Based on biochemical studies in vertebrate systems, it has been suggested that an integrin-Ilk-PINCH complex might be necessary for integrin-dependent cell adhesion (Li, 1999; Tu, 1999; Wu, 1999). Consistent with this view, a recent characterization of Drosophila Ilk revealed that Ilk colocalizes with ßPS integrin at muscle-attachment sites and is required for integrin function (Zervas, 2001). PINCH and Ilk display completely overlapping patterns of localization in Drosophila muscle, with both proteins prominently enriched at the muscle-attachment sites. PINCH and Ilk are also co-expressed in the visceral mesoderm and pharyngeal muscles. Thus, PINCH, Ilk and ßPS integrin are co-residents of the same cellular compartments in vivo (Clark, 2003).
To test directly whether PINCH is present in a molecular complex with Ilk in vivo, native immunoprecipitation studies were performed with embryo extracts prepared from a transgenic line carrying an Ilk::GFP genomic construct, that was previously shown to maintain wild-type Ilk activity based on its ability to rescue the ilk mutant phenotype (Zervas, 2001). In the anti-PINCH immunoprecipitate, a band of the expected size of ~75 kDa for the Ilk-GFP protein is detected with a mAb against GFP. This band is absent in anti-PINCH immunoprecipitates from wild-type embryos, indicating that the anti-GFP-reactive band is dependent on the presence of the Ilk::GFP transgene. The specificity of the co-precipitation between Drosophila PINCH and Ilk confirms that the two proteins are present in a common molecular complex in vivo (Clark, 2003).
The actin phenotypes described for stck mutants are similar to those reported for Drosophila Ilk mutants, in that the actin filament linkage appears to be unstable and actin filaments detach from the muscle membrane (Zervas, 2001). Since Ilk and PINCH associate in vivo, the stck mutant phenotype may arise as a result of Ilk mislocalization. his possibility was explored by examining the localization of an Ilk::GFP fusion protein in stck mutant embryos derived from stck17 germline clones (i.e. embryos that lack functional maternally-derived and zygotic PINCH protein). In stck mutant embryos, Ilk::GFP retains the capacity to localize at muscle-attachment sites. Thus, the phenotypes seen in a stck mutant cannot be attributed to mislocalization of Ilk (Clark, 2003).
Integrin-linked kinase (ILK) is an essential component of a multiprotein complex that links actin to the plasma membrane. This study has used a genetic approach to examine the molecular interactions that are essential for the assembly of this ILK-containing complex at Drosophila muscle attachment sites (MASs). Downstream of integrins, talin plays a decisive role in the recruitment of three proteins: ILK, Lim domain protein PINCH (steamer duck) and paxillin. The accumulation of ILK at MASs appears to follow an amplification mechanism, suggesting that numerous binding sites are generated by minimal levels of the upstream integrin and talin effectors. This property suggests that ILK functions as an essential hub in the assembly of its partner proteins at sites of integrin adhesion. PINCH stability, and its subcellular localization at MASs, depends upon ILK function, but that ILK stability and localization is not dependent upon PINCH. An in vivo structure-function analysis of ILK demonstrated that each ILK domain has sufficient information for its independent recruitment at embryonic MASs, whereas at later developmental stages only the kinase domain was effectively recruited. These data strengthen the view that the ILK complex is assembled sequentially at sites of integrin adhesion by employing multiple molecular interactions, which collectively stabilize the integrin-actin link (Zervas, 2011).
In cell culture models, focal adhesions are the prototype form of sites of integrin adhesions. Several lines of evidence suggest that the molecular assembly of focal adhesions is a multistep process, where different cytoplasmic proteins enter the adhesion sites in a defined sequential order. This study provides genetic evidence that supports a hierarchical model for formation of integrin adhesions, using the Drosophila embryonic MASs as a model system (Zervas, 2011).
This study reexamined whether integrins are essential for the localization of ILK to MASs. A previous study found that ILK was recruited independently of integrins. This conclusion has now been corrected: complete elimination of integrins resulted in loss of ILK recruitment. This result, therefore, reveals that the small amount of maternally provided integrin recruits unexpectedly high levels of ILK. This surprising finding has two important implications: 1) the recruitment of ILK and maintenance of cell-matrix adhesion have different requirements for the amount of integrin (low levels of integrin are sufficient to recruit ILK, but high levels are needed for functional adhesion), 2) the amount of ILK recruited by integrins is not set by the quantity of integrin at MASs, suggesting that there is an amplification mechanism so that a single integrin can recruit multiple ILK molecules (although the experiments do not reveal the relative number of molecules). A similar, but less extreme, lack of correlation between the levels of integrin and an intracellular integrin-associated protein at MASs has been found for talin. This apparent amplification could be explained by a multidocking scaffolding protein that is able to recruit, either directly or indirectly, several ILK molecules. Alternatively, ILK could have the ability to employ a variety of additional interactions with different binding partners in the integrin-containing junctions, so that even if integrins or other components are much reduced, multiple ILK molecules are recruited. The mechanism of the amplification is unknown, with a number of the integrin-associated proteins being potential components; the kindlin proteins are particular good candidates, because they bind both integrins and ILK (Karaköse, 2010), and Wech has been shown to bind both talin and ILK and to contribute to ILK recruitment (Löer, 2008; Zervas, 2011 and references therein).
Talin binding to integrins is one of the first molecular events, and is a prerequisite, in the formation of the integrin-cytoskeleton link. Therefore, whether talin is required to recruit components of the ILK-containing complex at sites of integrin adhesion was examined. As with integrins, it was essential to remove both the maternal and zygotic production of talin to reveal defects in ILK, PINCH and paxillin recruitment. Consequently, these results show an important function for talin in recruiting the ILK-containing complex. A similar role for talin in ILK recruitment at myotendinous junctions in mice has been reported. Currently there are no data to support a direct association between components of the ILK complex and talin, but Wech binds to talin and ILK suggesting that it could be the key linker between the two proteins (Löer, 2008), at least in muscles. Therefore, a model is favored whereby talin, in concert with integrins, recruits Wech and additional proteins. These proteins then in turn recruit ILK and paxillin, and, as shown here, ILK indirectly recruits PINCH (Zervas, 2011).
In vertebrate cells ILK and PINCH have mutually dependent functions. Their association is required to protect each other from a proteasome-dependent degradation and to facilitate each other's localization to focal adhesions. The direct interaction between ILK and PINCH has also been confirmed in Drosophila, and PINCH-mediated stabilization of ILK in mice has been confirmed genetically. In Drosophila, it was found that neither ILK protein stability nor its recruitment to MASs was affected in the absence of PINCH. This is in agreement with the persistent subcortical localization of ILK in the pinch-1-knockout mouse embryoid bodies, as well as in unc-97 mutants in C. elegans. By contrast, loss of ILK function reduced PINCH levels in Drosophila embryos. The instability of PINCH in the absence of ILK was overcome by overexpressing PINCH but, under these conditions, PINCH still was not localized to MASs. The isolated ILK ANKRs did localize to MASs in the absence of endogenous ILK, but were not able to recruit PINCH. Thus, rather than ILK recruiting PINCH by binding of its ANKRs to the LIM1 domain of PINCH, it appears to do so indirectly. This finding raises two important points. First, the interaction between the ILK ANKRs and LIM1 of PINCH might not be always maintained at sites of integrin adhesion. The finding that LIM1 is crucial for recruitment, but apparently not through interaction with ILK, suggests alternative interactions might be required. This is consistent with the observation that high levels of PINCH-GFP can be recruited to MASs, even when only the endogenous levels of ILK are present. Second, recent work in mammalian cells provided strong evidence that the primary function of PINCH was the recruitment of ILK through the ANKRs-LIM1 interaction (Stanchi, 2009). Notably, the recruitment of tensin and ILK at focal adhesions in cells lacking PINCH could be rescued by a direct fusion of the ILK kinase domain to integrin. However, the result suggests that, in Drosophila muscles, the reverse is true, and a primary function of ILK might be the stabilization and recruitment of PINCH (Zervas, 2011).
Finally, whether ILK and PINCH influence the subcellular localization and protein stability of paxillin was examined. Previous studies have demonstrated an interaction between the kinase domain of mammalian ILK and paxillin, and further suggested that this interaction controls ILK localization at focal adhesions. These studies identified key residues in ILK that contribute to paxillin interaction and ILK recruitment, such that ILK mutations at E359 and T384 impaired recruitment of ILK to focal adhesions. However, the ILK E359K mutation does not impair its ability to rescue the null phenotype or alter its recruitment in flies or mice. This study showed that mutating T384 in Drosophila ILK, equivalent to part of the paxillin-binding region in human ILK, also failed to impair the biological activity or recruitment of ILK, and neither ILK nor PINCH were required for paxillin recruitment. Thus, in MASs, the stability of paxillin and its subcellular localization does not depend on the ILK-PINCH complex (Zervas, 2011).
This study has examined the functions of the two main domains that constitute ILK (i.e. the ANKRs and the kinase domain), as well as specific binding sites that have been proposed to mediate interactions with phosphoinositides or paxillin and have been implicated in the regulation of the protein. From this work, it became evident that deletion of either the ANKRs or the kinase-like domain eliminates the essential functions of ILK in the developing organism. These two domains are required in the same molecule to execute the essential function of ILK. The proposed phosphoinositide-binding motif is dispensable for embryonic development but is required in the epithelial tissues of the wing, and therefore does contribute to ILK function. This suggests that the highest levels of ILK function are required in the wing. This is consistent with the finding that elimination of tensin only causes a wing-blister phenotype, even though it is also concentrated at MASs. By contrast, hypomorphic mutations in the gene encoding the αPS2 integrin subunit were found to cause muscle defects but not cause wing blisters, indicating that integrin adhesive sites are differentially sensitive to loss of different components of the integrin adhesion complex. The residue F436, which is located at the far end of the C-terminal region, was found to be crucial in vertebrate cells, but its mutation only mildly affected recruitment of the overexpressed protein and was dispensable for ILK function. It is possible that the F436A mutation reduces the binding affinity of ILK with Wech (Zervas, 2011).
Both the isolated ANKRs and the kinase domain are recruited to MASs, in contrast with the recruitment to focal adhesions seen in mammalian cells. This suggests that recruitment is more robust at MASs, so impaired ILK recruitment can still be visualized. It was surprising to find that the recruitment of the ANKRs of ILK did not involve an interaction with PINCH, as removing PINCH did not impair recruitment. Given that there is only one Drosophila pinch gene, in contrast with two in mammals, the ANKRs must be binding to another type of protein. One alternative candidate is Mlp84B, a protein that contains five LIM domains and is localized at MASs in an integrin-independent manner; it is known that the related zebrafish protein CSRP3 binds ILK (Zervas, 2011).
In summary, the results have demonstrated that, at the robust integrin adhesive sites that form in the developing muscles, ILK recruitment is much less sensitive to perturbation compared with recruitment in focal adhesions in cells in culture. The results show that integrins and talin are essential for the recruitment of ILK, and ILK in turn is essential for the recruitment of PINCH. How much of the function of ILK is mediated by PINCH, and whether it also functions independently through its interaction with parvin, will be the focus of future research (Zervas, 2011).
In an effort to deduce some conserved function among PINCH family members, the expression pattern of the Drosophila homologue of Unc-97, d-pinch (steamer duck), was examined. A developmental profile and expression pattern for d-pinch was determined by whole mount RNA in situ hybridization on 0-17-h embryos. D-pinch transcripts are first detected in stage 10 embryos, where it is expressed in the visceral and body wall muscle. By stage 13, the expression has increased, and some pharyngeal muscle staining is also detected. Of particular interest is the intense expression of the transcripts at the sites of gut constriction. In late-stage 16 embryos, when the myotendinous junction is just beginning to form, d-pinch transcripts are detected in the epidermal tendon cells, as well as the aforementioned muscle lineages. At this time of development, the heart musculature has differentiated as well; however, no d-pinch expression is seen in this muscle lineage (Hobert, 1999).
Like Unc-97-expressing cells, d-pinch-expressing cells are attached to extracellular matrix components via integrin receptors. The timing of d-pinch expression in these cells is consistent with the expression of integrin subunits as well. A very striking example of coexpression is the temporal pattern of d-pinch and the integrin subunit alphaPS1 in tendon cells. By late stage 16, high levels of alphaPS1 are detected in the tendon cells with no other epidermal expression of the transcript; it is at this embryonic stage that detect d-pinch transcript is first detected in tendon cells (Hobert, 1999).
The timing and tissue-specific expression of d-pinch suggests that it is involved in the terminal differentiation of muscles and tendon cells; one common feature of these cell types is their formation of adherens junctions. Integrin complexes are crucial for the stability of the junctions, as demonstrated by the dramatic muscle detachment phenotype seen in myospheroid/ßPS-integrin mutants. Based on the observation that the null Unc-97 phenotype is very severe and phenocopies mutations in the pat-3/ß-integrin gene, it is predicted that loss of d-pinch will also result in a severe disruption of muscle function (Hobert, 1999).
Drosophila PINCH displays five tandemly arrayed LIM domains that exhibit a high degree of sequence similarity to human PINCH1. Molecular and genomic analyses confirm that there is a single PINCH gene in Drosophila. Northern blots probed with a Drosophila pinch cDNA reveal a single transcript of 1.4 kb. Genefinder programs do predict a possible alternative start site that would use a different first exon; however, this would not affect the coding sequence and no existing Drosophila PINCH ESTs contain this alternative exon. Moreover, RT-PCR analysis of RNA from staged samples results in products identical in sequence to the original cDNA, further supporting the view that there is only one RNA species transcribed from the pinch locus. Northern analysis of developmentally staged RNA samples revealed that pinch expression parallels that of ßPS integrin. Specifically, pinch transcripts are maternally inherited and are expressed zygotically at the time of muscle differentiation. pinch RNA levels decrease during the larval stages, but increase again during pupal development, coincident with the terminal differentiation of the adult structures (Clark, 2003).
Since the stck mutants exhibited defects in the anchorage of actin filaments at the myotendinous junction, it was postulated that PINCH might be a constituent of these cell-substratum attachment sites. Indeed, by immunocytochemical analysis, PINCH protein in the developing somatic muscles, with prominent enrichment at the muscle-attachment sites. PINCH is also detected in other musculatures including the dorsal vessel (the heart equivalent in Drosophila). There is also prominent staining in the midgut epithelium. The affinity-purified serum also labels the chordotonal organs, but this appears to be due to crossreaction with another protein because this staining remains in stck17 maternal/zygotic mutants, whereas all muscle attachment site staining is absent (Clark, 2003).
pinch transcription is upregulated in pupae and adults, suggesting that PINCH may have functions during these later developmental stages as well. Moreover, the two stck alleles that encode PINCH were originally identified in a genetic screen for potential integrin effectors that relied on wing blister formation (Prout, 1997). Using mitotic recombination, it was confirmed that homozygous stck mutations cause wing blistering. This observation suggests that PINCH is expressed in the wing epithelium, and is required for integrin-dependent adhesion in this tissue, but neither the expression nor the subcellular localization of PINCH in wing epithelium had been described. To examine directly the expression and subcellular distribution of PINCH in the developing Drosophila wing, immunocytochemical analysis was performed with anti-PINCH and anti-ßPS integrin antibodies. PINCH expression was examined in wing discs dissected from wandering third instar larvae. PINCH was found associated with wing cell membranes; ßPS integrin displays a similar pattern at this stage of development. Later in development, when the wing epithelia have become apposed, ßPS integrin becomes enriched at basal junctions that form between the two layers. PINCH is enriched at this junction and is also associated with the cell cortex coincident with sites of integrin accumulation. Collectively, these findings support the view that PINCH is required for integrin function in both embryos and adults (Clark, 2003).
Alignment of the Drosophila pinch cDNA sequence with the deposited Drosophila genome sequence indicates that the pinch locus maps to 84E11-85A1. This assignment is in agreement with chromosome in situ hybridization data that placed pinch at 85A1-3. Several pre-existing mutations, which are generated from unrelated mutagenesis screens, map to the same cytological interval as pinch. One lethal complementation group, stck, is represented by two alleles (stck17 and stck18) that were isolated in a mutagenesis screen designed to identify gene products required for integrin function (Prout, 1997). Moreover, stck mutations were reported to enhance a phenotype associated with compromised integrin function (Prout, 1997). By DNA sequence analysis, it was found that both stck alleles contain mutations in the pinch locus that are predicted to disrupt the protein-coding region. stck17 contains a 571 bp internal deletion that removes DNA encoding the last two and a half LIM domains of PINCH, while stck18 has a two bp deletion that alters the reading frame in the fourth LIM domain (Clark, 2003).
The lethality associated with homozygous stck mutations can be rescued by introduction of a transgene that encodes wild-type PINCH. Further confirmation that PINCH is encoded by the stck locus comes from western immunoblot analysis of PINCH protein levels in stck mutants. Affinity-purified antiserum directed against a C-terminal PINCH epitope recognizes a single polypeptide with an apparent molecular mass of 31 kDa in wild-type embryos. Wild-type PINCH protein levels are significantly reduced in stck zygotic mutants and the protein is undetectable when maternal PINCH is also eliminated. Collectively, these data provide compelling evidence that Drosophila pinch is encoded by the stck locus (Clark, 2003).
The phenotypes associated with the two stck alleles described above have been characterized. When examined as hemizygous mutations, greater than 85% of the stck mutant embryos die, indicating a strong requirement for PINCH during embryonic development. Comparison of wild-type larvae and the few stck mutant larvae that survive to hatch revealed dramatic morphological differences. The stck mutant larvae are significantly shorter than wild-type larvae. Additionally, stck mutant larvae are nearly immobile, a phenotype that suggests impaired muscle function, and die within 24 hours of hatching (Clark, 2003).
pinch transcript is expressed prominently in the developing somatic muscles of Drosophila embryos (Hobert, 1999), therefore mutant embryos were examined more closely for any perturbations in somatic muscle patterning and development. Initial muscle patterning is not affected in stck mutants, indicating that PINCH is not required for muscle cell differentiation, fusion or migration. Defects in muscle morphology are first detected in stck mutants at embryonic stage 16. By comparing wild-type and mutant embryos that are stained with antibody directed against Mlp84B, a muscle-specific protein that is associated with the contractile apparatus and enriched at muscle-attachment sites, it is evident that the mutant muscles exhibit a distorted morphology. The embryonic musculature is less organized in stck mutants compared with their wild-type counterparts, and gaps are evident occasionally between adjacent muscle cells, indicating a failure of some muscle-attachment sites (Clark, 2003).
To evaluate whether the misshaped muscles have underlying cytoskeletal defects, the actin organization in stck mutant embryos was examined. In early stage 17 embryos, the actin filaments in the wild-type muscle cells are clearly organized into linear arrays that extend to the lateral borders of each muscle fiber. There is a clear enrichment of filamentous actin at the muscle termini, where the muscle cell membranes are attached to the tendon cell matrix. By contrast, the stck mutant muscles do not display such a high degree of actin filament organization. The actin filament bundles that comprise the myofibrils are buckled in appearance, and often do not extend to the segment boundaries. Additionally, many of the muscle attachments lack the enrichment in filamentous actin seen in wild-type animals. The significant alteration of myotendinous junction structure and composition suggests that the function of this specialized adhesive junction is probably compromised in stck mutant embryos. The disturbed cytoskeletal organization observed in the stck mutants progressively worsens as development proceeds, such that in late stage 17 mutant embryos, actin filament arrays are largely retracted to one end of the muscle, indicating a failure of at least one of the actin-membrane anchorage sites that normally tether the ends of the contractile machinery to the muscle cell membrane (Clark, 2003).
Both stck17 and stck18 alleles retain some PINCH-coding sequence. In particular, these mutant alleles could theoretically support the production of C-terminally truncated PINCH products that might retain partial function or have dominant-negative activity. In order to assess whether stck17 and stck18 behave as simple loss-of-function alleles, the cellular phenotypes of stck17 and stck18 hemizygotes were compared with embryos that carry a homozygous deletion of the stck locus (l(3)097) and a comparable terminal phenotype was observed. These findings illustrate that the stck17 and stck18 alleles disrupt PINCH function to a similar extent as occurs when PINCH function is completely eliminated by a gene deletion. Thus, stck17 and stck18 do not display any residual PINCH activity that ameliorates the mutant phenotype relative to what is observed in a molecular null. Moreover, neither stck17 nor stck18 heterozygotes display any cellular defects or loss of viability that might be anticipated if the stck17 and stck18 alleles produce a dominant-negative product (Clark, 2003).
Because pinch transcripts are maternally inherited, the phenotype was evaluated of animals in which both zygotically and maternally derived PINCH were eliminated by construction of germline clones. Analysis of maternal/zygotic stck mutants did not reveal additional phenotypes that were not evident in zygotic stck mutants; however, the disturbance in muscle morphology was evident at an earlier stage than for the zygotic mutants, with actin clumping apparent in some muscle cells by the end of stage 16, consistent with the time of onset of muscle contraction (Clark, 2003).
The Drosophila integrin subunits alphaPS2 and ßPS are also enriched at muscle-attachment sites, where they participate in the adhesion of the muscle termini to a specialized ECM, the tendon cell matrix. Using confocal microscopy, it was found that PINCH is precisely colocalized with ßPS integrin at muscle-attachment sites in the somatic muscle termini. PINCH and ßPS integrin proteins also display overlapping patterns of concentration in other tissues such as the visceral musculature, pharyngeal muscles and epithelial tissues (Clark, 2003).
Given the striking accumulation of PINCH and ßPS integrin at muscle-attachment sites, tests were performed to see whether PINCH depends on integrins to become properly distributed in the muscle. PINCH protein distribution was examined in embryos harboring null alleles of either ßPS integrin (myospheroid) or alphaPS2 integrin (inflated). The alphaPS2ßPS heterodimer is the integrin complex present on the muscle side of the myotendinous junction, and loss of either subunit prevents the localization of the other subunit. Compared with wild-type embryos in which PINCH displays a striking localization at muscle-attachment sites, PINCH is not enriched at the muscle termini of myospheroid or inflated mutants. The lack of PINCH staining in the myospheroid and inflated mutant embryos was not due to a failure in antibody penetration or disintegration of the muscle-attachment sites, since the nonspecific immunoreactivity of the chordotonal organs is still present, and the cytoskeletal protein PAK remains robustly localized at residual muscle-attachment sites in myospheroid embryos and in inflated embryos. The presence of PAK at the muscle borders in myospheroid mutants provides support for the conclusion that the loss of PINCH from muscle attachments in an integrin mutant is not due to a general defect in these junctions, and instead indicates a direct dependence of PINCH on integrins for its distribution in mature muscle. Some PINCH protein is detected concentrated at the muscle termini in younger myospheroid embryos. The most straightforward interpretation of these results is that PINCH requires integrins for its maintenance at muscle attachments, and not for its initial localization. However, although several groups have failed to detect maternally supplied ßPS integrin in myospheroid embryos at this time of development, it remains formally possible that some residual ßPS protein is present to recruit PINCH to the junctional complex at this earlier stage. In any case, these findings illustrate that, at a minimum, integrins are required for maintenance of PINCH at the junctional complexes (Clark, 2003).
In complementary experiments, ßPS integrin distribution was examined in wild-type and stck mutant embryos. Wild-type embryos show a striking accumulation of ßPS integrin at muscle-attachment sites. Although muscle morphology is perturbed in stck mutants, ßPS retains the capacity to localize at muscle-attachment sites when PINCH function is compromised by mutation. Thus, the appropriate targeting of ßPS integrin to the cell surface and their concentration at adhesive junctions can occur in the absence of PINCH (Clark, 2003).
Cell adhesion and migration are dynamic processes requiring the coordinated action of multiple signaling pathways, but the mechanisms underlying signal integration have remained elusive. Drosophila embryonic dorsal closure (DC) requires both integrin function and c-Jun amino-terminal kinase (JNK) signaling for opposed epithelial sheets to migrate, meet, and suture. PINCH, a protein required for integrin-dependent cell adhesion and actin-membrane anchorage, is present at the leading edge of these migrating epithelia and is required for DC. By analysis of native protein complexes, RSU-1, a regulator of Ras signaling in mammalian cells, has been identified as a novel PINCH binding partner that contributes to PINCH stability. Mutation of the gene encoding Drosophila RSU-1 results in wing blistering in Drosophila, demonstrating its role in integrin-dependent cell adhesion. Genetic interaction analyses reveal that both PINCH and RSU-1 antagonize JNK signaling during DC. These results suggest that PINCH and RSU-1 contribute to the integration of JNK and integrin functions during Drosophila development (Kadrmas, 2004).
To determine if PINCH contributes to DC, its localization was examined in stage 14 embryos. PINCH and ß-PS integrin colocalize in both the LE and the amnioserosa, consistent with PINCH's established role as an integrin effector. The amnioserosa is an extraembryonic tissue present on the dorsal surface of the embryo. Since it has been established that coordinated signaling between the amnioserosa and migrating epithelium is key to formation of LE focal complexes, PINCH could exert an effect in the LE epithelium, the amnioserosa, or both tissues. stck homozygous mutant embryos rescued with a PINCH:GFP transgene under the control of the endogenous PINCH promoter display PINCH-GFP at the LE of the advancing epithelial sheets. Within the LE, PINCH is precisely localized to areas of active phosphotyrosine signaling at triangular nodes corresponding to apical adherens junctions (Kadrmas, 2004).
Zygotic stck mutants proceed normally through DC with complete lethality arising at the embryo-to-larval transition. When maternal PINCH contribution is eliminated, only 12% of cuticles have wild-type morphology. Dorsal puckers and dorsal holes characteristic of aberrant DC are observed at a 36% and 23% frequency, respectively, indicating that maternally inherited PINCH is a key contributor to the process of DC. Moreover, in the absence of maternal PINCH, epithelial defects are observed in ventral patterning and head involution, indicating that PINCH may have additional functions in the developing embryo. Cuticles from embryos lacking both maternal and zygotic PINCH have the same array of phenotypes (Kadrmas, 2004).
PINCH is composed of five LIM domains, each of which could serve as a protein-binding interface. The SH2-SH3 adaptor protein, Nck2, has been reported to interact with mammalian PINCH. This association is intriguing because the Drosophila homologue of Nck2, Dreadlocks, interacts directly with Misshapen (Msn), a MAP4K in the JNK signaling cascade. As with other components of the JNK pathway, null mutations in msn result in embryonic lethality due to failure of DC. Although no direct binding of PINCH to Dreadlocks was observed in Drosophila, this study uncovered a link between PINCH's role in DC and the JNK cascade by testing for genetic interaction between stck and msn. Reduction of PINCH protein levels by introduction of a single copy of the loss-of-function allele, stck18, into the msn102 homozygous null background allows partial restoration of DC, suggesting that PINCH functions as a negative regulator of JNK signaling (Kadrmas, 2004).
Puckered (Puc) is a JNK phosphatase whose expression is up-regulated in response to JNK activation, setting up a negative feedback loop. During DC, JNK-regulated expression of a Puc-LacZ fusion reporter is restricted to the LE cells. In embryos lacking maternal PINCH, expression of the Puc-LacZ fusion protein is disorganized and present in an expanded number of cells, including those beyond the LE border. This phenotype is similar to Puc-LacZ expression observed in puc loss-of-function mutants and further supports a role for PINCH in the negative regulation of the JNK cascade (Kadrmas, 2004).
Thorax closure is a post-embryonic developmental process with features common to DC, including migration of epithelial sheets and a dependence on JNK signaling. Within the wing disc, cells of the stalk region are functionally similar to LE cells during DC. These cells comprise the eventual fusion site for adjacent imaginal discs and are active in JNK signaling. Spatially restricted JNK signaling in the stalk of wing disc can be visualized via a Puc-LacZ reporter, and PINCH expression overlaps with Puc-LacZ in this area of active JNK signaling. Therefore, as in DC, PINCH is properly positioned to act as a regulator of the JNK cascade (Kadrmas, 2004).
Although msn null mutations are embryonic lethal due to DC failure, flies homozygous for the hypomorphic allele msn3349 are semi-viable and a large proportion of the eclosing adults have thorax closure defects. These observations underscore the similarities between thorax closure and DC. In a stck18 heterozygous background, a greater percentage of msn3349 homozygotes are able to eclose, supporting the hypothesis that PINCH is a negative regulator of the JNK pathway in both dorsal and thorax closure (Kadrmas, 2004).
Drosophila PINCH was purified in complex with its binding partners using tandem affinity purification (TAP)–tagged PINCH (TAP-PINCH). stck homozygous mutant embryos rescued with a TAP:PINCH transgene driven by the endogenous stck promoter to wild-type levels afford material for purification of soluble, cytoplasmic TAP-PINCH complexes in the absence of endogenous PINCH protein. Three partners that copurified stoichiometrically with TAP-PINCH from embryos, as well as in complex with TAP-PINCH from cultured Drosophila S2R+ cells, were identified by mass spectrometric analysis. Consistent with what is observed in mammalian cells, ILK copurified with PINCH. The Drosophila homologue of the parvin/actopaxin family of proteins, CG32528, is also present in PINCH protein complexes. Parvin is known to bind ILK and actin in mammalian systems, but the isolated Parvin/ILK/PINCH complexes are the first to be described in Drosophila. Additionally, a novel 31-kD protein was identified as Drosophila CG9031. The CG9031 protein is 55% identical and 74% similar to human RSU-1, a leucine-rich repeat containing protein first identified as a suppressor of cell transformation by v-Ras and subsequently implicated in regulation of MAP kinase signaling, specifically the JNK and ERK cascades, when overexpressed in cultured cells. Despite its potent ability to act as a tumor suppressor, little is known about the mechanism of action of RSU-1. Its partnership with the PINCH protein allows placement of RSU-1 in a molecular pathway that is linked to integrins (Kadrmas, 2004).
To assess the specificity and nature of the interaction between PINCH and RSU-1, domain-mapping studies were performed in cell culture and in yeast two-hybrid assays. Drosophila RSU-1 copurifies with full-length His-tagged PINCH, but not with a truncated His-tagged PINCH containing only LIM1–3, confirming the specificity of the interaction and suggesting LIM4 and/or 5 is the site of binding. ILK, which binds LIM1 of PINCH, copurifies with both full-length and the truncated LIM1–3 version of His-tagged PINCH, serving as a positive control. Both PINCH and ILK are copurified with His-tagged RSU-1. Moreover, endogenous PINCH and RSU-1 can be coimmunoprecipitated. The site of RSU-1 binding to PINCH was further mapped using yeast two-hybrid analysis. Only cells expressing LIM5 bait/RSU-1 prey activated all three reporters, indicating LIM5 is the site of RSU-1 binding. Consistent with the view that they interact in vivo, PINCH:GFP and RSU-1 are prominently colocalized at integrin-rich muscle attachment sites in Drosophila embryos (Kadrmas, 2004). Drosophila RSU-1, which displays seven leucine-rich repeats with high sequence similarity to small GTPase regulators, is encoded by the CG9031 locus. A P-element insertion allele was characterized that disrupts the RSU-1 coding sequence. Flies homozygous for this mutation within CG9031 are viable and fertile, and lack RSU-1 protein as indicated by Western analysis with multiple anti-RSU-1 antibodies. PINCH and RSU-1 are both expressed in larval wing discs and similar to stck wing clones, the mutation within CG9031 produces flies with wing blisters at 60% penetrance. These data are consistent with PINCH and RSU-1 acting in concert to support integrin-dependent adhesion. The CG9031 gene was named icarus (ics) after the son of Daedalus who had unstable wings (Kadrmas, 2004).
Although elimination of RSU-1 function does not result in dorsal or thorax closure defects, the role of RSU-1 in these processes was evaluated by testing for genetic interactions between ics and msn. Similar to what occurs with reduction of stck dosage, homozygous mutation of ics suppresses DC defects observed in msn102 mutant embryos. Absence of RSU-1 also increases eclosure rates of msn3349 hypomorphs and completely suppresses the thorax defects present in msn3349 animals, suggesting that like PINCH, RSU-1 can function as a negative regulator of JNK signaling. To confirm that the suppression of msn DC defects by ics mutation is mediated by the JNK signaling cascade, RSU-1 was eliminated in basket (bsk) embryos that lack zygotic JNK, the terminal kinase in this cascade. Homozygous ics mutation suppresses the DC defects of bsk1 mutants, confirming that ics loss-of-function mutations affect DC by influencing the JNK cascade. Moreover, wing discs isolated from ics mutants display a 30% increase in active phospho-JNK relative to wild type, providing direct biochemical confirmation that RSU-1 influences JNK activation state in vivo. Although no localized accumulation of RSU-1 during DC was detected, RSU-1 is readily detected by Western analysis in stage 13 embryos that are undergoing DC. Thus, the temporal pattern of RSU-1 expression is consistent with genetic results that highlight its role in regulation of JNK-dependent morphogenesis (Kadrmas, 2004).
Analysis of PINCH and RSU-1 levels in wild-type versus stck or ics mutant embryos provided insight into the physiological significance of their association. In embryos mutant for both maternal and zygotic stck, RSU-1 is dramatically reduced relative to wild-type levels. Likewise, in ics embryos, PINCH levels are also decreased. These observations suggest that PINCH and RSU-1 are reciprocally dependent on each other for maximal expression and/or stability. The mechanism for coordinate regulation of RSU-1 and PINCH remains to be determined. Because the phenotypes associated with loss of RSU-1 represent a subset of stck phenotypes, the processes disturbed in ics mutants may be exquisitely sensitive to PINCH levels. Alternately, RSU-1 may have functions that are independent of its role in PINCH stabilization (Kadrmas, 2004).
The data are consistent with a model in which PINCH could modulate JNK signaling in two distinct ways. (1) PINCH is present at areas where JNK is active and antagonizes JNK signaling. This behavior is reminiscent of Drosophila Puc, a phosphatase regulator of the JNK cascade that establishes a negative feedback loop. PINCH has no intrinsic catalytic activity, but might recruit proteins that could alter the availability or activity of JNK signaling components. Like Puc, PINCH expression is up-regulated in response to constitutive JNK signaling. Availability of RSU-1 at these sites of active JNK signaling could independently regulate JNK signaling or modulate the effects of PINCH on JNK through regulation of PINCH stability. (2) PINCH and RSU-1 are required for integrin-dependent adhesion. PINCH links integrins to the actin cytoskeleton via ILK and Parvin, and these connections could influence both integrin-dependent adhesion and signaling. Integrin signaling, through a variety of tyrosine kinases and Rac, stimulates the JNK cascade; therefore, PINCH may also exert an influence on JNK signaling via integrin. The current findings illustrate that the cellular concentration of PINCH affects the level of RSU-1 and vice versa. Thus, modulation of the ratio of RSU-1 to PINCH could provide a mechanism to regulate JNK signaling during DC and thorax closure in Drosophila. It is hypothesized that PINCH/RSU-1 complexes fine-tune and integrate the JNK and integrin signaling cascades required during morphogenesis, highlighting the potential role of integrin-associated apical junctional complexes as signal coordination points for epithelial morphogenesis (Kadrmas, 2004).
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date revised: 20 January 2007
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