steamer duck: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - steamer duck
Synonyms - PINCH
Cytological map position - 85A1--3
Function - cytoskeletal crosslinker
Keywords - mesoderm, muscle attachment sites
Symbol - stck
FlyBase ID: FBgn0020249
Genetic map position -
Classification - LIM-only protein
Cellular location - cytoplasmic
|Recent literature||Wang, Y., Antunes, M., Anderson, A. E., Kadrmas, J. L., Jacinto, A. and Galko, M. J. (2015). Integrin adhesions suppress syncytium formation in the Drosophila larval epidermis. Curr Biol 25: 2215-2227. PubMed ID: 26255846
Integrins are critical for barrier epithelial architecture. Integrin loss in vertebrate skin leads to blistering and wound healing defects. However, how integrins and associated proteins maintain the regular morphology of epithelia is not well understood. This study found that targeted knockdown of the integrin focal adhesion (FA) complex components β-integrin, PINCH, and integrin-linked kinase (ILK) caused formation of multinucleate epidermal cells within the Drosophila larval epidermis. This phenotype was specific to the integrin FA complex and not due to secondary effects on polarity or junctional structures. The multinucleate cells resembled the syncytia caused by physical wounding. Live imaging of wound-induced syncytium formation in the pupal epidermis suggested direct membrane breakdown leading to cell-cell fusion and consequent mixing of cytoplasmic contents. Activation of Jun N-terminal kinase (JNK) signaling, which occurs upon wounding, also correlated with syncytium formation induced by PINCH knockdown. Further, ectopic JNK activation directly caused epidermal syncytium formation. No mode of syncytium formation, including that induced by wounding, genetic loss of FA proteins, or local JNK hyperactivation, involved misregulation of mitosis or apoptosis. Finally, the mechanism of epidermal syncytium formation following JNK hyperactivation and wounding appeared to be direct disassembly of FA complexes. In conclusion, the loss-of-function phenotype of integrin FA components in the larval epidermis resembles a wound. Integrin FA loss in mouse and human skin also causes a wound-like appearance. The results reveal a novel and unexpected role for proper integrin-based adhesion in suppressing larval epidermal cell-cell fusion-a role that may be conserved in other epithelia.
|Vakaloglou, K. M., Chrysanthis, G. and Zervas, C. G. (2016). IPP complex reinforces adhesion by relaying tension-dependent signals to inhibit integrin turnover. Cell Rep 14: 2668-2682. PubMed ID: 26972014
Cytoskeleton-mediated forces regulate the assembly and function of integrin adhesions; however, the underlying mechanisms remain unclear. The tripartite IPP complex, comprising ILK, Parvin, and PINCH, mediates the integrin-actin link at Drosophila embryo muscle attachment sites (MASs). This study demonstrate a developmentally earlier function for the IPP complex: to reinforce integrin-extracellular matrix (ECM) adhesion in response to tension. In IPP-complex mutants, the integrin-ECM linkage at MASs breaks in response to intense muscle contractility. Mechanistically, the IPP complex is required to relay force-elicited signals that decelerate integrin turnover at the plasma membrane so that the integrin immobile fraction is adequate to withstand tension. Epistasis analysis shows that alleviation of muscle contractility, downregulation of endocytosis, and enhanced integrin binding to the ECM are sufficient to restore integrin-ECM adhesion and maintain integrin-adhesome organization in IPP-complex mutants. These findings reveal a role for the IPP complex as an essential mechanosensitive regulatory switch of integrin turnover in vivo.
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 (Hobert, 1999) 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 (Prout, 1997), 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).
Cell adhesion to the extracellular matrix (ECM) is required for tissue architecture and can have dramatic effects on cell behavior. Integrins are transmembrane, heterodimeric receptors that comprise the primary recognition sites for binding to ECM. alpha- and ß-integrin subunits possess large extracellular domains that form a binding interface for specific ECM components. The cytoplasmic domains of integrins tether actin filaments, and recruit a wide array of proteins involved in signal transduction. Proteins that associate either directly or indirectly with integrin cytoplasmic tails may also contribute to activation of the ligand binding capacity of the integrins, thus modulating integrin adhesive function by an 'inside-out' signaling mechanism (Clark, 2003).
One cytoplasmic protein that has been postulated to play a role in integrin function is PINCH, a protein comprising five tandemly arrayed LIM domains (Rearden, 1994). LIM domains are double zinc-finger structures that serve as protein-binding interfaces; therefore, PINCH probably functions as a molecular scaffold that supports the assembly of a multi-protein complex at sites of integrin enrichment. In agreement with this notion, biochemical studies of human PINCH have identified Integrin-Linked Kinase (Ilk) as a binding partner for the first LIM domain of PINCH (Tu, 1999), and the SH2-SH3 adaptor protein NCK2 as a partner for the fourth LIM domain (Tu, 1998). Although the complete binding partner repertoire of PINCH remains to be elucidated, the colocalization of PINCH with integrins and its capacity to bind Ilk and NCK2 provided the first hints that PINCH might play a role in recruitment of regulatory factors to integrin-rich sites (Wu, 1999; Wu, 2001) and may thus contribute to integrin function (Clark, 2003).
Further support for the view that PINCH is essential for integrin function came from studies in which PINCH expression in C. elegans was compromised by RNA interference. Developing embryos that are deficient in PINCH display a paralyzed-at-twofold (PAT) phenotype, similar to that observed in integrin mutants (Hobert, 1999). In spite of the comparable developmental arrest when either integrin or PINCH function is compromised in the worm, this phenotypic description did not provide mechanistic insight into the relationship between PINCH and integrins. Recently, however, it was demonstrated that expression of a dominant-negative form of PINCH in tissue culture cells results in compromised cell adhesion (Zhang, 2002c). These findings are consistent with the view that PINCH is required for integrin-dependent cell adhesion. However, because the LIM domain is a conserved structural feature found in many modular proteins, it is essential that conclusions from studies using dominant-negative tools be confirmed using a loss-of-function strategy where specificity is insured (Clark, 2003).
Analysis of the cellular and developmental consequences of mutations in Drosophila pinch illustrates that PINCH is essential for integrin-dependent cell adhesion events in embryos and adults and reveals that PINCH is required to stabilize membrane-cytoskeletal linkages at sites of cell-substratum anchorage (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 (Hobert, 1999), 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 (J. L. Kadrmas, S. M. Pronovost and M. C. Beckerle, unpublished, cited in Clark, 2003). These latter results are consistent with findings reported previously for vertebrate PINCH and Ilk (Li, 1999; Tu, 1999). 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 (Hobert, 1999; Mackinnon, 2002). 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).
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 (Tu, 2001; Yamaji, 2001; Nikolopoulos, 2002). 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 (Zervas, 2001). Rather than employing an association with integrins, Ilk may rely on a protein such as Paxillin for its targeting to integrin-rich sites (Nikolopoulos, 2001). 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).
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).
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 (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).
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 LIM13, 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 LIM13 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).
C. elegans Unc-97 constitutes a novel component of muscular adherens junctions. Unc-97 and homologues from several other species define the PINCH family, a family of LIM proteins whose modular composition of five LIM domains implicates them as potential adapter molecules. unc-97 expression is restricted to tissue types that attach to the hypodermis, specifically body wall muscles, vulval muscles, and mechanosensory neurons. In body wall muscles, the UNC-97 protein colocalizes with the ß-integrin PAT-3 to the focal adhesion-like attachment sites of muscles. Partial and complete loss-of-function studies demonstrate that Unc-97 affects the structural integrity of the integrin containing muscle adherens junctions and contributes to the mechanosensory functions of touch neurons. The expression of a Drosophila homologue of unc-97 in two integrin containing cell types, muscles, and muscle-attached epidermal cells, suggests that unc-97 function in adherens junction assembly and stability has been conserved across phylogeny. In addition to its localization to adherens junctions Unc-97 can also be detected in the nucleus, suggesting multiple functions for this LIM domain protein (Hobert, 1999).
The X-linked unc-97 gene is defined by a single recessive allele, su110. Adult animals homozygous for the su110 mutation are limp, egg laying-defective, and show movement defects that vary from slow moving to paralyzed animals. This probably reflects the degree of disorganization of the vulval and body wall muscles. unc-97 mutants have shallow, easily disrupted muscle sarcomeres. Using polarized light microscopy it has also been found that mutant muscle cells are fragile, but if animals are handled carefully intact sarcomeres can be observed. When handled gently, unc-97(su110) muscle largely resembles wild-type muscle. Sarcomere dimensions and overall organization are similar between unc-97(su110) and wild-type muscle, but the dense bodies in su110 animals are never as clearly defined as in wild-type animals. The combination of coverslip pressure and slight rolling of an unc-97(su110) animal will also lead to the collapse of the sarcomere structure. Under similar conditions sarcomeres within wild-type muscle cells do not collapse. Anchorage of the sarcomere complex to the plasma membrane is therefore weak and easily disrupted in unc-97(su110) animals (Hobert, 1999).
The lability of the sarcomeres as observed by polarized light microscopy prompted an analysis of the structural integrity of the muscle attachment structures in more detail. To this end, the subcellular localization of two defined components of these muscle-hypodermal adherens junctions was determined. Immunhistochemistry with a ß-integrin/ PAT-3 monoclonal antibody revealed that the dense bodies do not for the most part show up as rows of discrete spots in unc-97 mutants, but instead appear primarily as diffuse stripes running parallel to the M line. Similarly, vinculin, which normally localizes exclusively to dense bodies, shows a diffuse stripe-like appearance in unc-97 mutants; the diffuse stripes do not always run in parallel, but occasionally fuse to one another. Due to the presence of M lines in the anti-PAT-3-stained animals, the fusion of these stripes can more easily be observed using anti-vinculin stained antibodies. These observations are consistent with UNC-97 playing a role in determining the structural integrity of adherens junctions, perhaps through clustering integrin or other dense body-associated proteins (Hobert, 1999).
The C. elegans proteoglycan perlecan, encoded by the unc-52 gene, localizes to the basement membrane underlying the muscle. Staining with anti-UNC-52 antibodies reveals staining at periodicities corresponding to the sites of the dense bodies and M-line structures. In unc-97(su110) mutant animals, the periodicity of the staining is abrogated; instead, UNC-52 appears more diffuse. The effect of the intracellular UNC-97 protein on the localization of the extracellular UNC-52 protein demonstrates that the correct localization of extracellular matrix components depends on the structural integrity of the muscular integrin complexes (Hobert, 1999).
To monitor the expression pattern of Unc-97, the transcriptional regulatory regions of the Unc-97 gene were fused to GFP and its expression was analyzed in transgenic C. elegans. A translational fusion protein, encoding all exons and introns of Unc-97 reveals a similar set of expressing cells. In both larvae and adult animals, Unc-97 is expressed in two different cell types, muscles and neurons. Strong expression is detectable in body wall muscle cells and vulval muscle cells, whereas no expression can be detected in pharyngeal muscles, intestinal muscle, or the anal depressor muscle. Weak expression can be observed in the anal sphincter muscle. In the nervous system, Unc-97 is expressed in the six mechanosensory receptor neurons, ALML/R, PLML/R, AVM, and PVM, that are responsible for sensing light touch. Intriguingly, a common theme of all the Unc-97-expressing cell types is their attachment via the extracellular matrix to the hypodermis. Unc-97 expression was examined at earlier developmental stages to determine its onset of expression in development. In embryos, Unc-97 expression can first be observed at mid-embryogenesis at ~300 min of development (Hobert, 1999).
The structural components that anchor myofibers to the extracellular matrix, such as integrin, vinculin, talin, and alpha-actinin are remarkably conserved in C. elegans and resemble the protein composition of adherens junctions in other systems, such as focal adhesions in tissue culture cells. It is thus likely that the fundamental mechanisms required to assemble these structure are conserved as well. Based on functional analysis of Unc-97 and the presence of highly related homologous molecules in flies and vertebrates, it is suggested that members of the PINCH family of LIM-only proteins are a critical component of muscle attachment and adherens junction assemblies across phylogeny. Indeed, the mouse Unc-97 homologue PINCH-1 has recently been shown to colocalize with ß1-integrins at focal adhesions. This interaction appears to be mediated by the ankyrin-repeat containing serine/threonine kinase Ilk, which by direct association with both ß1-integrins (Hannagan, 1996) and the mouse Unc-97 homologue PINCH-1 (Wu, C., personal communication to Hobert, 1999) appears to serve as a bridging molecule (Hobert, 1999).
Cellular attachment sites contain several LIM domain proteins, such as zyxin, paxillin, cysteine-rich proteins (CRP proteins: see Drosophila Mlp60A and Mlp84B) and others. In muscles, the CRP3/MLP and ALP proteins localize to Z-discs, attachment points of myofibers in vertebrates. Drosophila CRP homologues also localize to muscle attachment sites. However, the functional requirement for any LIM containing protein at these subcellular sites has until now only been reported for the CRP3/MLP protein, whose loss-of-function causes disorganization of cardiac myofibers. A similar requirement is demonstrated in this study for the LIM-only protein Unc-97 in body wall muscles in C. elegans. Since the C. elegans genome contains a gene highly related to the CRP proteins (T04C9.4), it is speculated that other LIM proteins besides Unc-97 will also be involved in adherens junction assembly (Hobert, 1999).
The su110 allele indicates that Unc-97 has an important role in maintaining sarcomere organization in growing and adult animals. Moreover, the RNAi (probable loss-of-function) experiment indicates an important regulatory role for Unc-97 during embryonic muscle development. However, it is unclear so far as to whether the embryonic defects caused by Unc-97 RNA interference reflect a requirement for Unc-97 in the initial stages of assembly of the adherens junctions or reflects a requirement for Unc-97 in stabilizing adherens junctions after they are formed to resist the mechanical stress they are exposed to upon elongation of the animal. Since Unc-97 expression clearly coincides with the onset of expression of adherens junction components and their assembly into these structures, a model is favored in which Unc-97 is involved in the initial assembly of the adherens junction components and keeps residing in these structures to ensure their stability (Hobert, 1999).
Although it is unclear how Unc-97 cooperates with ß-integrins and other focal adhesion components such as vinculin to determine adherens junction integrity, the modular organization of Unc-97 into five LIM domains implicates Unc-97 in binding to multiple proteins. Unc-97 might be a scaffold or adapter protein onto which various different components assemble to form a functional muscle attachment site. Since defective myofibrils cause as a secondary consequence the destabilization of muscle attachment sites, it is for example possible that Unc-97 serves as an anchor between myofibrils and membrane-anchored integrin components. In this model, dense body defects in Unc-97(su110) mutant animals would not arise from direct defects in the dense body structure per se, but would represent a secondary consequence of the myofibril disorganization (Hobert, 1999).
Analysis of Unc-97 localization in a living, nonfixed animal, using a rescuing, i.e., functionally intact Unc-97:: GFP reporter gene, lends support for an authentic in vivo dual subcellular localization of this LIM protein. However, at this point it is unclear whether the nuclear localization of Unc-97 is indeed functionally significant. In contrast to the localization of Unc-97 to adherens junctions, which correspond to the site of action of Unc-97 as inferred from the Unc-97 mutant phenotype, no such clear correlation exists to a possible nuclear function of Unc-97. However, the demonstration of a genetic interaction of Unc-97 with the LIM homeodomain transcription factor mec-3 could be explained on the basis of a physical interaction between these proteins and could thus reflect a functional requirement for Unc-97 in the nucleus. LIM- LIM interaction have been previously described and the direct interaction of Unc-97 with MEC-3 could affect the transcriptional activity of the MEC-3 transcription factor. Alternatively, it is also entirely possible that the genetic interaction of Unc-97 and mec-3 reflects an independent requirement for these genes in mechanosensory processes (Hobert, 1999).
Although further experiments will need to address the physiological significance of Unc-97 in the nucleus, there are several attractive hypotheses regarding a potential nuclear function of Unc-97. Unc-97 could be directly involved in gene regulatory events by interacting with specific transcription factors. The vertebrate LIM-only proteins CRP3/MLP and SLIM1/KyoT have been directly implicated in gene regulation via interaction with the transcription factors MyoD and RBP-J, respectively, whereas the LIM-only protein LMO2 assembles higher order transcriptional activation complexes by bridging other transcription factors that are directly involved in DNA binding . Alternatively, but not necessarily mutually exclusive, Unc-97 could represent a structural component of specific subnuclear domains. The localization of Unc-97 to discrete dots in muscle nuclei supports this hypothesis, although the nature of these dots is entirely unclear. Subnuclear domains of different types, such as speckles, coiled bodies, gems, and Kr bodies/nuclear domains have been described in various systems. Factors localizing to these domains are involved in distinct nuclear processes such transcriptional silencing and RNA processing (Hobert, 1999 and references therein).
Lastly, in regard to the dual subcellular localization of Unc-97 it is also tempting to speculate that Unc-97 transmits a signal from attachment sites to the nucleus. Although the question whether Unc-97 dynamically shuttles between attachment sites and the nucleus has not been addressed, focal adhesion-nuclear shuttling has been recently demonstrated for the LIM protein zyxin. The mouse Unc-97 homologue PINCH-1 has been shown to interact specifically with, and might serve as a substrate of, the integrin-linked kinase Ilk (Tu, 1999), a serine-threonine kinase implicated in integrin signal transduction. This observation might point to a potential role for Unc-97 in integrin-mediated signal transduction (Hobert, 1999).
Focal adhesions are multiprotein assemblages that link cells to the extracellular matrix. The transmembrane protein, integrin, is a key component of these structures. In vertebrate muscle, focal adhesion-like structures called costameres attach myofibrils at the periphery of muscle cells to the cell membrane. In Caenorhabditis elegans muscle, all the myofibrils are attached to the cell membrane at both dense bodies (Z-disks) and M-lines. Clustered at the base of dense bodies and M-lines, and associated with the cytoplasmic tail of beta-integrin, is a complex of many proteins, including UNC-97 (vertebrate PINCH). It has been shown that UNC-97 interacts with UNC-98, a 37-kD protein, containing four C2H2 Zn fingers, that localizes to M-lines. UNC-98 also interacts with the C-terminal portion of a myosin heavy chain. Multiple lines of evidence support a model in which UNC-98 links integrin-associated proteins to myosin in thick filaments at M-lines (Miller, 2006).
The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent OFT septation are critical events during vertebrate heart development. Four modified differential display (DD) screens were performed in the chick embryo to identify genes that may be involved in CNC and heart development. Full-length sequence of one of the DD clones has been obtained and identified as chick PINCH-1. This particularly interesting new cysteine-histidine-rich protein contains five protein-binding LIM domains (five double zinc fingers), a nuclear localization signal, and a nuclear export signal, allowing it to participate in integrin and growth factor signaling and possibly act as a transcription factor. Chick PINCH-1 is expressed in neural crest cells, both in the neural fold and cardiac OFT, and is also expressed in mesoderm derived-structures, including the myocardium, during avian embryogenesis. The normal expression pattern and overexpression in neural crest cell explants suggest that PINCH-1 may be a regulator of neural crest cell adhesion and migration (Martinsen, 2006).
Autoantibody eluted from aged human red blood cells was used to immunoscreen a human fetal liver expression library and led to the isolation of a cDNA encoding a novel 35.8 kDa protein with five LIM domains. An autoepitope homologous to the "senescent cell antigen" on the red blood cell membrane anion exchange protein is present in the first zinc finger of the third LIM domain. The gene for this novel protein is highly conserved in vertebrates, and its 4.6 kb mRNA is widely expressed in human tissues. Recombinant autoantigens such as the one reported here have potential applications in vitro for the purification, identification and quantitation of autoantibodies, and in vivo for the removal of autoantibodies, increasing red blood cell lifespan and reducing the need for transfusion (Rearden, 1994).
PINCH is a five LIM domain protein involved in the regulation of integrin-mediated cell adhesion. It has been shown that PINCH interacts with integrin-linked kinase and Nck2 (Drosophila homolog; Dreadlocks). A new isoform of PINCH is described that is called PINCH2. PINCH has therefore been renamed PINCH1. PINCH2 has an overall similarity of 92% to PINCH1 and contains five LIM domains like PINCH1. While protein and gene structure of the PINCH homologues are very similar and well conserved during evolution, differential expression pattern of the mRNAs is observed. Based on northern hybridization of mouse embryo RNA, PINCH1 is already detectable at E8.5. It is highly expressed during later stages of development and in all adult mouse tissues analyzed, with the highest levels in heart, lung, bladder, skin, and uterus. In contrast, significant PINCH2 expression starts at E14.5. In adult mice it is widely expressed, similar to PINCH1, but absent from spleen and thymus. In situ hybridization confirmed the Northern data and showed differential expression of PINCH1 and PINCH2 in embryonic intestine. PINCH2 localizes to focal adhesions in NIH 3T3 cells and to Z-disks in primary rat cardiomyocytes (Braun, 2003).
The distribution and abundance of PINCH in patients with breast, prostate, lung, colon, and skin carcinomas has been . Immunostaining for PINCH is increased in the cytoplasm of fibroblastoid cells in areas of the tumor-associated stroma in all carcinomatous tissues evaluated. The most intense stromal immunostaining for PINCH was noted at invasive edges, particularly in breast carcinomatous tissue. Immunoblotting of lysates from normal breast and breast carcinomatous tissue confirmed that PINCH protein expression is markedly increased in breast carcinomatous tissues. It is concluded that PINCH is up-regulated in tumor-associated stromal cells, particularly at invasive edges, and may be a marker for stroma manifesting the ability to facilitate invasion. Because of this and because PINCH functions as a "molecular switch" in signal transduction, PINCH may be a new target for drug discovery aimed at the tumor-associated stroma (Wang-Rodriguez, 2003).
PINCH2 belongs, together with PINCH1, to a family of focal adhesion proteins, the members of which are composed of five LIM domains. PINCH1 and PINCH2 interact, through their first LIM domain, with integrin-linked kinase and thereby link integrins with several signal transduction pathways. Despite their high similarity, PINCH1 and PINCH2 exert distinct functions during cell spreading and cell survival. To investigate the function of PINCH2 in vivo, PINCH2 was deleted in the mouse using the loxP/Cre system. In contrast to PINCH1-deficient mice, which die at the peri-implantation stage, PINCH2-null mice are viable, fertile and show no overt phenotype. Histological analysis of tissues that express high levels of PINCH2 such as bladder and kidney revealed no apparent abnormalities, but showed a significant upregulation of PINCH1, suggesting that the two PINCH proteins may have, at least in part, overlapping function in vivo. To further test this possibility, PINCH1-null mouse embryonic fibroblasts, which express neither PINCH1 nor PINCH2, were established. It was found that in fibroblasts with a PINCH1/2-null background, PINCH2 is able to rescue the spreading and adhesion defects of mutant fibroblasts to the same extent as PINCH1. Furthermore, the LIM1 domain only of either PINCH1 or PINCH2 can prevent ILK degradation despite their failure to localize to focal adhesions. Altogether these results suggest that PINCH1 and PINCH2 share overlapping functions and operate dependently and independently of their subcellular localization (Stanchi, 2005).
PINCH-1, a widely expressed protein consisting of five LIM domains and a C-terminal tail, is an essential focal adhesion protein with multiple functions including regulation of the integrin-linked kinase (ILK) level, cell shape, and survival signaling. The LIM1-mediated interaction with ILK regulates all these three processes. By contrast, the LIM4-mediated interaction with Nck-2, which regulates cell morphology and migration, is not required for the control of the ILK level and survival. Remarkably, a short 15-residue tail C-terminal to LIM5 is required for both cell shape modulation and survival, albeit it is not required for the control of the ILK level. The C-terminal tail not only regulates PINCH-1 localization to focal adhesions but also functions after it localizes there. These findings suggest that PINCH-1 functions as a molecular platform for coupling and uncoupling diverse cellular processes via overlapping but yet distinct domain interactions (Xu, 2005).
Integrin-linked kinase (Ilk) is a ubiquitously expressed protein serine/threonine kinase that has been implicated in integrin-, growth factor- and Wnt-signaling pathways. Ilk is a constituent of cell-matrix focal adhesions. Ilk Is recruited to focal adhesions in all types of cells examined upon adhesion to a variety of extracellular matrix proteins. By contrast, Ilk Is absent in E-cadherin-mediated cell-cell adherens junctions. PINCH is a protein consisting of five LIM domains, and has been identified as an Ilk binding protein. The Ilk-PINCH interaction requires the N-terminal-most ANK repeat (ANK1) of Ilk and one (the C-terminal) of the two zinc-binding modules within the LIM1 domain of PINCH. The Ilk ANK repeats domain, which is capable of interacting with PINCH in vitro, can also form a complex with PINCH in vivo. However, the efficiency of the complex formation or the stability of the complex is markedly reduced in the absence of the C-terminal domain of Ilk. The PINCH binding defective ANK1 deletion Ilk mutant, unlike the wild-type Ilk, is unable to localize and cluster in focal adhesions, suggesting that the interaction with PINCH is necessary for focal adhesion localization and clustering of Ilk. The N-terminal ANK repeats domain, however, is not sufficient for mediating focal adhesion localization of Ilk, since an Ilk mutant containing the ANK repeats domain but lacking the C-terminal integrin binding site fails to localize in focal adhesions. These results suggest that focal adhesions are a major subcellular compartment where Ilk functions in intracellular signal transduction, and provide important evidence for a critical role of PINCH and integrins in regulating Ilk cellular function (Li, 1999).
Integrin-linked kinase (ILK) is a ubiquitously expressed protein serine/threonine kinase that has been implicated in integrin-, growth factor- and Wnt-signaling pathways. ILK is a constituent of cell-matrix focal adhesions. ILK is recruited to focal adhesions in all types of cells examined upon adhesion to a variety of extracellular matrix proteins. By contrast, ILK is absent in E-cadherin-mediated cell-cell adherens junctions. PINCH, a protein consisting of five LIM domains, has been identified as an ILK binding protein. ILK-PINCH interaction requires the N-terminal-most ANK repeat (ANK1) of ILK and one (the C-terminal) of the two zinc-binding modules within the LIM1 domain of PINCH. The ILK ANK repeat domain, which is capable of interacting with PINCH in vitro, can also form a complex with PINCH in vivo. However, the efficiency of the complex formation or the stability of the complex is markedly reduced in the absence of the C-terminal domain of ILK. The PINCH binding defective ANK1 deletion ILK mutant, unlike the wild-type ILK, is unable to localize and cluster in focal adhesions, suggesting that the interaction with PINCH is necessary for focal adhesion localization and clustering of ILK. The N-terminal ANK repeats domain, however, is not sufficient for mediating focal adhesion localization of ILK, since an ILK mutant containing the ANK repeats domain but lacking the C-terminal integrin binding site fails to localize in focal adhesions. These results suggest that focal adhesions are a major subcellular compartment where ILK functions in intracellular signal transduction, and provide important evidence for a critical role of PINCH and integrins in regulating ILK cellular function (Lynch, 1999).
PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. These proteins interact in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and studies have detected clusters of PINCH that are colocalized with the alpha5beta1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways (Tu, 1999).
Integrin-linked kinase (ILK) is a multidomain focal adhesion (FA) protein that functions as an important regulator of integrin-mediated processes. A new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP) has been identified and characterized. CH-ILKBP is widely expressed and highly conserved among different organisms from nematodes to human. CH-ILKBP interacts with ILK in vitro and in vivo, and the ILK COOH-terminal domain and the CH-ILKBP CH2 domain mediate the interaction. CH-ILKBP, ILK, and PINCH, a FA protein that binds the NH2-terminal domain of ILK, form a complex in cells. CH-ILKBP localizes to FAs and associates with the cytoskeleton. Deletion of the ILK-binding CH2 domain abolishes the ability of CH-ILKBP to localize to FAs. Furthermore, the CH2 domain alone is sufficient for FA targeting, and a point mutation that inhibits the ILK-binding impairs the FA localization of CH-ILKBP. Thus, the CH2 domain, through its interaction with ILK, mediates the FA localization of CH-ILKBP. Overexpression of the ILK-binding CH2 fragment or the ILK-binding defective point mutant inhibits cell adhesion and spreading. These findings reveal a novel CH-ILKBP-ILK-PINCH complex and provide important evidence for a crucial role of this complex in the regulation of cell adhesion and cytoskeleton organization (Tu, 2001).
PINCH is a recently identified adaptor protein that comprises an array of five LIM domains. PINCH functions through LIM-mediated protein-protein interactions that are involved in cell adhesion, growth, and differentiation. The LIM1 domain of PINCH interacts with integrin-linked kinase (ILK), thereby mediating focal adhesions via a specific integrin/ILK signaling pathway. The PINCH LIM1 domain NMR structure has been solved and its binding to ILK has been characterized. LIM1 contains two contiguous zinc fingers of the CCHC and CCCH types and adopts a global fold similar to that of functionally distinct LIM domains from cysteine-rich protein and cysteine-rich intestinal protein families with CCHC and CCCC zinc finger types. Gel-filtration and NMR experiments demonstrate a 1:1 complex between PINCH LIM1 and the ankyrin repeat domain of ILK. A chemical shift mapping experiment has identified regions in PINCH LIM1 that are important for interaction with ILK. Comparison of surface features between PINCH LIM1 and other functionally different LIM domains indicates that the LIM motif might have a highly variable mode in recognizing various target proteins (Velyvis, 2001).
Integrin-linked kinase (Ilk) is a multidomain protein that plays important roles at cell-extracellular matrix (ECM) adhesion sites. A new LIM-domain containing protein (termed as PINCH-2) is described that forms a complex with Ilk. PINCH-2 is co-expressed with PINCH-1 (previously known as PINCH), another member of the PINCH protein family, in a variety of human cells. Immunofluorescent staining of cells with PINCH-2-specific antibodies shows that PINCH-2 localizes to both cell-ECM contact sites and the nucleus. Deletion of the first LIM (LIM1) domain of PINCH-2 abolishes the ability of PINCH-2 to form a complex with Ilk. The Ilk-binding defective LIM1-deletion mutant, unlike the wild type PINCH-2 or the Ilk-binding competent LIM5-deletion mutant, is incapable of localizing to cell-ECM contact sites, suggesting that Ilk binding is required for this process. Importantly, the PINCH-2-Ilk and PINCH-1-Ilk interactions are mutually exclusive. Overexpression of PINCH-2 significantly inhibits the PINCH-1-Ilk interaction and reduces cell spreading and migration. These results identify a novel nuclear and focal adhesion protein that associates with Ilk and reveals an important role of PINCH-2 in the regulation of the PINCH-1-Ilk interaction, cell shape change, and migration (Zhang, 2002a).
PINCH, integrin-linked kinase (Ilk) and calponin homology-containing Ilk-binding protein (CH-IlkBP) form a ternary complex that plays crucial roles at cell-extracellular matrix adhesion sites. To understand the mechanism underlying the complex formation and recruitment to cell-adhesion sites a combined structural, mutational and cell biological analysis was undertaken. Three-dimensional structure-based point mutations identified specific PINCH and Ilk sites that mediate the complex formation. Analyses of the binding defective point mutants revealed that the assembly of the PINCH-Ilk-CH-IlkBP complex is essential for their localization to cell-extracellular matrix adhesion sites. The formation of the PINCH-Ilk-CH-IlkBP complex precedes integrin-mediated cell adhesion and spreading. Furthermore, inhibition of protein kinase C, but not that of actin polymerization, inhibits the PINCH-Ilk-CH-IlkBP complex formation, suggesting that the PINCH-Ilk-CH-IlkBP complex likely serves as a downstream effector of protein kinase C in the cellular control of focal adhesion assembly. Finally, evidence is provided that the formation of the PINCH-Ilk-CH-IlkBP complex, while necessary, is not sufficient for Ilk localization to cell-extracellular matrix adhesion sites. These results provide new insights into the molecular mechanism underlying the assembly and regulation of cell-matrix adhesion structures (Zhang, 2002b).
PINCH co-localizes with Ilk in both focal adhesions and fibrillar adhesions. The molecular basis underlying the targeting of PINCH to the cell-matrix contact sites and the functional significance of the PINCH-Ilk interaction have been investigated. The N-terminal LIM1 domain, which mediates the Ilk binding, is required for the targeting of PINCH to the cell-matrix contact sites. The C-terminal LIM domains, although not absolutely required, play an important regulatory role in the localization of PINCH to cell-matrix contact sites. Inhibition of the PINCH-Ilk interaction, either by overexpression of a PINCH N-terminal fragment containing the Ilk-binding LIM1 domain or by overexpression of an Ilk N-terminal fragment containing the PINCH-binding ankyrin domain, retard cell spreading, and reduce cell motility. These results suggest that PINCH, through its interaction with Ilk, is crucially involved in the regulation of cell shape change and motility (Zhang, 2002c).
PINCH-1 is a widely expressed focal adhesion protein that forms a ternary complex with integrin-linked kinase (ILK) and CH-ILKBP/actopaxin/alpha-parvin (abbreviated as alpha-parvin herein) (see Drosophila Parvin). RNAi was used to investigate the functions of PINCH-1 and ILK in human cells. PINCH-1 and ILK, but not alpha-parvin, are shown to be essential for prompt cell spreading and motility. PINCH-1 and ILK, like alpha-parvin, are crucial for cell survival. Also, PINCH-1 and ILK are required for optimal activating phosphorylation of PKB/Akt, an important signaling intermediate of the survival pathway. Whereas depletion of ILK reduces Ser473 phosphorylation but not Thr308 phosphorylation of PKB/Akt, depletion of PINCH-1 reduces both the Ser473 and Thr308 phosphorylation of PKB/Akt. PINCH-1 and ILK function in the survival pathway not only upstream but also downstream (or in parallel) of protein kinase B (PKB)/Akt. This study also shows that, PINCH-1, ILK and to a less extent alpha-parvin are mutually dependent in maintenance of their protein, but not mRNA, levels. The coordinated down-regulation of PINCH-1, ILK, and alpha-parvin proteins is mediated at least in part by proteasomes. Finally, increased expression of PINCH-2, an ILK-binding protein that is structurally related to PINCH-1, prevented the down-regulation of ILK and alpha-parvin induced by the loss of PINCH-1 but failed to restore the survival signaling or cell shape modulation. These results provide new insights into the functions of PINCH proteins in regulation of ILK and alpha-parvin and control of cell behavior (Fukuda, 2003).
Many of the protein-protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. A widely expressed, Nck-related adaptor protein termed Nck-2 has been characterized. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-beta, and IRS-1. The association of Nck-2 with EGF receptors is regulated by EGF stimulation and involves largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributes to the complex formation. The association of Nck-2 with PDGF receptor-beta is dependent on PDGF activation and is mediated solely by the SH2 domain of Nck-2. Additionally, a stable association has been detected between Nck-2 and IRS-1 that is mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, evidence is provided indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain-containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways (Tu, 1998).
PINCH is an adaptor protein found in focal adhesions, large cellular complexes that link extracellular matrix to the actin cytoskeleton. PINCH, which contains an array of five LIM domains, has been implicated as a platform for multiple protein-protein interactions that mediate integrin signaling within focal adhesions. The LIM1 domain of PINCH functions in focal adhesions by binding specifically to integrin-linked kinase. Using NMR spectroscopy, it has been shown that the PINCH LIM4 domain, while maintaining the conserved LIM scaffold, recognizes the third SH3 domain of another adaptor protein, Nck2 (also called Nckbeta or Grb4), in a manner distinct from that of the LIM1 domain. Point mutation of LIM residues in the SH3-binding interface disrupts LIM-SH3 interaction and substantially impairs localization of PINCH to focal adhesions. These data provide novel structural insight into LIM domain-mediated protein-protein recognition and demonstrate that the PINCH-Nck2 interaction is an important component of the focal adhesion assembly during integrin signaling (Velyvis, 2003).
Weak protein-protein interactions (PPIs) are critical determinants of many biological processes. However, in contrast to a large growing number of well-characterized, strong PPIs, the weak PPIs are poorly explored. Genome wide, there exist few 3D structures of weak PPIs with, and none with. This study reports the NMR structure of an extremely weak focal adhesion complex between Nck-2 SH3 domain and PINCH-1 LIM4 domain. The structure exhibits a remarkably small and polar interface with distinct binding modes for both SH3 and LIM domains. Such an interface suggests a transient Nck-2/PINCH-1 association process that may trigger rapid focal adhesion turnover during integrin signaling. Genetic rescue experiments demonstrate that this interface is indeed involved in mediating cell shape change and migration. Together, the data provide a molecular basis for an ultraweak PPI in regulating focal adhesion dynamics during integrin signaling (Vaynberg, 2005).
Hic-5 is a focal adhesion LIM protein serving as a scaffold in integrin signaling. The protein comprises four LD domains in its N-terminal half and four LIM domains in its C-terminal half with a nuclear export signal in LD3 and is shuttled between the cytoplasmic and nuclear compartments. In this study, immunoprecipitation and in vitro cross-linking experiments showed that Hic-5 homo-oligomerized through its most C-terminal LIM domain, LIM4. Strikingly, paxillin, the protein most homologous to Hic-5, did not show this capability. Gel filtration analysis also revealed that Hic-5 differs from paxillin in that it has multiple forms in the cellular environment, and Hic-5 but not paxillin was capable of hetero-oligomerization with a LIM-only protein, PINCH, another molecular scaffold at focal adhesions. The fourth LIM domain of Hic-5 and the fifth LIM domain region of PINCH constituted the interface for the interaction. The complex included integrin-linked kinase, a binding partner of PINCH, which also interacts with Hic-5 through the region encompassing the pleckstrin homology-like domain and LIM domains of Hic-5. Of note, Hic-5 marginally affects the subcellular distribution of PINCH but directs its shuttling between the cytoplasmic and nuclear compartments in the presence of integrin-linked kinase. Uncoupling of the two signaling platforms of Hic-5 and PINCH through interference with the hetero-oligomerization resulted in impairment of cellular growth. Hic-5 is, thus, a molecular scaffold with the potential to dock with another scaffold through the LIM domain, organizing a mobile supramolecular unit and coordinating the adhesion signal with cellular activities in the two compartments (Mori, 2006).
Heart disease is a leading cause of death in newborn children and in adults. Efforts to promote cardiac repair through the use of stem cells hold promise but typically involve isolation and introduction of progenitor cells. This study shows that the G-actin sequestering peptide thymosin beta4 promotes myocardial and endothelial cell migration in the embryonic heart and retains this property in postnatal cardiomyocytes. Survival of embryonic and postnatal cardiomyocytes in culture is also enhanced by thymosin beta4. Thymosin beta4 forms a functional complex with PINCH and integrin-linked kinase (ILK), resulting in activation of the survival kinase Akt (also known as protein kinase B). After coronary artery ligation in mice, thymosin beta4 treatment results in upregulation of ILK and Akt activity in the heart, enhanced early myocyte survival and improved cardiac function. These findings suggest that thymosin beta4 promotes cardiomyocyte migration, survival and repair and the pathway it regulates may be a new therapeutic target in the setting of acute myocardial damage (Bock-Marquette, 2004).
Proteins at cell-extracellular matrix adhesions (e.g. focal adhesions) are crucially involved in regulation of cell morphology and survival. CH-ILKBP/actopaxin/alpha-parvin and affixin/beta-parvin (abbreviated as alpha- and beta-parvin, respectively), two structurally closely related integrin-linked kinase (ILK)-binding focal adhesion proteins, are co-expressed in human cells. Depletion of alpha-parvin dramatically increases the level of beta-parvin, suggesting that beta-parvin is negatively regulated by alpha-parvin in human cells. Loss of PINCH-1 or ILK, to which alpha- and beta-parvin bind, significantly reduces the activation of Rac, a key signaling event that controls lamellipodium formation and cell spreading. It was surprising to find that loss of alpha-parvin, but not that of beta-parvin, markedly stimulates Rac activation and enhances lamellipodium formation. Overexpression of beta-parvin, however, is insufficient for stimulation of Rac activation or lamellipodium formation, although it is sufficient for promotion of apoptosis, another important cellular process that is regulated by PINCH-1, ILK, and alpha-parvin. In addition, the interactions of ILK with alpha- and beta-parvin are mutually exclusive. Overexpression of beta-parvin or its CH(2) fragment, but not a CH(2) deletion mutant, inhibited the ILK-alpha-parvin complex formation. Finally, evidence is provided suggesting that inhibition of the ILK-alpha-parvin complex is sufficient, although not necessary, for promotion of apoptosis. These results identify Rac as a downstream target of PINCH-1, ILK, and parvin. Furthermore, they demonstrate that alpha- and beta-parvins play distinct roles in mammalian cells and suggest that the formation of the ILK-alpha-parvin complex is crucial for protection of cells from apoptosis (Zhang, 2004).
PINCH is a double zinc finger domain (LIM)-only adapter protein that functions to recruit the integrin-linked kinase (Ilk) to sites of integrin activation. Genetic studies have shown that PINCH and Ilk are required for integrin signaling. Since integrin activation is associated with Schwann cell migration, neurite outgrowth and regeneration, this study examined PINCH in the normal peripheral nervous system and after chronic constriction injury (CCI) in adult Sprague-Dawley rats. Immunohistochemistry identified PINCH immunoreactivity in cell bodies of dorsal root ganglia (DRG) neurons, axons, satellite cells, and Schwann cells. PINCH immunostaining was localized to the membrane of uninjured DRG cell bodies consistent with its localization at a site of integrin activation. In contrast, 5 days following CCI, PINCH immunostaining was diffuse throughout the DRG cell cytoplasm. Confocal microscopy of primary and transformed Schwann cells localized PINCH in cytoplasmic, perinuclear and nuclear areas. Examination of the PINCH sequence revealed a putative leucine-rich nuclear export signal (NES) and an overlapping basic nuclear localization signal (NLS). To demonstrate nuclear export of PINCH, rabbit anti-PINCH IgG was microinjected into Schwann cell nuclei and allowed to combine with PINCH contained within the nucleus. Immunofluorescence showed that the PINCH and anti-PINCH IgG complex rapidly translocated to the cytoplasm. Treatment with leptomycin B causes nuclear accumulation of PINCH, indicating that the CRM1 pathway mediates nuclear export of PINCH. Ilk activity in Schwann cells is enhanced by platelet-derived growth factor (PDGF) and tumor necrosis factor alpha. PINCH immunoprecipitates from PDGF- and TNFalpha-stimulated Schwann cells contained several high-molecular-weight threonine-phosphorylated proteins. Taken together, these results indicate that PINCH is an abundant shuttling/signaling protein in Schwann cells and DRG neurons (Campana, 2003).
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Bock-Marquette, I., Saxena, A., White, M. D., Dimaio, J. M. and Srivastava, D. (2004). Thymosin beta4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature 432(7016): 466-72. 15565145
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date revised: 12 January 2018
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