Adherens junctions (AJs) first form during cellularization (stage 5) in fly embryos as spot AJs, which later migrate apically and coalesce to form the ZA, a continuous belt-like AJ around the apical lateral cell boundary. The sdt mutations seem to specifically disrupt the fusion of apically localized spot AJs to form the ZA. In sdt zygotic mutants, AJ/ZA components Arm and DE-cad fail to form a smooth honeycomb pattern, but appear significantly fragmented. This failure of ZA formation and apicobasal polarity establishment results in an irregular and multilayered epithelial structure in later embryogenesis. Despite their initial normal distribution during gastrulation (stage 7 or 8), basolateral localization of both Discs Large (Dlg) and Scribbled (Scrib); these proteins mark the future septate junctions, is progressively compromised in later stages. Another indication that septate junctions fail to develop in sdt mutants is the cytoplasmic distribution of the septate junction marker Coracle at stage 14. All apicolateral and basolateral markers tested are mislocalized in sdt mutant epithelia (Hong, 2001).

Bazooka (Baz), DmPar-6 and atypical protein kinase C (aPKC) form a complex essential for both neuronal and epithelial polarity, and resemble Sdt in their epithelial expression and subcellular localization. As early as stage 7 these proteins all become mislocalized and form random aggregates around the apical cell boundary in epithelial cells. However, their apical localization in delaminating and dividing neuroblasts remains largely normal, not only in zygotic sdt mutant embryos but also in germline clone embryos in which both maternal and zygotic sdt contributions are removed. Likewise, the basal localization of Miranda (Mir) in neuroblasts is unaffected in sdt zygotic or germline clone embryos. Similar results are also seen in zygotic crb mutants, indicating that epithelial but not neuroblast polarity requires Sdt and Crb (Hong, 2001).

How do neuroblasts retain their normal apicobasal polarity in sdt mutants?. Unlike Crb and Dlt, the expression levels of Baz, DmPar-6 and aPKC are not significantly reduced in sdt mutants before stage 13. Moreover, Baz remains colocalized with Arm/DE-cad in the disrupted AJs, indicating a residual apicobasal polarity in sdt mutants. This is probably due to partial redundancy between Baz and Sdt, since removing zygotic copies of baz significantly enhances the null phenotype of sdt. This residual polarity retains Baz apically in sdt mutants, so that as a neuroblast delaminates, Baz is still concentrated in the apical stalk structure that expands into an apical crescent (Hong, 2001).

Crumbs, a transmembrane protein with EGF-like repeats, is an apical determinant for establishing apicobasal polarity in embryonic epithelia. The sdt and crb mutants exhibit nearly identical defects in epithelial apicobasal polarity, and crb and sdt function in a common genetic pathway. Precise colocalization of Sdt and Crb throughout embryonic development was found in all cells examined except for those in sensory organs: Sdt but not Crb is at the distal dendrites of sensory neurons. In epithelial cells Crb and Sdt are mutually dependent for their localization and stability. There is a marked reduction of the protein levels of Crb in sdt mutants and of Sdt in crb mutants after gastrulation. In both cases, instead of the normal apicolateral distribution, these proteins are found in sparse and discrete random spots. Dlt, which interacts with the Crb intracellular domain, is distributed similarly to Crb in sdt mutants, although its early expression and localization during cellularization is not affected even in sdt germline clone embryos. Conversely, overexpression of the Crb intracellular domain (Crb-intra) is sufficient to cause a 'dominant' phenotype, disrupting cell polarity, expanding the apical domain, and displacing endogenous Crb into cytoplasm. In such embryos a large fraction of Sdt, together with Crb and Crb-intra, is also displaced away from the membrane into the cytoplasm (Hong, 2001).

To test whether the mutual dependence of Sdt and Crb is due to their physical interaction, a GST::Crb-intra pull-down assay was carried out using in vitro translated ³⁵S-labelled Sdt. Both SdtA and SdtB bind specifically to Crb-intra. Two domains of Crb-intra are known: the C-terminal domain containing the EERLI motif, and the amino-terminal domain containing amino acids, such as Y10 and E16, that are conserved in Crb and its C. elegans homologs. Mutation in either domain abolishes the ability of Crb-intra to rescue the crb phenotype, but only the EERLI motif is required for inducing the dominant overexpression phenotype of Crb-intra. In the in vitro binding assay, binding of Sdt to Crb-intra is nearly abolished by removal of the C-terminal EERLI motif, but is not affected by Y10A and/or E16A mutations, indicating that Crb binds Sdt with its C-terminal EERLI motif (Hong, 2001).

The same C-terminal EERLI is also required for Dlt to bind to Crb. It will be of interest to determine whether Sdt and Dlt bind Crb cooperatively to form a tertiary complex, or whether they compete with each other for binding to the Crb intracellular domain. No direct binding between Dlt and Sdt was detected using the same in vitro GST pull-down assay. However, MAGUK proteins frequently show intramolecular auto-inhibition between different binding motifs so it remains possible that Sdt binds Dlt only after it first binds to Crb. Thus, whereas the Crb-Sdt (and possibly Dlt) pathway controls the epithelial but not neuroblast polarity, the Baz-DmPar-6-aPKC complex affects both epithelial and neuroblast polarity. Proteins in both pathways are expressed in epithelial cells with identical apico-lateral localization patterns but only Baz-DmPar-6-aPKC are expressed in neuroblasts. In epithelia, these two pathways are probably partially redundant in controlling apicobasal polarity. Their role in regulating ZA formation seems to be the key to their epithelial and cuticle phenotypes. Sdt and Crb colocalize in epithelia and together serve as apical determinants, but only partially overlap and are likely to serve different functions in sensory organs. A mutation in a human Crb homolog has been implicated as the cause of one form of retinitis pigmentosa, suggesting a potential role for the Crb family of proteins in the localization of a sensory transduction complex. The localization of Sdt to the dendritic tip and scolopale cells, the probable sites for sensory transduction, raises the possibility that Sdt may have a role in the development or function of a sensory transduction apparatus (Hong, 2001).

AP-2 complex-mediated endocytosis of Drosophila Crumbs regulates polarity via antagonizing Stardust

Maintenance of epithelial polarity depends on the correct localization and levels of polarity determinants. The evolutionarily conserved transmembrane protein Crumbs is crucial for the size and identity of the apical membrane, yet little is known about the molecular mechanisms controlling the amount of Crumbs at the surface. This study shows that Crumbs levels on the apical membrane depend on a well-balanced state of endocytosis and stabilization. The Adaptor Protein 2 (AP-2) complex binds to a motif in the cytoplasmic tail of Crumbs that overlaps with the binding site of Stardust, a protein known to stabilize Crumbs on the surface. Preventing endocytosis by mutations in AP-2 causes expansion of the Crumbs-positive plasma membrane and polarity defects, which can be partially rescued by removing one copy of crumbs. Strikingly, knocking-down both AP-2 and Stardust retains Crumbs on the membrane. This study provides evidence for a molecular mechanism, based on stabilization and endocytosis, to adjust surface levels of Crumbs, which are essential for maintaining epithelial polarity (Lin, 2015).

Data presented in this study, obtained using genetic and biochemical assays, has led to the conclusion that a delicate balance of stabilization and internalization controls proper Crb levels at the surface. This balance is mediated by binding of a conserved amino acid sequence of Crb to either Sdt or AP-2, suggesting a competitive binding between Sdt and AP-2. Such a mechanism, based on stabilization by PDZ-ligand interaction and AP-2-mediated endocytosis, has so far only be described for a few proteins, such as the NR2B subunit of the neuronal NMDA receptor and the glutamate transporter excitatory amino acid carrier 1 (EAAC1) in MDCK cells. In the latter case, AP-2 and the PDZ-domain of PDZK1 antagonistically regulate surface levels of EEAC1 by binding to two closely adjacent binding sites in EEAC1 (Lin, 2015).

Currently, it is only possible to speculate about the mechanisms that may determine which of the binding partners AP-2 or Sdt - binds to Crb. One possibility is that different binding affinities between Crb and its partners modulate the binding preference. Several arguments suggest that binding of AP-2 to Crb seems to be weaker than the Sdt-Crb interaction. First, AP-2 - Crb interactions could only be shown by liposome recruitment assays, but not by standard pull-down experiments, suggesting that the presence of membranes strengthen the interaction between the adaptor and its cargo. In fact, interaction of AP-2 with PtdIns4,5P2-containing membranes induces a conformational change of the AP-2 complex, thus facilitating binding to its cargo (Lin, 2015).

Second, binding of Sdt only depends on the C-terminal leucine and isoleucine residues of Crb. This is in agreement with recently published structural data using crystallography and fluorescence polarization, which show that the four C-terminal amino acids of human Crb1 interact with the PDZ domain of Pals1 by van der Waals contacts and charged interactions (Ivanova, 2015). In contrast, AP-2 binding to Crb requires the glutamine and arginine residues of Crb besides the leucine and isoleucine residues, at least under the in vitro conditions used in this study. The importance of more than one motif for strong binding of AP-2 is not unprecedented. Binding of the human immunodeficiency (HIV)-1 protein Nef to the α-σ2 -hemicomplex of AP-2 requires both a di-leucine- and a di-acidic-motif. Posttranslational modification of the ligand Crb itself could also contribute to a preferred binding to either Sdt/Pals1 or AP-2. It was recently documented that aPKC-mediated phosphorylation of threonine residues near the FBM of Crb abolishes the Crb- Moesin interaction, but not the Crb/Pals1 interaction (Wei, 2015; Lin, 2015 and references therein).

In the case of Drosophila Gliotactin, a transmembrane protein at the tricellular junction, phosphorylation of tyrosine residues is necessary for its endocytosis and ultimately lysosomal degradation, thus preventing overexpression, which would result in delamination, migration and finally apoptosis of cells (Lin, 2015).

Alternatively, the accessibility of the PDZ domain of Sdt/Pals1 to Crb could be modulated. In fact, a ~100-fold stronger binding of the PDZ domain of Pals1 to the Crb tail is achieved upon intra- or intermolecular interactions between the Src homology 3 (SH3)- and the guanylate kinase (GUK)-domain of Pals1. Similarly, the affinity of the PDZ domain of the postsynaptic density protein PSD-95 to its ligand is reduced upon phosphorylation of a tyrosine residue in a linker region between the third PDZ-domain and the subsequent SH3-domain, thereby weakening the intramolecular interaction between the PDZ- and the SH3 domain (Lin, 2015).

A striking observation was the high degree of phenotypic variability upon knock-down/knock -out of AP-2 in different epithelial tissues, and sometimes even in the same epithelium. This prevented study of the different aspects of the AP-2/Crb relation in just one epithelium. In the follicle epithelium, the phenotypes ranged from minor expansion of the apical surface to complete loss of polarity and overgrowth. Complete loss of AP-2α in wing discs leads to cell lethality, whereas mutant clones survived in eye discs. This variability might be due to the time point of induction of mitotic recombination, which could occur before or after establishment of epithelial polarity, or at time points of high or low Crb expression. Alternatively, additional AP-2-independent polarity regulators could act redundantly and in a tissue- and/or time-specific manner, thus modulating the severity of the phenotype, e.g. in the developing eye (Lin, 2015).

The results described in this study are compatible with the assumption that several phenotypes obtained by loss/reduction of AP-2 are a consequence of increased Crb levels on the plasma membrane, since similar phenotypes can be obtained upon overexpression of Crb. (1) The Crb-positive plasma membrane in AP-2α mutants cells is expanded both in the imaginal discs and the follicle epithelium, often at the expense of the lateral membrane. (2) Without functional AP-2α, the monolayered epithelium is often disrupted and becomes multilayered. (3) Surviving AP-2α³ clones in eye imaginal discs show strong Crb enrichment, similar as HEK293 cells, in which AP-2α is knocked-down by RNAi. (4) Assuming that a similar increase in Crb also occurs in AP-2α³ mutant cells induced in wing discs, their elimination could be a consequence of cell competition when next to wild-type cells (Lin, 2015).

Finally, the multi-layering phenotype of follicle epithelia lacking AP-2α can be partially suppressed by removing one copy of crb. A more direct insight into the relationship between Crb and AP-2 comes from an analysis of the garland cells, the functional equivalent of vertebrate podocytes. Podocytes are highly specialized epithelial cells in the kidney of vertebrates, which form long 'foot-processes' connected by slit diaphragms to form a filtration barrier in the renal glomerula. Interestingly, Crb2 is expressed in the kidney of both rats and zebrafish, where it localizes at the slit diaphragm of podocytes. crb2b mutant zebrafish show defects in the formation of the slit diaphragm as well as in arborization of the foot-processes. Furthermore, mutations in human Crb2 are linked to Steroid-resistant nephrotic syndrome (Ebarasi, 2015), a disease causing kidney failure due to defects in differentiation and function of podocytes. This study shows that AP-2α³ mutant garland cells are clearly impaired in Crb endocytosis. Whether loss of crb in garland cells affects their excretory function has to be determined (Lin, 2015).

A complex machinery is required to ensure proper Crb surface levels, which is key for the maintenance of apico-basal epithelial cell polarity. Crb levels can be regulated at multiple levels, including stabilization at the membrane via homophilic interactions of the extracellular domains in cis or trans, interactions of the cytoplasmic tail with scaffolding proteins, endocytosis, degradation and recycling by the retromer. The trafficking pathway offers multiple steps for regulation, and results presented here provide further mechanistic insight how binding of two counteracting PDZ motif-binding proteins, Sdt and AP-2, regulate proper Crb levels (Lin, 2015).

Given the finding that the balance between Crb stabilization and internalization/degradation is crucial for surface expression of Crb and hence polarity, future work will aim to identify the mechanisms that control this balance by regulating the rate of endocytosis, degradation and recycling by the retromer. Finding these regulators is challenging, but will give important insights into the mechanisms coordinating endocytosis and polarity, which is important to prevent tumorigenesis not only in Drosophila, but also in vertebrates (Lin, 2015).

GENE STRUCTURE

Between the Hong and Bachmann studies, four different std transcripts were detected. To obtain full-length complementary DNAs of stardust, an embryonic cDNA library was screened and a large transcription unit was identified that includes CG1617 and CG15341. Three cDNAs for this sdt candidate gene, sdt1, sdt2 and sdt3, differ at their 5' ends owing to alternative splicing, and code for two isoforms of potential Sdt protein: SdtA, with 1,292 amino acids; and SdtB, with 860 amino acids, lacking the 432 amino acids encoded by alternatively spliced exon 3. In vitro translation of sdt1 and sdt3 yielded products of the predicted size (Hong, 2001). std2 and std3 were not detected in the Bachmann study.

Different start sites were used for the generation of the multiple Std isoforms. Using cMAGUK1 as probe, the 4.5-kb cDNAcGUK1 was isolated, which starts about 25 kb downstream of the start site of cMAGUK1. A protein product (Sdt-GUK1) with Mr about 120K could be synthesized from cGUK1 in vitro. It differs from Sdt-MAGUK1 by 78 additional amino-terminal amino acids and by the lack of 284 internal amino acids, removing the PDZ, SH3 and Hook domains. Sequence analysis reveals two predicted proteins generated by the std gene. One protein, named Sdt-GUK2, is predicted to contain a complete GUK domain and a deletion of 271 amino acids compared with Sdt-MAGUK1 (corresponding with std1 of the Hong study). Thus, these data suggest that sdt transcripts encode two proteins, one with a MAGUK and a second with a GUK domain (Bachmann, 2001). Sdt-GUK2 was not detected in the Hong study.

PROTEIN STRUCTURE

Amino Acids - 1083 (GUK1 isoform); 1289 or 1292 (MAGUK1 isoform, coded for by std1); 860 (StdB isoform, coded for by std3)

Structural Domains

More than ten related but incomplete complementary DNAs were recovered and they were used to isolate additional cDNAs. The 5.7-kb cDNA cMAGUK1 defined by the Drosophila Genome Project detects three developmentally regulated transcripts of stardust of about 5, 6 and 7 kb, respectively. Data from in vitro transcription/translation and overexpression of an upstream activating sequence (UAS)-cMAGUK1 transgene in wild-type embryos suggest that the first AUG codon, which complies to the Kozak consensus sequence, is used as the start site, resulting in a protein product (Sdt-MAGUK1) of relative molecular mass about 140,000 (M_r ~140K). The protein contains a single PDZ (PSD-95, Discs Large, ZO-1) domain, an SH3 (Src homology region 3) domain and a GUK (guanylate kinase) domain, which, together with a Hook domain, classify Sdt-MAGUK1 as a member of the p55 subfamily of the MAGUK (membrane-associated guanylate kinase homologs) family of scaffolding proteins. The MAGUK domain exhibits a high degree of similarity to that of a predicted open reading frame (ORF) from Caenorhabditis elegans, to mouse Pals1 and to a human predicted ORF. Sdt-MAGUK1 also shares the putative Lin-7-binding domain (L27) with the mammalian proteins. No homology to the amino-acid sequence of exon 3 in SdtA was found (Bachmann, 2001; Hong, 2001).

EVOLUTIONARY HOMOLOGS

In Caenorhabditis elegans, three PDZ domain proteins, Lin-2, Lin-7, and Lin-10, are necessary for the proper targeting of the Let-23 growth factor receptor to the basolateral surface of epithelial cells. It has been demonstrated that homologs of Lin-2, Lin-7, and Lin-10 form a heterotrimeric complex in mammalian brain. Using Far Western overlay assay, additional proteins have been identified that can bind to the amino terminus of mLin-7 and the genes encoding these proteins were cloned using bacterial expression cloning. These proteins are called Pals, for proteins associated with Lin-7. These proteins, which include mammalian Lin-2, contain a conserved mLin-7 binding domain in addition to guanylate kinase, PDZ (postsynaptic density 95/discs large/zona occludens-1), and Src homology 3 domains. Using site-directed mutagenesis, the conserved residues among these proteins crucial for mLin-7 binding have been identified. Two of these proteins, Pals1 and Pals2, are newly described. Pals1, most closely related to Drosophila Stardust, consists of 675 amino acids and maps to mouse chromosome 12. Pals2 was found to exist in two splice forms of 539 and 553 amino acids and maps to mouse chromosome 6. Like mLin-2, Pals1 and Pals2 localize to the lateral membrane in Madin-Darby canine kidney cells. Pals proteins represent a new subfamily of membrane-associated guanylate kinases that allow for multiple targeting complexes containing mLin-7 (Kamberov, 2000).

Membrane-associated guanylate kinase (Maguk) proteins are scaffold proteins that contain PSD-95-Discs Large-zona occludens-1 (PDZ), Src homology 3, and guanylate kinase domains. A subset of Maguk proteins, such as mLin-2 and protein associated with Lin-7 (Pals)1, also contain two L27 domains: an L27C domain that binds mLin-7 and an L27N domain of unknown function. The L27N domain targets Pals1 to tight junctions by binding to a PDZ domain protein, Pals1-associated tight junction (PATJ) protein, via a unique Maguk recruitment domain. PATJ is a homolog of Drosophila Discs Lost, a protein that is crucial for epithelial polarity and that exists in a complex with the apical polarity determinant, Crumbs. PATJ and a human Crumbs homolog, CRB1, colocalize with Pals1 to tight junctions, and CRB1 interacts with PATJ, albeit indirectly, via binding the Pals1 PDZ domain. In agreement, a Drosophila homolog of Pals1 participates in identical interactions with Drosophila Crumbs and Discs Lost. This Drosophila Pals1 homolog represents Stardust, a crucial polarity gene in Drosophila. Thus, these data identify a new multiprotein complex that appears to be evolutionarily conserved and likely plays an important role in protein targeting and cell polarity (Roh, 2002).

In Drosophila, the Crumbs/Stardust/Discs-lost complex is required during the establishment of polarized epithelia. Embryos that lack a component of this complex or overexpress Crumbs exhibit defects in epithelial morphogenesis. A novel mammalian epithelial Crumbs isoform, Crumbs3 (CRB3) has been cloned. CRB3 exists in a complex at tight junctions (TJs) with Pals1 and PATJ, the mammalian homologs of Stardust and Discs lost, respectively. Overexpression of CRB3 leads to delayed TJ formation in MDCK epithelial cell monolayers and disruption of polarity in MDCK cysts cultured in collagen. Both phenomena require the last four residues of CRB3. Next, s dominant-negative Myc-Lin-2-Pals1 chimeric protein, where the PDZ domain of Lin-2 was replaced with that of Pals1, was expressed in MDCK cells. TJ and apical polarity defects are observed in these cells. Collectively, this suggests that the CRB-Pals1 interaction is important for formation of TJs and polarized epithelia. These results provide insight into the function of the mammalian Crumbs complex during TJ formation and epithelial polarization (Roh, 2003).

Drosophila Crumbs is a transmembrane protein that plays an important role in epithelial cell polarity and photoreceptor development. Overexpression of Crumbs in Drosophila epithelia expands the apical surface and leads to disruption of cell polarity. Drosophila Crumbs also interacts with two other polarity genes, Stardust and Discs Lost. Recent work has identified a human orthologue of Drosophila Crumbs, known as CRB1, that is mutated in the eye disorders, retinitis pigmentosa and Leber congenital amaurosis. CRB1 can form a complex with mammalian orthologues of Stardust and Discs Lost, known as protein associated with Lin-7 (Pals1) and Pals1 associated tight junction (PATJ), respectively. In the current report a full length cDNA has been cloned for a human paralogue of CRB1 called Crumbs3 (CRB3). In contrast to Drosophila Crumbs and CRB1, CRB3 has a very short extracellular domain but like these proteins it has a conserved intracellular domain that allows it to complex with Pals1 and PATJ. Mouse and human CRB3 have identical intracellular domains but divergent extracellular domains except for a conserved N-glycosylation site. CRB3 is localized to the apical surface and tight junctions but the conserved N linked glycosylation site does not appear to be necessary for CRB3 apical targeting. CRB3 is a specialized isoform of the Crumbs protein family that is expressed in epithelia and can tie the apical membrane to the tight junction (Markarova, 2003).

The Crumbs complex that also contains the cortical proteins Stardust and DPATJ (a homologue of PATJ), is crucial for the building of epithelial monolayers in Drosophila. Although loss of function of the Crumbs or Stardust genes prevents the stabilization of a belt of adherens junctions at the apico-lateral border of the cells, no phenotype has been described for the Dpatj gene and its role in epithelial morphogenesis and polarity remains unknown. Downregulated PATJ stable lines of Caco2 have been produced to clarify its role in epithelial morphogenesis. In PATJ knockdown cells, Pals1 (a Stardust homologue) is no longer associated with tight junctions whereas Crumbs3 (Crb3) is accumulated into a compartment spatially close to the apical membrane and related to early endosomes. Furthermore, occludin and ZO-3, two proteins of tight junctions are mislocalized on the lateral membrane indicating that PATJ plays a novel role in the building of tight junctions by providing a link between their lateral and apical components. Thus, PATJ stabilizes the Crb3 complex and regulates the spatial concentration of several components at the border between the apical and lateral domains (Michel, 2005).

Mutations in the human Crumbs homologue-1 (CRB1) gene cause retinal diseases including Leber's congenital amaurosis (LCA) and retinitis pigmentosa type 12. The CRB1 transmembrane protein localizes at a subapical region (SAR) above intercellular adherens junctions between photoreceptor and Muller glia (MG) cells. The Crb1-/- phenotype, as shown in Crb1-/- mice, is accelerated and intensified in primary retina cultures. Immuno-electron microscopy showed strong Crb1 immunoreactivity at the SAR in MG cells but barely in photoreceptor cells, whereas Crb2, Crb3, Patj, Pals1 and Mupp1 were present in both cell types. Human CRB1, introduced in MG cells in Crb1-/- primary retinas, was targeted to the SAR. RNA interference-induced silencing of the Crb1-interacting-protein Pals1 (protein associated with Lin7; Mpp5) in MG cells resulted in loss of Crb1, Crb2, Mupp1 and Veli3 protein localization and partial loss of Crb3. It is concluded that Pals1 is required for correct localization of Crb family members and its interactors at the SAR of polarized MG cells (van Rossum, 2006).

date revised: 20 May 2002

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