Integrin transmembrane receptors mediate cell adhesion through intracellular linker proteins that connect to the cytoskeleton. Of the numerous linker proteins identified, only a few, including Talin and Integrin-linked-kinase (ILK), are essential and evolutionarily conserved. The wech gene encodes a newly discovered and highly conserved regulator of integrin-mediated adhesion in Drosophila. Embryos deficient in wech have very similar phenotypes to integrin-null or Talin-null embryos, including muscle detachment from the body wall. The Wech protein contains a B-box zinc-finger and a coiled-coil domain, which is also found in RBCC/TRIM family members, and an NHL domain. In β-integrin or Talin mutants, Wech is mislocalized, whereas ILK localization depends on Wech. Evidence is provided that Wech interacts with the head domain of Talin and the kinase domain of ILK, and it is proposed that Wech is required to connect both core proteins of the linker complex during embryonic muscle attachment. Both the NHL and the B-box/coiled-coil domains of Wech are required for proper interaction with Talin and ILK. The single murine Wech orthologue is also colocalized with Talin and ILK in muscle tissue. It is proposed that Wech proteins are crucial and evolutionarily conserved regulators of the integrin-cytoskeleton link (Löer, 2008).
The formation of complex tissues in animals often involves stable adhesion between different cell layers and extracellular matrix substrates, which is, to a large extent, mediated by members of the integrin family of heterodimeric transmembrane receptors. Loss of integrins causes muscle detachment in flies and mice. Integrin mutations or aberrant expression can result in skin diseases, such as epidermolysis bullosa, and contribute to invasion and metastasis during cancerogenesis. A crucial part of the adhesive function of integrins is their ability to connect to the actin cytoskeleton. This involves a complex of adaptor proteins, which bind to the cytoplasmic tail of integrins and mediate the link to the cytoskeleton. Although many proteins have been identified that may contribute to the linker complex, in mammals, flies and worms only a few components have been identified as essential to the link. These include Talin, ILK, PINCH and Tensin, encoding an actin-capping protein, which coordinates signalling with cytoskeletal changes. Recent studies in mammalian cells and in Drosophila suggest that Talin is necessary for the initial formation of the integrin adhesion complex and reinforces it, possibly by recruiting other proteins, including ILK and Tensin. Absence of Talin causes defects that are almost identical to those seen in the absence of integrin, such as muscle detachment and failure of germ-band retraction during embryonic development. In contrast, absence of other components of the link causes only a subset of the defects. In different types of cells, integrins make diverse connections with the actin cytoskeleton; however, the molecular basis for the cell-type specific functions of integrins is still mostly unknown (Löer, 2008).
In a search for genes controlling integrin-mediated muscle attachment in Drosophila embryos, two P insertions were identified and one imprecise excision allele was generated, named wech, affecting a previously uncharacterized genetic locus. In homozygous embryos of the imprecise excision allele, wech66, wech transcript and protein levels were markedly reduced when compared with wild-type embryos. In wech66 germline clone embryos, Wech protein levels were reduced substantially and were barely detectable, indicating that wech66 is an amorphic allele. Molecular analysis, reversion of the phenotype by perfect excision of the P elements and genetic rescue experiments demonstrate that the lethality of wech alleles is linked to wech gene function (Löer, 2008).
Phenotypic analysis of homozygous wech66 mutants and of trans-heterozygous combinations with a deficiency indicates an essential role of wech in muscle attachment during Drosophila embryogenesis (wech is a German Rhineland term for 'detached' or 'gone'). During embryonic development, the somatic muscles attach to each other and to their anchoring points in the epidermis, the tendon cells, to generate a highly stereotyped pattern of 30 muscles in each abdominal hemi-segment. The cytoskeletal network forms bridges between the muscle and the tendon cell through dense hemi-adherens-type junctions formed between the tendon cell, the muscle and a thick layer of extracellular matrix material that is deposited in the space between them. This architecture provides the mechanical force that is required to resist muscle contraction during larval locomotion. The muscles start to attach to the tendon cells at embryonic stage 15 and during the last two stages of embryogenesis (stages 16 and 17) the attachment of the muscles to the tendon cells is elaborated by expansion of the hemi-adherens junctions, accumulation of tendon matrix and increased expression of β1-tubulin (Löer, 2008).
In late-stage embryos homozygous for wech66, it was found that muscles were detached from the body wall. This mutant phenotype became apparent at embryonic stage 16 and was enhanced during subsequent development, as the force of muscle contraction had increased. Myosin heavy chain (MHC) staining of stage 17 mutant embryos showed defects in most of the muscles at that point in development. When both the maternal and zygotic contributions of wech were removed, the detachment phenotype was more severe and became apparent at stage 15, indicating that wech has a maternal contribution, which is consistent with its ubiquitous expression during early stages of embryogenesis. Most of the muscles were multinucleated, indicating that wech mutants show no major defect in myoblast fusion. Analysis of DA1 (dorsal acute muscle 1) development in wech mutants further indicates that muscle differentiation and myoblast fusion are not affected. Rather, the data suggest a role of wech in muscle attachment, consistent with the late manifestation of the muscle detachment phenotype. Mutants for βPS integrin or Talin show phenotypes remarkably similar to wech mutants (Löer, 2008).
The wech gene encodes a multidomain protein containing a B-box zinc-finger domain and a coiled-coil domain characteristic of the RBCC/TRIM protein family. Members of this family usually contain a tripartite motif composed of a RING domain, one or two B-box motifs and a coiled-coil region. The Drosophila Wech protein does not contain a RING domain; however, it contains a carboxy-terminal NHL domain, which is also found in the Drosophila tumour suppressor proteins Brat and Mei-P26. In addition to Wech, brat and mei-P26 encode the only two other NHL-domain-containing family members in Drosophila. Molecular characterization of hypomorphic mutations in the brat gene suggests that the NHL domain carries the tumour suppressor function of this protein. Single-copy genes of wech orthologues are found in other invertebrates and in mammals, including mice, rats and humans. The Caenorhabditis Wech orthologue is named Lin-41 and is involved in the regulation of the progression from larval (L) stage 4 to the adult developmental programme (Löer, 2008).
To study the molecular function of Wech, an anti-Wech antibody was generated. Immunostaining indicates that Wech protein is expressed ubiquitously in all epithelial cells during early stages of embryogenesis. After germ-band retraction, Wech accumulated specifically in the muscle attachment sites. Co-immunostaining with the tendon and muscle-cell markers Short Stop and αPS2, respectively, indicate that Wech is highly localized in both cell types in a cortical localization at the attachment site. βPS Integrin and components of the cytoplasmic integrin-linked complex, Talin, ILK and Tensin, which bind to the cytoplasmic tail of βPS integrin, colocalized with Wech. Consistent with the expression of Wech in both the tendon and the muscle cells, the muscle detachment defect was rescued when Wech was re-supplied in both the tendon and muscle cells, using a combination of sr-GAL4 (tendon-cell-specific) and mef-GAL4 (muscle-cell-specific) in combination with UAS-wech-GFP. Whereas wech66 mutants are embryonic lethal, the rescued animals survived until the third instar-larval stage. Similarly, rescue could be obtained when Wech was ubiquitously expressed using hs-GAL4::UAS-wech-GFP or when the single driver lines, sr-GAL4 or mef-GAL4 were used, indicating that Wech expression rescues in either cell type. The latter finding is surprising and may be due to the residual maternal component in the muscles (Löer, 2008).
To test whether Wech is involved in integrin-mediated cell adhesion, the expression of Wech in βPS integrin (myospheroid, mys) mutants was analyzed. In zygotic mys mutants, the highly localized cortical accumulation of Wech at the attachment site failed and protein was also found in other parts of the cytoplasm at reduced levels. Consistently, a failure of cortical Wech localization was also found in zygotic and maternal mys mutants. These data indicate that βPS integrin is required for Wech localization. In contrast, βPS integrin was still properly localized in zygotic wech66 mutants and in maternal and zygotic wech germline clone embryos, in which Wech protein expression was markedly reduced. As Talin is known to interact through its head domain with integrin cytoplasmatic tails, Wech localization was analyzed in amorphic Talin (rhea2) mutants. In rhea mutants, Wech localization was markedly reduced, whereas in maternal and zygotic wech mutants, Talin seemed to be unchanged. This indicates that Talin is required for Wech localization. In contrast, Wech was required for proper ILK localization. In wech mutants, ILK localization at the attachment site was reduced, whereas Wech was still localized properly in ilk mutants. Consistent with these data, Tensin, which is known to require ILK for its localization, failed to accumulate at the attachment site in wech mutants. PINCH, which was shown to modulate ILK function by direct binding or by recruitment of an ILK-modifying factor, was still localized properly in wech mutants. In contrast to wech mutants, ILK was still concentrated at the muscle attachment sites in PINCH (steamer duck) mutant embryos. This suggests that Wech may be required to link an ILK-containing multiprotein complex to Talin, and PINCH may function as a molecular scaffold supporting the assembly of the ILK-containing linker multiprotein complex. The finding that the wech mutant phenotype is similar in severity to talin mutants and stronger than that of ilk mutants, suggests that other unknown factors, in addition to ILK, may depend on Wech function during muscle attachment (Löer, 2008).
To further analyse the putative role of Wech in integrin-mediated adhesion, biochemical co-immunoprecipitation analysis were performed. Extracts were used of mammalian HeLa cells expressing tagged fusion proteins of the Drosophila Wech, Talin, ILK or various subdomains of these proteins. Wech was found to interact with the protein kinase domain but not with the ankyrin-repeat domain of ILK. Furthermore, Wech interacts with the head domain of Talin. Co-immunoprecipitation analysis using Drosophila embryonic extracts further confirmed the interactions of Wech with Talin and ILK. To determine which protein domains of Wech may be required for its interaction with Talin and ILK at the muscle attachment sites, Myc-tagged Wech protein variants were generated with deletions of the N-terminal B-box zinc-finger and coiled-coil domains (Wech-δBCC) or the C-terminal NHL domain (Wech-δNHL. The deletion constructs were expressed in HeLa cells or in transgenic embryos and tested for interaction with ILK and Talin. In co-immunoprecipitation experiments using HeLa-cell extracts, it was found that the BCC domain of Wech is essential for binding to the protein kinase domain of ILK, whereas the head domain of Talin interacted with both the BCC and the NHL domains of Wech. In vitro, the interaction of the Talin head domain with the Wech BCC domain was, however, much stronger when compared with its interaction with the Wech NHL domain. When expressing the Wech protein deletion variants as Myc-tagged versions in the tendon cells using the strong sr-GAL4 driver, both deletion variants accumulated mainly in the cytoplasm, and colocalization with βPS Integrin, Talin or ILK was markedly reduced. This suggests that both the BCC and NHL domains of Wech may be required in vivo for its proper localization at the muscle attachment sites (Löer, 2008).
As mentioned above, single-copy genes of wech orthologues are found in flies, worms and mammals, including mice and humans. To investigate whether the murine Wech orthologue may be involved in integrin-mediated processes, an antibody was generated against the protein and its expression was studied in adult mice. The murine Wech protein was expressed in the sarcomeric Z-discs of adult muscles where it was colocalized with ILK, which has been identified recently as an architectural component of the Z-disc of heart muscles in zebrafish. Strong colocalization of murine Wech was also found with costameric Talin. Notably, only partial colocalization was detected with sarcolemmal β1 integrins. These results are consistent with the notion that Wech serves as a conserved adaptor, which is required for linking Talin and ILK in the integrin multiprotein complex (Löer, 2008).
In summary, this study has show that Wech is a crucial component for the physical link between integrins and the cytoskeleton in the Drosophila epidermal muscle attachment sites. It is proposed that Wech connects integrins and the cytoskeleton in the attachment sites by interacting with Talin and ILK, thereby linking the ILK-containing multiprotein adaptor complex to Talin and βPS integrin. The data suggest that Wech interacts with the head domain of Talin and the kinase domain of ILK. However, the involvement of other proteins in the physical connection between these core proteins cannot be excluded. The in vitro and in vivo experiments indicate that the Wech BCC and the NHL domains are essential for the functional interaction of Wech with Talin and ILK. In the murine muscles, the single murine Wech orthologue was also strongly colocalized with Talin and ILK. This suggests an evolutionarily conserved role of Wech proteins in the integrin-cytoskeleton link. In addition to Wech, only two other proteins in Drosophila contain an NHL domain, the tumour-suppressor proteins Brat and Mei-P26. The molecular characterization of brat mutations suggests that the NHL domain carries the tumour suppressor function of Brat. Whether the tumour suppressor functions of Brat and MeiP-26 involve the modulation of integrin-mediated adhesion through their NHL domains is not known. As a number of clinically relevant disorders are caused by integrin-related adhesive changes, including muscle dystrophies, the finding of a crucial component for integrin adhesive functions may have implications for understanding disease aetiologies (Löer, 2008).
Surprisingly, there is a difference in the distributions of Talin mRNA and protein during embryogenesis. In particular, high levels of mRNA in the nerve cord do not correspond to high protein levels. Early homogeneous staining for Talin mRNA is followed by prominent expression in the ventral nerve cord. Talin is expressed in a subset of ventral cord neurons (Brody, 2000 and 2002). Lower levels of mRNA are more broadly distributed, with enrichment in the gut and muscle attachment sites detected at stage 16. That this pattern is specific for this transcription unit was shown by testing with probes from four different parts of the transcript, including the C-terminal region to which antibodies were raised; deletion of the Talin gene (rhea79a) eliminates staining (Brown, 2002).
To examine the distribution of Talin protein, polyclonal and monoclonal antibodies were made against the C terminus of Talin. Five of the monoclonal antibodies recognize different epitopes on Drosophila Talin. All but one of the anti-Talin antibodies produced indistinguishable patterns of staining on fixed embryos. The one exception is a monoclonal antibody, J18, that crossreacts with an antigen expressed in three cells per hemisegment within the central nervous system (Brown, 2002).
Analysis of Talin protein distribution during embryogenesis shows that it is maternally deposited and is evenly distributed in the cytoplasm following cellularization. Talin becomes progressively concentrated at the membrane, first detected in the migrating primordial midgut cells and then at muscle attachment sites, where the muscles and epidermal cells are linked via integrins. Talin protein shows only a hint of the strong pattern of mRNA expressed in the nervous system. Consistent with the low level of protein in the nervous system, zygotic mutant rhea embryos do not have any defects in the structure of the nervous system, as assayed with glial and neuronal cell markers. The subcellular distribution of Talin at the muscle attachment sites was examined by immunoelectron microscopy (IEM). Talin is found within submembranous electron-dense material associated with the hemiadherens junctions at muscle attachment sites (Brown, 2002).
Most sites of Talin concentration at membranes correspond to sites of integrin concentration, and colocalization of the two proteins is seen at the edge of the epidermis, during dorsal closure, and at muscle attachments. No PS integrin staining lacking colocalized Talin was observed. However, Talin is concentrated at the membrane of some cells lacking integrin. This is clearest in the gonadal mesoderm, where recruitment of Talin to the cortex of the gonadal mesoderm cells occurs as the gonad condenses. To test whether recruitment of Talin to sites of integrin expression requires integrins, Talin distribution was examined in embryos lacking PS integrins. Embryos lacking the ßPS subunit show a loss of Talin concentration at muscle attachment sites. In embryos that lack the mesodermally expressed alphaPS2 integrin subunit, but that still contain epidermal PS1 (alphaPS1ßPS) integrin, Talin is lost from muscle ends but still concentrated at the basal ends of the attaching epidermal cells, the tendon cells. These results show that Talin is recruited from the cytoplasm by integrins in both cell layers of the muscle attachment site (Brown, 2002).
The absence of strong ßPS expression in the gonadal mesoderm suggests that a receptor different from and other than integrin recruits Talin to the membrane in this tissue. Consistent with this, removal of ßPS from the embryo does not alter Talin enrichment in the gonadal mesoderm, nor does removal of the other Drosophila ß integrin subunit, ßnu. This shows that recruitment of Talin in the gonadal mesoderm is not driven by integrins. An alternative candidate would be Layilin (Borowsky, 1998), but no clear ortholog of this protein is encoded in the Drosophila genome (Brown, 2002).
Organogenesis of the somatic musculature in Drosophila is directed by the precise adhesion between migrating myotubes and their corresponding ectodermally derived tendon cells. Whereas the PS integrins mediate the adhesion between these two cell types, their extracellular matrix (ECM) ligands have been only partially characterized. This study shows that the ECM protein Thrombospondin (Tsp), produced by tendon cells, is essential for the formation of the integrin-mediated myotendinous junction. Tsp expression is induced by the tendon-specific transcription factor Stripe, and accumulates at the myotendinous junction following the association between the muscle and the tendon cell. In tsp mutant embryos, migrating somatic muscles fail to attach to tendon cells and often form hemiadherens junctions with their neighboring muscle cells, resulting in nonfunctional somatic musculature. Talin accumulation at the cytoplasmic faces of the muscles and tendons is greatly reduced, implicating Tsp as a potential integrin ligand. Consistently, purified Tsp C-terminal domain polypeptide mediates spreading of PS2 integrin-expressing S2 cells in a KGD- and PS2-integrin-dependent manner. A model is proposed in which the myotendinous junction is formed by the specific association of Tsp with multiple muscle-specific PS2 integrin receptors and a subsequent consolidation of the junction by enhanced tendon-specific production of Tsp secreted into the junctional space (Subramanian, 2007).
The abnormal pattern of the somatic muscles in the tsp mutant embryo raised the possibility that the muscle-tendon integrin-mediated adhesion is defective in the mutant embryos. A hallmark of appropriate integrin-mediated adhesion is the accumulation of Talin at the cytoplasmic face of the hemiadherens junction, where it binds directly to the integrin cytoplasmic domain, modulating its ligand affinity and recruiting actin microfilaments to this site. A significant reduction of accumulated Talin levels in tsp mutant embryos is observed. Whereas Talin is still detected at the sites of muscle-muscle junctions, it was entirely missing at sites where individual muscles would normally form junctions with single tendon cells, in particular at the junction sites formed between the lateral transverse muscles and their corresponding tendon cells. The lack of Talin at these sites corresponds with the lack of ßPS-integrin staining and is consistent with the loss of appropriate myotendinous junction. Thus, in the absence of functional Tsp, individual myotubes fail to form integrin-mediated adherens junction with tendon cells (Subramanian, 2007).
The genetic locus that encodes Talin is rhea. The first two alleles, rhea1 and rhea2, were isolated in the wing blister screen (Prout, 1997). Two other alleles, rhea17 and rhea3, were isolated as mutations that dominantly enhance weak integrin mutations. The rhea1 and rhea2 alleles were mapped to 66D5-6. By locally hopping a P element in this region, which is not allelic to rhea, l(3)S1760, rhea79a was generated. The P element in this strain is inserted in the same position as l(3)S1760, within the coding region of the Drosophila ortholog of ergic-53, but is deleted for the ergic-53 coding region, leading to the initial suggestion that rhea encodes ergic-53. However, recombinational analysis placed rhea1 0.08 map units (25-50 kb) from the l(3)S1760 P element. The gene adjacent to ergic-53 was revealed to be Talin by the genome sequence. It was then found that Talin protein is reduced in rhea mutant embryos and imaginal disc clones and that Talin mRNA is absent in rhea79a (Brown, 2002).
To confirm that rhea is the Talin gene, the Talin-coding region was sequenced from rhea1 and rhea2. For each allele a small deletion was found that produces a frameshift in the Talin-coding sequence. For rhea1 the frameshift occurs after amino acid 1139, and the new reading frame terminates after two amino acids in the wrong frame. For rhea2 the frameshift occurs after amino acid 1279 and terminates after 31 out of frame amino acids. Inverse PCR was used to identify the proximal insertion site of the P element in rhea79a, which was found to be 1931 bp downstream of Talin, showing that the rhea79a deficiency deletes three genes, ergic-53, Talin, and CG6638. Each rhea allele has an aberration in the Talin coding sequence. A mutant deficient for Ergic53 complements rhea1, rhea2, and rhea17. Therefore, it is concluded that rhea encodes Talin (Brown, 2002).
Three aspects of the Talin mutant phenotype have been described (Prout, 1997). Clones of rhea/rhea cells in the wing do not attach to the other cell layer of the wing, causing a wing blister. The two initial alleles and two recently identified ones (rhea3 and rhea17) dominantly enhanced the wing blister phenotype of hypomorphic alleles of integrin genes (Prout, 1997). Finally, rhea mutant embryos have a detachment of the epidermis from the muscles, although the muscles remain attached end to end. The embryonic phenotype of Talin mutants was examined by EM, with particular attention to tendon cells. In wild-type embryos, tendon cells are spanned by microtubules that link basal hemiadherens junctions to tonofibrils that insert into the apical exoskeleton, thereby transferring the force of muscle contraction to the exoskeleton. In rhea/rhea cells, microtubules extended from apical tonofibrils toward the basal membrane, but mature basal attachment sites fail to form. Structural features of normal attachment sites, such as extensive folding of basal membranes and linkage of microtubules to the inner surface of basal membranes, are not generally present in rhea tendon cells. Also, in these rhea mutant tendon cells, microtubules are abnormally oriented, in some cases running parallel, rather than perpendicular, to the exoskeleton. Loss of Talin results in reduction of electron-dense material from the cytoplasmic face of hemiadherens junctions at muscle attachment sites. This suggests that Talin and/or the proteins it recruits make a significant contribution to this dense material (Brown, 2002).
These zygotic rhea mutant embryos still have some maternally deposited Talin. To analyze the phenotype resulting from the complete absence of Talin, the maternal contribution was removed by generating germline clones. Half of these rhea/rhea eggs receive a wild-type paternal allele, and the zygotic expression of Talin rescues the absence of maternal Talin in some, but not all, embryos. The number of hatching embryos varied from 33%–41%, rather than the expected 50% (depending on the allelic combination). Viable fertile adults developed from hatched embryos. Thus, maternal deposition of Talin protein is important, but not essential, for normal development (Brown, 2002).
Embryos lacking both maternal and zygotic talin have a stronger phenotype than those lacking either maternal or zygotic product, the most prominent features of which are a failure in germband retraction and strong muscle detachment. This phenotype is very similar to that of embryos lacking both maternal and zygotic ßPS. The similarities between the two phenotypes suggest that talin is essential for integrin function. Close examination of the muscle phenotype provides insight into the role of Talin in integrin-mediated adhesion. PS2 (alphaPS2ßPS) integrin localizes normally, demonstrating that integrins reach the cell surface and localize to the ends of muscles in the absence of talin. In detached muscles, actin staining is separate from PS2 integrin staining. This demonstrates that integrin is able to bind to the ECM, since, if it could not, it would be expected to remain on the surface of the detached muscle. Thus, a separation is seen between integrins and actin, not between integrins and the ECM, suggesting that the primary role of talin is to link integrins to the cytoskeleton, and not to stimulate their ligand binding. Talin does not appear to be required for condensation of the gonad, since this occurs in some mutant embryos. Condensation does fail in some embryos, but this could be a secondary effect caused by other morphogenetic defects. By examining different rhea alleles, it has been confirmed that this represents the null Talin phenotype. The phenotypes of mutant embryos from germline clones of rhea1 and rhea2 are indistinguishable, as are those of rhea79a, which deletes rhea and two flanking genes. Other data suggest, however, that the rhea1 and rhea2 mutations are weak dominant negatives. For example, producing rhea2/rhea79A embryos from maternal germline clones of rhea2 causes 65% (n = 153) failure of germ band retraction, while those of the rhea79A deficiency caused only 48% failure (Brown, 2002).
Further insight into the role of Talin was gained by looking at Talin and integrins in the imaginal disc epithelia. Just prior to pupariation it has been found that Talin and integrins colocalize into focal adhesion-like structures at the basal surface of the wing imaginal disc. Making clones of cells mutant for rhea results in loss of the staining of these structures with the Talin antibody. The clusters of Talin also fail to form in clones of cells lacking the ßPS integrin subunit. Clustering of integrins into these focal adhesion-like structures requires Talin function, as it does not occur in clones of cells lacking Talin. Therefore, clustering of integrins requires Talin, and clustering of Talin requires integrins. Loss of Talin does not grossly impair the rate of proliferation of the imaginal disc cells, since mutant clones are of a similar size to the wild-type twin spots. In addition, loss of Talin does not alter overall levels of integrin synthesis. Combining these results with those from the embryo suggests that Talin's role may be to promote integrin clustering, which, in turn, allows the establishment of a strong connection with the cytoskeleton (Brown, 2002).
The involvement of Talin in the transmission of integrin signals regulating gene expression was also examined. In Drosophila one signaling assay uses the enhancer trap 258, which is expressed in the midgut and fails to be repressed in the absence of PS1 integrin. The expression of this integrin target gene was examined in the absence of Talin. In embryos lacking maternal and zygotic Talin, midgut development was too disrupted to assay 258 expression. In the absence of the zygotic Talin, the midgut shows the characteristic phenotype of an integrin mutation: the gastric caeca fail to split from two initial evaginations into four slender tubes, the midgut does not elongate into a slender tube, and the proventriculus does not form properly. Despite these morphological defects, the 258 gene was repressed. The same result was obtained in more than 30 mutant midguts. While the possiblility cannot be ruled out that the small amount of maternal Talin left is sufficient for integrin signaling, but not for integrin adhesion, these results suggest that Talin is not required for integrin signaling to the nucleus (Brown, 2002).
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, a screen was conducted for autosomal mutations that produce blisters in somatic wing clones. Seventy-six independent mutations in 25 complementation groups were isolated, 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 (Prout, 1997).
Epithelial tubes that compose many organs are typically long lasting, except under specific developmental and physiological conditions when network remodeling occurs. Although there has been progress elucidating mechanisms of tube formation, little is known of the mechanisms that maintain tubes and destabilize them during network remodeling. This study describes Drosophila tendrils mutations that compromise maintenance of tracheal terminal branches, fine gauge tubes formed by tracheal terminal cells that ramify on and adhere tightly to tissues in order to supply them with oxygen. Homozygous tendrils terminal cell clones have fewer terminal branches than normal but individual branches contain multiple convoluted lumens. The phenotype arises late in development: terminal branches bud and form lumens normally early in development, but during larval life lumens become convoluted and mature branches degenerate. Their lumens, however, are retained in the remaining branches, resulting in the distinctive multi-lumen phenotype. Mapping and molecular studies demonstrate that tendrils is allelic to rhea, which encodes Drosophila talin, a large cytoskeletal protein that links integrins to the cytoskeleton. Terminal cells mutant for myospheroid, the major Drosophila ß-integrin, or doubly mutant for multiple edematous wings and inflated α-integrins, also show the tendrils phenotype, and localization of myospheroid ß-integrin protein is disrupted in tendrils mutant terminal cells. The results provide evidence that integrin-talin adhesion complexes are necessary to maintain tracheal terminal branches and luminal organization. Similar complexes may stabilize other tubular networks and may be targeted for inactivation during network remodeling events (Levi, 2006; full text of article).
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date revised: 25 August 2009
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