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 α subunit, α_PS1 or α_PS2, and a β_PS subunit. Mutations in the β_PS subunit and the α_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.     

Autosomal Mutations Affecting Adhesion between Wing Surfaces in Drosophila Melanogaster

Integrins are evolutionarily conserved transmembrane α,β 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 β(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.     

Ihog proteins contribute to integrin-mediated focal adhesions

Integrin expression forms focal adhesions, but how this process is physiologically regulated is unclear. Ihog proteins are evolutionarily conserved, playing roles in Hedgehog signaling and serving as trans-homophilic adhesion molecules to mediate cell-cell interactions. Whether these proteins are also engaged in other cell adhesion processes remains unknown. Here, we report that Drosophila Ihog proteins function in the integrin-mediated adhesions. Removal of Ihog proteins causes blister and spheroidal muscle in wings and embryos, respectively. We demonstrate that Ihog proteins interact with integrin via the extracellular portion and that their removal perturbs integrin distribution. Finally, we show that Boc, a mammalian Ihog protein, rescues the embryonic defects caused by removing its Drosophila homologs. We thus propose that Ihog proteins contribute to integrin-mediated focal adhesions.

     

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.     

Moleskin is essential for the formation of the myotendinous junction in Drosophila

It is the precise connectivity between skeletal muscles and their corresponding tendon cells to form a functional myotendinous junction (MTJ) that allows for the force generation required for muscle contraction and organismal movement. The Drosophila MTJ is composed of secreted extracellular matrix (ECM) proteins deposited between integrin-mediated hemi-adherens junctions on the surface of muscle and tendon cells. In this paper, we have identified a novel, cytoplasmic role for the canonical nuclear import protein Moleskin (Msk) in Drosophila embryonic somatic muscle attachment. Msk protein is enriched at muscle attachment sites in late embryogenesis and msk mutant embryos exhibit a failure in muscle–tendon cell attachment. Although the muscle–tendon attachment sites are reduced in size, components of the integrin complexes and ECM proteins are properly localized in msk mutant embryos. However, msk mutants fail to localize phosphorylated focal adhesion kinase (pFAK) to the sites of muscle–tendon cell junctions. In addition, the tendon cell specific proteins Stripe (Sr) and activated mitogen-activated protein kinase (MAPK) are reduced in msk mutant embryos. Our rescue experiments demonstrate that Msk is required in the muscle cell, but not in the tendon cells. Moreover, muscle attachment defects due to loss of Msk are rescued by an activated form of MAPK or the secreted epidermal growth factor receptor (Egfr) ligand Vein. Taken together, these findings provide strong evidence that Msk signals non-autonomously through the Vein-Egfr signaling pathway for late tendon cell late differentiation and/or maintenance.     

αPS1βPS integrin receptors regulate the differential distribution of Sog fragments in polarized epithelia

Bone morphogenetic proteins (BMPs) have important functions during epithelial development. In Drosophila, extracellular Short gastrulation (Sog) limits the action of the BMP family member Decapentaplegic (Dpp). We have shown that Integrin receptors regulate Sog activity and distribution during pupal wing development to direct placement of wing veins. Here, we show that Integrins perform a similar function in the follicular epithelium, impacting Dpp function during oogenesis and embryonic development. As reported for the wing, this effect is specific to mew, which codes for αPS1 integrin. Sog is subject to cleavage by metalloproteases, generating fragments with different properties. We also show that Integrins regulate the distribution of C‐ and N‐terminal Sog fragments in both epithelia, suggesting they may regulate the quality of BMP outputs. Our data indicate that αPS1βPS integrin receptors regulate the amount and type of Sog fragments available for diffusion in the extracellular space during oogenesis and pupal wing development. genesis 48:31–43, 2010. © 2009 Wiley‐Liss, Inc.     

Modulation of Integrin Activity is Vital for Morphogenesis

Cells can vary their adhesive properties by modulating the affinity of integrin receptors. The activation and inactivation of integrins by inside-out mechanisms acting on the cytoplasmic domains of the integrin subunits has been demonstrated in platelets, lymphocytes, and keratinocytes. We show that in the embryo, normal morphogenesis requires the α subunit cytoplasmic domain to control integrin adhesion at the right times and places. PS2 integrin (α_PS2β_PS) adhesion is normally restricted to the muscle termini, where it is required for attaching the muscles to the ends of other muscles and to specialized epidermal cells. Replacing the wild-type α_PS2 with mutant forms containing cytoplasmic domain deletions results in the rescue of the majority of defects associated with the absence of the α_PS2 subunit, however, the mutant PS2 integrins are excessively active. Muscles containing these mutant integrins make extra muscle attachments at aberrant positions on the muscle surface, disrupting the muscle pattern and causing embryonic lethality. A gain- of-function phenotype is not observed in the visceral mesoderm, showing that regulation of integrin activity is tissue-specific. These results suggest that the α_PS2 subunit cytoplasmic domain is required for inside-out regulation of integrin affinity, as has been seen with the integrin α_IIbβ₃.     

The function of PS integrins in Drosophila wing morphogenesis.

Integrins are found on many cell types during the development of most organisms. In Drosophila their functions can be analysed genetically. An analysis of lethal mutations in a PS integrin gene showed that the integrins were required for muscle attachment and for certain cell sheet migrations during embryogenesis. In this paper we use viable mutations in integrin component genes to look at integrin function in the later stages of development of one adult structure, the wing. We show that two known viable mutations, one which has its primary effect on the fly's escape response, the other on wing morphogenesis, are mutations in the beta and PS2alpha subunits, respectively, of the PS integrins. The mutation non-jumper (mys(mj42)) in the beta subunit leads to wasting of the thoracic jump muscles. Flies in which the dosage of this allele is reduced (and no wildtype copy is present) show defects also in wing morphogenesis. The two surfaces of the wing fail to connect properly, resulting in 'blistering' of the wing and the formation of extra crossveins. The mutation in the gene for the PS2alpha integrin subunit, inflated, also leads to a failure in wing surface apposition and consequent wing blistering. When the two mutations are combined, the mutant phenotype is greatly enhanced. Thus, one of the roles of the PS integrins in late Drosophila development is to ensure the correct apposition and patterning of the wing epithelia.     

The expression of PS integrins in Drosophila melanogaster imaginal disc cell lines

Summary Drosophila imaginal disc cell lines show a characteristic pattern of aggregation in culture, which appears to be due to cell-cell rather than cell-substrate interactions. We have examined the distribution of PS integrins in wing and leg cell lines, and find that these integrin homologues are expressed preferentially in aggregates. Cell sheets, small cell clumps and chains of cells express antigen at points of cell-cell contact only.     

Spatial regulation of cell adhesion in the Drosophila wing is mediated by Delilah, a potent activator of βPS integrin expression

In spite of our conceptual view of how differential gene expression is used to define different cell identities, we still do not understand how different cell identities are translated into actual cell properties. The example discussed here is that of the fly wing, which is composed of two main cell types: vein and intervein cells. These two cell types differ in many features, including their adhesive properties. One of the major differences is that intervein cells express integrins, which are required for the attachment of the two wing layers to each other, whereas vein cells are devoid of integrin expression. The major signaling pathways that divide the wing to vein and intervein domains have been characterized. However, the genetic programs that execute these two alternative differentiation programs are still very roughly drawn. Here we identify the bHLH protein Delilah (Dei) as a mediator between signaling pathways that specify intervein cell-fate and one of the most significant realizators of this fate, βPS integrin. Dei's expression is restricted to intervein territories where it acts as a potent activator of βPS integrin expression. In the absence of normal Dei activity the level of βPS integrin is reduced, leading to a failure of adhesion between the dorsal and ventral wing layers and a consequent formation of wing blisters. The effect of Dei on βPS expression is not restricted to the wing, suggesting that Dei functions as a general genetic switch, which is turned on wherever a sticky cell-identity is determined and integrin-based adhesion is required.     

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.     

Genetic analysis of the Drosophila alphaPS2 integrin subunit reveals discrete adhesive,morphogenetic and sarcomeric functions.

The integrin family of cell surface receptors mediates cell-substrate and cell-to-cell adhesion and transmits intracellular signals. In Drosophila there is good evidence for an adhesive role of integrins, but evidence for integrin signalling has remained elusive. Each integrin is an alphabeta heterodimer, and the Drosophila betaPS subunit forms at least two integrins by association with different alpha subunits: alphaPS1betaPS (PS1) and alphaPS2betaPS (PS2). The complex pattern of PS2 integrin expression includes, but is more extensive than, the sites where PS2 has a known requirement. In order to investigate whether PS2 integrin is required at these additional sites and/or has functions besides mediating adhesion, a comprehensive genetic analysis of inflated, the gene that encodes alphaPS2, was performed. We isolated 35 new inflated alleles, and obtained 10 alleles from our colleagues. The majority of alleles are amorphs (36/45) or hypomorphs (4/45), but five alleles that affect specific developmental processes were identified. Interallelic complementation between these alleles suggests that some may affect distinct functional domains of the alphaPS2 protein, which specify particular interactions that promote adhesion or signalling. One new allele reveals that the PS2 integrin is required for the development of the adult halteres and legs as well as the wing.     

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 α-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.     

Integrins modulate the Egfr signaling pathway to regulate tendon cell differentiation in the Drosophila embryo

Changes in the extracellular matrix (ECM) govern the differentiation of many cell types during embryogenesis. Integrins are cell matrix receptors that play a major role in cell-ECM adhesion and in transmitting signals from the ECM inside the cell to regulate gene expression. In this paper, it is shown that the PS integrins are required at the muscle attachment sites of the Drosophila embryo to regulate tendon cell differentiation. The analysis of the requirements of the individual alpha subunits, alphaPS1 and alphaPS2, demonstrates that both PS1 and PS2 integrins are involved in this process. In the absence of PS integrin function, the expression of tendon cell-specific genes such as stripe and beta1 tubulin is not maintained. In addition, embryos lacking the PS integrins also exhibit reduced levels of activated MAPK. This reduction is probably due to a downregulation of the Epidermal Growth Factor receptor (Egfr) pathway, since an activated form of the Egfr can rescue the phenotype of embryos mutant for the PS integrins. Furthermore, the levels of the Egfr ligand Vein at the muscle attachment sites are reduced in PS mutant embryos. Altogether, these results lead to a model in which integrin-mediated adhesion plays a role in regulating tendon cell differentiation by modulating the activity of the Egfr pathway at the level of its ligand Vein.     

Tenectin is a novel αPS2βPS integrin ligand required for wing morphogenesis and male genital looping in Drosophila

Morphogenesis of the adult structures of holometabolous insects is regulated by ecdysteroids and juvenile hormones and involves cell-cell interactions mediated in part by the cell surface integrin receptors and their extracellular matrix (ECM) ligands. These adhesion molecules and their regulation by hormones are not well characterized. We describe the gene structure of a newly described ECM molecule, tenectin, and demonstrate that it is a hormonally regulated ECM protein required for proper morphogenesis of the adult wing and male genitalia. Tenectin's function as a new ligand of the PS2 integrins is demonstrated by both genetic interactions in the fly and by cell spreading and cell adhesion assays in cultured cells. Its interaction with the PS2 integrins is dependent on RGD and RGD-like motifs. Tenectin's function in looping morphogenesis in the development of the male genitalia led to experiments that demonstrate a role for PS integrins in the execution of left-right asymmetry.     

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 β subunit of integrins. Integrins are αβ 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 α 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 α_PS2 integrins in the olfactory pathway in Drosophila. Received: 3 November 1999 / Accepted: 17 January 2000     

Tensin stabilizes integrin adhesive contacts in Drosophila

We report the functional characterization of the Drosophila ortholog of tensin, a protein implicated in linking integrins to the cytoskeleton and signaling pathways. A tensin null was generated and is viable with wing blisters, a phenotype characteristic of loss of integrin adhesion. In tensin mutants, mechanical abrasion is required during wing expansion to cause wing blisters, suggesting that tensin strengthens integrin adhesion. The localization of tensin requires integrins, talin, and integrin-linked kinase. The N-terminal domain and C-terminal PTB domain of tensin provide essential recruitment signals. The intervening SH2 domain is not localized on its own. We suggest a model where tensin is recruited to sites of integrin adhesion via its PTB and N-terminal domains, localizing the SH2 domain so that it can interact with phosphotyrosine-containing proteins, which stabilize the integrin link to the cytoskeleton.