: Biological Overview | References
Gene name - Paxillin
Cytological map position - 37D5-37D7
Function - cytoskeletal adaptor
Keywords - cytoskeleton, focal adhesion docking protein, regulates actin dynamics and cell adhesion, positively regulates Rac and negatively regulates Rho, muscle attachment site, mesoderm, autophagosome formation, wing, leg
Symbol - Pax
FlyBase ID: FBgn0041789
Genetic map position - chr2L:19400822-19426575
Classification - LIM domain protein
Cellular location - cytoplasmic
Paxillin is a prominent focal adhesion docking protein that regulates cell adhesion and migration. Although numerous paxillin-binding proteins have been identified and paxillin is required for normal embryogenesis, the precise mechanism by which paxillin functions in vivo has not yet been determined. An ortholog of mammalian paxillin in Drosophila (Dpax) has been identified and a genetic analysis of paxillin function during development was undertaken. Overexpression of Dpax disrupts leg and wing development, suggesting a role for paxillin in imaginal disc morphogenesis. These defects may reflect a function for paxillin in regulation of Rho family GTPase signaling since paxillin interacts genetically with Rac and Rho in the developing eye. Moreover, a gain-of-function suppressor screen identified a genetic interaction between Dpax and center divider cdi in wing development. Cdi belongs to the cofilin kinase family, which includes the downstream Rho target, LIM kinase (LIMK). Significantly, strong genetic interactions were detected between Dpax and Dlimk, as well as downstream effectors of Dlimk. Supporting these genetic data, biochemical studies indicate that paxillin regulates Rac and Rho activity, positively regulating Rac and negatively regulating Rho. Taken together, these data indicate the importance of paxillin modulation of Rho family GTPases during development and identify the LIMK pathway as a critical target of paxillin-mediated Rho regulation (Chen, 2005).
Regulation of cell adhesion to the surrounding extracellular matrix (ECM) plays an essential role in organizing tissues and organs during development, and dysregulation of cell adhesion has been implicated in tumor invasion and metastasis. The adhesion process is mediated predominantly through a class of heterodimeric transmembrane receptors called integrins. Engagement of integrin receptors transduces a cascade of signaling events that regulate cell migration, proliferation, and survival. The cytoplasmic domains of integrins associate with a group of dynamic effectors, which includes several actin-binding proteins, focal adhesion kinase (FAK), Src family kinases, p130CAS, and paxillin. Focal adhesions play an essential role in linking the ECM to the actin cytoskeleton (Chen, 2005).
Paxillin was originally identified as a tyrosine phosphorylated protein in v-src-transformed cells and subsequently as a target of cellular Src family kinases. Tyrosine phosphorylation of paxillin is also observed upon integrin-mediated cell adhesion and upon growth factor stimulation (Burridge, 1992). Molecular analysis revealed that paxillin is a multidomain protein which contains five leucine-rich motifs, known as LD repeats, and four tandemly arranged LIM domains that are conserved among mammalian, avian, and Drosophila paxillin (Schaller, 2001; Turner, 1994; Wheeler, 2001). The LD repeats allow paxillin to interact with actin-binding proteins, kinases, and the ARF family GTPase-activating proteins (ARF-GAPs). While a limited number of binding partners have been identified for the LIM domains, this region is crucial for mediating paxillin localization to focal adhesions (Brown, 1996; Schaller, 2001). The array of docking sites in paxillin mediate the recruitment and assembly of multiprotein complexes to focal adhesions and allow paxillin to function as an adapter to coordinate various signaling pathways (Schaller, 2001; Turner, 2000; Chen, 2005 and references therein).
The LD4 repeat of paxillin associates with several of the ARF-GAPs, which are known for their role in regulating membrane trafficking and organelle structure (Turner, 1999). The ARF-GAP protein p95PKL could link paxillin to Rho family GTPase signaling by binding to a protein complex containing the Rac-specific guanine nucleotide exchange factor PIX/COOL, the Rac effector target p21-activated serine/threonine kinase PAK, and the SH2-SH3 adaptor protein Nck (Manser, 1998; Turner, 1999; Chen, 2005 and references therein).
The Rho family GTPases, including Rho, Rac, and Cdc42, are known for their function in regulating actin cytoskeletal structure, cell motility, and morphogenesis. Activation of Rho induces focal adhesion and stress fiber formation, and activation of Rac and Cdc42 results in the formation of lamellipodia and filopodia, respectively. The recruitment of the PKL-PIX-PAK complex to focal adhesions by paxillin may stimulate the transition from Rho-mediated focal adhesions to Rac-mediated focal contacts to facilitate membrane protrusion and cell migration. Thus, these various features of the paxillin protein indicate that it can potentially play an important role in regulating signals from Rho GTPases to the actin cytoskeleton (Chen, 2005).
The in vivo function of paxillin has recently been addressed using a gene-targeting strategy in mice (Hagel, 2002). Targeted disruption of the paxillin gene in the mouse embryo results in multiple defects in the development of mesodermally derived tissues such as heart and somites. Analysis of focal adhesions and lamellipodia in embryo-derived cells lacking paxillin indicated that paxillin is not required for the formation of focal adhesions or lamellipodia; however, paxillin is likely to be important for maintenance of lamellipodia and is required for focal adhesion turnover (Webb, 2004). Consistent with these functions, paxillin-deficient cells also exhibit delayed cell spreading and reduced motility (Hagel, 2002; Chen, 2005 and references therein).
To further explore the role of paxillin in regulating signaling pathways that influence tissue morphogenesis during development, a genetic approach was used in Drosophila studies. Genetic analysis revealed that a Drosophila ortholog of mammalian paxillin modulates Rho and Rac GTPase signaling during Drosophila development. Significantly, paxillin antagonizes Rho signals to the actin cytoskeleton but promotes signals from Rac. This regulation may be crucial for development of imaginal disc structures such as the wing and leg, as overexpression of paxillin results in leg defects and wing blisters. In support of this hypothesis, a gain-of-function suppressor screening for Dpax-induced wing blisters identified a genetic interaction between Dpax and cdi, the Drosophila homolog of TESK, the cofilin kinase family member. Cofilin kinases include LIMKs, and LIMKs are important mediators of Rho signaling that promote actin polymerization by phosphorylating and inhibiting the actin-severing protein cofilin. Consistent with the results from the suppressor screening, Dlimk and other components in this pathway also interacted genetically with Dpax. One mechanism by which paxillin may control Rho signaling is at the level of Rac and Rho activation, as Rac and Rho are misregulated in paxillin-deficient mouse embryo fibroblasts (MEFs). Taken together, these data suggest that a conserved role for paxillin in regulating signals from Rho GTPases to the actin cytoskeleton is critical for normal tissue morphogenesis during development (Chen, 2005).
Paxillin is a scaffolding protein found in focal adhesions. Targeted disruption of paxillin in mice results in an early embryonic lethal phenotype with defects in multiple mesodermally derived structures. The recent completion of the Drosophila genome revealed the evolutionary conservation of many of the key molecules found in focal adhesions, including integrins, paxillin, vinculin, FAK, p130CAS (see CAS/CSE1 segregation protein), and ILK. The Drosophila paxillin is predominantly expressed in embryos, pupae, and male adults. In situ analysis of staged embryos reveals a restricted expression pattern of Dpax. In particular, Dpax is highly expressed in tissues undergoing cell shape changes or cell migration. Overexpression of Dpax in late larval stages results in a pupal lethal phenotype with few escapers bearing malformed phenotypes, suggesting that Dpax also plays an important role during later stages of development (Chen, 2005).
A loss-of-function mutant of Drosophila paxillin has not yet been reported. Therefore, the UAS/GAL4 system was employed to investigate the function of Dpax in the later stages of development. As has been reported for Drosophila FAK, overexpressing Dpax results in a blistered-wing phenotype. In mammals, paxillin is a substrate of FAK in transducing signals from integrins. FAK regulates focal adhesion disassembly and has been shown to be involved in Drosophila Wnt4-mediated cell movement during ovarian morphogenesis and is also required for border cell migration during oogenesis. The function of Dpax in oogenesis is not clear; however, Dpax is also highly expressed in the border cells (Chen, 2005).
The blistered-wing phenotype is also found in integrin mutant flies. In the prepupal stage, the wing is a single epithelial sheet, and integrins have been suggested to play a regulatory role. As development progresses this sheet folds into a dorsal and ventral side, and the integrins play an adhesive role at these later stages. Using drivers that are expressed at different stages of development, the studies suggest that paxillin could be important for both the regulatory and adhesive functions of the integrins. Such functions would be consistent with studies of mammalian systems in which paxillin functions downstream of multiple integrins and can regulate both inside out and outside in signaling. In addition, both paxillin and FAK are important for focal adhesion turnover. Thus, too much paxillin or FAK may increase the turnover of focal complexes and perturb the stable adhesion between two epithelia, thereby resulting in the blistering phenotype (Chen, 2005).
Using a gain-of-function screen for modifiers that can rescue the Dpax-induced wing blistering, Cdi/TESK was identified. Like LIMK, Cdi/TESK phosphorylates the actin-depolymerizing factor cofilin and stabilizes F-actin. Cdi/TESK is highly homologous to LIMK in the kinase domain; however, a recent study has demonstrated that Cdi/TESK functions downstream of Rac1 during spermatogenesis. Drosophila LIMK functions downstream of Rho1 in regulating disk morphogenesis. Dlimk and components in the Rho-LIMK pathway, including ssh, tsr, and bs/DSRF, also rescue the blistering phenotype. In addition, another regulator of SRF and actin, diaphanous, also shows genetic interactions with Dpax. Diaphanous is a direct effector of Rho which cooperates with LIMK to regulate SRF activation. All of these components play important roles in regulating F-actin synthesis. Taken together, these data indicate that it is possible that an increase in actin levels can prevent the increase in focal adhesion turnover caused by the excess level of paxillin, therefore suppressing the blistering phenotype. It is possible that simply overexpressing actin might be sufficient to rescue the blistering phenotype, although the results suggest that paxillin itself does not affect F-actin synthesis or actin organization. The ability of paxillin, however, to coimmunoprecipitate with LIMK and the increased cofilin phosphorylation in Pxl/ MEFs suggests that paxillin can modulate LIMK function. These data, combined with the genetic and biochemical evidence that paxillin can regulate Rho, suggest that paxillin could act at multiple points to regulate the Rho pathway (Chen, 2005).
Interestingly, while modulation of some components downstream of Rho is able to suppress the blistering phenotype, overexpression of other components such as ROK does not alter this phenotype. While this could reflect insufficient expression levels or more complex regulation of ROK, the data suggest that paxillin's regulation of the Rho pathway may involve either modulation of only certain downstream components or a lack of function for these components in the paxillin-induced phenotypes (Chen, 2005).
Rho GTPases play an important role in regulating actin cytoskeleton organization. Genetic and biochemical analysis reveal that paxillin activates Rac signaling but inactivates Rho signaling. Previous binding and localization studies suggest that mammalinan paxillin may regulate Rac through its indirect association with at least two Rac exchange factors. Pix/Cool is linked to paxillin via PKL/Git2, the ARF-GAP, and overexpression studies with mutants of paxillin and other members of this complex have led to the suggestion that paxillin may be important for recruiting this complex to focal contacts. A second binding partner, Crk, can also link paxillin to Rac activation via a nontraditional exchange factor, Dock180. Mislocalization of one or both complexes in Pxl/ mouse embryo fibroblasts (MEF)s could therefore lead to defects in Rac activation and subsequent defects in lamellipodium dynamics and migration. Both Pix/Cool and Crk localization were examined in rescued and Pxl/ MEFs and only a minor decrease was detected in Cool and Crk positive peripheral adhesions in Pxl/ cells. In MEFs, therefore, paxillin is not required for localization of these proteins to peripheral adhesions. This may be due to functional redundancy, as the paxillin family member Hic-5 can also bind the PKL-Pix complex and Crk can bind to other focal adhesion proteins, including p130Cas. In any case, mislocalization of these complexes is unlikely to account for the differences in Rac activation. In contrast, genetic studies of Drosophila have shown that deletion of a region encompassing the Drosophila homolog of Cool was able to suppress the Dpax-induced blistering. Thus, one potential mechanism by which paxillin may control Rac activation in Drosophila is through regulation of Pix/Cool. Since Rac and Rho have been shown to antagonize each other, it remains possible that in higher eukaryotes, paxillin could indirectly regulate Rac via regulation of Rho (Chen, 2005).
It is not clear how paxillin down-regulates Rho activity. Paxillin might be important for spatial regulation of Rho activity and/or controlling the activity or localization of a Rho GAP or GEF. Two Rho GAPs have been linked to mammalian paxillin. Graf is a Rho GAP that was originally identified as a Fak-binding partner, and a homolog of this protein has been identified in Drosophila studies. Since paxillin can interact with Fak, it is possible that loss of paxillin may somehow affect Graf localization or activation. While Fak localization to focal adhesions is less efficient in Pxl/ MEFs, the effects are minimal and thus this is unlikely to account for the enhanced Rho activity. It is worth noting that it has recently been reported that mammalian paxillin binds to the p120 RasGAP and competes with p120 RasGAP for binding to p190 RhoGAP. It has been suggested that paxillin inhibits Rho by promoting the formation of free p190 RhoGAP. The Drosophila ortholog of p190 RhoGAP does not bind to the Drosophila p120 RasGAP. In addition, only minor changes in p190 localization to the leading edge were detected in Pxl/ MEFs. Thus, paxillin may antagonize Rho function through multiple distinct regulatory mechanisms (Chen, 2005).
Taken together, these data suggest that while paxillin has the ability to interact with multiple proteins involved in diverse signaling pathways, a major function of this scaffolding protein in vivo is to regulate Rho family GTPases. Thus, misregulation of these GTPases is likely to account for the adhesion defects observed during development in mouse and Drosophila studies (Chen, 2005).
Integrin-linked kinase (ILK) is an essential component of a multiprotein complex that links actin to the plasma membrane. This study has used a genetic approach to examine the molecular interactions that are essential for the assembly of this ILK-containing complex at Drosophila muscle attachment sites (MASs). Downstream of integrins, talin plays a decisive role in the recruitment of three proteins: ILK, Lim domain protein PINCH (steamer duck) and paxillin. The accumulation of ILK at MASs appears to follow an amplification mechanism, suggesting that numerous binding sites are generated by minimal levels of the upstream integrin and talin effectors. This property suggests that ILK functions as an essential hub in the assembly of its partner proteins at sites of integrin adhesion. PINCH stability, and its subcellular localization at MASs, depends upon ILK function, but ILK stability and localization is not dependent upon PINCH. An in vivo structure-function analysis of ILK demonstrated that each ILK domain has sufficient information for its independent recruitment at embryonic MASs, whereas at later developmental stages only the kinase domain was effectively recruited. These data strengthen the view that the ILK complex is assembled sequentially at sites of integrin adhesion by employing multiple molecular interactions, which collectively stabilize the integrin-actin link (Zervas, 2011).
In multicellular organisms, tissue integrity depends upon the stable association of cells with the extracellular matrix (ECM). This requires a link between matrix ligands and the cytoskeleton, which is primarily mediated by the integrin family of surface receptors. Each integrin is composed of one α and one β subunit, and because these subunits have very short cytoplasmic tails it has been postulated that other scaffolding proteins assist integrins in the recruitment of downstream components that mediate the link to actin. The cytoskeletal molecules involved in this link, and the hierarchy of their interactions, are currently a subject of intense investigation, and genetic approaches in model organisms have provided novel insights into the function of these proteins. One such example is integrin-linked kinase (ILK), a protein initially identified by its ability to bind directly to the cytoplasmic tail of the β1 integrin subunit. ILK is a modular protein containing five tandem ankyrin repeats (ANKRs), a putative phosphoinositide-binding site and a kinase-like domain. ILK is required to maintain the molecular link between integrins and the sarcomeric actin filaments in Drosophila embryonic muscles and to assemble the link between integrins and the contractile apparatus of developing Caenorhabditis elegans muscles. Knockout of the single mouse gene showed that ILK is required for diverse developmental processes, with its earliest function being in epithelial polarization and normal actin distribution of the developing epiblast. In all three organisms, certain site-directed mutations within the kinase domain of ILK that should eliminate kinase activity did not show any defects, suggesting that the essential role of ILK is not the phosphorylation of target proteins. Instead, ILK binds to multiple proteins, suggesting that its primary function is as an adaptor. The recently published structure of the kinase domain of ILK supports the view that it is a binding adaptor rather than a kinase. Thus, genetic and structural data indicate that ILK is a pseudokinase. However, a clear picture of the essential adaptor function of ILK in assisting integrin function has yet to be developed (Zervas, 2011).
The large number of proteins that bind ILK suggests it is a central scaffolding molecule for the assembly of a protein complex. Interacting proteins have been identified by yeast two-hybrid screening and biochemical methods. Among these proteins are: PINCH, which contains five LIM domains and binds to the ANKRs of ILK through its first LIM domain; paxillin, which has four LIM domains and five leucine-rich motifs (LD) and binds to the ILK kinase domain (Nikolopoulos, 2001); and parvin, which has two calponin homology domains and binds to the ILK kinase domain. Both the ANKRs and kinase domains are essential for recruitment of ILK at focal adhesions in vertebrate cells in culture. Even before its association with integrins, ILK is in a cytoplasmic complex with PINCH and parvin and a fourth protein, Ras suppressor protein 1 (Rsu1), which binds the fifth PINCH LIM domain. So far the model of functional coordination between ILK, PINCH and parvin has not been fully supported by genetic studies. ILK is required for parvin (Pat-6) recruitment to sites of integrin adhesion in C. elegans but not the reverse, and PINCH is not required for ILK recruitment in either Drosophila or C. elegans (Zervas, 2011 and references therein).
This study combined genetic analysis and structure-function approaches to examine the mutual interactions of ILK and PINCH, as well as their interactions with talin and paxillin. Low levels of integrins were found to be sufficient to recruit substantial levels of ILK to the major sites of integrin adhesion in the embryo and larva, namely the muscle attachment sites (MASs), suggesting an amplification mechanism. Complete removal of talin resulted in the loss of ILK, PINCH and paxillin from MASs, in agreement with its crucial contribution to integrin function. Neither ILK nor PINCH was required for paxillin recruitment. Unexpectedly, PINCH stability and recruitment required both domains of ILK (i.e., PINCH-binding ANKRs and kinase domains). In embryos each ILK domain was recruited to the MASs, but recruitment of the isolated ANKRs was lost during development, indicating a change in mechanism. Finally, the function of a number of conserved ILK residues that have been shown to be important for interactions with other proteins were tested, but they were found to be dispensable. Collectively, the results indicate that ILK functions upstream of PINCH in the muscle and that the function of ILK is executed by simultaneous interactions of both the kinase and ANKR domains (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 (see Comparison of ILK recruitment mechanisms). These proteins then in turn recruit ILK and paxillin, and ILK indirectly recruits PINCH (Zervas, 2011).
In vertebrate cells ILK and PINCH have mutually dependent functions. Their association is required to protect each other from a proteasome-dependent degradation and to facilitate each other's localization to focal adhesions. The direct interaction between ILK and PINCH has also been confirmed in Drosophila, and PINCH-mediated stabilization of ILK in mice has been confirmed genetically. In Drosophila, it was found that neither ILK protein stability nor its recruitment to MASs was affected in the absence of PINCH. This is in agreement with the persistent subcortical localization of ILK in the pinch-1-knockout mouse embryoid bodies, as well as in unc-97 mutants in C. elegans. By contrast, loss of ILK function reduced PINCH levels in Drosophila embryos. The instability of PINCH in the absence of ILK was overcome by overexpressing PINCH but, under these conditions, PINCH still was not localized to MASs. The isolated ILK ANKRs did localize to MASs in the absence of endogenous ILK, but were not able to recruit PINCH. Thus, rather than ILK recruiting PINCH by binding of its ANKRs to the LIM1 domain of PINCH, it appears to do so indirectly. This finding raises two important points. First, the interaction between the ILK ANKRs and LIM1 of PINCH might not be always maintained at sites of integrin adhesion. The finding that LIM1 is crucial for recruitment, but apparently not through interaction with ILK, suggests alternative interactions might be required. This is consistent with the observation that high levels of PINCH-GFP can be recruited to MASs, even when only the endogenous levels of ILK are present. Second, recent work in mammalian cells provided strong evidence that the primary function of PINCH was the recruitment of ILK through the ANKRs-LIM1 interaction (Stanchi, 2009). Notably, the recruitment of tensin and ILK at focal adhesions in cells lacking PINCH could be rescued by a direct fusion of the ILK kinase domain to integrin. However, the result suggests that, in Drosophila muscles, the reverse is true, and a primary function of ILK might be the stabilization and recruitment of PINCH (Zervas, 2011).
Finally, whether ILK and PINCH influence the subcellular localization and protein stability of paxillin was examined. Previous studies have demonstrated an interaction between the kinase domain of mammalian ILK and paxillin, and further suggested that this interaction controls ILK localization at focal adhesions. These studies identified key residues in ILK that contribute to paxillin interaction and ILK recruitment, such that ILK mutations at E359 and T384 impaired recruitment of ILK to focal adhesions. However, the ILK E359K mutation does not impair its ability to rescue the null phenotype or alter its recruitment in flies or mice. This study showed that mutating T384 in Drosophila ILK, equivalent to part of the paxillin-binding region in human ILK, also failed to impair the biological activity or recruitment of ILK, and neither ILK nor PINCH were required for paxillin recruitment. Thus, in MASs, the stability of paxillin and its subcellular localization does not depend on the ILK-PINCH complex (Zervas, 2011).
This study has examined the functions of the two main domains that constitute ILK (i.e. the ANKRs and the kinase domain), as well as specific binding sites that have been proposed to mediate interactions with phosphoinositides or paxillin and have been implicated in the regulation of the protein. From this work, it became evident that deletion of either the ANKRs or the kinase-like domain eliminates the essential functions of ILK in the developing organism. These two domains are required in the same molecule to execute the essential function of ILK. The proposed phosphoinositide-binding motif is dispensable for embryonic development but is required in the epithelial tissues of the wing, and therefore does contribute to ILK function. This suggests that the highest levels of ILK function are required in the wing. This is consistent with the finding that elimination of tensin only causes a wing-blister phenotype, even though it is also concentrated at MASs. By contrast, hypomorphic mutations in the gene encoding the αPS2 integrin subunit were found to cause muscle defects but not cause wing blisters, indicating that integrin adhesive sites are differentially sensitive to loss of different components of the integrin adhesion complex. The residue F436, which is located at the far end of the C-terminal region, was found to be crucial in vertebrate cells, but its mutation only mildly affected recruitment of the overexpressed protein and was dispensable for ILK function. It is possible that the F436A mutation reduces the binding affinity of ILK with Wech (Zervas, 2011).
Both the isolated ANKRs and the kinase domain are recruited to MASs, in contrast with the recruitment to focal adhesions seen in mammalian cells. This suggests that recruitment is more robust at MASs, so impaired ILK recruitment can still be visualized. It was surprising to find that the recruitment of the ANKRs of ILK did not involve an interaction with PINCH, as removing PINCH did not impair recruitment. Given that there is only one Drosophila pinch gene, in contrast with two in mammals, the ANKRs must be binding to another type of protein. One alternative candidate is Mlp84B, a protein that contains five LIM domains and is localized at MASs in an integrin-independent manner; it is known that the related zebrafish protein CSRP3 binds ILK (Zervas, 2011).
In summary, the results have demonstrated that, at the robust integrin adhesive sites that form in the developing muscles, ILK recruitment is much less sensitive to perturbation compared with recruitment in focal adhesions in cells in culture. The results show that integrins and talin are essential for the recruitment of ILK, and ILK in turn is essential for the recruitment of PINCH. How much of the function of ILK is mediated by PINCH, and whether it also functions independently through its interaction with parvin, will be the focus of future research (Zervas, 2011).
In all metazoan organisms, the diversification of cell types involves determination of cell fates and subsequent execution of specific differentiation programs. During Drosophila myogenesis, identity genes specify the fates of founder myoblasts, from which derive all individual larval muscles. To understand how cell fate information residing within founders is translated during differentiation, this study focused on three identity genes, eve, lb, and slou, and how they control the size of individual muscles by regulating the number of fusion events. They achieve this by setting expression levels of Muscle protein 20 (Mp20), Paxillin (Pax), and M-spondin (mspo), three genes that regulate actin dynamics and cell adhesion and, as is shown in this study, modulate the fusion process in a muscle-specific manner. Thus, these data show how the identity information implemented by transcription factors is translated via target genes into cell-type-specific programs of differentiation (Bataillé, 2010).
The myoblast fusion is asymmetric and takes place between founder cells (FCs) and fusion competent myoblasts (FCMs). Previous reports originated the idea that FCMs are not 'naive' myoblasts and contribute to the modulation of fusion process. In contrast, the current results support a view that FCs rather than FCMs carry the instructive information and lead to the conclusion that FCMs do not play an active role in setting the number of fusion events. However, because the spatial distribution of FCMs seems to be nonuniform, it is conceivable that the local distribution of FCMs is coordinated with the requirements of FCs to facilitate fusion process (Bataillé, 2010).
The identity genes lb, slou, and eve are required to specify FCs at the origin of five muscles the DA1, DT1, SBM, VA2, and VT1. This study provides evidence that these identity genes are also required for setting the muscle-specific number of fusions and demonstrates how this identity information is executed. After specification step, FCs fuse, between the embryonic stage 12 and 15, with a determined number of FCMs to generate muscles with a specific number of nuclei. During this time period eve, lb, and slou continue to be expressed in subsets of developing muscles and the data show that they are sufficient to establish the muscle-specific fusion programs in DA1, SBM, and VT1 (11, 7, and 4 nuclei, respectively). Furthermore, slou in combination with other factors contributes to two other programs that end up with seven to eight fusion events in muscles DT1 and VA2. To regulate number of fusion events eve, lb, and slou act by modulating expression of genes involved in dynamics of actin cytoskeleton or cell adhesion. Starting from stage 13, they establish a muscle-specific combinatorial code of expression levels of three targets: Mp20, Pax, and mspo. The combination of expression of the targets leads to the muscle-specific control of the number of fusion events. This notion is supported by the fact that each of identity genes is able to impose at ectopic locations the combinatorial realisator code of Mp20, Pax, and mspo expression, and thus, ectopically execute its fusion program. Given that the code of Mp20, Pax, and mspo is not sufficient to explain fusion programs in all muscles, it is hypothesized that other identity gene targets exist that modulate fusion counting (Bataillé, 2010).
The data support a two-step model of myoblast fusion according to which a muscle precursor is formed between stage 12 and 13 by an initial fusion, and then, between stage 13 and 15, fuses with additional myoblasts until the muscle reaches its final size. The fact that Mp20, Pax, and mspo are expressed from stage 13 suggests that the transition point between the two steps depends not only on the timing of FCM migration but also on the activation of limiting factors such as the identity gene targets which modulate the number of additional fusions. Since no nuclear divisions were observed in FCs or in growing myotubes in any of the genetic contexts analyzed, it can confidently be said that the number of nuclei present in each muscle is determined only by the number of fusion events (Bataillé, 2010).
Specification of FCs requires combinatorial code of activities of identity genes. This study shows that the same identity genes play instructive roles in subsequent muscle-type-specific differentiation process. Importantly, the data enlighten the fact that the identity genes are not equivalent and have distinct, context-dependent mode of action. eve, lb, and slou are sufficient to set the fusion programs in DA1, SBM, and VT1 muscles; however, in VA2 and DT1 slou functions in a different way and seems not to have a decisive role in this process. Because the specification of the VA2 and DT1 FCs also involves functions of Poxm, Kr, and ap, it is hypothesized that they act together with slou in setting fusion programs of VA2 and DT1. This raises an important question about hierarchy of identity genes during execution of muscle identity programs and their roles in acquisition of specific properties of muscles such as number of nuclei, attachment points, and innervation (Bataillé, 2010).
The data presented in this study demonstrate that the number of fusion events in developing muscles is regulated by a muscle-specific combinatorial realisator code of identity gene targets. In contrast to the previously identified fusion genes acting in all muscles, the identified identity targets, Mp20, Pax, and mspo, display muscle-type specific expression and modulate fusion in a muscle-type-specific manner proportionally to the level of their expression. The loss and gain of function of each of them lead to subtle fusion phenotypes indicating that the range of fusion events controlled by these three candidates is limited. Indeed, the loss of function of Mp20 results in loss of two nuclei in a subset of muscles, whereas its overexpression induces the recruitment of maximum two FCMs. A similar range of defects in number of fusion events is observed in Pax and mspo mutant embryos indicating that they influence fusion process at the same level (Bataillé, 2010).
Mp20 encodes a cytoskeletal protein displaying restricted expression in adult muscles and sharing sequence homology with the lineage-restricted mouse proteins SM22alpha, SM22beta, and NP25. These proteins contain calponin-like repeats, and, in mammals, interact with F-actin and participate in the organization of the actin cytoskeleton. In Drosophila S2R cells, the RNAi knockdown of Mp20 induces a phenotype of round and nonadherent cells supporting its role in regulation of fusion process (Bataillé, 2010 and references therein).
The second candidate, Pax (DPxn37), is a scaffold protein that recruits structural and signaling molecules to the sites of focal adhesion. Pax has been shown to be involved in the actin cytoskeleton organization, cell adhesion, cell migration, and cell survival. In the developing Drosophila muscles, Pax protein localizes at muscle-tendon junctions suggesting that it may play a role in muscle attachment. The current analyses of Pax mutant embryos do not reveal muscle-tendon adhesion defects but show discrete myoblast fusion phenotypes, which correlate with differential muscle-specific expression of Pax. The role of Pax in modulating fusion is consistent with previously described implications of Pax interacting proteins, including ARF6 in myoblast fusion in both Drosophila and vertebrates, and FAK in vertebrates (Bataillé, 2010 and references therein).
Finally, mspo belongs to the F-Spondins, a conserved family of ECM proteins, which maintain cell-matrix adhesion in multiple tissues. In vertebrates, F-Spondins have context-dependent effects on axon outgrowth and cell migration. As Mp20, Pax, and Mspo are expressed in FC cells and growing myotubes, one possibility is that they modify the spreading and/or motility of FC protrusions required to attract FCMs. Alternatively, by modulating actin cytoskeleton, Mp20, Pax, and Mspo may also influence the stability of adhesion between the growing muscle and the FCM creating permissive conditions or blocking the progression of fusion process (Bataillé, 2010).
The muscle-type-specific regulation of fusion programs by the identity genes and their targets raises an intriguing question of how this regulation is executed from the mechanistic point of view. Because different levels of expression of Mp20, Pax, and mspo correlate with different fusion programs in both wild-type and genetically manipulated embryos, it was thought that by following kinetics of fusion in small and big muscles insights would be gained into how the fusion programs are modulated. It turns out that the rate of fusion is proportional to the size of muscle, meaning the number of fusion events, thus revealing that the identity genes acting via their targets set up the frequency of fusion events. Accordingly, loss and gain of function of identity genes and their targets identified here results in modulations of fusion programs by accelerating or slowing down the fusion rate. This finding provides insights into mechanistic understanding of muscle-type-specific regulation of fusion process and raises an important question about whether this mechanism is broadly conserved (Bataillé, 2010).
Autophagy is a conserved cellular process of macromolecule recycling that involves vesicle-mediated degradation of cytoplasmic components. Autophagy plays essential roles in normal cell homeostasis and development, the response to stresses such as nutrient starvation, and contributes to disease processes including cancer and neurodegeneration. Although many of the autophagy components identified from genetic screens in yeast are well conserved in higher organisms, the mechanisms by which this process is regulated in any species are just beginning to be elucidated. In a genetic screen in Drosophila melanogaster, a link was identified between the focal adhesion protein paxillin and the Atg1 kinase, which has been previously implicated in autophagy. In mammalian cells, paxillin was found to redistributed from focal adhesions during nutrient deprivation, and paxillin-deficient cells exhibit defects in autophagosome formation. Together, these findings reveal a novel evolutionarily conserved role for paxillin in autophagy (Chen, 2008).
This paper reports genetic interactions between paxillin and components implicated in developmental autophagy, including Atg1 and the ecdysone receptor, during wing maturation. Intriguingly, in te newly emerged adults, autophagy is also observed at the time of wing spreading. It was also found that over-expression of DSRF, which suppresses Pax-induced wing blisters, rescues Atg1-induced wing defects, thus further supporting a role of Atg1 in wing morphogenesis. EP3348, which strongly suppresses Pax-induced blistered wings, harbors a single transposable element inserted in the 5' untranslated region of the Drosophila Atg1 gene. It is notable that EP3348 suppresses Pax-induced wing defects more effectively than does Atg1-RNAi. Since Atg1 mutants exhibit a pupal lethal phenotype, but neither EP3348 nor expression of Atg1-RNAi result in lethality, it is likely that neither of them cause complete knockdown of Atg1 expression. The reason that EP3348 suppresses dPax-induced wing defects better than Atg1-RNAi is most likely due to differences in their effects on Atg1 expression. However, such differences may be subtle, and spatially restricted, as it was not possible to distinguish their effects on Atg1 expression by RTPCR of whole tissues (Chen, 2008).
It was also found that overexpression of Atg1 induces cell death and an aberrant actin cytoskeleton. When paxillin is coexpressed with Atg1, these phenotypes were enhanced, leading to increased lethality and defects in arista patterning. A deletion mutation, Df(2L)Pr.A16, which uncovers the Pax gene suppressed both Atg1- and dPax-induced defects. However, since Df(2L)Pr.A16 contains a large deletion, the possibility that the suppression effect is due to disruption of a gene that is independent of dPax cannot be ruled out. Most significantly, a marked decrease was found in the number of autophagosomes in both Pax mutant flies and paxillin-deficient mouse fibroblasts upon nutrient deprivation, thus revealing a novel requirement for paxillin in autophagy. It was also demonstrated that Atg1 can directly phosphorylate paxillin in vitro, suggesting that Atg1 may regulate the activity of paxillin. Thus far, however, it has not been possible to demonstrate that a kinase-deficient form of Atg1 detectably affects paxillin localization in mammalian cells, suggesting that the interaction of these two proteins in vivo may be more complex. Moreover, the fact that no genetic interactions were observed between other autophagy mutants and paxillin in vivo leaves open the formal possibility that the interaction between Atg1 and paxillin in Drosophila may not reflect roles for these proteins in autophagy (Chen, 2008).
Despite the identification of many autophagy-associated genes, the mechanism of autophagosome biogenesis, including the origin of membranes and how they are transported for vesicle formation, remains unclear. It has been proposed that autophagosomes form by the addition of membranes derived from ER or Golgi complex in the form of vesicle transport. Alternatively, autophagosomes could form by de novo synthesized membrane (Chen, 2008).
The precise function of Atg1 in this process is still unclear. In yeast two=hybrid screens, several proteins that directly interact with Atg1 have been identified, including Syntenin, a Rab5 GTPase.interacting protein, VAB-8, a kinesin subfamily like molecule, GABA receptor associated protein (GABARAP), and the Golgi-associated ATPase enhancer of 16kDa (GATE16). Significantly, several of these Atg1-interacting proteins are involved in membrane dynamics and vesicle trafficking, suggesting a conserved role for Atg1 in the regulation of membrane trafficking. Indeed, recent findings have implicated the mouse Atg1 ortholog, Ulk1/2, in endocytotic processes during neurogenesis (Chen, 2008).
Paxillin is a multidomain protein and functions as an essential regulatory molecule that couples integrins to the actin cytoskeleton in focal adhesions. Several paxillin-associated proteins are involved in regulation of integrin-mediated signaling associated with cell adhesion to extracellular matrix, motility and growth factor responses. The association of paxillin with Arf GAPs regulates paxillin localization, and the Arf small GTPases play a central role in membrane trafficking and cytoskeletal dynamics. While a role was found for paxillin in the regulation of membrane trafficking, whether this is related to its role in autophagy is not clear. Interestingly, the results indicate that integrin-mediated signaling is not required for paxillin's role in autophagy, suggesting that paxillin's role in the formation of autophagosomes is independent of signals from extracellular matrix. However, this aspect of the paxillin.autophagy relationship has only been examined thus far in Drosophila melanogaster, and so it remains to be determined whether the role of paxillin in autophagasome formation in mammalian cells is similarly integrin-independent (Chen, 2008).
Collective cell movement is a mechanism for invasion identified in many developmental events. Examples include the movement of lateral-line neurons in Zebrafish, cells in the inner blastocyst, and metastasis of epithelial tumors. One key model to study collective migration is the movement of border cell clusters in Drosophila. Drosophila egg chambers contain 15 nurse cells and a single oocyte surrounded by somatic follicle cells. At their anterior end, polar cells recruit several neighboring follicle cells to form the border cell cluster. By stage 9, and over 6 hr, border cells migrate as a cohort between nurse cells toward the oocyte. The specification and directionality of border cell movement are regulated by hormonal and signaling mechanisms. However, how border cells are held together while they migrate is not known. This study shows that negative-feedback loop controlling JNK activity regulates border cell cluster integrity. JNK signaling modulates contacts between border cells and between border cells and substratum to sustain collective migration by regulating several effectors including the polarity factor Bazooka and the cytoskeletal adaptor D-Paxillin. A role for the JNK pathway is anticipated in controlling collective cell movements in other morphogenetic and clinical models (Llense, 2008).
In an analysis of the mechanisms regulating the expression of puckered (puc), the gene encoding the Drosophila Jun N-terminal kinase (JNK) dual-specificity phosphatase (DSP) regulatory sequences (PG2) were uncovered directing its expression to border cells. PG2 expands across the first and second introns of puc, in which the pucB48 insertion is located. This expression is also observed in puc enhancer (pucB48) and protein trap lines (Llense, 2008).
JNKs represent a signaling hub with pivotal functions in cell proliferation, differentiation, and death. JNKs are inactivated by DSPs, and transcriptional induction of DSP expression is well documented as a negative-feedback mechanism. In Drosophila, this loop modulates JNK activity in processes such as epithelial expansion and overexpression of dominant-negative constructs relies on JNK signaling. Further, Puc overexpression leads to inhibition of JNK activity. Thus, Puc implements a negative-feedback loop in border cells (Llense, 2008).
Defects caused by the loss of JNK function in border cells included cluster dissociation and impaired motility. Instead of collectively following a leader cell, JNK-minus border cells autonomously disperse at the late step of migration, with most exhibiting long cellular extensions (LCEs) and actin-rich protrusions. JNK signaling does not affect polar cell specification or border cell recruitment (Llense, 2008).
Dissociation phenotypes are also observed in JNK-specific but not ERK-specific loss-of-function conditions for D-Fos, a major MAPK target, thereby ruling out potential interference via ERK. Indeed, reduced D-Fos suppresses border cell migration defects induced by elevated JNK activity (Llense, 2008).
Does JNK act in a linear pathway or does it target multiple independent effectors simultaneously to produce a multifaceted phenotype? Cells that migrate as part of a group cling firmly to each other while adhering transiently to the substrate. So, during migration, border cells show apicobasal polarity and remain attached to one another and to polar cells. Cell contacts are enriched in the adherens junctions (AJs) components, DE-Cadherin and Armadillo (β-Catenin). In electron microscopy (EM) preparations, border cells are tightly bound, whereas interfaces between border and nurse cells exhibit multiple interdigitations (Llense, 2008).
In JNK-minus conditions, namely after Puc overexpression or in bsk (JNK) clones, cell polarity is disrupted and only remnants of apical markers, such as Bazooka (Baz), are present. Adhesion is impaired, and DE-Cadherin and Armadillo are downregulated. Reduction of JNK activity also resulted in β-Integrin accumulation at ectopic actin-rich protrusions. These also accumulate MyoVI, consistent with its role in force generation. In summary, upon depletion of JNK activity, border cells lose apicobasal polarity and progress into a mesenchymal phenotype. Indeed, EM preparations show that border-border cell contacts are less tight than wild-type cell contacts and cell membranes detach from each other at multiple sites. The end result is a cluster with multiple leading edges and residual cell-cell contacts (Llense, 2008).
How does the JNK pathway become activated in border cells? Rho, Rac, and Cdc42 GTPases are potential candidates. Loss of Rac completely abolishes border cell migration. However, phenotypes for RhoA and Cdc42 expression of dominant-negative forms -- RhoADN and Cdc42DN -- closely resemble JNK-minus induced dissociation. Furthermore, in Cdc42DN, polarity, cell contacts, and redistribution of substrate adhesion and motor markers are similarly affected. Most importantly, reporters of JNK activity such as Jun phosphorylation and the expression of the pucB48 transgene are also downregulated. Null cdc42 MARCM clones display the same phenotype, although frequency and penetrancy were very low. Therefore, a role for other GTPases, such as RhoA, in JNK activation cannot be ruled out (Llense, 2008).
Border cell clusters deficient for Baz (BazRNAi) resemble JNK loss of function (which leads to Baz downregulation) and exhibit dissociation and downregulation of DE-Cadherin. Thus, Baz, a critical landmark of epithelial polarity, could serve as an effector for the control of border-border cell contacts. To test this, Baz was overexpressed in cells lacking JNK activity (or expressing Cdc42DN); Baz was strongly rescued cluster integrity and DE-Cadherin expression (Llense, 2008).
Epithelial cells use a specialized repertoire of integrin receptors to mediate contacts and migration. However, border cells lacking β-Integrin were still able to adopt a leading migratory position, although the effect of complete removal of integrins from the cluster has not been reported (Llense, 2008).
Interestingly, β-Integrin antibodies reveal a rosette staining in border cell clusters that colocalize with AJ markers. Thus, β-Integrin could participate in the stabilization or strengthening of cell contacts, as shown for amnioserosa and larval epithelial cells in Drosophila, mammalian keratinocytes, and carcinoma cell clusters. Furthermore, β-Integrin, after JNK inactivation, strikingly accumulates at the front of LCEs suggesting a second function in cell invasiveness, as observed in leukocytes (Llense, 2008).
Direct evidence for β-Integrin involvement in border cell migration was obtained by RNAi in a sensitized JNK-minus condition. The expression of β-Integrin dsRNAs in border cells reduced β-Integrin levels but did not cause migration or integrity defects. However, in the presence of Puc, β-Integrin RNAi led to a strong enhancement of cluster dissociation and prevented the full extension of LCEs, which become mostly blunted. Moreover, an adhesion dominant negative (diβ) integrin chimera showed weak, but reproducible, dissociation phenotypes. Thus, β-Integrin turns out to participate in, first, the stabilization of border-border cell contacts and, second, the promotion of LCEs extension. The integrin countereceptors that facilitate border cell attachment and invasiveness are not yet known (Llense, 2008).
D-Paxillin was present in border cell contacts but was downregulated in JNK-minus conditions. Genomic-profiling analyses of JNK mutants suggests a transcriptional control of D-Paxillin expression. However, other options, such as subcellular relocation after phosphorylation, could also explain why D-Paxillin may be absent from JNK-minus border cells. Expression in border cells of two different D-Paxillin dsRNA lines was found to result in JNK loss-of-function-like dissociation, DE-Cadherin downregulation and β-Integrin accumulation at LCEs. Expression of a Talin RNAi line does not produce any migration phenotype, although it impairs follicle epithelia integrity (Llense, 2008).
In migratory leukocytes, PKA-mediated integrin phosphorylation prevents Paxillin accumulation at the leading front. Paxillin-integrin interactions in lateral positions lead to the inhibition of Rac, whose activation is thus spatially limited to the leading edge where it induces lamellipodia. Consequently, D-Paxillin might stabilize β-Integrin in border-border cell contacts. Its absence, in JNK-minus conditions, would lead in lateral and trailing cells to Rac activation, dissociation of border-border cell contacts, and extension of β-Integrin-rich ectopic lamellipodia. Indeed, the PKA-RII subunit is expressed in border cells, and border cells mutant for PKA show migration defects (Llense, 2008).
Interestingly, D-Paxillin overexpression rescued the border cell defect resulting from loss of JNK activity (or expression of Cdc42DN). DE-Cadherin relocated to border-border cell contacts, and β-Integrin expression was partially eliminated from residual LCEs. D-Paxillin overexpression alone had no effects (Llense, 2008).
It was further asked whether the control of cell polarity and cytoskeletal adaptor proteins by JNK were related. Paxillin expression was strongly reduced in baz mutant conditions, whereas Baz expression was only slightly affected by interference in Paxillin expression (Llense, 2008).
JNK signaling regulates border cells clustering by controlling at least two key elements, cell polarity (Baz) and cytoskeletal adaptor proteins (D-Paxillin), and as a consequence cell-cell contacts and cell-substrate attachments. Interestingly, the overexpression of Hindsight (Hnt), a target and negative regulator of JNK, results in similar defects to those caused by inhibition of JNK. Because re-expression of a variety of proteins (Baz, D-Paxillin, DE-Cadherin, and Armadillo) can rescue the dissociation phenotype and given that each time rescue is achieved, DE-Cadherin and Armadillo expression are restored, a plausible explanation for the effects observed with JNK-minus and Hnt overexpression is that there is an overall loss of multiple cell-cell adhesion complexes. The restoration of any of them would provide sufficient cell-cell adhesion to enable the cluster to move as a collective (Llense, 2008).
The individual migratory abilities of JNK-minus border cells could be partially explained by the observed β-Integrin relocalization to LCEs (border-nurse cell contacts). Alternatively, border cells could have lost their capacity to respond to positional gradients leading to random outward movements. Border cells use PVF and EGF to guide their migration. Blocking PVR and EGFR does not reduce the ability of border cells to extend protrusions but abolishes their directionality, with protrusions now extending in all directions. However, in these conditions, border cell clusters do not dissociate, thereby ruling out the possibility that dissociation in JNK mutants is due only to loss of directional guidance. A directionality index (DI) can be calculated. A DI of 0 indicates equal numbers of protrusions extending forward and backward. A DI of 1 indicates that cells only extend protrusions in the direction of migration. This study found a DI of 0.59 for wild-type clusters. In the absence of JNK, however, clusters show a DI ranging from -0.2 to 0, suggesting that JNK-minus border cells are blind to positional cues. This fact accounts for recently described synergistic effects of JNK and PVR signaling on border cells (Llense, 2008).
This model makes a significant prediction: JNK hyperactivation should increase adhesiveness and eventually block migration. Accordingly, ut was observed that the overexpresssion of a constitutively active form of Hep, the overexpresssion of a constitutively active form of Misshapen, or loss of function clones of puc resulted in nonmigratory and strongly compacted clusters. Occasionally, the death of a number of border cells was observed (Llense, 2008).
So far, the molecular and cellular study of collective versus individual migration both in developmental and cancer models has mainly focused on the analysis of structural elements. The identification of the JNK cascade as a key determinant of migratory responses in border cells could have an important impact in the understanding of collective movements. Border cell migration could serve as a good model for studying migratory transitions, thus impacting on the understanding of cancer metastasis and invasiveness, during which so little is known about the signaling mechanisms controlling migratory behavior (Llense, 2008).
Paxillin is a focal adhesion scaffolding protein, which has been proposed to play a role in focal adhesion dynamics. A cDNA clone of the Drosophila homologue of paxillin has been isolated. Comparison of the Drosophila paxillin sequence with those of vertebrate paxillins shows strong conservation of the LIM domains and LD repeats. Using the Drosophila genomic sequence, two partial curated transcripts were identied and the structure of the paxillin gene was deduced. No homologues of other members of the paxillin family such as HIC-5 or leupaxin are to be found in the Drosophila genome. Surprisingly paxillin mRNA is expressed in a restricted pattern during embryogenesis. In particular it is strongly expressed in cells and tissues undergoing cell shape changes or cell migration. Many of the sites of expression are also known to be sites of integrin function or FAK expression. The data support a role for paxillin as an adapter and/or signaling protein during developmental processes involving integrin-mediated adhesion (Wheeler, 2001).
Integrin-dependent cell adhesion and spreading are critical for morphogenesis, tissue regeneration, and immune defense but also tumor growth. However, the mechanisms that induce integrin-mediated cell spreading and provide mechanosensing on different extracellular matrix conditions are not fully understood. By expressing β3-GFP-integrins with enhanced talin-binding affinity, integrin activation, clustering, and substrate binding was experimentally uncoupled from its function in cell spreading. Mutational analysis revealed that Tyr747, located in the first cytoplasmic NPLY(747) motif, induced spreading and paxillin adapter recruitment to substrate- and talin-bound integrins. In addition, integrin-mediated spreading, but not focal adhesion localization, is affected by mutating adjacent sequence motifs known to be involved in kindlin binding. On soft, spreading-repellent fibronectin substrates, high-affinity talin-binding integrins form adhesions, but normal spreading was only possible with integrins competent to recruit the signaling adapter protein paxillin. This proposes that integrin-dependent cell-matrix adhesion and cell spreading are independently controlled, offering new therapeutic strategies to modify cell behavior in normal and pathological conditions (Pinon, 2014).
Search PubMed for articles about Drosophila Paxillin
Bataillé, L., et al. (2010). Downstream of identity genes: muscle-type-specific regulation of the fusion process. Dev. Cell 19(2): 317-28. PubMed ID: 20708593
Brown, M. C., et al. (1996). Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding. J. Cell Biol. 135: 1109-1123. PubMed ID: 8922390
Burridge, K., Turner, C. E. and Romer, L. H. (1992). Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J. Cell Biol. 119: 893-903. PubMed ID: 1385444
Chen, G. C., et al. (2005). Regulation of Rho and Rac signaling to the actin cytoskeleton by Paxillin during Drosophila development. Molec. Cell. Biol. 25: 979-987. 15657426
Chen, G. C., et al. (2008). Genetic interactions between Drosophila melanogaster Atg1 and paxillin reveal a role for paxillin in autophagosome formation. Autophagy 4(1): 37-45. PubMed ID: 17952025
Hagel, M., et al. (2002). The adaptor protein paxillin is essential for normal development in the mouse and is a critical transducer of fibronectin signaling. Mol. Cell. Biol. 22: 901-915. PubMed ID: 11784865
Karaköse, E., Schiller, H. and Fässler, R. (2010). The kindlins at a glance. J. Cell Sci. 123: 2353-2356. PubMed ID: 20592181
Llense, F. and Martin-Blanco, E. (2008). JNK signaling controls border cell cluster integrity and collective cell migration. Curr. Biol. 18(7): 538-44. PubMed ID: 18394890
Löer, B., et al. (2008). The NHL-domain protein Wech is crucial for the integrin-cytoskeleton link. Nat. Cell Biol. 10(4): 422-8. PubMed ID: 18327251
Manser, E., et al. (1998). PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol. Cell 1: 183-192. PubMed ID: 9659915
Nikolopoulos, S. N. and Turner, C. E. (2001). Integrin-linked Kinase (ILK) binding to paxillin LD1 motif regulates ILK localization to focal adhesions. J. Biol. Chem. 276, 23499-23505. PubMed ID: 11304546
Pinon, P., Parssinen, J., Vazquez, P., Bachmann, M., Rahikainen, R., Jacquier, M. C., Azizi, L., Maatta, J. A., Bastmeyer, M., Hytonen, V. P. and Wehrle-Haller, B. (2014). Talin-bound NPLY motif recruits integrin-signaling adapters to regulate cell spreading and mechanosensing. J Cell Biol 205: 265-281. PubMed ID: 24778313
Schaller, M. D. (2001). Paxillin: a focal adhesion-associated adaptor protein. Oncogene 20: 6459-6472. PubMed ID: 11607845
Stanchi, F., et al. (2009). Molecular dissection of the ILK-PINCH-parvin triad reveals a fundamental role for the ILK kinase domain in the late stages of focal-adhesion maturation. J. Cell Sci. 122: 1800-1811. PubMed ID: 19435803
Turner, C. E. and Miller, J. T. (1994). Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak-binding region. J. Cell Sci. 107: 1583-1591. PubMed ID: 7525621
Turner, C. E., et al. (1999). Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: a role in cytoskeletal remodeling. J. Cell Biol. 145: 851-863. PubMed ID: 10330411
Turner, C. E. (2000). Paxillin interactions. J. Cell Sci. 113: 4139-4140. PubMed ID: 11069756
Webb, D. J., et al. (2004). FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat. Cell Biol. 6: 154-161. PubMed ID:
Wheeler, G. N. and Hynes, R. O. (2001). The cloning, genomic organization and expression of the focal contact protein paxillin in Drosophila. Gene 262: 291-299. PubMed ID: 11179695
Zervas, C. G., et al. (2011). A central multifunctional role of integrin-linked kinase at muscle attachment sites. J. Cell Sci. 124(Pt 8): 1316-27. PubMed ID: 21444757
date revised: 23 July 2014
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