Integrin linked kinase
Integrins play a crucial role in cell motility, cell proliferation and cell survival. The evolutionarily conserved LIM protein PINCH is postulated to act as part of an integrin-dependent signaling complex. The molecular architecture of PINCH (Particularly Interesting New Cysteine-Histidine rich protein), which consists exclusively of multiple LIM domains suggests that it may function as a platform for the docking and/or productive juxtaposition of proteins involved in integrin signaling. In order to evaluate the role of PINCH in integrin-mediated cellular events, function of PINCH in Drosophila melanogaster was directly tested in vivo. The steamer duck (stck) alleles, that were first identified in a screen for potential integrin effectors, represent mutations in Drosophila pinch. stck mutants die during embryogenesis, revealing a key role for PINCH in development. Muscle cells within embryos that have compromised PINCH function display disturbed actin organization and cell-substratum adhesion. Mutation of stck also causes failure of integrin-dependent epithelial cell adhesion in the wing. Consistent with the idea that PINCH could contribute to integrin function, PINCH protein colocalizes with ßPS integrin at sites of actin filament anchorage in both muscle and wing epithelial cells. Furthermore, it is shown that integrins are required for proper localization of PINCH at the myotendinous junction. Integrin-linked kinase (Ilk), is also essential for integrin function. Drosophila PINCH and Ilk are complexed in vivo and are coincident at the integrin-rich muscle-attachment sites in embryonic muscle. Interestingly, Ilk localizes appropriately in stck mutant embryos, therefore the phenotypes exhibited by the stck mutants are not attributable to mislocalization of Ilk. These results provide direct genetic evidence that PINCH is essential for Drosophila development and is required for integrin-dependent cell adhesion (Clark, 2003).
The genetic analysis of PINCH function has led to four main conclusions: (1) Drosophila PINCH is encoded by the stck locus and is essential for embryonic development and maintenance of tissue architecture; (2) PINCH is necessary for stable actin-membrane anchorage in muscle and contributes to integrin-dependent adhesion in muscle cells and epithelial cells; (3) integrins are required for the stable association of PINCH with muscle-attachment sites; and (4) the lethal stck mutant phenotype cannot be attributed to mislocalization of the PINCH-binding partner, Ilk, whose recruitment to muscle-attachment sites appears normal in stck mutant embryos (Clark, 2003).
Genetic analyses of the roles of integrins in Drosophila have clearly highlighted the importance of integrins for adhesion and signaling in vivo. Drosophila PINCH is colocalized with integrins in both muscle and epithelial cells. Integrins retain the capacity to accumulate at muscle-attachment sites in stck mutants, illustrating that PINCH does not have an obligatory role in the proper processing and membrane targeting of integrins in vivo. The integrin staining in stck mutants does lack the high degree of order and lateral registration observed in wild-type embryos. In the Drosophila system, it is difficult to distinguish whether this modest disorganization simply reflects the underlying disturbance of the musculature or if it is revealing some contribution of PINCH to maintenance of spatially restricted integrin localization. In C. elegans embryos in which PINCH function is compromised by unc-97 mutation, both integrin and vinculin spread laterally beyond their normal zones of accumulation in dense plaques, suggesting a role for PINCH in clustering of adhesive junction components in this system (Clark, 2003).
Interestingly, PINCH depends on the presence of integrins for its stable accumulation at muscle-attachment sites. Several other proteins, including Talin, Ilk, Myosin II and Short stop colocalize with ßPS integrin at Drosophila muscle-attachment sites. These proteins display variable levels of dependence on integrins for their localization. Like Talin, a well-established integrin effector, PINCH depends on the presence of integrins for its concentration at muscle-attachment sites. The reliance of PINCH and Talin on integrins for their spatially restricted accumulation in muscle emphasizes their connection to the integrin receptors (Clark, 2003).
Integrins must establish links to both extracellular determinants and to intracellular cytoskeletal elements in order to support strong adhesion. Examination of the cellular defects in stck mutant muscle suggests that PINCH contributes to the stabilization of actin-membrane linkages at integrin-rich adhesion sites. In a stck mutant muscle cell, the actin filaments lose their linear organization and eventually accumulate in clumps at one end of the cell. These defects are interpreted to mean that a primary consequence of disturbed PINCH function is a destabilization of the linkage between the actin cytoskeleton and the muscle membrane; it appears that the actin-membrane attachments in stck mutants lack the mechanical strength to remain intact during cyclic muscle contraction. Because integrin functionality relies on the ability of the receptors to establish a transmembrane link between the cytoskeletal elements and the extracellular matrix, reduced substratum attachment strength and/or stability might also be expected to occur if membrane cytoskeletal linkages were compromised. Consistent with this prediction, loss of adhesion is evident in the stck17-/- wing cell clones and, to some extent, in muscles of stck mutant embryos (Clark, 2003).
The molecular architecture of PINCH suggests that it may function as a platform for the docking and/or productive juxtaposition of protein partners. Ilk, a binding partner of PINCH, is thus a candidate to collaborate with PINCH in the stabilization of integrin-cytoskeletal linkages. Consistent with the view that PINCH and Ilk cooperate to promote stable actin anchorage at sites of integrin-mediated adhesion, the phenotypes that result from compromised function of either protein in Drosophila are very similar (Zervas, 2001; Clark, 2003). Moreover, PINCH and Ilk are colocalized in Drosophila embryos and are recovered in a protein complex isolated from embryos by immunoprecipitation. Drosophila PINCH also interacts directly with Ilk using two-hybrid methods. These results are consistent with findings for vertebrate PINCH and Ilk. PINCH and Ilk also colocalize at actin-membrane anchorage sites in C. elegans muscle, and elimination of either gene product was shown to produce a paralyzed at twofold stage (PAT) phenotype similar to that seen for ß-integrin mutants. Collectively, results in both invertebrate and vertebrate systems illustrate that the capacity to form a PINCH/Ilk complex has been conserved through evolution (Clark, 2003 and references therein).
Given the fact that Ilk and PINCH colocalize, co-precipitate and have similar loss of function phenotypes, it is possible that disturbed PINCH function could adversely affect Ilk localization and that such mislocalization might account for the stck mutant phenotype. To explore this possibility the localization of Ilk was examined in stck mutant embryos; Ilk was found to be unperturbed in its ability to accumulate at muscle-attachment sites, even when a dramatic lethal phenotype is evident in stck mutant embryos. As noted above, ßPS integrin also accumulates at muscle-attachment sites in stck mutant embryos. These findings illustrate that the proper localization of integrin and Ilk is not sufficient to stabilize actin membrane linkages at sites of integrin-dependent adhesion, and define PINCH as a critical component of the molecular machinery necessary for the tethering of actin to the integrin-rich membranes (Clark, 2003).
The demonstration that single ilk and stck mutants both display deficiencies in integrin-dependent processes illustrates that neither PINCH nor Ilk is sufficient on its own to support full integrin function. It is possible that PINCH acts as a positive regulator of Ilk function, either by modulating Ilk function by direct binding or by recruitment of an Ilk-modifying factor. Alternatively, Ilk may activate some PINCH function that is crucial for stabilization of actin-membrane linkages. Finally, a PINCH-Ilk protein complex may be a key component of the platform necessary for the recruitment of other proteins required to achieve stable actin-membrane associations. In this regard, it is of interest that PINCH and Ilk can be recovered in a complex with the Ilk-binding partner, CH-IlkBP, a calponin domain-containing protein related to Affixin and Actopaxin that could provide the link to actin filaments. Because the localization of Drosophila PINCH is dependent on integrins, the establishment of PINCH-Ilk complexes at muscle-attachment sites is not be supported in the absence of integrin function. This dependence of PINCH localization on integrins could provide a means to couple integrin adhesive function to its role in cytoskeletal anchorage (Clark, 2003).
In vertebrate cells, PINCH and Ilk appear to be mutually dependent on each other for their localization to integrin-rich focal adhesions (Zhang, 2002b). However, as noted above, despite their ability to interact with each other, PINCH and Ilk show distinct requirements for their recruitment to specific subcellular domains in Drosophila. In particular, it is shown that PINCH requires functional integrins for its localization to muscle-attachment sites, whereas it has previously been demonstrated that Drosophila Ilk fails to bind integrins directly and localizes normally in an integrin mutant. Rather than employing an association with integrins, Ilk may rely on a protein such as Paxillin for its targeting to integrin-rich sites. Although Drosophila PINCH requires integrins for its stable accumulation at muscle-attachment sites, there is no evidence that PINCH can associate directly with integrin cytoplasmic domains, therefore additional proteins probably act as a bridge (Clark, 2003 and references therein).
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).
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 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 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).
Integrin-linked kinase (ILK), PINCH (Steamer Duck in Drosophila) and Parvin constitute the tripartite IPP complex that maintains the integrin-actin link at embryonic muscle attachment sites (MASs) in Drosophila. This study shows that parvin null mutants in Drosophila exhibit defects in muscle adhesion, similar to ILK and PINCH mutants. Furthermore, the identical muscle phenotype of the triple mutant, which for the first time in any organism removed the entire IPP-complex function, genetically demonstrated that Parvin, ILK and PINCH function synergistically. This is consistent with the tight localization of the tripartite complex at sites of integrin adhesion, namely MASs in the developing embryo and focal-contact-like structures in the wing epithelium. Parvin contains tandem unconventional calponin-homology (CH) domains separated by a linker sequence, and a less-well conserved N-terminal region. In vivo structure-function analysis revealed that all the domains are essential for Parvin function, whereas recruitment at integrin adhesion sites is mediated by two localization signals: one located within the CH2 domain as previously reported, and a second novel signal within the CH1 domain. Interestingly, this site is masked by the linker region between the two CH domains, suggesting a regulatory mechanism to control Parvin localization. Finally, whereas in muscles only ILK controls the stability and localization of both PINCH and parvin, in the wing epithelium the three proteins mutually depend on each other. Thus molecular differences exist in the assembly properties of IPP complex in specific tissues during development, where differential modulation of the integrin connection to the cytoskeleton is required (Vakaloglou, 2012).
During animal development cells assemble into tissues and establish specific adhesion sites with the surrounding extracellular matrix (ECM). ECM enables the stable attachment of cells and their separation in distinct layers permitting tissue morphogenesis. The integrin family of transmembrane proteins mediate physical contact of cells with the ECM. This connection requires direct binding of the extracellular domain of both the α- and the β-subunit of integrin to their ECM ligands, whereas their small cytoplasmic tails bind a network of proteins to form the integrin adhesome and mediate the link to the actin cytoskeleton (Geiger, 2011). The molecular composition of the integrin adhesome network is diverse, highly dynamic and largely determined by the cell type and the physical strength required by the local developmental microenvironment (Vakaloglou, 2012).
The tripartite IPP complex containing integrin-linked kinase (ILK), PINCH and parvin is central to the integrin adhesome network. In invertebrates, the IPP-complex components are encoded by single genes, in contrast to the two PINCH genes and three parvin genes (α-, β- and 7gamma;-parvin) in mammals. The early evolutionary appearance of ancestral IPP-complex components suggest that the IPPb complex may have been one of the first molecular machines used by integrins (Vakaloglou, 2012 and references therein).
ILK plays a central role in the formation of the IPP complex. It contains five tandem ankyrin repeats (ANKRs) followed by a kinase-like domain, and interacts with the β1 and β3 integrin cytoplasmic tails). The ANKRs of ILK bind to PINCH, an adaptor protein containing five LIM domains, whereas the kinase-like domain of ILK binds to parvin, which in turn binds to actin (Legate, 2006). Thus, a simple model of IPP-complex function envisages either direct or indirect anchoring of ILK to integrins, with parvin mediating the link to actin. This model is supported by studies showing linear assembly of IPP complex in invertebrates. In Caenorhabditis elegans, ILK is required for both parvin and PINCH subcellular localization but not the reverse, and in Drosophila ILK is similarly required for PINCH stability and recruitment, although parvin has not been tested (Vakaloglou, 2012).
In mammalian cells, genetic elimination of one component of the IPP complex results in a significant decrease of the other two members, although some compensation has been observed for parvins and PINCH. Moreover, distinct IPP complexes are formed within cells containing different parvin and PINCH members, resulting in different functional properties. However, it is unknown whether removing all IPP components enhances the defects of single deletions, and thus whether all components function together or have other individual functions (Vakaloglou, 2012).
The parvin family was initially identified by sequence similarity to α-actinin. Recent detailed analysis of parvin CH2-domain interactions with the ILK kinase domain and the leucine aspartate (LD)-rich repeats of paxillin suggested that parvin contributes to the assembly of focal adhesions in mammalian cells. Genetic ablation of α-parvin in mice uncovered its function in vascular morphogenesis as a negative regulator of RhoA/ROCK signaling. However, due to functional compensation by β-parvin, earlier developmental functions of the parvin family were hard to study, possibly explaining why deletion of α-parvin does not phenocopy defects in early embryo development observed with genetic elimination of ILK or PINCH-1. Expression of, γ-parvin is restricted to the hematopoietic system, where it is not essential (Vakaloglou, 2012).
Previously found that ILK is required to link the actin cytoskeleton to the integrin-containing junctions at embryonic muscle attachment sites (MASs) in Drosophila, and PINCH was subsequently shown to be required for the same process. To date, there have been no genetic studies of the single Drosophila parvin gene, resulting in a currently incomplete characterization of IPP-complex function in flies. This paper reports a detailed functional characterization of parvin and the IPP complex in Drosophila development by generating a series of specific mutants and analyzing their effects on protein function and localization, in both the MASs of developing embryos and the wing epithelium (Vakaloglou, 2012).
This study has established the essential role of parvin in integrin-mediated adhesion and has demonstrated that parvin participates together with ILK and PINCH in the maintenance of the integrin-actin link in vivo. These conclusions are supported by: (1) the identical muscle phenotype of parvin mutants to mutants of ILK, PINCH and the triple mutant; (2) colocalization of all three proteins at sites of integrin adhesion; (3) the fact that ILK is necessary and sufficient for parvin stability and recruitment to MASs; (4) the suppression of dominant negative effects of overexpressed parvin within muscle by ILK; and (5) interdependence of all three IPP-complex components for their stability and subcellular localization at the basal side of wing epithelia, unlike the central role of ILK in IPP-complex assembly at the embryonic MASs. The results suggest that depending on the cell context, discrete molecular events coordinate the assembly of adhesion complexes into functional attachment structures (see Genetic hierarchy of integrin-actin linker complex assembly at MASs and in wing epithelium) (Vakaloglou, 2012).
parvin mutants were generated to complete the functional characterization of the entire IPP complex in Drosophila development. parvin null mutants shared similar phenotypic abnormalities with ILK and PINCH mutants as well as the integrin hypomorphic mutations described previously, including muscle defects due to actin filament retraction from the muscle ends in the late embryo. The muscle defects and lethality associated with parvin mutants was completely rescued by moderate expression of a wild-type parvin transgene mainly in somatic muscles and tendon cells, indicating that parvin has essential functions in these tissues. This study generated the first in vivo triple null mutant in an animal for all proteins that comprise the IPP complex. The identical loss-of-function phenotype at muscles in the triple versus single mutants strongly suggests that these three proteins work together as a complex by reinforcing the integrin-actin link at MASs in the Drosophila embryo. Although loss of parvin function at the muscles causes defective adhesion, high levels of parvin is also detrimental for the developing organism, suggesting that unlike overexpression of other IPP-complex members, the amount and subcellular localization of parvin needs to be tightly regulated (Vakaloglou, 2012).
parvin recruitment at MASs appears to depend exclusively on its interaction with ILK. In the absence of ILK, endogenous parvin protein levels are drastically reduced. Although endogenous parvin protein levels also decrease in the absence of PINCH, most likely due to moderate reduction of the parvin mRNA levels, this can be completely rescued by coexpression of ILK. Previously, it was shown that removal of PINCH does not affect the levels of a genomic ILK-GFP transgene. However, its effect on endogenous ILK levels remains undetermined due to the lack of a specific antibody against Drosophila ILK. Therefore, even a slight reduction of endogenous ILK levels in pinch mutants can not be excluded, providing an alternative explanation for both the moderate reduction of parvin and its complete restoration upon ILK overexpression. Overexpressed parvin in muscle cells remains in the cytoplasm, suggesting that the number of available binding sites for parvin at MASs is limited. However, simultaneous overexpression of parvin and ILK results in accumulation of parvin at MASs, indicating that ILK is necessary and sufficient for subcellular localization of parvin. High levels of cytoplasmic uncomplexed parvin in muscle cells induce dominant lethality, which is reversed upon coexpression of ILK. Previous studies in cells have shown that an imbalance in the amount of α- and β-parvin complexed with ILK induces apoptosis. Taken together these results suggest that the stoichiometry of ILK and parvin is crucial. Indeed, in Drosophila embryos and S2R+ cells, all components of the IPP complex are co-purified in stoichiometric amounts (Kadrmas, 2004) (Vakaloglou, 2012).
Parvin is known to be recruited to sites of integrin adhesion by direct interaction of its CH2 domain with the kinase domain of ILK. The current study also showed that the CH2 domain of Drosophila parvin is sufficient for localization to MASs. In addition, a novel ability of the parvin CH1 domain to localize at integrin junctions was uncovered. The close correlation between the levels of ILK and the stability and localization of the CH1 domain strongly suggest their direct interaction. Based on the similar negative effect of the E356K mutation of ILK on the recruitment of each CH domain, it is predicted that they bind to the same site within the kinase domain of ILK, although with different affinity. An alternative explanation that is favored less is the existence of a factor-X that recruits the CH1 domain at MASs, whereas ILK mediates factor-X recruitment. One candidate could be the fly homolog of αPIX, dPix, which is localized at MASs and binds to the CH1 domain of β-parvin in mammalian cells (Vakaloglou, 2012).
The data suggests that the CH1 domain localization signal is masked by the linker region. Although no direct biochemical evidence was provided supporting such an intramolecular interaction, a similar interaction has been reported in filamin, another CH-domain-containing protein. Given the strong possibility that both CH domains of parvin interact with ILK, the linker might function as a regulatory module ensuring that only high affinity interactions between parvin and ILK occur, thus controlling recruitment of parvin at MASs. The functional significance of parvin CH1-domain interaction with ILK requires further investigation involving the identification of specific mutations within the CH1 domain that could disrupt the binding on ILK (Vakaloglou, 2012).
Proteins containing tandem CH domains possess the ability to bind to filamentous actin (Gimona, 2002). Mammalian parvins show a differential ability to bind to F-actin. Although human α-parvin binds to actin filaments, β-parvin does not, despite 73% sequence identity. In mammalian cells, both α and β-parvin are largely associated with focal adhesions rather than the actin cytoskeletal filaments. In the living fly embryo, it was found that parvin-GFP expressed from its own promoter was similarly not colocalized with F-actin in the muscle cells, but instead accumulated heavily at MASs. By contrast, the two CH domains of Short stop colocalized with filamentous actin. These findings reflect differences in affinity for actin as supported by in vitro dissociation constants (ShotCH 0.022μM and α-parvin 8μM). Thus, within muscles parvin does not appear to have sufficient actin binding activity to co-distribute with actin filaments. Given that several proteins are known to bind to both CH domains in regions overlapping the putative actin binding sites, the role of parvin as an actin-binding protein In vivo is questionable. Hence, interaction of the CH domains with F-actin appears to be either less favored or to occur only in a highly regulated manner at sites of integrin adhesion (Vakaloglou, 2012).
Studies of mammalian parvins have suggested possible biochemical functions for Drosophila parvin. To gain further insight into the mechanisms by which parvin functions in integrin-mediated adhesion, the functional requirement of each protein domain was assessed. It was demonstrated that both the CH1 and CH2 domains are essential to strengthen the integrin-actin link and their importance coincides with their capacity to mediate an interaction with ILK that is responsible for their individual localization. Moreover, both CH domains are required in the same molecule. Hence, it is likely that these parvin domains link ILK and other proteins together to maintain the integrin-actin link. This study also found that the less well conserved N-terminal region of parvin is not essential for integrin-mediated adhesion at MASs, similar to pat-6 N-terminal domain in C. elegans. However, later in development the N-terminus becomes indispensable, suggesting a different set of essential molecular interactions for parvin depending on the developmental context (Vakaloglou, 2012).
In mammalian cells all three proteins of the IPP complex are mutually dependent for their stability and recruitment at sites of integrin adhesion. However, genetic studies both in C. elegans and Drosophila have shown that in muscle cells ILK behaves as a master component for complex assembly. Several studies have pointed out that the two well established models for stable adhesion in Drosophila, MASs and the basal side of the wing epithelium, exhibit molecular differences in assembly properties of the integrin adhesion complex, as well as differential sensitivity in genetically induced perturbations. In agreement with these studies, this study showed that in the wing epithelium the hierarchy of genetic interactions among the IPP-complex members differs and that, like in mammalian cells, the three proteins are interdependent for their stability and subcellular localization. This finding highlights the existence of diverse molecular strategies capable of mediating distinct adhesion properties in the developing organism and therefore underlines the importance of in vivo analysis of the same molecular machine of integrin adhesion junctions in various morphogenetic processes (Vakaloglou, 2012).
Parvin is a putative F-actin binding protein important for integrin-mediated cell adhesion. This study used overexpression of Drosophila Parvin to uncover its functions in different tissues in vivo. Parvin overexpression caused major defects reminiscent of metastatic cancer cells in developing epithelia, including apoptosis, alterations in cell shape, basal extrusion and invasion. These defects were closely correlated with abnormalities in the organization of F-actin at the basal epithelial surface and of integrin-matrix adhesion sites. In wing epithelium, overexpressed Parvin triggered increased Rho1 protein levels, predominantly at the basal side, whereas in the developing eye it caused a rough eye phenotype and severely disrupted F-actin filaments at the retina floor of pigment cells. Genes were identified that suppressed these Parvin-induced dominant effects, depending on the cell type. Co-expression of both ILK and the apoptosis inhibitor DIAP1 blocked Parvin-induced lethality and apoptosis and partially ameliorated cell delamination in epithelia, but did not rescue the elevated Rho1 levels, the abnormal organization of F-actin in the wing and the assembly of integrin-matrix adhesion sites. The rough eye phenotype was suppressed by coexpression of either PTEN or Wech, or by knock-down of Xrp1. Two main conclusions can be drawn from these studies: (1) high levels of cytoplasmic Parvin are toxic in epithelial cells; (2) Parvin in a dose dependent manner affects the organization of actin cytoskeleton in both wing and eye epithelia, independently of its role as a structural component of the ILK-PINCH-Parvin complex that mediates the integrin-actin link. Thus, distinct genetic interactions of Parvin occur in different cell types and second site modifier screens are required to uncover such genetic circuits (Chountala, 2012).
Parvin proteins are highly conserved and participate in the assembly and function of the integrin adhesome. This study employed the UAS/Gal4 system to investigate additional functions of Parvin upon overexpression in a tissue specific manner and to identify novel genetic interactions in the wing and the eye. Drosophila Parvin promotes apoptosis when overexpressed in vivo, similar to mammalian β-Parvin in HeLa cells. Expression of β-Parvin in breast cancer cells was recently shown to inhibit tumor progression and cell proliferation suggesting that this study of the cellular and molecular changes associated with Parvin overexpression in Drosophila may be relevant to cancer pathology. At the cellular level it was demonstrated that overexpressed Parvin induced alterations in the organization of the actin cytoskeleton, disruption of cell-matrix adhesion, cell invasion and cell delamination. Mechanistically, it was shown that overexpressed Parvin causes JNK activation and enhanced MMP1 levels. A functional link between Parvin and subcellular distribution of Rho1 was revealed. Interestingly, it was shown that these Parvin-induced signaling effects are not dependent on its interaction with ILK (Chountala, 2012).
Among the three counterparts of the ILK/PINCH/Parvin-complex, only overexpression of full-length Parvin induced ectopic apoptosis and excessive lethality in the larval and pupae developmental stages. Nevertheless, in the wing imaginal discs overexpression of other components of the integrin adhesome such as tensin and paxillin also results in apoptosis and lethality, including activation of the JNK pathway and modulation of Rho1 activity, respectively. Overexpression of Parvin increases Rho1 protein levels predominantly at the basal side of the wing epithelium, although loss of Parvin did not cause a reciprocal reduction of Rho1 levels. Given the previous reports that mammalian Parvins interact with two regulators of the small GPTases family, the GEF α pix and the CdGAP respectively, one hypothesis would be that high levels of Parvin sequester these factors and interfere with their interaction with Rho1. As a consequence, Rho1 is released from the apicolateral side where normally is enriched. The elevated Rho1 levels in the basal compartment of the epithelium could explain the formation of ectopic actin accumulation in accordance with previous studies. As already described Rho1 is able to induce JNK-dependent apoptosis and F-actin organization defects in the wing epithelia cells. Therefore, it is plausible that the elevated JNK activity observed upon Parvin overexpression is caused by aberrant elevation of Rho1 basaly. Taking these findings together, it is proposed that Parvin-induced cellular defects in the wing epithelia are mediated by increased levels of Rho1, however, a putative role of additional unidentified factors that are activated downstream of Parvin independently of Rho1 cannot be ruled out (Chountala, 2012).
It was recently showed that coexpression of ILK together with Parvin-GFP in the mesoderm is sufficient to completely rescue Parvin-induced lethality and control Parvin subcellular localization (Vakaloglou, 2012), suggesting that coupling of Parvin to ILK could have a protective effect in epithelia viability. Rescue experiments were performed to investigate whether Parvin function in the wing epithelium is mechanistically linked to its interaction with ILK, by coexpressing Parvin with ILK. Expression of ILK alone did not completely rescue the dominant effects of Parvin overexpression in the developing wing epithelia, had a mild suppressive effect on the rough eye phenotype and did not change the subcellular distribution of Parvin-GFP in the wing epithelial cells. Both the JNK activity and the increase in Rho1 protein levels were also not affected by ILK coexpression. Even when high levels of ILK are present, the putative interaction of Parvin with GTPase regulators is not disturbed and the imbalance of Rho1 subcellular distribution is maintained. That is not unexpected given that both αpix and CdGAP interact with the N-terminus region of Parvin, whereas ILK binds on the C-terminus. These findings demonstrate that the functional interplay between Parvin and ILK depends on the cell context and that Parvin interacts with other proteins and perform additional roles. In addition to functioning as a structural element of the integrin-actin link, it also acts as a dosage dependent modulator of actin cytoskeleton organization and cell homeostasis in the developing epithelia, via modulating the subcellular distribution of Rho1 (Chountala, 2012).
Because overexpression of Parvin caused extensive apoptosis in the wing epithelium, to mechanistically uncouple the Parvin-induced cellular defects from Parvin-induced apoptosis, rescue experiments were performed by coexpressing Parvin and DIAP1, which blocks apoptosis by inhibiting both the initiator caspase DRONC and the effector caspases DriCe and Dcp-1. DIAP1 alone did not efficiently suppress the cellular defects of Parvin in the wing. Both ILK and DIAP1 had to be coexpressed to completely rescue the lethality, presumably by coupling the reduction of excessive cytoplasmic Parvin by ILK and the inhibition of DRONC-mediated apoptosis by DIAP1. Coexpression of ILK and DIAP1 rescue both cell apoptosis and cell extrusion in the wing poutch cells, but not in the hinge and notum. These findings were not entirely unexpected, given previous documentation of regional differences within the wing imaginal disc regarding the differential requirement of actin regulators for epithelial integrity. However, consistent with these results from ILK rescue experiments, coexpression of DIAP1 or both ILK and DIAP1 did not ameliorate either the irregular organization of F-actin or the disorganized integrin-matrix adhesion sites and did not change the elevated levels of Rho1 in the basal side of the wing epithelium. These results demonstrate that the Parvin-induced cellular defects are not a simple consequence of apoptosis, but rather a distinct feature of Parvin function (Chountala, 2012).
Overexpression of Parvin in the eye generates a rough eye phenotype. At the cellular level the basal actin cytoskeleton in the eye retina is severely disrupted, suggesting that a cause of the abnormal eye development could be initiated by abnormalities in the cell shape of pigment cells, as in the case of the wing epithelium. Because the Parvin-induced eye phenotype was sensitive to the copy number of Parvin transgenes and to temperature, a modifier screen was performed to uncover novel genetic interactors. It was found that elevated levels of Wech and PTEN antagonize the Parvin-induced dominant effects in the developing eye and completely suppress the rough eye phenotype, whereas high levels of ILK has only minimal suppression activity (Chountala, 2012).
Wech is an ILK binding protein and it is not clear why it could suppress Parvin-induced dominant defects at elevated levels rather than ILK itself, which directly binds to Parvin and rescues lethality completely in the mesoderm (Vakaloglou, 2012) and significantly in enGal4 expressing cells. The lack of data regarding Wech function in the eye, preclude further analysis at this point. The second surprising result of this study was the ability of high levels of PTEN to suppress the rough eye phenotype induced by Parvin overexpression. UAS::PTEN overexpression under GMRGal4 has been reported to induce a rough-eye phenotype by inhibiting cell-cycle progression in proliferating cells and inducing apoptosis in a cell-context dependent manner. In the current experiments expression of the same UAS::PTEN lines obtained from two different donors did not result in eye roughening. One possible explanation could be the use of longGMRGal4 in these experiments, because previous studies drove expression of UAS::PTEN with GMRGal4. In addition, previous reports suggested that expression by longGMRGal4 driver in the developing eye follows a more strict pattern in the photoreceptor cells. Taken these data and previous reports together, it is speculated that Parvin and PTEN have antagonistic functions within the eye epithelium and coexpression of both proteins counterbalance their induced dominant effects upon overexpression. Currently insufficient data is available to point a specific pathway that could be modified by Parvin and PTEN and leads to rough eye phenotype. However, the recent report that Parvin is associated with PKB (Kimura, 2010) together with previous data suggesting that Parvin may facilitates the recruitment of PKB at plasma membrane Fukuda, 2009, suggests that Parvin could antagonized the negative effects of PTEN on PKB activation by reducing PIP3 levels (Chountala, 2012).
The third suppressor gene found was Xrp1. Xrp1 contains an AT-hook motif that is found in nuclear proteins with DNA binding activity. Currently, sufficient information is available to speculate on putative functional interaction between Parvin-induced signaling and nuclear activity. However, previous studies on Xrp1 point on its role as a p53-dependent negative regulator of cell proliferation following genotoxic stress. Among the genes that enhanced the Parvin-induced rough eye were all of the integrin subunits known to be expressed in the eye, including αPS1, αPS2 and βPS, the cytoskeletal regulators ZASP52 and the transgelin homolog encoded by CG14996 of unknown function (Chountala, 2012).
In conclusion, the findings revealed novel cell context-dependent roles for Parvin in the whole organism. Besides its known function as a structural component of the IPP-complex that mediate the integrin-actin link, it was demonstrated that Parvin can also affect cell-matrix adhesion, organization of actin cytoskeleton and cell homeostasis, by regulating Rho1 and JNK levels in an ILK-independent manner. These findings are relevant to situations where cell homeostasis is altered ranging from the physiological renewal of tissues to cancer pathology. In addition, the modifier genetic screen revealed novel interactors that affect Parvin function in a living organism. These in vivo data provide the first insight into genetic circuits influenced by Parvin and offer a framework for additional detailed studies to elucidate how these genetic networks interact (Chountala, 2012).
Many kinases that act in specific processes during development have been found to be ubiquitously expressed. By contrast, the mRNA distribution of Ilk is both temporally and spatially regulated. At the cellular blastoderm stage, Ilk expression is low, and it becomes stronger during gastrulation, mainly in the presumptive mesoderm. Its expression continues to increase through stage 13 within the somatic mesoderm, the midgut endoderm, and the surrounding visceral mesoderm. High levels of Ilk expression are maintained in the somatic and visceral muscles through the end of stage 16, when the embryos are almost fully developed (Zervas, 2001).
The finding that Ilk mRNA is expressed mainly in mesodermal tissues was confirmed by the localization of Ilk-GFP protein. However, low levels of Ilk are found to be distributed throughout the embryo. Some of these correspond to sites of integrin expression, such as at the leading edge of the epidermis and amnioserosa during dorsal closure. Strong expression of Ilk-GFP in the visceral mesoderm is first detected at stage 12, and it accumulates steadily during embryogenesis, following the level of mRNA expression. In midstage 16 embryos, Ilk-GFP is particularly strong in the midgut constrictions and the pharyngeal muscles. Low levels of Ilk-GFP were found in the ventral nerve cord and throughout the epidermis. The most striking feature of Ilk localization during embryogenesis is its tight localization at muscle attachment sites, where PS integrins are strongly expressed. These data show that high levels of Ilk-GFP are found at the places where integrins are found and the two proteins are tightly colocalized. There is not strong expression of Ilk at epidermal sites where wingless signaling through ß-catenin is particularly active, but low levels of Ilk are detectable throughout the embryo, so this expression pattern does not exclude the possibility of the suggested interaction between Ilk and ß-catenin/T cell factor signaling occurring in Drosophila (Zervas, 2001).
The colocalization of Ilk-GFP with integrins raises the question as to whether this is due to the binding of Ilk to the cytoplasmic tail of the ßPS subunit. The distribution of Ilk-GFP was examined in embryos mutant for the ßPS subunit and it was found that Ilk-GFP is still concentrated at the muscle attachments. In addition, no interaction between Drosophila Ilk and the ßPS subunit cytoplasmic domain could be detected by two-hybrid analysis, although the interaction between human ILK and human ß1 integrin could be reproduced. Weak interaction is seen between Drosophila Ilk and the ß1 integrin cytoplasmic tail, but not between human ILK and ßPS, indicating that the differences between the cytoplasmic tails (11 of 47 amino acids) have caused the loss of this interaction. Thus, no evidence could be provided for a direct interaction between Drosophila Ilk and the PS integrins, either in yeast or in the Drosophila embryonic muscles (Zervas, 2001).
The striking colocalization observed between Ilk and the PS integrins was not particularly surprising given that ILK was identified by its ability to bind integrins and their colocalization at focal contacts. It was more surprising to find that Ilk is localized normally to the muscle ends in the absence of integrins. This refutes one obvious possible mechanism for Ilk localization: apparently it is not recruited to the muscle ends by binding the cytoplasmic domain of ßPS. In cell culture, there is evidence to suggest that the NH2 terminus of Ilk may also play a part in localization (Li, 1999), and this is currently being testing in flies. Consequently, although no data supporting a direct interaction between Ilk and ßPS in vivo could be presented, the fact that loss of Ilk funtion gives a phenotype similar to that engendered from the loss of integrins strongly suggests that they interact. The only place where Ilk and integrins colocalize where Ilk does not cause an integrin-like phenotype is in the leading edge of the epidermis during dorsal closure. There are several possible explanations for this, the simplest being that the function of Ilk in these cells is redundant. It is worth noting that even the function of integrins in this process is not clear but is probably different from their assembly of strong adhesive junctions, as seen in muscles and wings (Zervas, 2001).
Tissue-specific stem cells are maintained by both local secreted signals and cell adhesion molecules that position the stem cells in the niche microenvironment. In the Drosophila midgut, multipotent intestinal stem cells (ISCs) are located basally along a thin layer of basement membrane that composed of extracellular matrix (ECM), which separates ISCs from the surrounding visceral musculature: the muscle cells constitute a regulatory niche for ISCs by producing multiple secreted signals that directly regulate ISC maintenance and proliferation. This study shows that integrin-mediated cell adhesion, which connects the ECM and intracellular cytoskeleton, is required for ISC anchorage to the basement membrane. Specifically, the alpha-integrin subunits including alphaPS1 encoded by mew and alphaPS3 encoded by scb, and the beta-integrin subunit encoded by mys are richly expressed in ISCs and are required for the maintenance, rather than their survival or multiple lineage differentiation. Furthermore, ISC maintenance also requires the intercellular and intracellular integrin signaling components including Talin, Integrin-linked kinase (Ilk), and the ligand, Laminin A. Notably, integrin mutant ISCs are also less proliferative, and genetic interaction studies suggest that proper integrin signaling is a prerequisite for ISC proliferation in response to various proliferative signals and for the initiation of intestinal hyperplasia after loss of adenomatous polyposis coli (Apc). These studies suggest that integrin not only functions to anchor ISCs to the basement membrane, but also serves as an essential element for ISC proliferation during normal homeostasis and in response to oncogenic mutations (Lin, 2013).
To assess the function of Ilk during development, mutations were sought in the Ilk gene. The region containing the Ilk gene was characterized genetically as part of the studies of the nearby gene Ecdysone-induced protein 78C (Eip78C). A genetic screen for lethal mutations uncovered by the deficiency Df(3L)Pc-14d, which deletes 78C2;D1, has identified two new lethal complementation groups: l(3)78Ca and l(3)78Cb. By mapping the genes within a genomic clone, it was found that Eip78C is proximal to Ilk, and deficiency mapping has shown that Eip78C is distal to l(3)78Cb, and proximal to l(3)78Ca, making the latter the best candidate for the Ilk locus. The Ilk coding region was sequenced from the DNA of flies containing the l(3)78Ca mutation and it was compared to the sequence of the gene in the strain that was mutagenized in the genetic screen. Sequences from three independent PCR amplifications of the l(3)78Ca mutant DNA have a single change, the nucleotide transition, which changes W211 to a stop codon. Therefore, this mutant gene will produce a truncated form of Ilk that lacks the kinase domain, which is also the region in human ILK (but not Drosophila Ilk) that binds to the integrin cytoplasmic tail by yeast two-hybrid interaction (Zervas, 2001).
A second allele, ilk2, was isolated in a screen for genes required for integrin-mediated adhesion in the adult wing. A single allele was isolated, which is homozygous lethal and causes a dominant wing blister phenotype. This mutation is lethal over Ilk1, although a few adult escapers (<5%) are seen. Genetic and cytological analysis reveals that ilk2 is associated with a reciprocal translocation between the second and third chromosomes. The dominant phenotype of ilk2 is not shared by other Ilk mutations (Ilk1 and Ilk deficiencies), so it is not certain whether the phenotype is caused by the aberration in the Ilk gene or a second site mutation (Zervas, 2001).
Embryos homozygous for Ilk1 die at the end of embryogenesis. To check whether the Ilk mutant embryos have defects similar to the pattern defects caused by the loss of the Wnt signal through ß-catenin (armadillo), or the reduced cuticle caused by loss of PKB, the cuticle secreted by the epidermis was examined. The cuticle of Ilk mutant embryos is completely normal with, for example, no indication of the dramatic pattern changes observed when ß-catenin is defective. The development of the midgut was also found to be normal, ruling out a requirement for Ilk in the ß-catenin signaling that occurs in the visceral mesoderm (Zervas, 2001).
Ilk is required for muscle adhesion in embryos. Having observed a relatively mild phenotype for Ilk, which is detectable later in development than the PS integrin phenotype, it was of interested to establish with certainty that this mutation completely removes Ilk function. A test was performed to see whether Ilk1 is an amorphic (null) allele by comparing the homozygous Ilk1 phenotype to that of Ilk1/Df(3L)Pc-14d embryos; the phenotypes were found to be identical, demonstrating that Ilk1 is an amorphic allele. Embryos transheterozygous for the two overlapping deficiencies Df(3L)Pc-14d and Df(3L)ME-107, and therefore completely deficient for Ilk, also have an equivalent muscle phenotype. Another possibility is that some Ilk mRNA or protein that is deposited in the egg during oogenesis persists until late stages of embryogenesis, masking a complete loss of function phenotype. The maternal product was removed by making germ-line clones of the Ilk1 mutation. It was found that embryos lacking both maternal and zygotic Ilk have a modestly more severe muscle phenotype, with clumping of the actin first visible a little earlier, at the end of stage 16. These embryos also have normal cuticles and do not display any additional defects compared with embryos lacking zygotic Ilk function. Therefore, maternal contribution of Ilk does not significantly compensate for the loss of Ilk synthesized during embryogenesis (Zervas, 2001).
Because Ilk has been proposed to act as an effector for integrin signaling, it was of interest to test whether it is required for this role in Drosophila. Two genes expressed in the Drosophila midgut that are targets of integrin signaling have been identified, providing the first transcriptional assay for integrin signaling in this organism. One of these targets, 258, was tested in embryos lacking Ilk function (also in the absence of maternal product) and 258 was found to be expressed normally, demonstrating that Ilk is not required for this integrin-signaling pathway. Complete loss of integrin function causes additional defects in midgut morphogenesis and dorsal closure, which are not seen in the Ilk mutant embryos (Zervas, 2001).
Overexpression of Ilk in cell culture has been proposed to affect several signaling molecules, including GSK3ß (Delcommenne, 1998); therefore, it was of interest to test whether this also occurs in a whole organism. The Gal4 system was used to drive additional expression of Ilk in the wing, on top of the endogenous protein levels, since this tissue shows clear phenotypes for the different signaling pathways. A UAS::Ilk construct was prepared and a variety of GAL4 drivers was used. No phenotype was detected: neither wing blisters indicative of an effect on integrins nor differentiation defects that might have revealed an effect on ß-catenin signaling. It was demonstrated that this construct does express functional Ilk by using it to rescue the embryonic lethality of Ilk1/Df(3L)Pc-14d flies. Expression of UAS::Ilk primarily in the mesoderm with the driver 24B::Gal4 (which is also expressed in the epidermal tendon cells in the embryo) is sufficient to rescue the embryonic lethality of the Ilk mutation, and viable adults were obtained. The surviving adults have blistered wings, perhaps due to insufficient expression of UAS::Ilk by 24B in the wings. Several points can be concluded from these experiments: the UAS::Ilk construct is functional, the lethality of the Ilk1 mutation is rescued by expression of the Ilk protein alone; Ilk is only required in the embryo in those cells where the 24B driver is expressed, and additional expression of Ilk does not perturb signaling through ß-catenin in the wing (Zervas, 2001).
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date revised: 25 June 2011
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