Gene name - crumbs
Cytological map position -
Function - cell polarity
Symbol - crb
Genetic map position -
Classification - trans-membrane, EGF and laminin A repeats
Cellular location - cell surface
|Recent literature||Sherrard, K.M. and Fehon, R.G. (2015). The transmembrane protein Crumbs displays complex dynamics during follicular morphogenesis and is regulated competitively by Moesin and aPKC. Development 142(10):1869-78. PubMed ID: 25926360
The transmembrane protein Crumbs (Crb) functions in apical polarity and epithelial integrity. To better understand its role in epithelial morphogenesis, this study examined Crb localization and dynamics in the late follicular epithelium of Drosophila. Crb was unexpectedly dynamic during middle-to-late stages of egg chamber development, being lost from the marginal zone (MZ) in stage 9 before abruptly returning at the end of stage 10b, then undergoing a pulse of endocytosis in stage 12. The reappearance of MZ Crb was necessary to maintain an intact adherens junction and MZ. Although Crb has been proposed to interact through its juxtamembrane domain with Moesin (Moe), a FERM domain protein that regulates the cortical actin cytoskeleton, the functional significance of this interaction is poorly understood. This study found that whereas the Crb juxtamembrane domain was not required for adherens junction integrity, it was necessary for MZ localization of Moe, aPKC and F-actin. Furthermore, Moe and aPKC functioned antagonistically, suggesting that Moe limits Crb levels by reducing its interactions with the apical Par network. Additionally, Moe mutant cells lost Crb from the apical membrane and accumulated excess Crb at the MZ, suggesting that Moe regulates Crb distribution at the membrane. Together, these studies reveal reciprocal interactions between Crb, Moe and aPKC during cellular morphogenesis.
|Wei, Z., Li, Y., Ye, F. and Zhang, M. (2015). Structural basis for the phosphorylation-regulated interaction between the cytoplasmic tail of cell polarity protein crumbs and the actin-binding protein moesin. J Biol Chem 290: 11384-11392. PubMed ID: 25792740
The type I transmembrane protein Crumbs (Crb) plays critical roles in the establishment and maintenance of cell polarities in diverse tissues. As such, mutations of Crb can cause different forms of cancers. The cell intrinsic role of Crb in cell polarity is governed by its conserved, 37-residue cytoplasmic tail (Crb-CT) via binding to moesin and protein associated with Lin7-1 (PALS1). However, the detailed mechanism governing the Crb.moesin interaction and the balance of Crb in binding to moesin and PALS1 are not well understood. This paper reports the 1.5 A resolution crystal structure of the moesin protein 4.1/ezrin/radixin/moesin (FERM).Crb-CT complex of mouse, revealing that both the canonical FERM binding motif and the postsynaptic density protein-95/Disc large-1/Zonula occludens-1 (PDZ) binding motif of Crb contribute to the Crb.moesin interaction. It was further demonstrated that phosphorylation of Crb-CT by atypical protein kinase C (aPKC) disrupts the Crb.moesin association but has no impact on the Crb.PALS1 interaction. The above results indicate that, upon the establishment of the apical-basal polarity in epithelia, apical-localized aPKC can actively prevent the Crb.moesin complex formation and thereby shift Crb to form complex with PALS1 at apical junctions. Therefore, Crb may serve as an aPKC-mediated sensor in coordinating contact-dependent cell growth inhibition in epithelial tissues.
Vichas, A., Laurie, M.T. and Zallen, J.A.
(2015). The Ski2-family
helicase Obelus regulates Crumbs alternative splicing and cell polarity.
J Cell Biol 211: 1011-1024. PubMed ID: 26644515
Alternative splicing can have profound consequences for protein activity, but the functions of most alternative splicing regulators are not known. This study shows that Obelus, a conserved Ski2-family helicase, is required for cell polarity and adherens junction organization in the Drosophila melanogaster embryo. In obelus mutants, epithelial cells display an expanded apical domain, aggregation of adherens junctions at the cell membrane, and microtubule-dependent defects in centrosome positioning. Through whole-genome transcriptome analysis, it was found that Obelus is required for the alternative splicing of a small number of transcripts in the early embryo, including the pre-mRNA that encodes the apical polarity protein Crumbs. In obelus mutants, inclusion of an alternative exon results in increased expression of a Crumbs isoform that contains an additional epidermal growth factor-like repeat in the extracellular domain. Overexpression of this alternative Crumbs isoform recapitulates the junctional aggregation and centrosome positioning defects of obelus mutants. These results indicate that regulation of Crumbs alternative splicing by the Obelus helicase modulates epithelial polarity during development.
|Lin, Y. H., Currinn, H., Pocha, S. M., Rothnie, A., Wassmer, T. and Knust, E. (2015). AP-2 complex-mediated endocytosis of Drosophila Crumbs regulates polarity via antagonizing Stardust. J Cell Sci [Epub ahead of print]. PubMed ID: 26527400
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.
|Flores-Benitez, D. and Knust, E. (2015). Crumbs is an essential regulator of cytoskeletal dynamics and cell-cell adhesion during dorsal closure in Drosophila. Elife 4 [Epub ahead of print]. PubMed ID: 26544546
The evolutionarily conserved Crumbs protein is required for epithelial polarity and morphogenesis. This study identified a novel role of Crumbs as a negative regulator of actomyosin dynamics during dorsal closure in the Drosophila embryo. Embryos carrying a mutation in the FERM (protein 4.1/ezrin/radixin/moesin) domain-binding motif of Crumbs die due to an overactive actomyosin network associated with disrupted adherens junctions. This phenotype is restricted to the amnioserosa and does not affect other embryonic epithelia. This function of Crumbs requires DMoesin, the Rho1-GTPase, class-I p21-activated kinases and the Arp2/3 complex. Data presented here point to a critical role of Crumbs in regulating actomyosin dynamics, cell junctions and morphogenesis.
|Whitney, D. S., Peterson, F. C., Kittell, A. W., Egner, J. M., Prehoda, K. E. and Volkman, B. F. (2016). Binding of Crumbs to the Par-6 CRIB-PDZ module is regulated by Cdc42. Biochemistry 55: 1455-1461. PubMed ID: 26894406
Par-6 is a scaffold protein that organizes other proteins into a complex required to initiate and maintain cell polarity. Cdc42-GTP binds the CRIB module of Par-6 and alters the binding affinity of the adjoining PDZ domain. Allosteric regulation of the Par-6 PDZ domain was first demonstrated using a peptide identified in a screen of typical carboxyl-terminal ligands. Crumbs, a membrane protein that localizes a conserved polarity complex, was subsequently identified as a functional partner for Par-6 that likely interacts with the PDZ domain. This study shows by nuclear magnetic resonance that Par-6 binds a Crumbs carboxyl-terminal peptide and reports the crystal structure of the PDZ-peptide complex. The Crumbs peptide binds Par-6 more tightly than the previously studied carboxyl peptide ligand and interacts with the CRIB-PDZ module in a Cdc42-dependent manner. The Crumbs:Par-6 crystal structure reveals specific PDZ-peptide contacts that contribute to its higher affinity and Cdc42-enhanced binding. Comparisons with existing structures suggest that multiple C-terminal Par-6 ligands respond to a common conformational switch that transmits the allosteric effects of GTPase binding.
|Nguyen, M.B., Vuong, L.T. and Choi, K.W. (2016).
Ebi modulates wing growth by ubiquitin-dependent downregulation of Crumbs in Drosophila. Development 143: 3506-3513. PubMed ID: 27702784
Notch signaling at the dorsoventral (DV) boundary is essential for patterning and growth of wings in Drosophila. The WD40 domain protein Ebi has been implicated in the regulation of Notch signaling at the DV boundary. This study shows that Ebi regulates wing growth by antagonizing the function of the transmembrane protein Crumbs (Crb). Ebi physically binds to the extracellular domain of Crb (Crbext), and this interaction is specifically mediated by WD40 repeats 7-8 of Ebi and a laminin G domain of Crbext. Wing notching resulting from reduced levels of Ebi is suppressed by decreasing the Crb function. Consistent with this antagonistic genetic relationship, Ebi knockdown in the DV boundary elevates the Crb protein level. Furthermore, Ebi is required for downregulation of Crb by ubiquitylation. Taken together, the study proposes that the interplay of Crb expression in the DV boundary and ubiquitin-dependent Crb downregulation by Ebi provides a mechanism for the maintenance of Notch signaling during wing development.
|Nemetschke, L. and Knust, E. (2016). Drosophila Crumbs prevents ectopic Notch activation in developing wings by inhibiting ligand-independent endocytosis. Development 143(23): 4543-4553. PubMed ID: 27899511
Many signalling components are apically restricted in epithelial cells, and receptor localisation and abundance is key for morphogenesis and tissue homeostasis. Hence, controlling apicobasal epithelial polarity is crucial for proper signalling. Notch is a ubiquitously expressed, apically localised receptor, which performs a plethora of functions; therefore, its activity has to be tightly regulated. This study shows that Drosophila Crumbs, an evolutionarily conserved polarity determinant, prevents Notch endocytosis in developing wings through direct interaction between the two proteins. Notch endocytosis in the absence of Crumbs results in the activation of the ligand-independent, Deltex-dependent Notch signalling pathway, and does not require the ligands Delta and Serrate or γ-secretase activity. This function of Crumbs is not due to general defects in apicobasal polarity, as localisation of other apical proteins is unaffected. These data reveal a mechanism to explain how Crumbs directly controls localisation and trafficking of the potent Notch receptor, and adds yet another aspect of Crumbs regulation in Notch pathway activity. Furthermore, the data highlight a close link between the apical determinant Crumbs, receptor trafficking and tissue homeostasis.
Crumbs protein is essential for the biogenesis of the adherens junction and the establishment of apical polarity in ectodermally derived epithelial cells. The adherens junction is a multiprotein complex that attaches one cell to another in an epithelial layer. The junction is not spread randomly between the cells, but is found in a belt-like, zonular structure encircling the apical side of the cell.
The apical side of the cell faces the outside of the embryo, in opposition to the inward facing basal side. Placement of the adherens junction is critical because it signals to the cell which side is out and which side is in, preventing the mixing of apical cell membrane tissue with the biochemically distinct basal cell membrane, and thereby assuring cell polarity.
DE-cadherin (shotgun) and Armadillo comprise the two main constituents of the adherens junction. DE-cadherin is a homophilic adhesion transmembrane molecule that links the outside of the cell with the inside. Armadillo, the Drosophila homolog of beta-catenin, is a molecule that links the adherens junction with the cell's cytoskeleton.
crumbs mutants fail to establish adherens junctions and thus fail to establish epithelial cell polarity. Crumbs protein delimits the apical border, thus establishing the proper position for the border's placement . The defect in crumbs minus mutants results in a misdistribution of Armadillo and DE-cadherin, resulting in a disruption of tissue integrity. Curiously, despite the lack of adherens junction formation in such mutants, there is no accompanying loss of membrane polarity. This supports the view that membrane polarity exists prior to the formation of adherens junctions, and establishes the pattern of proper placement of the junction (Grawe, 1996).
Crumbs protein is distributed over the entire apical cell surface of epithelial cells and accumulates at the outer margin of the apical membrane where neighboring cells are in contact. However, no Crumbs protein is detected at the zonula adherens. This suggests that the polarizing activity of Crumbs arises from a direct or indirect binding of the Crumbs protein to adherens junction material at the outer rim of the marginal zone. The retention of adherens junction material in direct contact with the marginal zone would facilitate the formation of the zonula adherens from patches of adherens junction material that assemble through interaction with Crumbs protein (Tepass, 1996).
A conserved motif in Crumbs is required for E-cadherin localisation and zonula adherens formation in Drosophila. Expression of just the short membrane-bound cytoplasmic domain is sufficient to rescue major defects associated with the loss of crumbs function. The cytoplasmic domain of Crumbs is highly conserved in two putative crumbs homologs in C. elegans. To assess the significance of conserved residues, various point mutations and deletions were introduced into this region. Two functional domains were revealed: an amino-terminal region and the carboxy-terminal amino acids EERLI. Both are necessary for rescue of the crumbs phenotype. The EERLI motif interacts with Discs Lost (now redefined as Drosophila Patj), a cytoplasmic protein containing PDZ domains. Overexpression of the Crumbs cytoplasmic domain induces a transition from the single-layered epithelium to a multilayered tissue. This transition is associated with redistribution of the Drosophila homolog of the cell adhesion molecule E-cadherin, and depends on the presence of the EERLI motif (Klebes, 2000).
Two C. elegans genes that encode transmembrane proteins with multiple EGF-like repeats and short cytoplasmic domains were detected in the database. The cytoplasmic domains of both proteins, called CeCrb1 and CeCrb2, also consist of 37 amino acids, nine of which are conserved in all three proteins. A transgene (CD2-IntraCE), encoding the cytoplasmic domain of CeCrb1 fused to the rat transmembrane protein CD2 (which provides a transmembrane domain and a tag) was expressed in wild-type Drosophila embryos. The phenotypic consequences were compared with those induced by overexpression of a corresponding Drosophila fusion protein (CD2-IntraWT). Both CD2-Intra proteins induce the same phenotype, which is indistinguishable from that caused by the expression of Myc-IntraWT: the epidermis became multilayered and DE-cadherin and phosphotyrosine-containing epitopes are mislocalized. This shows that the functionally important regions responsible for inducing formation of a multilayered epidermis are conserved in the cytoplasmic domain of CeCrb1 (Klebes, 2000).
Data presented here suggest a model in which the Drosophila Crb protein organizes the assembly of an apically localized protein scaffold in epithelial cells that is required for the proper formation and localisation of the ZA. This scaffold includes the protein Dlt and probably other, as yet unidentified, proteins, its assembly depends on the carboxy-terminal segment of Crb. The model further suggests that the Crb-mediated control of DE-cadherin localization depends on interaction between the Crb cytoplasmic domain and the PDZ protein Dlt. Neither DE-cadherin nor Dlt are localized in crb mutant embryos, whereas both proteins are sequestered by mislocalized Crb. However Dlt remains apically localized after overexpression of DE-cadherin. The interaction of Crb with Dlt depends on Crb's carboxy-terminal motif, EERLI. This motif is also necessary for misdistribution of DE-cadherin upon Crb overexpression and for the rescue of crb mutant embryos. The presence of four PDZ domains in Dlt makes it an ideal partner for recruiting other proteins into a hypothetical Crb-dependent, membrane-associated protein network. PDZ domains have been shown to act as versatile organizers of multiprotein complexes. In many cases, the binding site of the interacting protein, often a transmembrane protein, is localized at its carboxyl terminus and ends with a hydrophobic amino-acid residue. Class I PDZ domains bind a conserved S/T-X-V motif (where X is any amino acid), whereas class II domains recognize ligands that carry a hydrophobic amino-acid residue at the -2 position. Since the Dlt-binding site in Crb differs from these motifs, the first PDZ domain of Dlt, which binds to Crb in vitro, may belong to a different class. The presence of the ERLI motif in both C. elegans homologs and the similarities between the phenotypes produced by overexpression of CD2-IntraWT and CD2-IntraCE in the Drosophila embryo suggest that this region might mediate comparable interactions in the nematode. Not surprisingly, a protein similar to Drosophila Dlt has also been detected in the C. elegans database, pointing to the possible conservation of additional components of the postulated protein network (Klebes, 2000).
Data indicate that the EERLI motif is necessary, but not sufficient, to rescue the phenotype of crb mutant embryos. Rescue also requires an intact amino-terminal region of the cytoplasmic domain. It is tempting to speculate that the region containing the mutated amino acids may be involved in additional protein-protein interactions. A comparable situation is provided by a group of transmembrane proteins, including glycophorin C, beta-neurexin and syndecans, that have been identified, respectively, as ligands for the class II PDZ proteins p55, CASK and syntenin. The cytoplasmic tails of these proteins terminate in the tetrapeptides EYFI, EYYV and EFYA, respectively, and show additional conservation in their amino-terminal regions. For glycophorin C it has been demonstrated that the 12-residue sequence immediately adjacent to the membrane binds directly to protein 4.1, a member of the 4.1 superfamily, which includes, among others, the so-called ERM proteins (ezrin, radixin, moesin). The latter proteins provide a linkage between cell-surface receptors and the spectrin/actin cytoskeleton. The 12-residue sequence of glycophorin C that binds protein 4.1 includes a Gly8-Thr9-Tyr10 motif, which is Gly8-Ser9-Tyr10 in beta-neurexin and all syndecans. The corresponding region of Drosophila Crb also contains a Gly8-Thr9-Tyr10 motif at an equivalent position (Gly-His/Lys-Tyr in the C. elegans proteins); mutating Tyr10 to alanine completely abolishes the rescuing function. It is unlikely that the Drosophila protein 4.1 homolog, encoded by coracle, is a partner of Crb in wild-type embryos. Coracle is associated with septate junctions, which are localized basally to the ZA, and colocalizes with Discs Large, a PDZ-domain protein, and beta-neurexin IV. The amino-terminal region conserved between Drosophila and the two C. elegans homologs extends further, to Gly8-X9-Tyr10-X(11-15)-Glu16. The data clearly show that Glu16, which is also a charged amino acid in syndecans, glycophorin C and neurexin, is also of crucial importance for the rescuing function (Klebes, 2000).
There is a further indication of possible involvement of the amino-terminal region of the Crb cytoplasmic domain in interactions with other, as yet unknown, proteins closely associated with the plasma membrane. All CD2 fusion proteins used in this study fail to rescue crb mutant embryos, even those containing the full-length cytoplasmic domain. Whereas the Myc fusion proteins contain the Crb transmembrane domain, immediately followed by the cytoplasmic portion, CD2 fusion proteins contain the CD2 transmembrane domain and provide a spacer of 45 amino acids between the membrane and the cytoplasmic tail of Crb. This spacing could prevent interactions between the cytoplasmic segment of Crb and a hypothetical partner localized at the membrane. At present, however, it cannot be determined whether it is this spacer, the lack of the Crb transmembrane domain, or some other feature of the CD2 fusion protein that is responsible for the lack of rescuing function (Klebes, 2000).
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 localisation, 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. Because the molecular nature of the stardust gene is not yet known, no information can be obtained about its relationship with Crb expression at present (Klebes, 2000).
The formation of tubular structures from epithelial sheets is a key process of organ formation in all animals, but the cytoskeletal rearrangements that cause the cell shape changes that drive tubulogenesis are not well understood. Using live imaging and super-resolution microscopy to analyze the tubulogenesis of the Drosophila salivary glands, this study found that an anisotropic plasma membrane distribution of the protein Crumbs, mediated by its large extracellular domain, determines the subcellular localization of a supracellular actomyosin cable in the cells at the placode border, with myosin II accumulating at edges where Crumbs is lowest. This study shows that Crumbs directs aPKC anisotropy which negatively regulate myosin II, probably through Rok. Laser ablation shows that the cable is under increased tension, implying an active involvement in the invagination process. Crumbs anisotropy leads to anisotropic distribution of aPKC, which in turn can negatively regulate Rok, thus preventing the formation of a cable where Crumbs and aPKC are localized (Roper, 2012).
Myosin II has emerged as a key player in morphogenesis because of its ability to form contractile structures together with F-actin that can directly alter the shapes of cells. Different pools of myosin II within epithelial cells undergoing morphogenesis have been observed, namely apical junctional myosin, apical medial myosin, and in addition myosin organized into supracellular structures termed myosin cables or purse-strings. All three myosin II pools have been shown to be important for epithelial morphogenesis, but how much the activities of the pools depend on each other and how their specific assembly is regulated is much less clear (Roper, 2012).
Using the formation of the invagination of the salivary glands in the fly embryo as a model allowed me to analyze a morphogenetic process in which all three different pools of myosin are present. Upon specification of the gland placode, myosin II levels are drastically upregulated in the secretory cells of the placode, and myosin accumulates at cortical regions and medially within the apical 'dome' of each cell. In addition, a supracellular myosin cable surrounding the placode is formed in a process by which parts of existing structures (remnants of parasegmental cables) are joined together with a newly specified dorsal section of the cable (Roper, 2012).
Compared to mesoderm invagination in the fly, a well-studied process that depends on both apical medial and cortical myosin assemblies, the invagination of the tubes of the salivary gland topologically rather resembles wound healing or dorsal closure processes, as the surrounding epidermis is drawn in from around the placode to cover the patch where cells are invaginating into the embryo (Roper, 2012).
All three processes have in common that the patch of cells 'disappearing' from the plane of the epithelium is surrounded by a contractile actomyosin cable. In contrast to wound healing and dorsal closure, the cable in the case of salivary gland tubulogenesis is assembled within the cells on the inside, whereas it is assembled in the surrounding epithelial cells in the former two instances. Thus, the signal for cable assembly is provided by the 'inside' cells in the salivary gland placode (Roper, 2012).
The laser ablation data presented in this study clearly demonstrate that the cable around the placode is under increased tension, even when compared to other myosin enriched edges. The tension is in magnitude comparable to the tension determined for shorter supracellular myosin cables observed during germband extension in the fly embryo. This increased tension indicates active involvement in the invagination process. Previous modeling studies on sea urchin invagination have shown that a contractile apical ring surrounding a placode could be a driving force for invagination. Interestingly, upon laser ablation the cable around the salivary gland placode was very quickly repaired, suggesting a continuous signal to assemble myosin at the outermost surface of the placode. This fast repair precluded laser ablation as a means of probing function of the cable in the invagination in contrast to medial and junctional myosin (Roper, 2012).
Crumbs, the transmembrane component of the apical protein complex, shows a very striking anisotropic localization at the border of the placode, that is complementary to the accumulation of myosin II forming the cable. The data strongly support a model whereby Crumbs intracellular tails at cell edges facing toward the inside of the placode recruit aPKC, which can act as a negative regulatory factor impinging on Rok, thus preventing cable assembly at edges containing high levels of Crumbs tails. This leaves active Rok at the cell edges forming the placode boundary, where it acts to recruit myosin into the cable.
Interestingly, only the presence or artificial introduction of cortical anisotropy of Crumbs and downstream aPKC has this effect. The central cells of the placode that are not forming the boundary all show strongly upregulated levels of Crumbs, aPKC, Rok, and myosin II, but in these cells a high density of Crumbs tails does not preclude accumulation of junctional membrane-proximal myosin. Thus, the change in density of Crumbs tails, not the overall concentration, is instructive in this system (Roper, 2012).
Crumbs has previously been shown to have an effect on salivary gland morphogenesis through a proposed regulation of the apical membrane domain and has been implicated in tracheal pit invagination through regulation of phospho-Moesin). Also, members of the Crumbs polarity complex have been shown to be able to interact with the Par3 (Bazooka)/Par-6/aPKC complex (e.g., Par-6 can bind to Crumbsintra; aPKC can phosphorylate Crumbsintra. Also, an anticorrelation between localization of Crb and aPKC compared to Lgl and myosin has been described in the denticle belts of the fly epidermis (Kaplan, 2010). This work now describes a potential link from Crumbs through aPKC to Rok and myosin II, which would link the interaction of two different polarity factors directly with the coordination of morphogenesis through myosin II at a molecular level (Roper, 2012).
The large extracellular domain of Crumbs has long posed an enigma with regard to its role. Crumbs' function in epithelial polarity can mostly be mediated by its intracellular domain (Klebes, 2000). Only for photoreceptor morphogenesis, the extracellular domain appears required within the fly, though its molecular role is unclear. The protein domains present in the extracellular domain, namely EGF repeats and lamG domains, are both found in many classical and nonclassical cadherins. Data presented in this study suggest that Crumbs could be organized in the plasma membrane through homophilic interactions of the extracellular domains between molecules on neighboring cells: Crumbs shows highly anisotropic localization within the wild-type placode but also within wild-type cells bordering a crumbs mutant clone (Chen, C. L., 2010) or in clusters of Crumbs expressing cells in a null mutant embryo and within cells at the edge of an ectopic step change in Crumbs expression levels. Also in vitro, Crumbs accumulates at contact zones between expressing cells. The extracellular domain appears the ideal candidate to mediate this anisotropy, which is supported by the following findings: (1) the CrbTMextra-GFP shows anisotropic localization at borders with cells not expressing the construct; (2) endogenous Crumbs in wild-type cells is induced to localize in an anisotropic fashion when neighboring cells are depleted of endogenous Crumbs and only express the intracellular domain; and (3) the Crbintra-FLAG shows uniform expression in cells. These observations exclude that another transmembrane protein that interacts with the intracellular domain of Crumbs in equal stoichiometry could mediate the anisotropy, though it cannot formally exclude that another extracellular factor might act as an intermediary between two Crumbs extracellular domains. These data are strongly supported by recent evidence from zebrafish, where vertebrate Crumbs isoforms appear to mediate homophilic interactions to promote orderly arrays of photoreceptors. Also, recent data analyzing the establishment of polarity in the Drosophila follicular epithelium suggest a role for cis-interaction of Crumbs molecules within a single cell (Fletcher, 2012). Thus, a clear role for the Crumbs extracellular domain in organizing plasma membrane domains through homophilic interactions in cis and in trans is prominently emerging (Roper, 2012).
Data presented in this study describe a link between the transmembrane protein Crumbs and myosin II structures actively engaged in controlling morphogenesis. Crumbs' ability to interact in trans allows the step change in Crumbs levels between placode and surrounding cells to be translated into a subcellular asymmetry, the anisotropic localization of Crumbs. This mechanism provides the cells at the border of the salivary gland placode with the means of sensing this positional information and allows them to turn the positional information into a morphogenetic readout: myosin cable formation. In the future it will be interesting to determine if the arrangement of Crumbs and myosin II described in this study is conserved during topologically similar processes of tube invagination, such as, for instance, the side budding of branches during lung or mammary gland morphogenesis (Roper, 2012).
There are two RNAs produced by addition of different polyA tracts, but the source of this variation is not known (Tepass, 1990).
cDNA clone length - 7226 bases
Bases in 5' UTR -113 plus
Exons - five
Bases in 3' UTR - 505
crumbs encodes a large transmembrane protein with 30 EGF-like repeats and four laminin A G-domain-like repeats in its extracellular domain, suggesting its participation in protein-protein interactions. There is an N-terminal CAX pepeat. The cytoplasmic region consists of 28 amino acids (Tepass, 1990 and Knust, 1993).
Proteins encoded by the tumor suppressor fat gene, the neurogenic slit gene and crumbs gene of Drosophila contain domains homologous with modules identified previously in laminin A. These proteins of Drosophila have a number of features in common: they have large extracellular regions containing laminin A modules linked to epidermal growth factor-like domains, and they are all involved in cell-cell interactions that are crucial for correct morphogenesis of ectodermal tissues (development of midline neuroepithelial, organization of epithelial tissues etc.). Patthy has suggested that the laminin A-type modules of these proteins play important roles in interactions controlling ectodermal differentiation (Patthy, 1992).
date revised: December 10 2002
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