A family of laminin-related proteins controlling ectodermal differentiation in Drosophila.

It is shown that the 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 neuroepithelia, organizationof epithelial tissues etc.). It is suggested that the laminin A-type modules of these proteins play important roles in the interactions that control ectodermal differentiation.     

Crumbs regulates polarity and prevents light-induced degeneration of the simple eyes of Drosophila, the ocelli

The evolutionary conserved transmembrane protein Crumbs (Crb) regulates morphogenesis of photoreceptor cells in the compound eye of Drosophila and prevents light-dependent retinal degeneration. Here we examine the role of Crb in the ocelli, the simple eyes of Drosophila. We show that Crb is expressed in ocellar photoreceptor cells, where it defines a stalk membrane apical to the adherens junctions, similar as in photoreceptor cells of the compound eyes. Loss of function of crb disrupts polarity of ocellar photoreceptor cells, and results in mislocalisation of adherens junction proteins. This phenotype is more severe than that observed in mutant photoreceptor cells of the compound eye, and resembles more that of embryonic epithelia lacking crb. Similar as in compound eyes, crb protects ocellar photoreceptors from light induced degeneration, a function that depends on the extracellular portion of the Crb protein. Our data demonstrate that the function of crb in photoreceptor development and homeostasis is conserved in compound eyes and ocelli and underscores the evolutionarily relationship between these visual sense organs of Drosophila. The data will be discussed with respect to the difference in apico-basal organisation of these two cell types.     

Drosophila laminin A chain sequence, interspecies comparison, and domain structure of a major carboxyl portion.

Recent studies ascribed some biological actions of cell adhesion and cell outgrowth to the carboxyl-most 1200 amino acids of vertebrate laminin A chains. Here we report a 6.1-kilobase pair nucleotide cDNA sequence encoding 1951 amino acids and the carboxyl end of a Drosophila laminin A chain. It corresponds to the mouse laminin A domains G, I, II, and III, but may represent a different type of laminin A chain. The arrangement of the cysteine-rich repeats of domain III resembles that of B2 chains. However, it has more amino acid identity with a portion of the mouse laminin A chain domain IIIb than with other laminin repeats. Domains I and II are consistent with an interrupted coiled-coil alpha-helical model of the long arm of laminin but are poorly conserved. The G domain contains five subdomains which are individually related to subdomains of vertebrate laminin A chains. The results indicate that laminin G subdomains should be considered individually, rather than merely as parts of a G-globule. A sequence of hydroxyamino acids contributes to a spacer between two of the subdomains. Stretches of hydroxyamino acids may be indicative of junctions between domains of extracellular Drosophila proteins.     

Interactions between the crumbs,lethal giant larvae and bazooka pathways in epithelial polarization

Several protein complexes that are involved in epithelial apicobasal polarity have been identified. However, the mechanism by which these complexes interact to form an integrated polarized cell morphology remains unclear. Crumbs (Crb) and Lethal giant larvae (Lgl) are components of distinct complexes that regulate epithelial polarization in Drosophila melanogaster, but may not interact directly as they localize to the apical and basolateral membrane, respectively. Nevertheless, a genetic screen identifies marked functional interactions between crb and lgl. These interactions extend to other genes within the crb (stardust, sdt) and lgl (discs large, dlg; scribble, scrib) pathways. Our findings suggest that the crb and lgl pathways function competitively to define apical and basolateral surfaces. They also suggest that in the absence of lgl pathway activity, the crb pathway is not required to maintain epithelial polarity. Moreover, we show that crb and lgl cooperate in zonula adherens formation early in development. At later stages, epithelial cells in these mutants acquire normal polarity, indicating the presence of compensatory mechanisms. We find that bazooka (baz) functions redundantly with crb/sdt to support apical polarity at mid- to late-embryogenesis. Despite regaining cell polarity, however, epithelial cells in crb and lgl pathway mutants fail to re-establish normal overall tissue architecture, indicating that the timely acquisition of polarized cell structure is essential for normal tissue organization.     

Laminin A chain: expression during Drosophila development and genomic sequence.

A Drosophila laminin A chain gene was characterized as a 14 kb genomic nucleotide sequence which encodes an open reading frame of 3712 amino acids in 15 exons. Overall, this A chain is similar to its vertebrate counterparts, especially in its N- and C-terminal globular domains, but the sequence that forms the laminin A short arm is quite different and larger. Laminin messages appear in newly formed mesoderm and are later prominently expressed in hemocytes, which also synthesize basement membrane collagen IV. The composition of Drosophila basement membranes changes with development. A novel method of tandemly fused RNA probes showed that developmental increases of laminin mRNAs were primarily associated with periods of morphogenesis, and preceded those of collagen IV, a protein strongly expressed during growth. The ratio of A:B1:B2 mRNAs varied little during embryogenesis, with less mRNA for A than B chains. Staining of embryos with antibodies confirmed and extended the information provided by in situ hybridization. Homologs of the G-subdomains of this A chain, which occur in interacting regions of agrin, perlecan, laminin and sex steroid binding protein, may be involved in protein associations.     

Drosophila laminin: characterization and localization

Drosophila laminin was isolated from the medium of Drosophila Kc cell cultures. It was purified by velocity sedimentation, gel filtration, and chromatography. Drosophila laminin is a disulfide-linked molecule consisting of three chains with apparent molecular masses of 400, 215, and 185 kD. In electron micrographs, it has the cross-shaped appearance with globular domains characteristic of vertebrate laminin with closely similar dimensions. The amino acid composition and lectin-binding properties of Drosophila laminin are given. Polyclonal antibodies to Drosophila laminin were prepared and their specificity was established. In developing embryos immunofluorescence staining was detected between 6 and 8 h of development; and in sections of 8-9-h and older embryos immunostaining was seen at sites where basement membranes are present surrounding internal organs, muscles, underlying the hypodermal epithelium, and in the nervous system. Basement membrane staining was also seen in larva and adults. Cells from Drosophila embryos dissociated at the cellular blastoderm stage were grown in culture and some specific, differentiated cells synthesized laminin after several hours of culture as shown by immunofluorescence. The significance of the evolutionary conservation of the structure of this basement membrane component is discussed.     

Drosophila substrate adhesion molecule: sequence of laminin B1 chain reveals domains of homology with mouse

Laminin, a substrate adhesion molecule in vertebrates, is a large glycoprotein complex in basement membranes that promotes cell adhesion, cell migration, and neurite outgrowth. Here we report on the cloning of the genes encoding the three subunits of Drosophila laminin. Sequence analysis of cDNA clones encoding the Drosophila B1 chain reveals a multidomain structure similar to that of its mouse homolog. The Drosophila sequence has only 25% amino acid identity with the mouse sequence in domains I, II, and IV. However, in one of the putative collagen-binding regions (domain VI) and the two cysteine-rich domains of EGF-like repeats (domains III and V), the amino acid identity between these two evolutionarily distant species jumps to 55%. Moreover, the number, length, and unique amino acid sequences of each of the 13 EGF-like repeats are highly conserved between Drosophila and mouse, suggesting that each may serve a unique function in protein-protein interactions.     

Role of Crumbs proteins in the control of epithelial cell and photoreceptor morphogenesis

Degeneration of retina can have many causes and among the genes involved, CRB1 has been shown to be associated with Retinitis pigmentosa (RP) group 12 and Leber congenital amaurosis (LCA), two dramatic pathologies in young patients. CRB1 belongs to a family of genes conserved from Caenorhabditis elegans to human. In Drosophila melanogaster, for example, crb is essential both for the formation of the adherens junctions in epithelial cells of ectodermal origin during gastrulation and for the morphogenesis of photoreceptors in the eye. Crumbs is a transmembrane protein with a short cytoplasmic domain that interacts with scaffold proteins, Stardust and Discs lost, and with the apical cytoskeleton made of moesin and betaheavy-spectrin. The extracellular domain of Crumbs is essential for its function in photoreceptors but so far there are no known proteins interacting with it. In human, there are three known crb homologues, CRB1, 2 and 3, and CRB1 is expressed in the retina and localizes to the adherens junctions of the rods. Based on the model drawn from Drosophila, CRB1 could be involved in maintaining the morphology of rods to ensure a normal function of the retina. This is supported by the fact that the homologues of the known partners of Crumbs are also conserved in human and expressed in the retina. Understanding the precise molecular mechanism by which CRB1 acts will help to find new therapies for patients suffering from RP12 and LCA.     

Domains in proteins and proteoglycans of the extracellular matrix with functions in assembly and cellular activities

Most proteins of the extracellular matrix (ECM), such as the glycoproteins, collagens and proteoglycans, consist of many structurally autonomous domains that are often functionally distinct. Consequently these proteins are designated as mosaic proteins. Related domains are often found in several different ECM proteins. Domains which are of importance for assembly have been identified by fragmentation and other approaches. Triple-stranded coiled-coil domains in laminin and probably also in tenascin and thrombospondin are responsible for chain selection, a process which may be important for the formation of tissue specific isoforms. Globular domains at the C-terminus of collagenous domains are essential for the registration of the three chains and triple-helix formation. Fibrillar assemblies of these triple helices with constituent globular domains serve important assembly functions in many collagens including collagens IV and VI. Many other domains with more specialized functions in assembly have been identified in laminin, fibronectin and other ECM proteins. Cys-rich domains with either distant or close homology with epidermal growth factor are repeated manifold in rod-like regions of a number of ECM proteins including laminin, tenascin and thrombospondin. They may serve as spacer elements but as suggested for laminin some domains of this type may also function as signals for cellular growth and differentiation. Another important cellular function common to many ECM proteins is cell attachment. Several cell attachment sites have been localized in structurally unrelated domains of the same or of different ECM proteins.     

Primary structure of the Drosophila laminin B2 chain and comparison with human, mouse, and Drosophila laminin B1 and B2 chains

Laminin, a major component of basement membranes, is a large glycoprotein consisting of three disulfide-bonded subunits, A, B1, and B2. We have isolated and sequenced a Drosophila laminin B2 chain cDNA clone that spans 5737 nucleotides. The deduced amino acid sequence predicts that the mature and nonglycosylated polypeptide has a chain length of 1606 residues (Mr = 178,665). This B2 chain contains 100 half-cystine residues, most of which are located in two cysteine-rich domains, and 11 N-X-S or N-X-T sequences which are potential sites of N-linked glycosylation. The predicted secondary structure reveals the presence of six structurally distinct domains, of which two are mainly alpha-helical, two are cysteine-rich with homologous repeats, and two are globular regions. The Drosophila B2 chain is 40.3 and 41.1% identical to the human and mouse B2 chains, respectively, and 29.6, 30.0, and 29.4% identical to the Drosophila, human, and mouse B1 chains, respectively.     

Role of the Crumbs complex in the regulation of junction formation in Drosophila and mammalian epithelial cells

The formation of a belt-like junctional complex separating the apical from the lateral domain is an essential step in the differentiation of epithelial cells. Thus protein complexes regulating this event are of first importance for the development of cell polarity and physiological functions of epithelial tissues. In Drosophila, the discovery of a gene, crb, controlling the coalescence of the spots of zonula adherens (ZA) into a adhesive ring around the cells was a major step. We know now that Crumbs, the product of crb is an apical transmembrane protein conserved in mammals and that it interacts by its cytoplasmic domain with two cortical modular proteins, Stardust (Sdt) and Discs lost (Dlt) that are also essential for the correct assembly of the ZA. These two proteins are also conserved in mammals and it is most likely that the Crumbs complex plays a similar role in very different species. Recently, we have shown that Crumbs interacts with the cortical cytoskeleton made of DMoesin and beta heavy-Spectrin and this connection could explain in part the role of Crumbs in building the ZA. Future work will help to understand several aspects of the Crumbs complex that are still unknown, like the role of the large extracellular domain or the precise function of Sdt and Dlt in the building of the ZA. Finding an answer to these questions will help to find new therapies for Retinitis pigmentosa and other retina degeneration in which CRB1, the human homologue of crb, has been involved.     

Crb apical polarity proteins maintain zebrafish retinal cone mosaics via intercellular binding of their extracellular domains

Cone photoreceptors are assembled by unknown mechanisms into geometrically regular mosaics in many vertebrate species. The formation and maintenance of photoreceptor mosaics are speculated to require differential cell-cell adhesion. However, the molecular basis for this theory has yet to be identified. The retina and many other tissues express Crumbs (Crb) polarity proteins. The functions of the?extracellular domains of Crb proteins remain to be understood. Here we report cell-type-specific expression of the crb2a and crb2b genes at the cell membranes of photoreceptor inner segments and Müller cell apical processes in the zebrafish retina. We demonstrate that the extracellular domains of Crb2a and Crb2b mediate a cell-cell adhesion function, which plays an essential role in maintaining the integrity of photoreceptor layer and cone mosaics. Because Crb proteins are expressed in many types of epithelia, the Crb-based cell-cell adhesion may underlie cellular patterning in other epithelium-derived tissues as well.     

Perlecan domain V of Drosophila melanogaster. Sequence, recombinant analysis and tissue expression.

The C-terminal domain V of the basement membrane proteoglycan perlecan was previously shown to play a major role in extracellular matrix and cell interactions. A homologous sequence of 708 amino-acid residues from Drosophila has now been shown to be 33% identical to mouse perlecan domain V. It consists of three laminin G-type (LG) and epidermal growth factor-like (EG) modules but lacks the EG3 module and a link region found in mammalian perlecans. Recombinant production of Drosophila perlecan domain V in mammalian cells yielded a 100-kDa protein which was folded into a linear array of three globular LG domains. Unlike the mouse counterpart, domain V from Drosophila was not modified by glycosaminoglycans and endogenous proteolysis, due to the absence of the link region. It showed moderate affinities for heparin and sulfatides but did not bind to chick alpha-dystroglycan or to various mammalian basement membrane proteins. A single RGD sequence in LG3 of Drosophila domain V was also incapable of mediating cell adhesion. Production of a proteoglycan form of perlecan (approximately 450 kDa) in one Drosophila cell line could be demonstrated by immunoblotting with antibodies against Drosophila domain V. A strong expression was also found by in situ hybridization and immunohistology at various stages of embryonic development and expression was localized to several basement membrane zones. This indicates, as for mammalian species, a distinct role of perlecan during Drosophila development.     

Structure of the C-terminal laminin G-like domain pair of the laminin alpha2 chain harbouring binding sites for alpha-dystroglycan and heparin

The laminins are large heterotrimeric glycoproteins with fundamental roles in basement membrane architecture and function. The C-terminus of the laminin alpha chain contains a tandem of five laminin G-like (LG) domains. We report the 2.0 A crystal structure of the laminin alpha2 LG4-LG5 domain pair, which harbours binding sites for heparin and the cell surface receptor alpha-dystroglycan, and is 41% identical to the laminin alpha1 E3 fragment. LG4 and LG5 are arranged in a V-shaped fashion related by a 110 degrees rotation about an axis passing near the domain termini. An extended N-terminal segment is disulfide bonded to LG5 and stabilizes the domain pair. Two calcium ions, one each in LG4 and LG5, are located 65 A apart at the tips of the domains opposite the polypeptide termini. An extensive basic surface region between the calcium sites is proposed to bind alpha-dystroglycan and heparin. The LG4-LG5 structure was used to construct a model of the laminin LG1-LG5 tandem and interpret missense mutations underlying protein S deficiency.     

wing blister, a new Drosophila laminin alpha chain required for cell adhesion and migration during embryonic and imaginal development

We report the molecular and functional characterization of a new alpha chain of laminin in Drosophila. The new laminin chain appears to be the Drosophila counterpart of both vertebrate alpha2 (also called merosin) and alpha1 chains, with a slightly higher degree of homology to alpha2, suggesting that this chain is an ancestral version of both alpha1 and alpha2 chains. During embryogenesis, the protein is associated with basement membranes of the digestive system and muscle attachment sites, and during larval stage it is found in a specific pattern in wing and eye discs. The gene is assigned to a locus called wing blister (wb), which is essential for embryonic viability. Embryonic phenotypes include twisted germbands and fewer pericardial cells, resulting in gaps in the presumptive heart and tracheal trunks, and myotubes detached from their target muscle attachment sites. Most phenotypes are in common with those observed in Drosophila laminin alpha3, 5 mutant embryos and many are in common with those observed in integrin mutations. Adult phenotypes show blisters in the wings in viable allelic combinations, similar to phenotypes observed in integrin genes. Mutation analysis in the eye demonstrates a function in rhabdomere organization. In summary, this new laminin alpha chain is essential for embryonic viability and is involved in processes requiring cell migration and cell adhesion.     

The Drosophila tissue polarity gene starry night encodes a member of the protocadherin family.

The tissue polarity genes control the polarity of hairs, bristles and ommatidia in the adult epidermis of Drosophila. We report here the identification of a new tissue polarity gene named starry night (stan). Mutations in this essential gene alter the polarity of cuticular structures in all regions of the adult body. The detailed polarity phenotype of stan on the wing suggested that it is most likely a component of the frizzled (fz) pathway. Consistent with this hypothesis, stan appears to be downstream of and required for fz function. We molecularly cloned stan and found that it encodes a huge protocadherin containing nine cadherin motifs, four EGF-like motifs, two laminin G motifs, and seven transmembrane domains. This suggests that Stan functions in signal reception, perhaps together with Fz.     

Domains of laminin

Extracellular matrix molecules are often very large and made up of several independent domains, frequently with autonomous activities. Laminin is no exception. A number of globular and rod-like domains can be identified in laminin and its isoforms by sequence analysis as well as by electron microscopy. Here we present the structure-function relations in laminins by examination of their individual domains. This approach to viewing laminin is based on recent results from several laboratories. First, some mutations in laminin genes that cause disease have affected single laminin domains, and some laminin isoforms lack particular domains. These mutants and isoforms are informative with regard to the activities of the mutated and missing domains. Second, laminin-like domains have now been found in a number of other proteins, and data on these proteins may be informative in terms of structure-function relationships in laminin. Finally, a large body of data has accumulated on the structure and activities of proteolytic fragments, recombinant fragments, and synthetic peptides from laminin. The proposed activities of these domains can now be confirmed and extended by in vivo experiments. © 1996 Wiley-Liss, Inc.     

Laminins and other strange proteins.

Laminins are large multidomain proteins of the extracellular matrix (ECM) with important functions in the development and maintenance of cellular organization and supramolecular structure, in particular in basement membranes. Each molecule is composed of three polypeptide chains, A (300-400 kDa) and B1 and B2 (180-200 kDa), which together form the characteristic cross-shaped laminin structure with three short arms and one long arm. Many different domains have been identified in laminin by sequence analysis, structural investigations, and functional studies. Each short arm is formed by homologous N-terminal portions of one of the three chains. Structurally, each short arm contains two or three globular domains which are connected by rows of manyfold-repeated Cys-rich "EGF-like" domains. In all three chains this region is followed by a long heptad repeat region similar to those found in many alpha-helical coiled-coil proteins. These parts of the three laminin chains constitute a triple-stranded coiled-coil domain, which forms the extended rodlike structure of the long arm. This is the only domain in the protein which is made up of more than one chain and consequently serves the function of chain assembly. The two B chains are terminated by the coiled-coil domain, but the A chain contains an additional C-terminal segment which accounts for five globular domains located at the tip of the long arm. Several important functions of laminin have been assigned to individual domains in either the short arms or terminal regions of the long arm.(ABSTRACT TRUNCATED AT 250 WORDS)