A possible direct interaction between Sdt and Crumbs was tested using the yeast two-hybrid system. A fusion protein consisting of the MAGUK domain of Sdt and the transactivation domain of GAL4 was co-expressed in yeast cells with various forms of the Crb cytoplasmic domain, fused to the DNA-binding domain of GAL4. The full-length intracellular domain of Crb and those with mutations in a region close to the membrane (Crb-intraWT, Crb-intraY10A and Crb-intra E16A) showed strong interaction with the MAGUK domain of Sdt. In contrast, lack of the four C-terminal amino acids ERLI (Crb-intraDERLI) completely abolishes the interaction. This suggests that binding of the MAGUK domain of Sdt depends on the four C-terminal amino acids of Crb (Bachmann, 2001).
Polarized cells contain numerous membrane domains, but it is unclear how the formation of these domains is coordinated to create a single integrated cell architecture. Genetic screens of Drosophila embryos have identified three complexes, each containing one of the PDZ domain proteins -- Stardust (Sdt), Bazooka (Baz) and Scribble (Scrib) -- that control epithelial polarity and formation of zonula adherens. These complexes can be ordered into a single regulatory hierarchy that is initiated by cell adhesion-dependent recruitment of the Baz complex to the zonula adherens. The Scrib complex represses apical identity along basolateral surfaces by antagonizing Baz-initiated apical polarity. The Sdt-containing Crb complex is recruited apically by the Baz complex to counter antagonistic Scrib activity. Thus, a finely tuned balance between Scrib and Crb complex activity sets the limits of the apical and basolateral membrane domains and positions cell junctions. These data suggest a model in which the maturation of epithelial cell polarity is driven by integration of the sequential activities of PDZ-based protein complexes (Bilder, 2003).
Formation of multiprotein complexes is a common theme to pattern a cell, thereby generating spatially and functionally distinct entities at specialised regions. Central components of these complexes are scaffold proteins, which contain several protein-protein interaction domains and provide a platform to recruit a variety of additional components. There is increasing evidence that protein complexes are dynamic structures and that their components can undergo various interactions depending on the cellular context. However, little is known so far about the factors regulating this behaviour. One evolutionarily conserved protein complex, which can be found both in Drosophila and mammalian epithelial cells, is composed of the transmembrane protein Crumbs/Crb3 and the scaffolding proteins Stardust/Pals1 and DPATJ/PATJ, respectively, and localises apically to the zonula adherens. In vitro analysis shows that, similar as in vertebrates, the single PDZ domain of Drosophila Par-6 can bind to the four C-terminal amino acids (ERLI) of the transmembrane protein Crumbs. To further evaluate the binding capability of Crumbs to Par-6 and the MAGUK protein Stardust, analysis of the PDZ structural database and modelling of the interactions between the C-terminus of Crumbs and the PDZ domains of these two proteins were performed. The results suggest that both PDZ domains bind Crumbs with similar affinities. These data are supported by quantitative yeast two-hybrid interactions. In vivo analysis performed in cell cultures and in the Drosophila embryo show that the cytoplasmic domain of Crumbs can recruit Par-6 and DaPKC to the plasma membrane. The data presented here are discussed with respect to possible dynamic interactions between these proteins (Kempkens, 2006).
Homology modelling and energy calculations indicate that the PDZ domains of Sdt and Par-6 both show a high affinity for Crb. According to a classification of PDZ domains, the PDZ domains of Par-6 and Sdt fall into different categories: n (neutral) – h (hydrophic) for Par-6 and Sp (small and polar) – h (hydrophobic) for Sdt. The distinction is based on the nature of the amino acids in two critical positions, those immediately following the second β-strand and at the beginning of the second α-helix. However, this classification, based exclusively on the amino acids at two positions, does not adequately describe the complexity of the PDZ family and is not sufficient to predict the specificity of binding. This is borne out by the results presented in this study (Kempkens, 2006).
The crystal structure used as a template in this study (D. melanogaster Par-6, 1RZX.PDB) includes a canonical type I ligand (ESLV), and this can easily be replaced in the structure by the C-terminal tetrapeptide of Crb (ERLI). Although better binding of the Crb C-terminus to Par-6 than to Sdt is predicted, this conclusion should be treated with caution, since the predicted structure of the Crb-Sdt complex is based solely on homology modelling. The analysis of interactions between Crb and PDZ domains showed minor van der Waals clashes (<0.3 kcal/mol), as well as strong polar and hydrophobic ligand/PDZ-domain interactions. An important component of the interaction is electrostatic. This could favour fast association (Kon) and dissociation rates (Koff), which would facilitate flexible regulation of complex formation. These values, which are slightly better for Par-6, could account for the affinity and specificity observed in the experimental interaction studies. The predicted Kd for interaction between the domains considered in this study fall in the usual range for experimental determinations of PDZ-ligand binding affinities reported by many authors. The theoretical determination of binding interactions by FoldX has been tested for many domain-peptide interactions (i.e. SH3, SH2, PDZ, Ras-Rab, etc.), is validated by available experimental data, and shows a high degree of accuracy. In this sense, the theoretical cut-off for non-binders can be reasonably approximated to that used for experimental procedures, normally 1E−4M. Thus, all putative ligands with interaction energies below 1E−4M are considered to be non-binders (Kempkens, 2006).
It has been shown that the PDZ domain of vertebrate Par-6 can bind not only to the C-terminus of CRB3, but also to the C-terminus of the N-type Ca2+ channel and the C-terminus of neurexin, and that the PDZ domains of vertebrate and Drosophila Par-6 proteins can interact with an internal region in the N-terminal portion of Pals1/Sdt. These data support the view that PDZ domains are promiscuous and can bind different ligands, thanks to the plasticity of the carboxylate-binding loop. This conformational flexibility can be modulated by interaction with other proteins. For example, binding of Cdc42 to Par-6 significantly enhances its affinity for the C-terminal ligand, while it has no effect on the interaction with the internal binding sequence of Pals1. To what extent other components facilitate loop rearrangement to accommodate internal ligands to the Par-6 PDZ binding pocket in vivo remains to be determined. Interestingly, although the PDZ domains of both Par-6 and Sdt bind to the C-terminus of Crb, only the PDZ domain of Par-6 interacts with the internal sequence in Sdt (Kempkens, 2006).
Since the binding affinities of the PDZ domains of Sdt and Par-6 for the C-terminus of Crb are in the same range, and all three proteins are expressed in the same cells, other factors must be considered that might influence their interactions. These could include, for example, (1) temporal differences in expression, (2) protein modifications, and (3) interactions with other factors. Par-6 is present in the embryo before Sdt and Crb appear, since it is expressed maternally. In the genetic hierarchy controlling cell polarity and ZA assembly, components of the Baz/Par-6/DaPKC complex appear to form the top tier, with Baz acting very early during cellularisation to establish the apical domain in an adherens junction-independent manner. At this stage, Par-6 is diffusely distributed in the cytoplasm. It becomes localised apically only after cellularisation is complete. Localisation of Par-6 requires an interaction with the monomeric GTPase Cdc42, which, like its vertebrate homologue, binds Par-6 via a conserved CRIB (Cdc42/Rac interactive binding) domain. Thus, overexpression of a dominant-negative form of Cdc42 prevents apical localisation of Par-6, and a mutant version of Par-6 that cannot interact with Cdc42 (Par-6-ΔP) remains diffusely distributed in the cytoplasm (Kempkens, 2006).
Binding of the PDZ domain of Sdt to the C-terminus of Crb may also depend on additional contacts mediated by the other interaction domains of Sdt. Sdt is a member of the MAGUK protein family, with two L27 domains, one SH3, one PDZ and one guanylate kinase (GUK) domain. The L27 domains of Pals1 and Sdt bind to PATJ/DPATJ and mLin-7/DLin-7. No partner(s) for the SH3 and GUK domains of Sdt have been described so far. Recently, a direct interaction was shown to occur between the SH3/Hook domain of MPP5/Pals1 and the GUK domain of another MAGUK family member, MPP4, in mouse photoreceptor cells. Furthermore, yeast two-hybrid experiments have uncovered an interaction between the SH3 and the Hook/GUK domains of Sdt. In other members of the MAGUK family, such as human Discs large (hDlg), CASK and p55, these two domains participate in intra- as well as intermolecular interactions, with the former being preferred, at least in vitro. Currently, the data do not allow a decision as to whether the observed SH3-Hook/GUK interaction is intra- or intermolecular, nor to what extent this might influence the capacity of the PDZ domain to bind to Crb in vivo. Similarly, at present it is only possible to speculate on the possible influence on the binding specificity/activity of the phosphorylation of the intracellular domain of Crb, which has been demonstrated by in vitro assays, but also occurs under certain in vivo conditions. Future experiments will contribute to the elucidation of the dynamic interactions among these proteins within the cell and help in the understanding of how they establish and maintain the polarised phenotype (Kempkens, 2006).
The formation and maintenance of cell polarity is essential for epithelial morphogenesis. Patj (the Drosophila homolog of mammalian Patj) is multi-PDZ domain protein that localizes to the apical cell membrane and form a protein complex with cell polarity proteins, Crumbs (Crb) and Stardust (St). Whereas Crb and Sdt are known to be required for the organization of adherens junctions (AJs) and rhabdomeres in differentiating photoreceptors, the in vivo function of Dpatj as a member of the Crb complex in developing eye has been unclear due to the lack of loss-of-function mutations specifically affecting the patj gene. Genetic analysis of hypomorph, null, and RNA inerference reveals distinct dual functions of Patj in developing and mature photoreceptors. The C-terminal region (PDZ domains 2-4) of Patj is not essential for development of the animal but is required to prevent late-onset photoreceptor degeneration. In contrast, the N-terminal region of Patj is essential for animal viability and photoreceptor morphgenesis during development. The localization and maintenance of Crb and Sdt in the apical photoreceptor membrane are strongly affected by reduced level of Patj. Patj is necessary for proper positioning of AJs and the integrity of photoreceptors in the developing retina as well as for the maintenance of adult photoreceptors. This study provides evidence that Drosophila Patj has domain-specific early and late functions in regulating the localization and stability of the Crb-Sdt complex in photoreceptor cells (Nam, 2006).
Dynamic changes take place in developing photoreceptors to reorganize the apical cell membrane during pupal stage. Crb and Sdt are required for growth and maintenance of rhabdomeres and AJs during this time of photoreceptor morphogenesis. Patj binds Sdt to form a conserved heterotrimeric Crb complex (Roh, 2002), but its function in vivo has been unclear. In this study, a key question whether Patj is an essential member of the Crb complex was addressed in vivo (Nam, 2006).
This study made three new observations that demonstrate important functions of Patj: (1) hypomorphic patj shows severe late-onset degeneration of photoreceptor cells in adult eye although the mutant eyes develop relatively normally; (2) analysis of patj null mutant patj RNAi demonstrate that Patj is essential for early development of the animal and for morphogenesis of AJ and apical membrane domains of photoreceptor cells during pupal development (Nam, 2006).
Consistent with these results on the late-onset degeneration of photoreceptor cells in hypomorphic adult eyes, (3) degeneration of adult photoreceptor cells was found. However, despite the findings on the phenotypes of the hypomorphic mutant, it is worth noting that there are important differences between these two studies. First, a second study on patj function (Richard, 2005) is limited to the analysis of a viable hypomorphic condition patj that shows no obvious defects in the pattern of AJs and rhabdomeres in the eyes until approximately 70% late pupal development. In contrast, the current study with the newly generated null mutant and RNAi reveals important developmental functions of Patj in the eye as well as animal viability. Second, the hypomorpic mutant is not only deleted in the PDZ2-4 domain portion of the dpatj gene but also deficient for JTBR and partially CG32327 that are adjacent to the 3' end of patj. In this study, it was shown that patj RNAi causes similar phenotypes of patj null mutant in the eye, suggesting that JTBR and CG32327 do not affect eye development and, thus, have no detectable influence on analysis of hypomorphic and null mutants (Nam, 2006).
The complete loss of patj causes several significant defects in early pupal eyes at approximately 45% pupal development. (1) Both Crb and Sdt are strongly reduced or mislocalized in the absence of Patj, although the loss of patj shows slightly weaker phenotypes than those of crb and sdt mutants. Furthermore, and in contrast to the hypomorphic mutant, (2) null clones or the eyes expressing patj RNAi reveal striking basolateral displacement or expansion of AJ markers such as Arm and Baz. These results suggest that the N- and C-terminal region of Patj protein may have distinct functions in the photoreceptor cells. Because the hypomorphic mutant shows relatively normal development of photoreceptors until later pupal stage or eclosion, the PDZ2-4 domains are not required during early photoreceptor development but are necessary for the maintenance of fully differentiated photoreceptors during very late pupal and adult stage. Of interest, a significant level of Crb and Sdt are apically localized in 45% pupal photoreceptors in hypomorphic mutant eyes (Richard, 2005) but are lost later in adult eyes (Richard, 2005), suggesting that PDZ2-4 domains are required to maintain the Crb complex in late pupal and adult stages. In contrast, the N-terminal region of Patj containing MRE and PDZ1 is crucial for morphogenesis of AJ and rhabdomere during the first half of pupal stage. The MRE motif of Patj interacts with the L27 domain of Sdt. Thus, it is likely that the N-terminal region expressed in hypomorphic mutants might be sufficient for interaction with Sdt, allowing normal development. Conversely, Sdt cannot bind Patj in the null mutant, resulting in the failure of normal development as in crb or sdt mutants (Nam, 2006).
Recently, it has been shown that reduction of Patj by RNAi in MDCK epithelial cell culture leads to delayed tight junction formation and defects in cell polarization. Similarly, mammalian Patj is required for localizing tight junction proteins and stabilizing the Crb3 complex in human intestinal cells. Thus, the findings on the important function of Drosophila Patj in developing photoreceptors are consistent with the results from these mammalian studies, and further provide evidence for the developmental function of Patj in the organization of apical cell membrane in the in vivo animal model. Mouse Patj is closely related to Drosophila Patj, but it has 10 PDZ domains in contrast to the presence of 4 PDZ domains in Drosphila Patj. It will be interesting to see whether the mammalian Patj also show distinct functions of the N-terminal MRE and the multiple PDZ domains in organizing cell polarity and maintaining the stability of the Crb complex, respectively (Nam, 2006).
Mutations in the human CRB1 gene cause retinal diseases such as retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis. The current results that Patj has dual functions in photoreceptor morphogenesis and maintenance in developing and adult animals, respectively, suggest that like CRB1, human Patj may be a target for early- and late-onset retinal diseases. It has been shown that the extracellular domain of Crb is required for preventing photoreceptor degeneration in ageing adult fly eyes. Results from this study and Richard (2005) indicate that PDZ2-4 domains of Drosophila Patj are required for blocking late-onset photoreceptor degeneration. It is currently unknown whether the requirement of Crb extracellular domain and the intracellular function of Drosophila Patj PDZ2-4 domains are related events in the maintenance of adult photoreceptors (Nam, 2006).
The establishment of apicobasal polarity in epithelial cells is a prerequisite for their function. Drosophila photoreceptor cells derive from epithelial cells, and their apical membranes undergo elaborate differentiation during pupal development, forming photosensitive rhabdomeres and associated stalk membranes. Crumbs (Crb), a transmembrane protein involved in the maintenance of epithelial polarity in the embryo, defines the stalk as a subdomain of the apical membrane. Crb organizes a complex composed of several PDZ domain-containing proteins, including Patj (formerly known as Discs lost). Taking advantage of a Patj mutant line in which only a truncated form of the protein is synthesized, Patj was demonstrated to be necessary for the stability of the Crb complex at the stalk membrane and is crucial for stalk membrane development and rhabdomere maintenance during late pupal stages. Moreover, Patj protects against light-induced photoreceptor degeneration (Richard, 2006).
This study presents evidence to show that Patj plays an important role in stabilizing the Crb complex in photoreceptor cells (PRCs). A truncated form of Patj, consisting of the N-terminal L27 and the first PDZ domain, is produced in Patj mutant eyes. It is further shown that the truncated Patj protein fails to stabilize Crb and Sdt at the stalk membrane during late pupal development and in adult eyes. It has been demonstrated that Crb is required for proper localization of Sdt and Patj at the stalk membrane of PRCs and that, in sdtXP96, the maintenance of Crb and Patj is compromised. These data together with the results presented in this study support the view that lack of any component of the Crb complex leads to mislocalization and/or dysfunction of the whole complex in the Drosophila eye (Richard, 2006).
To understand how the Crb complex may ensure a proper morphogenesis of photoreceptor cells, two major events during photoreceptor development have to be considered. In the first half of pupal development, stabilization of the ZAs is essential to maintain adhesion between PRCs during the tremendous cell shape changes that take place later when the cells undergo elongation. Furthermore, from 37% pupal development onward, the apical membrane differentiates into the rhabdomere and the stalk (Richard, 2006).
In crb and sdt mutants, the rhabdomeres are shorter and thicker, suggesting a failure to stabilize adhesion in early stages of pupal development, which in turn prevents proper elongation. This interpretation is consistent with the observation that, in eyes lacking crb function, the continuity of the ZA is interrupted at early stages of pupal development. Patj mutants do not exhibit any obvious defects in ZA development or PRC elongation, which can be explained by the fact that components of the Crb complex are still correctly localized until 70% pupal development. This timing contrasts with crb or sdt mutant PRCs, in which the integrity of the Crb complex is lost at an early stage of pupal development. Several explanations may account for this different behavior of Patj mutant PRCs. (1) The N-terminal portion of Patj may still retain some function during early pupal development, which stabilizes the Crb complex and, hence, the ZA. (2) Alternatively, Patj does not play a major function for the stability of the complex at early stages. (3) Finally, additional factors may interact with Patj and stabilize the Crb complex at early stages of development, and these interactions still occur with a truncated Patj. In fact, recent in vitro studies have suggested direct interactions between Patj and either Drosophila Par-6 or Drosophila PKC, two members of the other apically localized protein complex, which is essential for epithelial cell polarity in the embryo. However, it can be excluded that the suggested interaction between the third PDZ domain of Patj and the N-terminal domain of DmPar-6 plays any role in the stabilization of the complex during the first half of pupal development. The truncated Patj protein studied here lacks the third PDZ domain, yet it remains localized at the apical membrane at this stage (Richard, 2006).
The other major aspect of PRC maturation -- the differentiation of the apical membrane into rhabdomere and stalk -- is affected in crb, sdtXP96, and Patj mutant eyes, suggesting that all three components are necessary for this process. These mutations result in a shortening of the stalk membrane. The weaker phenotype of the Patj mutant relative to that of crb mutants is probably due to the hypomorphic nature of the former. Separation of the apical membrane of PRCs into two distinct domains, the rhabdomere and the stalk, becomes manifest at approximately 55% pupal development and coincides with the restriction of Crb and its associated proteins to this region. No other mutant affecting the length of stalk membrane has been described to date, although some mutants affect individual aspects of the crb or Patj morphogenetic phenotype, displaying thicker (bifocal; DSec61), malformed (Glued; WASp) or missing (overexpression of amphiphysin) rhabdomeres. Thus, the regulation of stalk membrane development seems to be a unique function for members of the Crb complex (Richard, 2006).
One phenotype of Patj mutants observed in this study, the progressive resorption of rhabdomeric microvilli, has not been described to date for any other mutant of the Crb complex. This raises the question whether Patj is involved in other processes in addition to those that are controlled by crb and sdt. The rhabdomere is composed of microvilli, each of which is supported by actin filaments. Rhabdomere morphogenesis and integrity depend on constant renewal of the membrane and on a highly organized actin cytoskeleton. Thus, it is not surprising that mutations in proteins involved in endo- or exocytosis, such as dynamin, Rab1, Rab6, Rab11, Sec6, Sec61, or Sunglasses, affect the integrity of the rhabdomere. It has been suggested that the addition of new membrane occurs at the base of the rhabdomere in Drosophila, while shedding occurs at the distal tip in tipulids. The further analysis of the function of these genes, the subcellular distribution of the respective proteins and their possible interactions with members of the Crb complex will be required to determine any involvement of the Crb complex in these processes. Rhabdomere integrity is also affected in eyes lacking proteins involved in actin structure and remodeling, such as NinaC, Chaoptin, Glued, Moesin or Rac1 but also in mutants for rhodopsin itself, which plays a structural role in addition to its function in signal transduction. In this scenario, Patj could help to stabilize the cytoskeleton and thereby maintain the integrity of the rhabdomere. Alternatively, the four PDZ domains in Patj may mediate the assembly of additional proteins. The identification and functional characterization of these proteins will shed light on the process by which Patj controls the stability of PRCs (Richard, 2006).
At present, the possibility that the defects observed in pigment cells in Patj mutant eyes contribute to the mutant phenotype observed in PRCs cannot be excluded. In vertebrate eyes, the pigment epithelium plays an active role in the renewal of rhodopsin, and defects in the pigment epithelium can lead to degeneration of PRCs. It is not yet known whether pigment cells in the Drosophila eye have a comparable function, although they certainly serve to insulate the PRCs of individual ommatidia from the light impinging on their neighbors. The accumulation and fusion of pigment granules in Patj mutant eyes may point to a defect in vesicular biogenesis and/or secretion. Whether this defect also affects interactions with the PRCs, in other words, whether pigment cells play an active role in the maintenance of the rhabdomeres or PRC function, and if so, whether Patj is involved in this process, is not known (Richard, 2006).
Finally, the results of this study demonstrate that Patj, like Crb, protects PRCs from the deleterious effects of excess light. The degeneration of PRCs observed in Patj mutant eyes may be a direct consequence of the failure to stabilize the components of the Crb complex at the stalk membrane. Previously published data have shown that the absence of crb in Drosophila eyes leads to retinal degeneration under similar lighting conditions. Similarly, mice deficient for CRB1 display signs of retinal degeneration upon exposure to light, which are reminiscent of defects seen in patients bearing mutations in the CRB1 gene. However, the penetrance of degeneration observed in crb eyes is much higher than that observed in Patj eyes, although the cellular features of degeneration observed in both mutants are similar. Taking into account that the crb clones were produced in a white background, whereas Patj eyes are red (due the transgenes that were introduced), it is not unlikely that the pigments could play a protective role in the latter case, as also shown previously for white mutants. Preliminary experiments suggest that the presence of pigments in crb mutant ommatidia indeed slows down the light-dependent degeneration (Richard, 2006).
Taken together, these results extend the knowledge of the genes involved in controlling retinal morphogenesis and preventing light-dependent PRC degeneration in the fly. Mutations in human CRB1 lead to RP12 and LCA, two severe forms of retinal dystrophy, raising the question whether mutations in the homologues of the other members of the complex might result in similar phenotypes. Understanding the molecular mechanisms leading to the mutant phenotype in the fly will certainly contribute to unraveling the pathogenesis of these retinal dystrophies in humans (Richard, 2006).
Affinity-purified rabbit antibodies against a glutathione S-transferase (GST) fusion of the C-terminal 200 amino acids common to SdtA and SdtB clearly outlines epithelial cells in wild-type embryos, but not in zygotic XN05 or XP96 mutant embryos. The speckles seen in mutant as well as wild-type embryos are background nonspecific staining. From the onset of gastrulation (stage 5 or 6), Sdt is expressed around the apical cell boundaries and the apical region of the lateral cortex. In late gastrulation, coincident with formation of the zonula adherens (ZA), Sdt starts to form a solid continuous belt around the apical boundary of epithelial cells, and is also enriched at the apical surface. Sdt distribution overlaps with adherens junction markers Armadillo (Arm) and DE-cadherin (DE-cad), although Sdt seems to localize slightly apical to adherens junctions as well. When septate junctions form at the basolateral side of epithelial cells (embryonic stage 13 or 14), Sdt retains its apical localization and association with the ZA, and does not overlap with the septate junction marker Coracle (Cor). The tracheal system, foregut and hindgut, which are part of the primary epithelia, also express Sdt at their apical surface. Apical lateral subcellular distribution of Sdt persists throughout embryonic development in epithelia, although the expression gradually decrease in stages 16 and 17. Sdt is also expressed in sensory organs. For example, in antennomaxillary complexes and chordotonal organs, strong Sdt staining is seen on the dendritic tips of sensory neurons and scolopale lumen formed by support scolopale cells (Hong, 2001).
In the fly embryo, sdt transcripts can be detected from the cellular blastoderm onwards, where they are restricted to the anlage of the ectoderm (prospective ectoderm) and excluded from that of the mesoderm. Later on, sdt transcripts are present in epithelial derivatives of the ectoderm, that is, the epidermis, the foregut and hindgut, the tracheae, the salivary glands and the Malpighian tubules, where they are localized apically. At no time is sdt RNA detectable in neuroblasts or other cells of the central nervous system. Both the tissue distribution and the subcellular localization of sdt transcripts are similar to those of crb transcripts (Bachmann, 2001).
An antiserum was raised against two synthetic peptides of the Sdt GUK domain, which therefore should recognize both Sdt-MAGUK1 and Sdt-GUK1/2. It is specific for Sdt, because no staining was observed in several sdt alleles. Apical staining is detected from gastrulation (stage 7) onwards in all epithelia derived from the ectoderm. Hence, Sdt shows the same temporal and spatial expression pattern as Crb. Sdt is colocalized with Crb in the SAC, apical to the ZA. In crb mutants, Sdt protein is lost from the plasma membrane, whereas some residual ZA components show a scattered distribution on all membranes. Sdt in crb mutants thus behaves similarly to Crb protein in sdt mutants. Overexpression of Crb results in the depletion of Sdt from the apical surface (Bachmann, 2001).
The mutations bazooka (baz) and sdt belong to a group in which mutant embryos show severe abnormalities in the differentiation of the larval cuticles, including the genes crumbs (crb) and shotgun (shg). During cellularization, the formation of cell membranes begins with the generation of cleavage furrows extending from the periphery of the embryo in a radial direction, thus establishing the normal boundaries between cells in the ectoderm. New plasma membranes are assembled until a monolayer cell sheet has formed that shares many features with epithelial cell monolayers, including epithelial cell functions and polarized membrane transport. A major redistribution of Arm and PY occurs during cellularization and early gastrulation. At mid-cellularization, staining becomes stronger on the apical aspect than in the more basal part of the lateral domain. By the end of cellularization, Arm and PY staining are strongly reduced at the basal part of the cleavage furrow that separates nuclei of cells in the blastoderm. bazooka and stardust have been identified as X-linked genes required for the formation of the ZA in the embryo. Mutations in either of these genes leads to a zygotic embryonic lethal phenotype, which is characterized by either severe malformation or an absence of the embryonic epidermis (Wieschaus, 1984 and Tepass, 1993).
Morphogenetic movements of epithelia during development underlie the normal elaboration of the final body plan. The tissue integrity critical for these movements is conferred by anchorage of the cytoskeleton by adherens junctions, initially spot and later belt-like, zonular structures, which encircle the apical side of the cell. Loss-of-function mutations in the Drosophila genes crumbs and stardust lead to the loss of cell polarity in most ectodermally derived epithelia, followed in some, such as the epidermis, by extensive apoptosis. Both mutants fail to establish proper zonulae adherentes in the epidermis. These results suggest that the two genes are involved in different aspects of this process. Further, they are compatible with the hypothesis that crumbs delimits the apical border, where the zonula adherens usually forms and where Crumbs protein is normally most abundant. In contrast, stardust seems to be required at an earlier stage for the assembly of the spot adherence junctions. In both mutants, the defects observed at the ultrastructural level are preceded by a misdistribution of Armadillo and DE-cadherin, the homologs of beta-catenin and E-cadherin, respectively, which are two constituents of the vertebrate adherens junctions. Strikingly, expansion of the apical membrane domain in epidermal cells by overexpression of crumbs also abolishes the formation of adherens junctions and results in the disruption of tissue integrity, but without loss of membrane polarity. This result supports the view that membrane polarity is independent of the formation of adherens junctions in epidermal cells (Grawe, 1996).
The zonula adherens (ZA) belongs to a family of actin-associated cell junctions called adherens junctions. Antibodies specific to cellular junctions and nascent plasma membranes have been used to study the formation of the zonula adherens in relation to the establishment of basolateral membrane polarity. The same approach was then used as a test system to identify X-linked zygotically active genes required for ZA formation. ZA formation begins during cellularization; the basolateral membrane domain is established at mid-gastrulation. By creating deficiencies for defined regions of the X chromosome, genes have been identified that are required for the formation of the ZA and the generation of basolateral membrane polarity. Embryos mutant for both stardust (sdt) and bazooka (baz) fail to form a ZA. In addition to the failure to establish the ZA, the formation of the monolayered epithelium is disrupted after cellularization, resulting in the formation of a multilayered sheet of cells by mid-gastrulation. Electron microscope analysis of mutant embryos reveals a conversion of cells exhibiting epithelial characteristics into cells exhibiting mesenchymal characteristics. To investigate how mutations that affect an integral component of the ZA itself influence ZA formation, embryos with reduced maternal and zygotic supply of wild-type Armadillo protein were studied. These embryos, like embryos mutant for both sdt and baz, exhibit an early disruption of ZA formation. These results suggest that early stages in the assembly of the ZA are critical for the stability of the polarized blastoderm epithelium (Müller, 1996).
The mutations baz and sdt belong to a group in which mutant embryos show severe abnormalities in the differentiation of the larval cuticles, including the genes crumbs (crb) and shotgun (shg). Although the similarity in the late phenotypes of these mutants shows that the respective genes are all required for the same process, i.e., epithelial differentiation, it is difficult to determine whether all these genes act in a common pathway. Nevertheless, the genes crb and sdt show an interesting genetic interaction. Using chromosomal duplications, it has been shown that the phenotype of crb (null) embryos can be rescued by an additional copy of sdt but not vice versa (Tepass, 1993). Based on these findings, a model has been proposed that positions sdt downstream of crb in a regulatory hierarchy (Tepass, 1993). This model is complicated by the fact that sdt regulates Crb protein distribution (Tepass, 1993). A more attractive model might be that sdt functions in a parallel pathway, and, in sufficient dosage, bypasses the requirement for crb (Muller, 1996).
It is equally complicated to arrange sdt and baz in a linear pathway, given that the double mutant of zygotic null alleles shows a stronger phenotype than the single mutants. The product of the baz gene is provided maternally and zygotically. Although maternal baz may rescue the hemizygous baz phenotype to a certain extent, it is difficult to explain the enhancement of the presumed null phenotype of sdt that occurs when baz is removed, assuming the two genes function in a strict pathway. In summary, it is suggested that although baz, crb and sdt are important for the same process, it is most likely that they act in different, but related pathways (Muller, 1996)
Hemizygous baz single mutants show normal Arm and NT staining up to stage 10 of embryogenesis. Even at this rather late stage, the ZA is disordered only locally while large regions of the epidermis are still normal. Similarly, sdt single mutant embryos exhibit abnormal ZA morphology only late in development. The effects of the baz;sdt double mutant are much more severe. In postgastrula embryos, adherens junction plaques are strongly reduced and often absent; these plaques are normally present at the apical-lateral junction, indicating the presence of the ZA. The relatively weak zygotic phenotype of baz can be explained by a strong maternal component for the expression of baz. baz null embryos were produced to analyze the ZA phenotype of embryos that lack both maternal and zygotic baz activity. baz null embryos show early disruption of ZA formation like that seen in baz;sdt zygotic double mutants. In particular, the concentration of Arm in the apical region of cell contact at the beginning of gastrulation is absent in both baz null mutants and the double mutants. In addition to these early alterations in ZA formation, two morphological features of both mutant phenotypes are very similar: (1) when cellularization is complete, cell shapes in the mutant embryos are aberrant; instead of the highly columnar cell shape in the epithelium of wild-type embryos, the cells have irregular outlines and some appear bottle-shaped; (2) the regular hexagonal pattern of Arm staining seen on the surface of wild-type embryos is distorted in the double mutants; the apical cell surfaces appear to vary in size, indicating that the apical domains are expanded in some cells and constricted in others. No ZAs are found in baz null mutant embryos. Germ-line null armadillo mutants exhibit a phenotype similar to baz;sdb mutants, showing that Armadillo is also required for ZA formation. It is suggested that early stages in the assembly of the ZA are critical for the assembly and/or stability of the polarized blastoderm epithelium (Muller, 1996).
Mutations in 17 genes (26 deficiencies) were characterized that interact with Armover and/or Armunder. Interaction strength varies from deficiency to point mutation, suggesting that several genes in the original deficiencies could have contributed to, or modified, the interaction. Only for 7 of the 17 genes have interactions been identical between the point mutation and the corresponding starting deficiency. The 17 genes were sorted into four groups. Group 2 consists of genes required for cell adhesion: This group includes shotgun (which encodes DE-cadherin), as expected. Also uncovered were fat (ft) and dachsous (ds). These two genes encode nonclassical cadherin characterized by a huge extracellular domain containing up to 35 cadherin repeats and a bipartite Arm binding site. Interactions with these two mutants are similar to those observed with shotgun (DE-cadherin), the only difference being that ft interacts more weakly than shg with Armover. In addition to genes encoding cadherins (classical and nonclassical), interactions have been observed with some of the genes known to be essential (directly or indirectly) for the assembly or maintenance of adherens junctions -- stardust (sdt), discs-large (dlg), and crumbs (crb). These interact in the same direction as shg; however, the suppression of Armunder is always weaker and only dlgM52 enhances Armover to the same extent as zw3M11 (Greaves, 1999).
Crb is the earliest zygotically expressed apical transmembrane protein, but nothing is known about the cis-regulatory sequences that target it to the apical face of the cell nor the mechanisms and proteins required for this process. Nothing is known about the function of the large extracellular domain; its overexpression in a secreted or membrane-anchored form (lacking the cytoplasmic domain) does not induce any mutant phenotype. Embryos devoid of maternal Dlt fail to localize Crb. Since the blastoderm epithelium of these embryos itself lacks cell polarity, however, all other defects, including improper Crb localization, could be regarded as secondary effects. In embryos mutant for stardust, Crb is first expressed apically, but during germ band extension it is no longer detectable, making stardust a likely regulator for the maintenance of apical localization of Crb. In agreement with this, stardust mutant embryos develop a phenotype nearly identical to that of crb mutant embryos (Klebes, 2000).
To identify X chromosomal genes required for salivary gland development in the Drosophila embryo, embryos hemizygous for EMS-induced lethal mutations were screened to find mutations causing gross morphological defects in salivary gland development. The parental strain carried a lac Z transgene on the second chromosome, which was specifically expressed in the salivary glands so the mutations could be unambiguously identified. Embryos from 3,383 lines were tested for salivary gland abnormalities following lacZ staining. From 63 lines exhibiting aberrant salivary gland phenotypes, 52 stable lines were established containing mutations affecting salivary gland development. From these, 39 lines could be assigned to nine complementation groups: armadillo, brinker, folded gastrulation, giant, hindsight, Notch, runt, stardust and twisted gastrulation (Lammel, 2000).
The identified X chromosomal genes with respect to their possible contributions to salivary gland development is discussed here. For mutations in giant and Notch, severe defects or the absence of the salivary glands have been described previously. In giant mutant larvae, the gnathocephalic structures are affected, which correspond to the labial segment. This is the region where most of the salivary gland anlage originates. giant is required for the expression of Sex combs reduced (Scr), the master regulator for salivary gland development. Scr expression is absent in the labial lobe in giant mutations. The small glands seen in the absence of giant may form from remaining cells of PS 2 still expressing Scr protein. Notch is involved in the formation of epidermal cells, and in its absence neural precursor cells are formed instead. As a result, in Notch mutants a coherent sheet of epidermal cells is found only in the most dorsal position, outside the neurogenic ectoderm. Only a few ventrolateral epidermal cells are left, which may fail to form a salivary gland anlage. Similar explanations may hold for mutations such as stardust, which affect other general aspects of epithelial development, since in the presence of these mutations the maintenance of coherent epidermal sheets is disrupted (Lammel, 2000 and references therein).
Apicobasal cell polarity is crucial for morphogenesis of photoreceptor rhabdomeres and adherens junctions (AJs) in the Drosophila eye. Crumbs (Crb) is specifically localized to the apical membrane of photoreceptors, providing a positional cue for the organization of rhabdomeres and AJs. The Crb complex consisting of Crb, Stardust (Sdt) and Discs-lost (Dlt) colocalizes with another protein complex containing Par-6 and atypical protein kinase C (aPKC) in the rhabdomere stalk of photoreceptors. Loss of each component of the Crb complex causes age-dependent mislocalization of Par-6 complex proteins, and ectopic expression of Crb intracellular domain is sufficient to recruit the Par-6 complex. The absence of Par-6 complex proteins results in severe mislocalization and loss of Crb complex. Dlt directly binds to Par-6, providing a molecular basis for the mutual dependence of the two complexes. These results suggest that the interaction of Crb and Par-6 complexes is required for the organization and maintenance of apical membranes and AJs of photoreceptors (Nam, 2003).
The strong dependence of Crb localization on Sdt and Dlt suggests that Crb may be destabilized or may not be targeted to the membrane in the absence of Sdt or Dlt. It is intriguing that Sdt and Dlt are lost only partially in the absence of Crb. The findings of a direct interaction between Dlt and Par-6 suggest that Sdt-Dlt can still be targeted to the membrane in the absence of Crb through the binding of Dlt to the Par-6 complex. However, it is important to note that Dlt is essentially lost in sdt mutant clones and vice versa. This raises an intriguing possibility that Dlt or Sdt are dependent on each other in vivo to be targeted to the apical membrane via binding to either Crb or Par-6. This mutual dependency between Dlt and Sdt may explain why Dlt and Sdt are lost in the absence of the other, rather than being associated with the Par-6 complex (Nam, 2003).
The interaction between the Crb and Par-6 complexes is mediated by the PDZ3 region of Dlt and the N-terminal domain of Par-6. The N-terminal domain of Par-6 is also used for binding aPKC. Therefore, a potential function of Dlt is to bind Par-6 in competition with aPKC or to facilitate the interaction of Par-6 with aPKC or other Par-6 binding proteins. Mutant analysis indicates that loss of Dlt and Sdt in sdt- clones causes mislocalization of both Crb and Par-6 complex proteins. This suggests that Sdt-Dlt interaction provides a scaffold to recruit Crb complex to the Par-6 complex and enhance the stability of these two complexes rather than functioning as a competitor for aPKC (Nam, 2003).
Proteins in Crb and Par-6 complexes consist of multiple functional domains which may be involved in diverse protein-protein interactions. A recent study has shown that in mammalian cell culture systems the PDZ domain of Par-6 binds not only Par-3 but also the N terminus of Pals1. These results suggest that the crosstalk between the Crb and Par-6 complexes is mediated by multiple domain-specific interactions. Evidence from genetic analysis using mutants suggests that the crosstalk between the two complexes is mutually required for normal organization of apical membranes and AJs in vivo, and also provides a basis for partial redundancy of these complexes in the organization of photoreceptor cell polarity. Interestingly, when either Crb or Sdt is lost, mislocalization or elimination of other associated components including Par-6 complex proteins becomes more severe in the age-dependent manner. This suggests that the Crb complex may be required for the maintenance rather than the formation of the Par-6 complex. The age-dependent degenerative phenotype may be related to the requirement of extensive apical membrane growth to make rhabdomeres and AJs along the growing axis of photoreceptors during pupal stage. Loss of any one component of the Crb complex is likely to be increasingly more detrimental as the process of membrane reorganization proceeds. In crb- or sdt- mutants, significant fractions of Par-6 complex proteins remain in the membrane despite the age-dependent and progressive mislocalization of apical markers. By contrast, loss of Par-6 or aPKC results in mislocalization of Dlt from the apical membrane. This suggests that the Par-6 complex plays essential functions for membrane localization of Crb complex proteins. Furthermore, both Par-6 and aPKC seem to be important for survival and/or proliferation of retinal cells because mutant clones were very small compared with adjacent twin spots and often completely disrupted, probably due to cell death. This is consistent with the findings of frequent apoptosis in aPKC- or par-6- embryos (Nam, 2003).
An important distinction of Par-6 complex in the photoreceptors from other epithelia is the localization of Baz. Baz localizes with Crb complex in the subapical membrane or both the subapical region and AJ in the Drosophila embryonic epithelia. Vertebrate Par-3 also localizes to the apical tight junction in vertebrate epithelial cells. By contrast, Baz in the photoreceptors is specifically positioned in the AJs basal to the all other proteins in the Crb/Par-6 complexes. Baz and Arm are recruited together to ectopic membrane sites by misexpression of CrbJM, suggesting that Baz is an integral component of AJ. However, Baz is not recruited by CrbPBM, whereas Par-6 and aPKC can be ectopically recruited by CrbPBM rather than CrbJM. Therefore, Baz appears to be recruited to AJ independently of Par-6/aPKC (Nam, 2003).
Intriguingly, despite its specific localization to AJs, loss of Baz results in most severe disruption of AJ as well as the more apical Dlt domain. It has been proposed that the Par-6/aPKC cassette is recruited to the site of cell-cell contact and then moves along the most apical zone of the developing cell-cell contact. In this process, an important step for cell polarity formation is to tether the cytoplasmic Par-6/aPKC complex to the site of cell-cell contact at the membrane, which is mediated by the interaction of Par-3 and a membrane protein JAM. Therefore, the results that baz mutation causes loss of Dlt and AJs support the crucial role of Baz in the initial step of cell polarization. However, the distinct localization of Baz from Par-6 and aPKC in the photoreceptors suggests that the mode of Baz localization varies in different systems. In photoreceptors, Baz may be targeted to the membrane with Par-6 but be sorted out from Par-6 in subsequent steps of polarization to remain in the AJs, whereas Par-6-aPKC-Baz cassette remains together in the complex in other epithelia. In contrast to Baz, aPKC localizes to both rhabdomere stalk and AJ, suggesting that Baz and Par-6 are completely separated during polarization while aPKC is not sorted from both Par-6 and Baz. The critical function of Baz in the localization of Crb complex in the rhabdomere stalk is consistent with the requirement of Baz for Crb localization in embryonic epithelia. However, the requirement of Baz in the embryo appears to be dependent on the stage of development since Crb distribution in the absence of Baz becomes normal in late embryos. On the contrary, such stage-dependent recovery of Crb complex localization has not been observed in baz- photoreceptor cells (Nam, 2003).
Recent studies have shown that mutations in human CRB1 cause RP12 and LCA, severe recessive retinal diseases, emphasizing the importance of Crb family proteins in the eyes of mammals including humans. The Drosophila Crb and human CRB1 are localized in analogous subcellular membrane domains of photoreceptors, the rhabdomere stalk and the inner segment in Drosophila and human photoreceptors, respectively. Besides similar subcellular localization, Crb and human CRB1 are functionally conserved. Age-dependent photoreceptor defects in the crb mutant also provide analogy to age-dependent retinal degeneration in RP12/LCA patients. These studies here imply that hCRB1 may function as a protein complex with homologs of Sdt and Dlt and such a complex may interact with a homologous Par-6 complex. Whether such homologous human genes are the targets of inherited retinal diseases such as RP remains to be studied (Nam, 2003).
Bachmann, A., Schneider, M., Theilenberg, E., Grawe, F. and Knust, E. (2001). Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity. Nature 414(6864): 638-43. 11740560
Bilder, D., Schober, M. and Perrimon, N. (2003). Integrated activity of PDZ protein complexes regulates epithelial polarity. Nat. Cell Biol. 5(1): 53-8. 12510194
Hong, Y., Stronach, B., Perrimon, N., Jan, L. Y. and Jan, Y. N. (2001). Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts. Nature 414(6864): 634-8. 11740559
Grawe, F., et al. (1996). The Drosophila genes crumbs and stardust are involved in the biogenesis of adherens junctions. Development 122(3): 951-959. 8631272
Greaves, S., et al. (1999). A screen for identifying genes interacting with Armadillo, the Drosophila homolog of ß-catenin. Genetics 153: 1753-1766.
Kamberov, E. et al. Molecular cloning and characterization of Pals, proteins associated with mLin-7. J. Biol. Chem. 275: 11425-11431. 10753959
Kempkens, O., et al. (2006). Computer modelling in combination with in vitro studies reveals similar binding affinities of Drosophila Crumbs for the PDZ domains of Stardust and DmPar-6. Eur. J. Cell Biol. 85(8): 753-67. Medline abstract: 16697075
Klebes, A. and Knust, E. (2000). A conserved motif in Crumbs is required for E-cadherin localisation and zonula adherens formation in Drosophila. Curr. Biol.
Lammel, U. and Saumweber, H. (2000). X-linked loci of Drosophila melanogaster causing defects in the morphology of the embryonic salivary glands. Dev. Genes Evol. (2000) 210: 525-535. 11180803
Makarova, O., Roh, M. H., Liu, C. J., Laurinec, S. and Margolis, B. (2003). Mammalian Crumbs3 is a small transmembrane protein linked to protein associated with Lin-7 (Pals1). Gene 302: 21-29. 12527193
Michel, D., et al. (2005). PATJ connects and stabilizes apical and lateral components of tight junctions in human intestinal cells. J. Cell Sci. 118: 4049-4057. 16129888
Müller, H.-A. and Wieschaus, E. (1996). armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila. J. Cell Biol. 134: 149-163
Nam, S.-C. and Choi, K.-W. (2003). Interaction of Par-6 and Crumbs complexes is essential for photoreceptor morphogenesis in Drosophila. Development 130: 4363-4372. 12900452
Nam, S. C. and Choi, K. W. (2006). Domain-specific early and late function of Patj in Drosophila photoreceptor cells. Dev. Dyn. 235(6): 1501-7. Medline abstract: 16518799
Richard, M., Grawe, F. and Knust, E. (2006). Patj plays a role in retinal morphogenesis and protects against light-dependent degeneration of photoreceptor cells in the Drosophila eye. Dev. Dyn. 235(4): 895-907. Medline abstract: 16245332
Roh, M. H., et al. (2002). The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost. J. Cell Biol. 157(1): 161-72. 11927608
Roh, M. H., et al. (2003). The Crumbs3-Pals1 complex participates in the establishment of polarity in mammalian epithelial cells. J. Cell Sci. 116: 2895-2906. 12771187
Schonbaum, C. P., et al. (1992). The Drosophila melanogaster stranded at second (sas) gene encodes a putative epidermal cell surface receptor required for larval development. Dev. Biol. 151: 431-445.
Tepass, U. and Knust, E. (1993). Crumbs and stardust act in a genetic pathway that controls the organization of epithelia in Drosophila melanogaster. Dev. Biol. 159(1): 311-26.
van Rossum, A. G., et al. (2006). Pals1/Mpp5 is required for correct localization of Crb1 at the subapical region in polarized Muller glia cells. Hum. Mol. Genet. 15(18): 2659-72. Medline abstract: 16885194
Weischaus, E., Nusslein-Volhart, C. and Jurgens, G. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster III. Zygotic loci on the X chromosome and the fourth chromosome. Roux's Arch. Dev. Biol. 193: 296-307
date revised: 10 July 2007
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