Position-specific Integrins Research Articles

Integrins regulate DLG/FAS2 via a CaM kinase II-dependent pathway to mediate synapse elaboration and stabilization during postembryonic development.

Calcium/calmodulin dependent kinase II (CaMKII), PDZ-domain scaffolding protein Discs-large (DLG), immunoglobin superfamily cell adhesion molecule Fasciclin 2 (FAS2) and the position specific (PS) integrin receptors, including betaPS and its alpha partners (alphaPS1, alphaPS2, alphaPS3/alphaVolado), are all known to regulate the postembryonic development of synaptic terminal arborization at the Drosophila neuromuscular junction (NMJ). Recent work has shown that DLG and FAS2 function together to modulate activity-dependent synaptic development and that this role is regulated by activation of CaMKII. We show that PS integrins function upstream of CaMKII in the development of synaptic architecture at the NMJ. betaPS integrin physically associates with the synaptic complex anchored by the DLG scaffolding protein, which contains CaMKII and FAS2. We demonstrate an alteration of the FAS2 molecular cascade in integrin regulatory mutants, as a result of CaMKII/integrin interactions. Regulatory betaPS integrin mutations increase the expression and synaptic localization of FAS2. Synaptic structural defects in betaPS integrin mutants are rescued by transgenic overexpression of CaMKII (proximal in pathway) or genetic reduction of FAS2 (distal in pathway). These studies demonstrate that betaPS integrins act through CaMKII activation to control the localization of synaptic proteins involved in the development of NMJ synaptic morphology.

Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane.

Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK-green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.

Genetic analysis of olfC demonstrates a role for the position-specific integrins in the olfactory system of Drosophila melanogaster.

Genetic analysis of olfC provides evidence for a role for integrins in the development and/or function of the olfactory system of Drosophila. The olfC gene was identified on the basis of mutations that result in specific defects in behavioural responses to acetate esters, and has been mapped to the cytogenetic interval 7D1;D5-6 on the X chromosome. The myospheroid (mys) gene maps to this region and encodes a beta subunit of integrins. Integrins are alpha(beta) heterodimers which are present on the cell surface and have been implicated in a variety of signalling roles. Mutations in mys fail to complement the olfactory deficits of olfC mutants. These defects can be rescued by misexpression of the mys+ gene under control of a hsp70 promoter. Mutations that affect the alpha subunit of the position-specific integrin PS2 show a dominant interaction with olfC. These results suggest that olfC is allelic to mys and functions together with alphaPS2 integrins in the olfactory pathway in Drosophila.

A role for PS integrins in morphological growth and synaptic function at the postembryonic neuromuscular junction of Drosophila.

A family of three position-specific (PS) integrins are expressed at the Drosophila neuromuscular junction (NMJ): a beta subunit ((betaPS), expressed in both presynaptic and postsynaptic membranes, and two alpha subunits (alphaPS1, alphaPS2), expressed at least in the postsynaptic membrane. PS integrins appear at postembryonic NMJs coincident with the onset of rapid morphological growth and terminal type-specific differentiation, and are restricted to type I synaptic boutons, which mediate fast, excitatory glutamatergic transmission. We show that two distinctive hypomorphic mutant alleles of the beta subunit gene myospheroid (mys(b9) and mys(ts1)), differentially affect betaPS protein expression at the synapse to produce distinctive alterations in NMJ branching, bouton formation, synaptic architecture and the specificity of synapse formation on target cells. The mys(b9) mutation alters betaPS localization to cause a striking reduction in NMJ branching, bouton size/number and the formation of aberrant 'mini-boutons', which may represent a developmentally arrested state. The mys(ts1) mutation strongly reduces betaPS expression to cause the opposite phenotype of excessive synaptic sprouting and morphological growth. NMJ function in these mutant conditions is altered in line with the severity of the morphological aberrations. Consistent with these mutant phenotypes, transgenic overexpression of the betaPS protein with a heat-shock construct or tissue-specific GAL4 drivers causes a reduction in synaptic branching and bouton number. We conclude that betaPS integrin at the postembryonic NMJ is a critical determinant of morphological growth and synaptic specificity. These data provide the first genetic evidence for a functional role of integrins at the postembryonic synapse.

DFak56 Is a Novel Drosophila melanogaster Focal Adhesion Kinase

The mammalian focal adhesion kinase (FAK) family of nonreceptor protein-tyrosine kinases have been implicated in controlling a multitude of cellular responses to the engagement of cell surface integrins and G protein-coupled receptors. We describe here a Drosophila melanogaster FAK homologue, DFak56, which maps to band 56D on the right arm of the second chromosome. Full-length DFak56 cDNA encodes a phosphoprotein of 140 kDa, which shares strong sequence similarity not only with mammalian p125(FAK) but also with the more recently described mammalian Pyk2 (also known as CAKbeta, RAFTK, FAK2, and CADTK) FAK family member. DFak56 has intrinsic tyrosine kinase activity and is phosphorylated on tyrosine in vivo. As is the case for FAK, tyrosine phosphorylation of DFak56 is increased upon plating Drosophila embryo cells on extracellular matrix proteins. In situ hybridization and immunofluorescence staining analysis showed that DFak56 is ubiquitously expressed with particularly high levels within the developing central nervous system. We utilized the UAS-GAL4 expression system to express DFak56 and analyze its function in vivo. Overexpression of DFak56 in the wing imaginal disc results in wing blistering in adults, a phenotype also observed with both position-specific integrin loss of function and position-specific integrin overexpression. Our results imply a role for DFak56 in adhesion-dependent signaling pathways in vivo during D. melanogaster development.

Cloning and Characterization of Dfak56, a Homolog of Focal Adhesion Kinase, in Drosophila melanogaster

The focal adhesion kinase (FAK) protein-tyrosine kinase plays important roles in cell adhesion in vertebrates. Using polymerase chain reaction-based cloning strategy, we cloned a Drosophila gene that is homologous to the vertebrate FAK family of protein-tyrosine kinases. We designated this gene Dfak56 and characterized its gene product. The overall protein structure and deduced amino acid sequence of Dfak56 show significant similarity to those of FAK and PYK2. Dfak56 has in vitro autophosphorylation activity at tyrosine residues. Expression of the Dfak56 mRNA and the protein was observed in the central nervous system and the muscle-epidermis attachment site in the embryo, where Drosophila position-specific integrins are localized. The results suggest that like FAK in vertebrates, Dfak56 functions downstream of integrins. Dfak56 was tyrosine-phosphorylated upon integrin-dependent attachment of the cell to the extracellular matrix. We conclude that the Dfak56 tyrosine kinase is involved in integrin-mediated cell adhesion signaling and thus is a functional homolog of vertebrate FAK.

Kakapo, a gene required for adhesion between and within cell layers in Drosophila, encodes a large cytoskeletal linker protein related to plectin and dystrophin.

Mutations in kakapo were recovered in genetic screens designed to isolate genes required for integrin-mediated adhesion in Drosophila. We cloned the gene and found that it encodes a large protein (>5,000 amino acids) that is highly similar to plectin and BPAG1 over the first 1,000-amino acid region, and contains within this region an alpha-actinin type actin-binding domain. A central region containing dystrophin-like repeats is followed by a carboxy domain that is distinct from plectin and dystrophin, having neither the intermediate filament-binding domain of plectin nor the dystroglycan/syntrophin-binding domain of dystrophin. Instead, Kakapo has a carboxy terminus similar to the growth arrest-specific protein Gas2. Kakapo is strongly expressed late during embryogenesis at the most prominent site of position-specific integrin adhesion, the muscle attachment sites. It is concentrated at apical and basal surfaces of epidermal muscle attachment cells, at the termini of the prominent microtubule bundles, and is required in these cells for strong attachment to muscles. Kakapo is also expressed more widely at a lower level where it is essential for epidermal cell layer stability. These results suggest that the Kakapo protein forms essential links among integrins, actin, and microtubules.

A screen to identify Drosophila genes required for integrin-mediated adhesion.

Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. We have used FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F1 generation and screened for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, 3 are PS integrin genes, 2 are blistered and bloated, and the remaining 9 appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin alpha subunit genes. Mutations in 8 loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the strongest integrin mutations, cause a "tail-up" embryonic lethal phenotype, and mutant alleles of 1 of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function.

Requirements for the cytoplasmic domain of the alphaPS1, alphaPS2 and betaPS integrin subunits during Drosophila development.

The integrins are a family of transmembrane heterodimeric proteins that mediate adhesive interactions and participate in signaling across the plasma membrane. In this study we examine the functional significance of the cytoplasmic domains of the alphaPS1, alphaPS2 and betaPS subunits of the Drosophila Position Specific (PS) integrin family by analyzing the relationship between cytoplasmic domain structure and function in the context of a developing organism. By examining the ability of ssPS molecules lacking the cytoplasmic domain to rescue embryonic abnormalities associated with PS integrin loss, we find that although many embryonic events require the betaPS cytoplasmic domain, this portion of the molecule is not required for at least two processes requiring PS integrins: formation of midgut constrictions and maintaining germband integrity. Furthermore, our studies demonstrate that mutant proteins affecting four highly conserved amino acid residues in the cytoplasmic tail function with different efficiencies during embryonic development, suggesting that interaction of PS integrins with cytoplasmic ligands is developmentally modulated during embryogenesis. We have also examined the ability of alphaPS1 and alphaPS2 to function without their cytoplasmic domains. By analyzing the ability of transgenes producing truncated alphaPS molecules to rescue abnormalities associated with integrin loss, we find that the cytoplasmic tail of alphaPS2 is essential for both embryonic and postembryonic processes, while this portion of alphaPS1 is not required for function in the wing and in the retina. Furthermore, temperature-shift experiments suggest roles for the alphaPS2 cytoplasmic domain in signaling events occurring in the developing wing.

Autosomal mutations affecting adhesion between wing surfaces in Drosophila melanogaster.

Integrins are evolutionarily conserved transmembrane alpha,beta heterodimeric receptors involved in cell-to-matrix and cell-to-cell adhesions. In Drosophila the position-specific (PS) integrins mediate the formation and maintenance of junctions between muscle and epidermis and between the two epidermal wing surfaces. Besides integrins, other proteins are implicated in integrin-dependent adhesion. In Drosophila, somatic clones of mutations in PS integrin genes disrupt adhesion between wing surfaces to produce wing blisters. To identify other genes whose products function in adhesion between wing surfaces, we conducted a screen for autosomal mutations that produce blisters in somatic wing clones. We isolated 76 independent mutations in 25 complementation groups, 15 of which contain more than one allele. Chromosomal sites were determined by deficiency mapping, and genetic interactions with mutations in the beta PS integrin gene myospheroid were investigated. Mutations in four known genes (blistered, Delta, dumpy and mastermind) were isolated. Mutations were isolated in three new genes (piopio, rhea and steamer duck) that affect myo-epidermal junctions or muscle function in embryos. Mutations in three other genes (kakapo, kiwi and moa) may also affect cell adhesion or muscle function at hatching. These new mutants provide valuable material for the study of integrin-dependent cell-to-cell adhesion.

Development of the Indirect Flight Muscle Attachment Sites inDrosophila:Role of the PS Integrins and thestripeGene

Using markers that are expressed at muscle attachment sites, we have examined the early pupal development (first 36 hr) of Indirect Flight Muscle (IFM) attachments in the fruit flyDrosophila melanogaster.Expression of theDrosophilahomologs of vertebrate integrins, the Position-Specific (PS) antigens, is known to differentially mark epidermal (PS1α) and muscle (PS2α) components of the developing IFM attachment sites. During myogenesis, PS2α is detected transiently in imaginal myoblasts that fuse with persistent larval muscles to give rise to the Dorsal Longitudinal Muscles (DLMs), but not in myoblasts that fusede novoto give rise to the DorsoVentral Muscles. The integrins are not expressed at attachment sites when the muscle fibers first make their appearance (12–20 hr). Following muscle–epidermal contact, PS1 and PS2 are detected at muscle attachment sites. PS1 expression is at the muscle ends and also in the long epidermal processes that connect the developing muscle fibers to their sites of attachment in the epidermis, while PS2 expression is restricted to the muscle ends. Epidermal cells that will contribute to the adult attachment sites are defined as early as the third larval instar. Both anterior and posterior sites of attachment of the IFMs are marked by the expression of reporter β-galactosidase activity in a P-element line B14.0, which is an insertion at thestripelocus. B14.0 (stripe) is seen in distinct domains in the wing and leg imaginal discs which give rise to the thoracic cuticle. The expression is maintained during pupal development. The B14.0 (stripe) expressing epidermal cells contact the developing muscle fibers, leading to the formation of the myotendon junction. We show that the dorsal and ventral attachment sites of one group of IFMs, the DVMs arise from two different imaginal discs (wing and leg, respectively), which may explain the differential effect of mutations such asbendlesson these muscles. Attachment sites for the other group of IFMs, the DLMs, on the other hand, arise from one imaginal disc (wing). B14.0 (stripe) expression defines epidermal cells of the adult attachment sites and is likely to function during early events leading to the formation of muscle–epithelial contacts. The PS integrins are detected at later stages, suggesting a role in the stabilization and maturation of the muscle–epidermal contacts into myotendon junctions.

Functions for PS Integrins in Tissue Adhesion, Migration, and Shape Changes during Early Embryonic Development in Drosophila

The position-specific (PS) integrins of Drosophila are a family of cell surface heterodimers that function as receptors for extracellular matrix molecules. In this study we used time-lapse videomicroscopy to examine living embryos lacking both maternal and zygotic expression of the genes that encode the α PS1, α PS2, and β PS integrin subunits. We demonstrate roles for these molecules as early as gastrulation. Abnormalities in mutant embryos include: separation and twisting of the embryonic germband, abnormal shape and migration of midgut primordia, irregular visceral mesoderm, detachment or amnioserosa cells, rupture of the cuticle along the dorsal midline, lack of midgut constriction, and detachment of somatic muscles. These observations suggest multiple roles for PS integrins in the adhesion of cells and in the formation, organization, and migration of embryonic tissues. We also show that although α PS1β PS and α PS2β PS are often expressed in adjacent embryonic tissues, this distribution does not necessarily reflect equivalent requirements. Furthermore, the complete loss of both α subunits does not produce all of the phenotypes observed in embryos lacking β PS. This suggests that α PS1β PS and α PS2β PS are not required in all embryonic processes utilizing PS integrins.

Drosophila integrins and their ligands

The major advance during the past year was the identification of ligands for two of the previously known position-specific integrins in Drosophila. At the same time, two new Drosophila integrin subunits (one α and one β) were discovered, and significant progress was made on developmental genetic analyses of integrin functions, shedding light on the roles of integrins in Drosophila development.

Null mutations in the alpha PS2 and beta PS integrin subunit genes have distinct phenotypes.

The two Drosophila position-specific (PS) integrins are expressed on complementary sides of sites where different cell layers adhere to each other, such as the attachments of the embryonic muscles to the epidermis. While there is suggestive evidence that the PS integrin-mediated adhesion is via the extracellular matrix, it is also possible that it occurs through the direct interaction of the two integrins, alpha PS1 beta PS and alpha PS2 beta PS. To help distinguish between these possibilities a comparison between the phenotypes caused by the absence of the beta PS subunit and the absence of one of the PS alpha subunits, alpha PS2, has been made. Two pieces of evidence are provided that prove that the alpha PS2 subunit is encoded by the locus inflated (if). Firstly, three new if alleles have been isolated, each of which is associated with a molecular lesion in the alpha PS2 gene, and each of which results in the complete loss of if activity. Secondly, a 39 kb fragment of genomic DNA that encompasses the alpha PS2 gene completely rescues if mutations when introduced into the germline by P-element-mediated transformation. A comparison of the null inflated phenotype with that of the locus that encodes the beta PS subunit, myospheroid (mys), reveals that while the beta PS subunit is required for the adhesion of the epidermis along the dorsal midline, the alpha PS2 subunit is not. In if mutant embryos, the muscles remain attached to the other cell layers significantly longer than in a mys mutant embryo. This shows that the alpha PS2 beta PS integrin only contributes part of the adhesive activity at the sites of PS integrin adhesion, and rules out a model where PS integrin function occurs solely by the direct interaction of the two PS integrins.

Integrins and morphogenesis

The Drosophila position specific (PS) integrins consist of two cell surface heterodimers, PS1 (alpha PS1 beta PS) and PS2 (alpha OS2 beta PS), which are expressed on complementary sides of attachments between cell layers and are essential for these attachments. Current evidence suggests that the PS integrins bind to components of the extracellular matrix, similar to the majority of vertebrate integrins, but specific Drosophila ligands have not yet been identified. In the embryo PS1 is found on the surface of the epidermis and endoderm, while PS2 is restricted to the mesoderm. The integrins are concentrated at the sites where the somatic muscles attach to the epidermis and at the interface between the visceral mesoderm and the endoderm. In myospheroid mutant embryos, which lack the beta PS subunit, the adhesion between the mesoderm and the other cell layers fails. The PS integrins are also required for the adhesion of the dorsal to the ventral surface of the wing during metamorphosis. PS1 is expressed on the basal surface of the dorsal cells and PS2 is expressed on the ventral cells. Loss of PS integrin function in the wing results in balloon shaped wings because of the failure of the two surfaces of the wing blade to adhere to each other. These and other aspects of the phenotypes of mutations in the genes encoding the PS integrins indicate that integrins play an important role in the adhesion of different cell layers to each other and thus an essential role in the morphogenesis of the organism. The use of extracellular matrix receptors in this role may aid in keeping the different cell layers distinct.

Cloning and characterization of αPS1, a novel Drosophila melanogaster integrin

The Drosophila position-specific integrins (PS integrins or PS antigens) comprise two heterodimeric complexes, α PS1 β PS and α PS2 β PS. With the cloning of α PS1 described here, we complete the characterization of the primary structure of the three PS integrin subunits. We have purified the α PS1 subunit, obtained peptide sequence and isolated genomic and cDNA clones. The encoded α PS1 protein contains pattern of the cleaved alpha integrins, three putative metal binding domains and shows the other characteristic features of alpha integrins. Regions of sequence variation indicate that α PS1 is distinct from all other alpha chains. The transcript analysis shows that the patterns of both α PS1 mRNA and protein expression are the same, suggesting that the gene is controlled transcriptionally. We compare the gene structures of the Drosophila α PS1, α PS2, the human α PS1and α PS2 (p150,95) and the C. elegans F54G8.3 integrins. We find several positions and phases of introns conserved which, supported by conservation also in the amino acid sequence, indicates that they all derive from a common ancestral gene.

Analyses of PS integrin functions during Drosophila development.

The Drosophila position-specific (PS) antigens are homologues of the vertebrate integrins, a family of transmembrane proteins that function in cell-matrix and cell-cell adhesion. The common beta subunit of PS integrins (PS beta) is encoded by the lethal(l)myospheroid gene (mys) and is required during wing, eye and muscle development. By expressing PS beta protein at defined developmental periods, we have shown that PS integrins are required throughout pupation, but not earlier, for normal development of wings. In contrast, the key requirement for PS integrins in eye development occurs only in the late pupa. Furthermore, PS integrins are apparently not required for the differentiation of the ommatidial cells; only for their organization. These results are consistent with roles for PS integrins in the interactions between the wing epithelia during the two phases of pupal wing expansion and in maintaining the attachment of a fully formed fenestrated membrane to the basement membrane of the retina. We have also examined the functional significance of alternative splicing of the transcript of the mys gene using P element-mediated transformation to introduce transgenes producing only one of the two spliced forms of PS beta. We find that either form is sufficient to rescue postembryonic mys phenotypes in the wing, eye and muscle but that both of the two splice forms are necessary to rescue the mys embryonic defects. This result indicates a requirement for the alternative splicing of mys during embryogenesis. The location of the alternative exons suggests that the two forms of the PS beta integrin subunit may interact with alternative alpha subunits and/or ligands.

Integrins hold Drosophila together

The Drosophila position-specific (PS) integrins are members of the integrin family of cell surface receptors and are thought to be receptors for extracellular matrix components. Each PS integrin consists of an alpha subunit, alpha PS1 or alpha PS2, and a beta PS subunit. Mutations in the beta PS subunit and the alpha PS2 subunit have been characterised and reveal that the PS integrins have an essential role in the adhesion of different cell layers to each other. The PS integrins are especially required for the function of the cell-matrix-cell junctions, where the muscles attach to the epidermis and where one surface of the developing wing adheres to the other. These junctions are similar to vertebrate focal adhesions and hemidesmosomes, which also contain integrins. Integrin-mediated cell to cell adhesion via the extracellular matrix provides a way for tissues to adhere to each other without intermingling of their cells.

Requirement for integrins during Drosophila wing development

The position-specific (PS) integrins of Drosophila are highly homologous to vertebrate integrins, most of which are cell-surface receptors for extracellular matrix components. Integrins are heterodimers, each consisting of noncovalently associated alpha- and beta-subunits. As for the subfamilies of vertebrate integrins, the same beta-subunit is found in both Drosophila PS integrins, combined with a specific alpha-subunit to generate either a complete functional PS1 or PS2 integrin. Both alpha- and beta-subunits are large transmembrane proteins (relative molecular masses greater than 100,000). Either one or both of these two PS integrins are expressed in most fly tissues during development. A particularly intriguing pattern of expression is found in the mature wing imaginal disc, where the PS1 integrin is expressed primarily on the presumptive dorsal wing epithelium, and the PS2 integrin is found almost exclusively on the ventral epithelium. Immediately after pupariation, the central wing pouch evaginates, folding along its centre to appose the epithelia that will secret the dorsal and ventral surfaces of the adult wing blade. Here we report the results of a genetic analysis indicating that both of the PS integrins are required to maintain the close apposition of the dorsal and ventral wing epithelia during morphogenesis. Also, we conclude that the integrins are not necessary for the maintenance of the cell lineage restriction between the two presumptive wing surfaces in the developing imaginal disc.