There is growing evidence that Notch pathway activation can result in consequences on cell morphogenesis and behaviour, both during embryonic development and cancer progression. In general, Notch is proposed to co-ordinate these processes by regulating expression of key transcription factors. However, many Notch-regulated genes identified in genome-wide studies are involved in fundamental aspects of cell behaviour, suggesting a more direct influence on cellular properties. By testing the functions of 25 such genes it was confirmed that 12 are required in developing adult muscles consistent with roles downstream of Notch. Focusing on three, Reck, rhea/talin and trio, their expression was varified in adult muscle progenitors, and Notch-regulated enhancers in each were identified. Full activity of these enhancers requires functional binding sites for Su(H), the DNA-binding transcription factor in the Notch pathway, validating their direct regulation. Thus, besides its well-known roles in regulating the expression of cell-fate determining transcription factors, Notch signalling also has the potential to directly affect cell morphology/behaviour by modulating expression of genes such as Reck, rhea/talin and trio. This sheds new light on functional outputs of Notch activation in morphogenetic processes (Pezeron, 2014).
Transient (short-term) cell adhesion underlies dynamic processes such as cell migration, whereas stable (long-term) cell adhesion maintains tissue architecture. Ongoing adhesion complex turnover is essential for transient cell adhesion, but it is not known whether turnover is also required for maintenance of long-term adhesion. This study used fluorescence recovery after photobleaching to analyze the dynamics of an integrin adhesion complex (IAC) in a model of long-term cell-ECM adhesion, myotendinous junctions (MTJs), in fly embryos and larvae. The turnover of components of the IAC used fluorescently tagged constructs of β position-specific (βPS) integrin (βPS-integrin-YFP) and of the core structural IAC components Talin (Talin-GFP) and Tensin (Tensin-GFP), as well as a viable line with a GFP inserted in the genomic ilk (integrin-linked kinase) gene (ILK-GFP). IAC was found to undergo turnover in MTJs, and this process was found to be mediated by clathrin-dependent endocytosis. Moreover, the small GTPase Rab5 can regulate the proportion of IAC components that undergo turnover. Also, altering Rab5 activity weakened MTJs, resulting in muscle defects. In addition, growth of MTJs was concomitant with a decrease in the proportion of IAC components undergoing turnover. It is proposed that IAC turnover is tightly regulated in long-term cell-ECM adhesions to allow normal tissue growth and maintenance (Yuan, 2010).
This is the first study of the turnover of integrin adhesions in live animals. The MTJs analyzed are long-lasting cell-ECM adhesions that form during late embryonic stages and last throughout larval life (about 5 days at room temperature). Although MTJs grow and undergo remodeling at larval stages, they must nonetheless support ongoing muscle attachment during this time. Overall, these results show considerable IAC dynamics in the MTJs. The lowest levels of IAC turnover measured were in 3rd instar larval muscles and even at that stage the mobile fraction of IAC components ranged from as low as 5% for homozygous talin-GFP to as high as 24% for homozygous integrin-YFP (Yuan, 2010).
Surprisingly, it was found that a significant proportion of the βPS-integrin in MTJs is mobile. Previous studies in cell culture suggested that integrins are mostly immobile within the range of the life-time of focal contacts (10 to 30 minutes), whereas other components of the IAC are highly dynamic and have a half-life on the order of 2-7 minutes. In the MTJs, the proportion of βPS-integrin that is mobile is in line with other components of the IAC, such as talin, tensin and ILK. Although this suggests that some differences exist between the turnover mechanisms of stable and transient adhesions, major mechanistic similarity was found between turnover in MTJs and focal contacts. For instance, both processes require dynamin-mediated endocytosis and are regulated by the Rab family of small GTPases. This study establishes the MTJ as a useful model to analyze turnover in the context of stable cell-ECM adhesion (Yuan, 2010).
Mobile fractions of various IAC components were measured to assess their dynamics at the MTJs. In the case of integrins, the mobile fraction could be a measurement of turnover (assembly and disassembly) of the IAC or, alternatively, of lateral diffusion. FRAP experiments on whole and partial MTJs demonstrate that lateral mobility is not a significant factor contributing to the integrin dynamics that were measured. For the cytoplasmic components of the IAC, the mobile fractions could measure one or more of three processes: turnover, the assembly and disassembly of the IAC; diffusion of IAC molecules within the cytoplasm; or exchange, the process in which cytoplasmic IAC components bind to and depart from the already assembled adhesion complex. For example, a recent study found that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion and a fast-diffusing cytoplasmic pool. Although it is likely that all three processes listed could contribute to the dynamics of various IAC cytoplasmic components, it is proposed that the mobile fraction observed in the MTJ is mainly due to IAC assembly and disassembly, rather than diffusion and exchange. This is suggested based on two observations. First, the fluorescence recovery of IAC components reaching their mobile fractions was measured in the range of minutes and seconds rather than milliseconds. Studies in cell culture show that the dynamics of IAC components near the adhesion site are dominated by binding kinetics rather than by free diffusion and occur on a similar timescale. Second, if the mobile fraction of ILK represented only the binding kinetics of ILK with other IAC components, then an increase in the stability of integrin at the MTJ would not reduce the mobile fraction of ILK. However, it was observed that the mobile fractions of both ILK and βPS-integrin significantly decline upon blockage of endocytosis. Nevertheless, it is still possible that ILK undergoes exchange; this might account for some of the 20% of the ILK protein that remained in the mobile fraction when clathrin-mediated endocytosis was inhibited (Yuan, 2010).
Rab5 concentrates at MTJs and can regulate the size of the mobile fraction of IAC molecules that are undergoing turnover. This is consistent with published results showing that other Rab proteins, such as Rab21, regulate adhesion. In migrating cells, overexpression of Rab21 leads to increased integrin adhesion, whereas decreased expression of Rab21 leads to reduced adhesion. Intriguingly, MTJ defects are conferred by the expression of either Rab5-DN, which decreases the mobile fraction, or Rab5-CA, which increases the mobile fraction. It is not clear why the expression of either DN or CA versions of Rab5 gave rise to a nearly identical phenotype. However, the findings are consistent with previous work in flies showing that overexpression of integrins gives rise to muscle-detachment phenotypes identical to those found in integrin null mutants. By extension, a small reduction or a small increase in the amount of immobile ECM-ligand-bound integrin conferred by expression of Rab5-DN or Rab5-CA could lead to a similar muscle defect. These observations emphasize the importance of precisely regulating the level of Rab5 activity at the MTJ for the maintenance of muscle attachment. It is likely that maintenance of the MTJ necessitates a careful balance between the process of integrin internalization and IAC disassembly, and the process of integrin trafficking to the MTJ and IAC assembly. Any deviation from the required balance between adhesion complex assembly and disassembly leads to muscle detachment (Yuan, 2010).
At the end of muscle morphogenesis (stage 16 of embryogenesis), IACs in muscles exhibit high rates of turnover similar to those observed in migrating cells. One possible explanation is that, because muscle morphogenesis involves dynamic processes, such as cell migration and tissue rearrangement, it requires extensive IAC turnover. The high levels of turnover observed at the immediate conclusion of muscle morphogenesis are therefore a lingering after-effect of this phase of myogenesis. Another likely explanation is that a certain amount of turnover persists in the newly formed MTJ to allow growth and remodeling to take place during larval development. Moreover, it is predicted that the substantial levels of turnover observed in late embryonic and early larval stages are generally unsustainable in mature MTJs. Furthermore, it is conjectured that a gradual reduction in the level of turnover, similar to observations in the MTJs, is a general feature of cell adhesion complexes undergoing the transformation from a transient to a stable and long-lasting adhesion (Yuan, 2010).
In addition to supporting stabilization of the adhesion junctions, it is speculated that the reduction in the proportion of integrin and IAC components that undergo turnover plays an active role in MTJ growth. Shifting a greater proportion of the integrins in MTJs from the mobile to the immobile fraction could result in an increase in the size and overall strength of the MTJs, so that they can support the strain placed on muscle-tendon attachment as muscles grow. The question arises as to whether MTJs in adults, which form during pupal stages and last even longer, also exhibit IAC turnover. Adult muscles do not undergo further growth, but could potentially undergo remodeling of the MTJs, for example in response to increased mechanical stress. Integrin turnover in the adult might also contribute to the repair of MTJs in response to accrued mechanical damage. Because of the presence of an exoskeleton in the adults, it is not currently possible to analyze integrin turnover using FRAP, but the data show that depletion of integrin and other IAC components in adult muscles gives rise to muscle defects, consistent with ongoing adhesion complex turnover (Yuan, 2010).
Based on these data, it is proposed that, in order to maintain the MTJs, the level of IAC turnover in the fully assembled muscle must be limited to within a specific range. This level of turnover necessitates equilibrium between IAC disassembly and IAC assembly. There are three generalized models for the turnover of the IAC: in one case, the entire complex is disassembled and assembled as a set unit; the second is that some of the IAC remains assembled and that only integrin molecules are internalized; the third is a mixture of both. The experiments show that an increase or decrease in the mobile fraction of integrin is correlated with a similar increase or decrease in the turnover of other IAC components. Especially striking in this regard are the coordinated developmental changes in the mobile fractions of individual IAC components that occur during larval stages. This suggests that the turnover of multiple IAC components is co-regulated, which makes it unlikely that only integrins recycle while the rest of the complex remains intact (Yuan, 2010).
Previous work has implicated focal adhesion kinase (FAK) and Src family kinases in regulating the dynamics of integrin-mediated adhesion. However, expressing a dominant-negative version of Src in fly muscles induced early muscle defects, whereas disrupting FAK did not affect IAC turnover. An important future goal is to identify the mechanism by which turnover is controlled in order to gain further insight into how IAC dynamics are modulated during development (Yuan, 2010).
It is hypothesized that modulating the levels of integrin turnover in the context of a stable long-term adhesive contact, such as the MTJ, provides a way for tissues to respond to changes in the external environment without wholesale disassembly and assembly of the adhesive contact. The ongoing existence of MTJs in a dynamic state enables expansion, contraction, remodeling and changes in the molecular components of the adhesion complex. This provides a flexibility that is vitally important for long-term tissue maintenance (Yuan, 2010).
Integrin-linked kinase (ILK) is an essential component of a multiprotein complex that links actin to the plasma membrane. This study has used a genetic approach to examine the molecular interactions that are essential for the assembly of this ILK-containing complex at Drosophila muscle attachment sites (MASs). Downstream of integrins, talin plays a decisive role in the recruitment of three proteins: ILK, Lim domain protein PINCH (steamer duck) and paxillin. The accumulation of ILK at MASs appears to follow an amplification mechanism, suggesting that numerous binding sites are generated by minimal levels of the upstream integrin and talin effectors. This property suggests that ILK functions as an essential hub in the assembly of its partner proteins at sites of integrin adhesion. PINCH stability, and its subcellular localization at MASs, depends upon ILK function, but ILK stability and localization is not dependent upon PINCH. An in vivo structure-function analysis of ILK demonstrated that each ILK domain has sufficient information for its independent recruitment at embryonic MASs, whereas at later developmental stages only the kinase domain was effectively recruited. These data strengthen the view that the ILK complex is assembled sequentially at sites of integrin adhesion by employing multiple molecular interactions, which collectively stabilize the integrin-actin link (Zervas, 2011).
In cell culture models, focal adhesions are the prototype form of sites of integrin adhesions. Several lines of evidence suggest that the molecular assembly of focal adhesions is a multistep process, where different cytoplasmic proteins enter the adhesion sites in a defined sequential order. This study provides genetic evidence that supports a hierarchical model for formation of integrin adhesions, using the Drosophila embryonic MASs as a model system (Zervas, 2011).
This study reexamined whether integrins are essential for the localization of ILK to MASs. A previous study found that ILK was recruited independently of integrins. This conclusion has now been corrected: complete elimination of integrins resulted in loss of ILK recruitment. This result, therefore, reveals that the small amount of maternally provided integrin recruits unexpectedly high levels of ILK. This surprising finding has two important implications: 1) the recruitment of ILK and maintenance of cell-matrix adhesion have different requirements for the amount of integrin (low levels of integrin are sufficient to recruit ILK, but high levels are needed for functional adhesion), 2) the amount of ILK recruited by integrins is not set by the quantity of integrin at MASs, suggesting that there is an amplification mechanism so that a single integrin can recruit multiple ILK molecules (although the experiments do not reveal the relative number of molecules). A similar, but less extreme, lack of correlation between the levels of integrin and an intracellular integrin-associated protein at MASs has been found for talin. This apparent amplification could be explained by a multidocking scaffolding protein that is able to recruit, either directly or indirectly, several ILK molecules. Alternatively, ILK could have the ability to employ a variety of additional interactions with different binding partners in the integrin-containing junctions, so that even if integrins or other components are much reduced, multiple ILK molecules are recruited. The mechanism of the amplification is unknown, with a number of the integrin-associated proteins being potential components; the kindlin proteins are particular good candidates, because they bind both integrins and ILK (Karaköse, 2010), and Wech has been shown to bind both talin and ILK and to contribute to ILK recruitment (Löer, 2008; Zervas, 2011 and references therein).
Talin binding to integrins is one of the first molecular events, and is a prerequisite, in the formation of the integrin-cytoskeleton link. Therefore, whether talin is required to recruit components of the ILK-containing complex at sites of integrin adhesion was examined. As with integrins, it was essential to remove both the maternal and zygotic production of talin to reveal defects in ILK, PINCH and paxillin recruitment. Consequently, these results show an important function for talin in recruiting the ILK-containing complex. A similar role for talin in ILK recruitment at myotendinous junctions in mice has been reported. Currently there are no data to support a direct association between components of the ILK complex and talin, but Wech binds to talin and ILK suggesting that it could be the key linker between the two proteins (Löer, 2008), at least in muscles. Therefore, a model is favored whereby talin, in concert with integrins, recruits Wech and additional proteins. These proteins then in turn recruit ILK and paxillin, and, 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 transmembrane receptors mediate cell adhesion through intracellular linker proteins that connect to the cytoskeleton. Of the numerous linker proteins identified, only a few, including Talin and Integrin-linked-kinase (ILK), are essential and evolutionarily conserved. The wech gene encodes a newly discovered and highly conserved regulator of integrin-mediated adhesion in Drosophila. Embryos deficient in wech have very similar phenotypes to integrin-null or Talin-null embryos, including muscle detachment from the body wall. The Wech protein contains a B-box zinc-finger and a coiled-coil domain, which is also found in RBCC/TRIM family members, and an NHL domain. In β-integrin or Talin mutants, Wech is mislocalized, whereas ILK localization depends on Wech. Evidence is provided that Wech interacts with the head domain of Talin and the kinase domain of ILK, and it is proposed that Wech is required to connect both core proteins of the linker complex during embryonic muscle attachment. Both the NHL and the B-box/coiled-coil domains of Wech are required for proper interaction with Talin and ILK. The single murine Wech orthologue is also colocalized with Talin and ILK in muscle tissue. It is proposed that Wech proteins are crucial and evolutionarily conserved regulators of the integrin-cytoskeleton link (Löer, 2008).
The formation of complex tissues in animals often involves stable adhesion between different cell layers and extracellular matrix substrates, which is, to a large extent, mediated by members of the integrin family of heterodimeric transmembrane receptors. Loss of integrins causes muscle detachment in flies and mice. Integrin mutations or aberrant expression can result in skin diseases, such as epidermolysis bullosa, and contribute to invasion and metastasis during cancerogenesis. A crucial part of the adhesive function of integrins is their ability to connect to the actin cytoskeleton. This involves a complex of adaptor proteins, which bind to the cytoplasmic tail of integrins and mediate the link to the cytoskeleton. Although many proteins have been identified that may contribute to the linker complex, in mammals, flies and worms only a few components have been identified as essential to the link. These include Talin, ILK, PINCH and Tensin, encoding an actin-capping protein, which coordinates signalling with cytoskeletal changes. Recent studies in mammalian cells and in Drosophila suggest that Talin is necessary for the initial formation of the integrin adhesion complex and reinforces it, possibly by recruiting other proteins, including ILK and Tensin. Absence of Talin causes defects that are almost identical to those seen in the absence of integrin, such as muscle detachment and failure of germ-band retraction during embryonic development. In contrast, absence of other components of the link causes only a subset of the defects. In different types of cells, integrins make diverse connections with the actin cytoskeleton; however, the molecular basis for the cell-type specific functions of integrins is still mostly unknown (Löer, 2008).
In a search for genes controlling integrin-mediated muscle attachment in Drosophila embryos, two P insertions were identified and one imprecise excision allele was generated, named wech, affecting a previously uncharacterized genetic locus. In homozygous embryos of the imprecise excision allele, wech66, wech transcript and protein levels were markedly reduced when compared with wild-type embryos. In wech66 germline clone embryos, Wech protein levels were reduced substantially and were barely detectable, indicating that wech66 is an amorphic allele. Molecular analysis, reversion of the phenotype by perfect excision of the P elements and genetic rescue experiments demonstrate that the lethality of wech alleles is linked to wech gene function (Löer, 2008).
Phenotypic analysis of homozygous wech66 mutants and of trans-heterozygous combinations with a deficiency indicates an essential role of wech in muscle attachment during Drosophila embryogenesis (wech is a German Rhineland term for 'detached' or 'gone'). During embryonic development, the somatic muscles attach to each other and to their anchoring points in the epidermis, the tendon cells, to generate a highly stereotyped pattern of 30 muscles in each abdominal hemi-segment. The cytoskeletal network forms bridges between the muscle and the tendon cell through dense hemi-adherens-type junctions formed between the tendon cell, the muscle and a thick layer of extracellular matrix material that is deposited in the space between them. This architecture provides the mechanical force that is required to resist muscle contraction during larval locomotion. The muscles start to attach to the tendon cells at embryonic stage 15 and during the last two stages of embryogenesis (stages 16 and 17) the attachment of the muscles to the tendon cells is elaborated by expansion of the hemi-adherens junctions, accumulation of tendon matrix and increased expression of β1-tubulin (Löer, 2008).
In late-stage embryos homozygous for wech66, it was found that muscles were detached from the body wall. This mutant phenotype became apparent at embryonic stage 16 and was enhanced during subsequent development, as the force of muscle contraction had increased. Myosin heavy chain (MHC) staining of stage 17 mutant embryos showed defects in most of the muscles at that point in development. When both the maternal and zygotic contributions of wech were removed, the detachment phenotype was more severe and became apparent at stage 15, indicating that wech has a maternal contribution, which is consistent with its ubiquitous expression during early stages of embryogenesis. Most of the muscles were multinucleated, indicating that wech mutants show no major defect in myoblast fusion. Analysis of DA1 (dorsal acute muscle 1) development in wech mutants further indicates that muscle differentiation and myoblast fusion are not affected. Rather, the data suggest a role of wech in muscle attachment, consistent with the late manifestation of the muscle detachment phenotype. Mutants for βPS integrin or Talin show phenotypes remarkably similar to wech mutants (Löer, 2008).
The wech gene encodes a multidomain protein containing a B-box zinc-finger domain and a coiled-coil domain characteristic of the RBCC/TRIM protein family. Members of this family usually contain a tripartite motif composed of a RING domain, one or two B-box motifs and a coiled-coil region. The Drosophila Wech protein does not contain a RING domain; however, it contains a carboxy-terminal NHL domain, which is also found in the Drosophila tumour suppressor proteins Brat and Mei-P26. In addition to Wech, brat and mei-P26 encode the only two other NHL-domain-containing family members in Drosophila. Molecular characterization of hypomorphic mutations in the brat gene suggests that the NHL domain carries the tumour suppressor function of this protein. Single-copy genes of wech orthologues are found in other invertebrates and in mammals, including mice, rats and humans. The Caenorhabditis Wech orthologue is named Lin-41 and is involved in the regulation of the progression from larval (L) stage 4 to the adult developmental programme (Löer, 2008).
To study the molecular function of Wech, an anti-Wech antibody was generated. Immunostaining indicates that Wech protein is expressed ubiquitously in all epithelial cells during early stages of embryogenesis. After germ-band retraction, Wech accumulated specifically in the muscle attachment sites. Co-immunostaining with the tendon and muscle-cell markers Short Stop and αPS2, respectively, indicate that Wech is highly localized in both cell types in a cortical localization at the attachment site. βPS Integrin and components of the cytoplasmic integrin-linked complex, Talin, ILK and Tensin, which bind to the cytoplasmic tail of βPS integrin, colocalized with Wech. Consistent with the expression of Wech in both the tendon and the muscle cells, the muscle detachment defect was rescued when Wech was re-supplied in both the tendon and muscle cells, using a combination of sr-GAL4 (tendon-cell-specific) and mef-GAL4 (muscle-cell-specific) in combination with UAS-wech-GFP. Whereas wech66 mutants are embryonic lethal, the rescued animals survived until the third instar-larval stage. Similarly, rescue could be obtained when Wech was ubiquitously expressed using hs-GAL4::UAS-wech-GFP or when the single driver lines, sr-GAL4 or mef-GAL4 were used, indicating that Wech expression rescues in either cell type. The latter finding is surprising and may be due to the residual maternal component in the muscles (Löer, 2008).
To test whether Wech is involved in integrin-mediated cell adhesion, the expression of Wech in βPS integrin (myospheroid, mys) mutants was analyzed. In zygotic mys mutants, the highly localized cortical accumulation of Wech at the attachment site failed and protein was also found in other parts of the cytoplasm at reduced levels. Consistently, a failure of cortical Wech localization was also found in zygotic and maternal mys mutants. These data indicate that βPS integrin is required for Wech localization. In contrast, βPS integrin was still properly localized in zygotic wech66 mutants and in maternal and zygotic wech germline clone embryos, in which Wech protein expression was markedly reduced. As Talin is known to interact through its head domain with integrin cytoplasmatic tails, Wech localization was analyzed in amorphic Talin (rhea2) mutants. In rhea mutants, Wech localization was markedly reduced, whereas in maternal and zygotic wech mutants, Talin seemed to be unchanged. This indicates that Talin is required for Wech localization. In contrast, Wech was required for proper ILK localization. In wech mutants, ILK localization at the attachment site was reduced, whereas Wech was still localized properly in ilk mutants. Consistent with these data, Tensin, which is known to require ILK for its localization, failed to accumulate at the attachment site in wech mutants. PINCH, which was shown to modulate ILK function by direct binding or by recruitment of an ILK-modifying factor, was still localized properly in wech mutants. In contrast to wech mutants, ILK was still concentrated at the muscle attachment sites in PINCH (steamer duck) mutant embryos. This suggests that Wech may be required to link an ILK-containing multiprotein complex to Talin, and PINCH may function as a molecular scaffold supporting the assembly of the ILK-containing linker multiprotein complex. The finding that the wech mutant phenotype is similar in severity to talin mutants and stronger than that of ilk mutants, suggests that other unknown factors, in addition to ILK, may depend on Wech function during muscle attachment (Löer, 2008).
To further analyse the putative role of Wech in integrin-mediated adhesion, biochemical co-immunoprecipitation analysis were performed. Extracts were used of mammalian HeLa cells expressing tagged fusion proteins of the Drosophila Wech, Talin, ILK or various subdomains of these proteins. Wech was found to interact with the protein kinase domain but not with the ankyrin-repeat domain of ILK. Furthermore, Wech interacts with the head domain of Talin. Co-immunoprecipitation analysis using Drosophila embryonic extracts further confirmed the interactions of Wech with Talin and ILK. To determine which protein domains of Wech may be required for its interaction with Talin and ILK at the muscle attachment sites, Myc-tagged Wech protein variants were generated with deletions of the N-terminal B-box zinc-finger and coiled-coil domains (Wech-δBCC) or the C-terminal NHL domain (Wech-δNHL. The deletion constructs were expressed in HeLa cells or in transgenic embryos and tested for interaction with ILK and Talin. In co-immunoprecipitation experiments using HeLa-cell extracts, it was found that the BCC domain of Wech is essential for binding to the protein kinase domain of ILK, whereas the head domain of Talin interacted with both the BCC and the NHL domains of Wech. In vitro, the interaction of the Talin head domain with the Wech BCC domain was, however, much stronger when compared with its interaction with the Wech NHL domain. When expressing the Wech protein deletion variants as Myc-tagged versions in the tendon cells using the strong sr-GAL4 driver, both deletion variants accumulated mainly in the cytoplasm, and colocalization with βPS Integrin, Talin or ILK was markedly reduced. This suggests that both the BCC and NHL domains of Wech may be required in vivo for its proper localization at the muscle attachment sites (Löer, 2008).
As mentioned above, single-copy genes of wech orthologues are found in flies, worms and mammals, including mice and humans. To investigate whether the murine Wech orthologue may be involved in integrin-mediated processes, an antibody was generated against the protein and its expression was studied in adult mice. The murine Wech protein was expressed in the sarcomeric Z-discs of adult muscles where it was colocalized with ILK, which has been identified recently as an architectural component of the Z-disc of heart muscles in zebrafish. Strong colocalization of murine Wech was also found with costameric Talin. Notably, only partial colocalization was detected with sarcolemmal β1 integrins. These results are consistent with the notion that Wech serves as a conserved adaptor, which is required for linking Talin and ILK in the integrin multiprotein complex (Löer, 2008).
In summary, this study has show that Wech is a crucial component for the physical link between integrins and the cytoskeleton in the Drosophila epidermal muscle attachment sites. It is proposed that Wech connects integrins and the cytoskeleton in the attachment sites by interacting with Talin and ILK, thereby linking the ILK-containing multiprotein adaptor complex to Talin and βPS integrin. The data suggest that Wech interacts with the head domain of Talin and the kinase domain of ILK. However, the involvement of other proteins in the physical connection between these core proteins cannot be excluded. The in vitro and in vivo experiments indicate that the Wech BCC and the NHL domains are essential for the functional interaction of Wech with Talin and ILK. In the murine muscles, the single murine Wech orthologue was also strongly colocalized with Talin and ILK. This suggests an evolutionarily conserved role of Wech proteins in the integrin-cytoskeleton link. In addition to Wech, only two other proteins in Drosophila contain an NHL domain, the tumour-suppressor proteins Brat and Mei-P26. The molecular characterization of brat mutations suggests that the NHL domain carries the tumour suppressor function of Brat. Whether the tumour suppressor functions of Brat and MeiP-26 involve the modulation of integrin-mediated adhesion through their NHL domains is not known. As a number of clinically relevant disorders are caused by integrin-related adhesive changes, including muscle dystrophies, the finding of a crucial component for integrin adhesive functions may have implications for understanding disease aetiologies (Löer, 2008).
Integrins are heterodimeric adhesion receptors that link the extracellular matrix (ECM) to the cytoskeleton. Binding of the scaffold protein, talin, to the cytoplasmic tail of β-integrin causes a conformational change of the extracellular domains of the integrin heterodimer, thus allowing high-affinity binding of ECM ligands. This essential process is called integrin activation. This study reports that the Z-band alternatively spliced PDZ-motif-containing protein (Zasp) cooperates with talin to activate α5β1 integrins in mammalian tissue culture and αPS2βPS (Inflated/Myospheroid) integrins in Drosophila. Zasp is a PDZ-LIM-domain-containing protein mutated in human cardiomyopathies previously thought to function primarily in assembly and maintenance of the muscle contractile machinery. Notably, Zasp is the first protein shown to co-activate α5β1 integrins with talin and appears to do so in a manner distinct from known αIIbβ3 integrin co-activators (Bouaouina, 2012).
Combining in vivo studies in Drosophila and activation assays in mammalian cell culture, this study shows that the muscle-specific protein Zasp cooperates with talin head to enhance integrin activation. This conclusion is based on the similarity in phenotypes of Zasp-deficient and talinR367A-mutant Drosophila, genetic rescue of the Zasp null phenotype by talin head over-expression, suppression of lethality associated with integrin activating mutations in Zasp heterozygous flies, enhanced mobility of βPS integrins in Zasp-deficient muscles and integrin activation in CHO cells. Notably, Zasp potentiates talin head-mediated activation of α5β1 but not αIIbβ3 integrins, making it distinct from other known integrin coactivators. Zasp is mutated in cardiomyopathies and myofibrillar myopathies and knockout of Zasp in mice, zebrafish or Drosophila leads to severe muscle defects. The ability of muscles to transmit intracellular actomyosin-mediated contractility to neighboring cells and tissues requires adhesion to the ECM and assembly of cytoskeletal complexes that link adhesion receptors to the contractile apparatus. The in vivo data in Drosophila, in particular the increased integrin mobility in Zasp and talinR367A mutant myotendinous junctions, demonstrate that Zasp regulates integrin function in muscles and is required for myotendinous junction maturation. The partial rescue of Zasp mutants by the overexpression of the talin head domain, and the attenuation of lethality in βPS mutants by removing one allele of Zasp or talin, indicate that Zasp regulates integrin activation in Drosophila. Thus, in addition to its previously recognized role in the assembly and maintenance of the muscle contractile machinery, Zasp may also serve to coordinate muscle adhesion through modulation of integrin activation (Bouaouina, 2012)
Talin< serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. Previous studies have shown that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. This study describes results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head was shown to attenuate the assembly and reinforcement of the integrin adhesion complex. Importantly, evidence is provided that this mutation blocks integrin clustering in vivo. It is proposed that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development (Ellis, 2014: PubMed).
Drosophila vinculin is more harmful when hyperactive than absent, and can circumvent integrin to form adhesion complexes
Vinculin is a highly conserved protein involved in cell adhesion and mechanotransduction, and both gain and loss of its activity causes defective cell behaviour. This study examined how altering vinculin activity perturbs integrin function within the context of Drosophila development. Whereas loss of vinculin produced relatively minor phenotypes, gain of vinculin activity, through a loss of head-tail autoinhibition, caused lethality. The minimal domain capable of inducing lethality is the talin-binding D1 domain, and this appears to require talin-binding activity, as lethality was suppressed by competition with single vinculin-binding sites from talin. Activated Drosophila vinculin triggered the formation of cytoplasmic adhesion complexes through the rod of talin, but independently of integrin. These complexes contain a subset of adhesion proteins but no longer link the membrane to actin. The negative effects of hyperactive vinculin were segregated into morphogenetic defects caused by its whole head domain and lethality caused by its D1 domain. These findings demonstrate the crucial importance of the tight control of the activity of vinculin (Maartens, 2016).
Cell adhesion is mediated by multiprotein complexes that link transmembrane receptors to the cytoskeleton. These complexes are assembled at discrete sites of the membrane, and both loss and gain of adhesion protein activity causes cellular and developmental defects, which have pathological consequences (Maartens, 2016).
The first step in building a cell-matrix adhesion is the binding of transmembrane integrin receptors to extracellular matrix (ECM) components. This is followed by recruitment of cytoplasmic adhesion proteins, for example talin (also known as Rhea in flies), which occurs through the cytoplasmic tail of integrin. Talin is a crucial component of the link as it can simultaneously bind integrins (with its FERM-domain head) and actin (with an actin-binding site at the C-terminus of its long rod domain). Talin feeds back to promote integrin activation and is required for the recruitment of numerous cytoplasmic adhesion proteins. Of particular interest is the force-dependent recruitment of vinculin. In vitro work has established that stretching the rod of talin exposes previously hidden vinculin-binding sites (VBSs, single helices within the α-helical bundles that make up the rod) that can then bind vinculin. Consistent with this model, the recruitment of vinculin to adhesions in cell culture is particularly sensitive to myosin II inhibition (Maartens, 2016 and references therein).
A series of four-helical bundles (seven in vertebrates, six in invertebrates) make up the head domain of vinculin, which is linked by a partially disordered proline-rich region to the five-helical bundle of the tail. Interaction sites for vinculin ligands have been mapped across the protein. A key ligand is talin, and the interaction has been narrowed to the first two four-helical bundles of the head, the D1 domain (also known as Vh1): the VBSs in talin bind to the first four-helical bundle of the D1 domain, transforming it into a five-helical bundle. This first bundle of D1 retains most of the VBS-binding activity of the D1 domain in a two-hybrid assay, suggesting it is the minimal talin-binding site, but the second bundle is also capable of binding some ligands, and the entire D1 domain is generally used as a minimal head domain. Vinculin is notable among integrin-associated proteins for also localising to cell–cell adhesions, and this is mediated through an interaction of the head with either α- or β-catenin. The flexible neck of vinculin binds proteins of the CAP and vinexin family, among other ligands, and the tail binds to actin, the scaffolding protein paxillin, and the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), and also promotes dimerisation. By simultaneously binding talin and actin, vinculin provides an additional link to the cytoskeleton, giving extra mechanical support to the adhesion. A strengthening role is consistent with the relatively milder effects of losing vinculin compared to losing talin in cells in culture and in developing animals (Maartens, 2016 and references therein).
Although vinculin has many binding partners, the full-length protein has little binding activity due to a head-tail interaction stabilising the inactive conformation. Constructs that relieve this head-tail autoinhibition are hyperactive, and dramatically increase the size and stability of focal adhesions associated with activated integrins, as well as making the recruitment of adhesion proteins no longer sensitive to myosin II inhibition. The talin-binding D1 domain alone is sufficient to produce these effects, and, reciprocally, reducing the ability of the D1 domain to bind talin eliminates them. The vinculin tail adds additional activity: it is required for hyperactive vinculin to produce traction forces and reorient adhesions in response to polarised forces. A key aspect of vinculin function is therefore its activation status, and its effects on cell behaviour might be caused by its action on talin as well as direct or indirect recruitment of proteins to adhesions. Although the impact of hyperactive vinculin on cellular behaviour has been well documented, the impacts of these changes on cells within the organism have yet to be addressed. A mutant that produces hyperactive vinculin in mouse has a milder version of the defects caused by absence of vinculin, but vinculin levels are also strongly reduced, making it difficult to separate loss- and gain-of-function effects (Maartens, 2016).
To probe further how vinculin contributes to adhesion, this study has used Drosophila to compare loss- and gain-of-function effects during development. Vinculin hyperactivity was found to be far more deleterious to the organism than inactivity, and a new function was discovered for vinculin in bringing adhesion proteins together independently of the usual integrin cue. The D1-talin-rod interaction is crucial for the formation of these cytoplasmic adhesion subcomplexes, supporting a model where hyperactive vinculin ectopically activates talin in the cytoplasm by mimicking the effect of force on talin. Finally, this study dissected the negative effects of hyperactive vinculin into two discrete activities: morphogenetic defects caused by its head domain, and lethality caused by its D1 domain (Maartens, 2016).
Whereas flies can tolerate the loss of vinculin, this study has discovered that excessive vinculin activity is lethal, and causes defects in muscle development. Both of these deleterious effects appear to require binding a VBS-containing protein such as talin. Talin is also required for a new role of Drosophila vinculin: inducing the formation of cytoplasmic aggregates that are adhesion subcomplexes. These subcomplexes are not linked to integrins or the cytoskeleton, and demonstrate that adhesion protein complexes can form without any input from integrin (Maartens, 2016).
Flies lacking vinculin displayed defects in the adult musculature, similar to the reported mild defects in larval musculature. Other tissues appeared normal, and attempts to identify additional impairment in the athletic abilities of the flies were not successful, so the fly phenotype remains weaker than the phenotypes observed in mice, zebrafish or nematodes lacking vinculin. Redundancy with other adhesion proteins (such as talin) might explain the relatively mild phenotype of this highly conserved protein (Maartens, 2016 and references therein).
The contrast between the consequences of loss and gain of vinculin activity are striking. In general, overexpressed integrin-associated proteins do not induce lethality in Drosophila (for example, talin , tensin, and ILK). Two integrin-associated proteins cause lethality when the wild-type form is overexpressed: focal adhesion kinase and parvin. The lethality of vinculin relied on a reduction of its autoinhibition, but this does not seem general to Drosophila adhesion proteins: disruption of talin autoinhibition has mild effects, and whereas expression of tensin fragments does cause some phenotypes, expression of fragments of other adhesion proteins does not (see above references). The severe effects of hyperactive vinculin fit with the very strong intermolecular interactions that keep it in the closed state. Activating mutations of vinculin have not been reported in the human population, as expected if, as in flies, they cause dominant lethality (Maartens, 2016).
Expressing vinc-CO or vinc-Head in the developing musculature led to developmental defects. These could arise as a result of hyperactive vinculin in the aggregates or at the adhesion site. The defects are distinct from integrin loss phenotypes, and this might reflect recruitment of additional proteins contributing to muscle formation to the aggregates or the adhesion. Cytoskeletal machinery is crucial for muscle fusion and muscle pathfinding to tendon cell targets, and an interaction between hyperactive vinculin complexes and more general cytoskeletal factors might explain the muscle defects. Sequestration of Z-disc proteins to ectopic intracellular aggregates has been implicated in the muscle phenotypes associated with myofibrillar myopathy, and a similar effect may be stimulated by hyperactive vinculin. (Maartens, 2016).
Cytoplasmic aggregate formation appears to be unique to Drosophila vinculin. Gallus vinc-D1 did not generate cytoplasmic aggregates, even though it appeared to interact with Drosophila talin (coexpressing Drosophila VBSs blocked its recruitment and lethality). In vertebrate cell culture, hyperactive vinculin is recruited to integrin adhesions at the membrane, but cytoplasmic talin-containing aggregates have not been reported. The interaction between vertebrate vinc-Head and talin rod in vitro requires prior stretching of the rod by force, consistent with the in vivo interaction relying on prior talin stretching at the adhesion. In contrast, mitochondrial targeting experiments indicate that activated Drosophila vinculin can bind to un-stretched talin in the cytoplasm. A prediction from these results is that vertebrate vinculin should not recruit talin to the mitochondria, whether active or not. However, in vertebrate cells, talin is recruited by mitochondrially targeted vinc-CO and even full-length vinculin (albeit very weakly). However, in these cases targeted vinculin constructs pull the mitochondria to the membrane, so that vinculin and talin become associated with integrins and actin (no such association was found in targeting experiments carried out in this study). Thus, it seems feasible that, in these experiments, the association of vinc-CO is with stretched talin at the adhesion site, rather than with cytoplasmic talin as occurs in Drosophila (Maartens, 2016).
Several lines of evidence show that hyperactive Drosophila vinculin formed aggregates by binding to cytoplasmic talin. In the absence of talin, no aggregates were formed, and the rod of talin was a sufficient platform for aggregation, with longer sections supporting more aggregates, presumably due to an increase in the number of VBSs available per talin molecule. VBS coexpression blocked aggregate formation, suggesting that direct binding between vinculin and talin was important, and indeed the minimal vinculin fragment capable of forming aggregates was the talin-binding D1 domain. Hyperactive, but not wild-type, vinculin was capable of recruiting talin to the mitochondrial surface. Vinc-CO recruitment of talin to the aggregates was not altered by the loss of integrins, ruling out an alternative hypothesis whereby an initial stretching of talin at the adhesion is a first step in the formation of the cytoplasmic aggregates (Maartens, 2016).
An interesting feature of the vinculin–talin interaction is its reciprocity: just as hyperactive vinculin appears to bind closed talin, isolated VBSs can bind closed vinculin on the mitochondria, consistent with the capacity of vertebrate VBSs to dislodge the head from the tail in vitro. Thus, the interaction between vinculin and talin in Drosophila need only require activation of one partner. An open question is whether there are normal signals, mimicked by the 'T12' mutation, that open Drosophila vinculin so that it can force talin into an extended conformation. Recently, it has been found that expression of a mutant talin with a deletion of domains R2-R3, which contain four VBSs, causes very similar effects to expressing vinc-CO. Binding of activated-vinculin thus alleviates some form of internal negative regulation within talin, which might in part be due to regulation of the central actin-binding domain encompassing R4-R8 (Maartens, 2016).
Gallus vinc-D1 demonstrated that hyperactive vinculin could induce lethality in Drosophila without forming aggregates. How it does so remains an open question, but the idea is favored that it is caused by the action of the D1 domain of vinculin on talin at the adhesion sites. The vinculin head stabilises talin into a stretched conformation in cells and in vitro, and this relies on prior stretching of talin. Furthermore, vinculin is required for talin to extend fully away from the plasma membrane. Thus, lethality could arise from hyperactive vinculin binding to stretched talin and the failure of vinculin to release when force is reduced. Cycles of stretching and relaxation might be crucial for normal talin function or relaxation of talin might be required for its dynamic turnover. Alternatively, hyperactive vinculin might stimulate too much adhesion, stabilising integrin adhesions and reducing turnover in dynamic morphogenetic events. Elevated integrin expression has been shown to hinder cell migration in the Drosophila ovary, and vinculin stimulation of integrin activation might affect similar processes. The lethality caused by vinc-D1 constructs occurs without defects in muscle morphogenesis. Assessing whether the muscle defects of vinc-Head and vinc-CO also contribute to lethality would require a method to block the lethality of vinc-D1 without impairing the muscle phenotypes of vinc-Head or vinc-CO, which is currently lacking (Maartens, 2016).
Although this study has examined vinculin D1 from only two species, it is speculated that vertebrate vinculin has lost the ability to bind to closed talin, and might have become more tightly closed by the addition of an eighth four-helix bundle that occurred during the evolution of the deuterostome lineage. Thus, vertebrate cells might be even more sensitive to the consequences of aberrant association between vinculin and talin (Maartens, 2016).
The results suggest that certain proteins have the ability to act as a switch, triggering assembly of an integrin adhesion complex. Integrins are well known to have this switch ability: engagement with the ECM and clustering triggers the formation of adhesion sites. When Drosophila vinculin loses autoinhibition, it triggers the assembly of an adhesion complex, and this process can occur entirely independently of integrins. In contrast to integrins, however, the full complement of adhesion proteins is not recruited, suggesting that additional mechanisms are required (for instance, membrane proximity, application of force, or signalling). This raises the question of how the additional proteins are recruited to the cytoplasmic aggregates, and whether the pathways involved are similar to those utilised by constitutively active vinculin at adhesions and by integrins and talin in normal adhesions. Recruitment requires talin, but the relative contributions of vinculin and talin have yet to be established (Maartens, 2016).
Integrin-independent interactions of adhesion proteins have been demonstrated by fluorescence correlation analysis wherein adhesion components self-assembled in the cytosol. However, these 'building blocks' were composed of three or four protein species, never assembled into larger structures and did not include a talin-vinculin interaction. Nevertheless, the above work shows how interactions between the component parts of the adhesion need not necessarily rely on a direct or even indirect link to integrins, consistent with the current work. A key role of integrins might be to trigger the assembly of the cytoplasmic adhesion-complex-specific sites in the membrane, rather than being a necessary part of this link (Maartens, 2016).
From an evolutionary perspective, certain cytoplasmic components like vinculin and talin predate the integrins. It is tempting to propose that integrins co-opted pre-existing cytoplasmic complexes, using them to strengthen their adhesion to the ECM at discrete sites along the cell surface. This evolutionary change may also have required mechanisms to restrict the spontaneous formation of adhesion-like complexes in the cytoplasm. The strong head–tail interaction of vinculin could be one such mechanism (Maartens, 2016).
Surprisingly, there is a difference in the distributions of Talin mRNA and protein during embryogenesis. In particular, high levels of mRNA in the nerve cord do not correspond to high protein levels. Early homogeneous staining for Talin mRNA is followed by prominent expression in the ventral nerve cord. Talin is expressed in a subset of ventral cord neurons (Brody, 2000 and 2002). Lower levels of mRNA are more broadly distributed, with enrichment in the gut and muscle attachment sites detected at stage 16. That this pattern is specific for this transcription unit was shown by testing with probes from four different parts of the transcript, including the C-terminal region to which antibodies were raised; deletion of the Talin gene (rhea79a) eliminates staining (Brown, 2002).
To examine the distribution of Talin protein, polyclonal and monoclonal antibodies were made against the C terminus of Talin. Five of the monoclonal antibodies recognize different epitopes on Drosophila Talin. All but one of the anti-Talin antibodies produced indistinguishable patterns of staining on fixed embryos. The one exception is a monoclonal antibody, J18, that crossreacts with an antigen expressed in three cells per hemisegment within the central nervous system (Brown, 2002).
Analysis of Talin protein distribution during embryogenesis shows that it is maternally deposited and is evenly distributed in the cytoplasm following cellularization. Talin becomes progressively concentrated at the membrane, first detected in the migrating primordial midgut cells and then at muscle attachment sites, where the muscles and epidermal cells are linked via integrins. Talin protein shows only a hint of the strong pattern of mRNA expressed in the nervous system. Consistent with the low level of protein in the nervous system, zygotic mutant rhea embryos do not have any defects in the structure of the nervous system, as assayed with glial and neuronal cell markers. The subcellular distribution of Talin at the muscle attachment sites was examined by immunoelectron microscopy (IEM). Talin is found within submembranous electron-dense material associated with the hemiadherens junctions at muscle attachment sites (Brown, 2002).
Most sites of Talin concentration at membranes correspond to sites of integrin concentration, and colocalization of the two proteins is seen at the edge of the epidermis, during dorsal closure, and at muscle attachments. No PS integrin staining lacking colocalized Talin was observed. However, Talin is concentrated at the membrane of some cells lacking integrin. This is clearest in the gonadal mesoderm, where recruitment of Talin to the cortex of the gonadal mesoderm cells occurs as the gonad condenses. To test whether recruitment of Talin to sites of integrin expression requires integrins, Talin distribution was examined in embryos lacking PS integrins. Embryos lacking the ßPS subunit show a loss of Talin concentration at muscle attachment sites. In embryos that lack the mesodermally expressed alphaPS2 integrin subunit, but that still contain epidermal PS1 (alphaPS1ßPS) integrin, Talin is lost from muscle ends but still concentrated at the basal ends of the attaching epidermal cells, the tendon cells. These results show that Talin is recruited from the cytoplasm by integrins in both cell layers of the muscle attachment site (Brown, 2002).
The absence of strong ßPS expression in the gonadal mesoderm suggests that a receptor different from and other than integrin recruits Talin to the membrane in this tissue. Consistent with this, removal of ßPS from the embryo does not alter Talin enrichment in the gonadal mesoderm, nor does removal of the other Drosophila ß integrin subunit, ßnu. This shows that recruitment of Talin in the gonadal mesoderm is not driven by integrins. An alternative candidate would be Layilin (Borowsky, 1998), but no clear ortholog of this protein is encoded in the Drosophila genome (Brown, 2002).
Tissue-specific stem cells are maintained by both local secreted signals and cell adhesion molecules that position the stem cells in the niche microenvironment. In the Drosophila midgut, multipotent intestinal stem cells (ISCs) are located basally along a thin layer of basement membrane that composed of extracellular matrix (ECM), which separates ISCs from the surrounding visceral musculature: the muscle cells constitute a regulatory niche for ISCs by producing multiple secreted signals that directly regulate ISC maintenance and proliferation. This study shows that integrin-mediated cell adhesion, which connects the ECM and intracellular cytoskeleton, is required for ISC anchorage to the basement membrane. Specifically, the alpha-integrin subunits including alphaPS1 encoded by mew and alphaPS3 encoded by scb, and the beta-integrin subunit encoded by mys are richly expressed in ISCs and are required for the maintenance, rather than their survival or multiple lineage differentiation. Furthermore, ISC maintenance also requires the intercellular and intracellular integrin signaling components including Talin, Integrin-linked kinase (Ilk), and the ligand, Laminin A. Notably, integrin mutant ISCs are also less proliferative, and genetic interaction studies suggest that proper integrin signaling is a prerequisite for ISC proliferation in response to various proliferative signals and for the initiation of intestinal hyperplasia after loss of adenomatous polyposis coli (Apc). These studies suggest that integrin not only functions to anchor ISCs to the basement membrane, but also serves as an essential element for ISC proliferation during normal homeostasis and in response to oncogenic mutations (Lin, 2013).
Organogenesis of the somatic musculature in Drosophila is directed by the precise adhesion between migrating myotubes and their corresponding ectodermally derived tendon cells. Whereas the PS integrins mediate the adhesion between these two cell types, their extracellular matrix (ECM) ligands have been only partially characterized. This study shows that the ECM protein Thrombospondin (Tsp), produced by tendon cells, is essential for the formation of the integrin-mediated myotendinous junction. Tsp expression is induced by the tendon-specific transcription factor Stripe, and accumulates at the myotendinous junction following the association between the muscle and the tendon cell. In tsp mutant embryos, migrating somatic muscles fail to attach to tendon cells and often form hemiadherens junctions with their neighboring muscle cells, resulting in nonfunctional somatic musculature. Talin accumulation at the cytoplasmic faces of the muscles and tendons is greatly reduced, implicating Tsp as a potential integrin ligand. Consistently, purified Tsp C-terminal domain polypeptide mediates spreading of PS2 integrin-expressing S2 cells in a KGD- and PS2-integrin-dependent manner. A model is proposed in which the myotendinous junction is formed by the specific association of Tsp with multiple muscle-specific PS2 integrin receptors and a subsequent consolidation of the junction by enhanced tendon-specific production of Tsp secreted into the junctional space (Subramanian, 2007).
The abnormal pattern of the somatic muscles in the tsp mutant embryo raised the possibility that the muscle-tendon integrin-mediated adhesion is defective in the mutant embryos. A hallmark of appropriate integrin-mediated adhesion is the accumulation of Talin at the cytoplasmic face of the hemiadherens junction, where it binds directly to the integrin cytoplasmic domain, modulating its ligand affinity and recruiting actin microfilaments to this site. A significant reduction of accumulated Talin levels in tsp mutant embryos is observed. Whereas Talin is still detected at the sites of muscle-muscle junctions, it was entirely missing at sites where individual muscles would normally form junctions with single tendon cells, in particular at the junction sites formed between the lateral transverse muscles and their corresponding tendon cells. The lack of Talin at these sites corresponds with the lack of ßPS-integrin staining and is consistent with the loss of appropriate myotendinous junction. Thus, in the absence of functional Tsp, individual myotubes fail to form integrin-mediated adherens junction with tendon cells (Subramanian, 2007).
Myofibril stability is required for normal muscle function and maintenance. Mutations that disrupt myofibril stability result in individuals who develop progressive muscle wasting, or muscular dystrophy, and premature mortality. This study presents investigations of the Drosophila l(2)thin [l(2)tn] mutant. The "thin" phenotype exhibits features of the human muscular disease phenotype in that tn mutant larvae show progressive muscular degeneration. Loss-of-function and rescue experiments determined that l(2)tn is allelic to the tn locus [previously annotated as both CG15105 and another b-box affiliate (abba)]. tn encodes a TRIM (tripartite motif) containing protein highly expressed in skeletal muscle and is orthologous to the human limb-girdle muscular dystrophy type 2H disease gene Trim32. Thin protein is localized at the Z-disk in muscle, but l(2)tn mutants showed no genetic interaction with mutants affecting the Z-line-associated protein muscle LIM protein 84B. l(2)tn, along with loss-of-function mutants generated for tn, showed no relative mislocalization of the Z-disk proteins alpha-Actinin and muscle LIM protein 84B. In contrast, tn mutants had significant disorganization of the costameric orthologs beta-integrin, Spectrin, Talin, and Vinculin, and the initial description for the costamere, a key muscle stability complex, in Drosophila is presented. These studies demonstrate that myofibrils progressively unbundle in flies that lack Thin function through progressive costamere breakdown. Due to the high conservation of these structures in animals, this study demonstrates a previously unknown role for TRIM32 proteins in myofibril stability (LaBeau-DiMenna, 2012).
The genetic locus that encodes Talin is rhea. The first two alleles, rhea1 and rhea2, were isolated in the wing blister screen (Prout, 1997). Two other alleles, rhea17 and rhea3, were isolated as mutations that dominantly enhance weak integrin mutations. The rhea1 and rhea2 alleles were mapped to 66D5-6. By locally hopping a P element in this region, which is not allelic to rhea, l(3)S1760, rhea79a was generated. The P element in this strain is inserted in the same position as l(3)S1760, within the coding region of the Drosophila ortholog of ergic-53, but is deleted for the ergic-53 coding region, leading to the initial suggestion that rhea encodes ergic-53. However, recombinational analysis placed rhea1 0.08 map units (25-50 kb) from the l(3)S1760 P element. The gene adjacent to ergic-53 was revealed to be Talin by the genome sequence. It was then found that Talin protein is reduced in rhea mutant embryos and imaginal disc clones and that Talin mRNA is absent in rhea79a (Brown, 2002).
To confirm that rhea is the Talin gene, the Talin-coding region was sequenced from rhea1 and rhea2. For each allele a small deletion was found that produces a frameshift in the Talin-coding sequence. For rhea1 the frameshift occurs after amino acid 1139, and the new reading frame terminates after two amino acids in the wrong frame. For rhea2 the frameshift occurs after amino acid 1279 and terminates after 31 out of frame amino acids. Inverse PCR was used to identify the proximal insertion site of the P element in rhea79a, which was found to be 1931 bp downstream of Talin, showing that the rhea79a deficiency deletes three genes, ergic-53, Talin, and CG6638. Each rhea allele has an aberration in the Talin coding sequence. A mutant deficient for Ergic53 complements rhea1, rhea2, and rhea17. Therefore, it is concluded that rhea encodes Talin (Brown, 2002).
Three aspects of the Talin mutant phenotype have been described (Prout, 1997). Clones of rhea/rhea cells in the wing do not attach to the other cell layer of the wing, causing a wing blister. The two initial alleles and two recently identified ones (rhea3 and rhea17) dominantly enhanced the wing blister phenotype of hypomorphic alleles of integrin genes (Prout, 1997). Finally, rhea mutant embryos have a detachment of the epidermis from the muscles, although the muscles remain attached end to end. The embryonic phenotype of Talin mutants was examined by EM, with particular attention to tendon cells. In wild-type embryos, tendon cells are spanned by microtubules that link basal hemiadherens junctions to tonofibrils that insert into the apical exoskeleton, thereby transferring the force of muscle contraction to the exoskeleton. In rhea/rhea cells, microtubules extended from apical tonofibrils toward the basal membrane, but mature basal attachment sites fail to form. Structural features of normal attachment sites, such as extensive folding of basal membranes and linkage of microtubules to the inner surface of basal membranes, are not generally present in rhea tendon cells. Also, in these rhea mutant tendon cells, microtubules are abnormally oriented, in some cases running parallel, rather than perpendicular, to the exoskeleton. Loss of Talin results in reduction of electron-dense material from the cytoplasmic face of hemiadherens junctions at muscle attachment sites. This suggests that Talin and/or the proteins it recruits make a significant contribution to this dense material (Brown, 2002).
These zygotic rhea mutant embryos still have some maternally deposited Talin. To analyze the phenotype resulting from the complete absence of Talin, the maternal contribution was removed by generating germline clones. Half of these rhea/rhea eggs receive a wild-type paternal allele, and the zygotic expression of Talin rescues the absence of maternal Talin in some, but not all, embryos. The number of hatching embryos varied from 33%–41%, rather than the expected 50% (depending on the allelic combination). Viable fertile adults developed from hatched embryos. Thus, maternal deposition of Talin protein is important, but not essential, for normal development (Brown, 2002).
Embryos lacking both maternal and zygotic talin have a stronger phenotype than those lacking either maternal or zygotic product, the most prominent features of which are a failure in germband retraction and strong muscle detachment. This phenotype is very similar to that of embryos lacking both maternal and zygotic ßPS. The similarities between the two phenotypes suggest that talin is essential for integrin function. Close examination of the muscle phenotype provides insight into the role of Talin in integrin-mediated adhesion. PS2 (alphaPS2ßPS) integrin localizes normally, demonstrating that integrins reach the cell surface and localize to the ends of muscles in the absence of talin. In detached muscles, actin staining is separate from PS2 integrin staining. This demonstrates that integrin is able to bind to the ECM, since, if it could not, it would be expected to remain on the surface of the detached muscle. Thus, a separation is seen between integrins and actin, not between integrins and the ECM, suggesting that the primary role of talin is to link integrins to the cytoskeleton, and not to stimulate their ligand binding. Talin does not appear to be required for condensation of the gonad, since this occurs in some mutant embryos. Condensation does fail in some embryos, but this could be a secondary effect caused by other morphogenetic defects. By examining different rhea alleles, it has been confirmed that this represents the null Talin phenotype. The phenotypes of mutant embryos from germline clones of rhea1 and rhea2 are indistinguishable, as are those of rhea79a, which deletes rhea and two flanking genes. Other data suggest, however, that the rhea1 and rhea2 mutations are weak dominant negatives. For example, producing rhea2/rhea79A embryos from maternal germline clones of rhea2 causes 65% (n = 153) failure of germ band retraction, while those of the rhea79A deficiency caused only 48% failure (Brown, 2002).
Further insight into the role of Talin was gained by looking at Talin and integrins in the imaginal disc epithelia. Just prior to pupariation it has been found that Talin and integrins colocalize into focal adhesion-like structures at the basal surface of the wing imaginal disc. Making clones of cells mutant for rhea results in loss of the staining of these structures with the Talin antibody. The clusters of Talin also fail to form in clones of cells lacking the ßPS integrin subunit. Clustering of integrins into these focal adhesion-like structures requires Talin function, as it does not occur in clones of cells lacking Talin. Therefore, clustering of integrins requires Talin, and clustering of Talin requires integrins. Loss of Talin does not grossly impair the rate of proliferation of the imaginal disc cells, since mutant clones are of a similar size to the wild-type twin spots. In addition, loss of Talin does not alter overall levels of integrin synthesis. Combining these results with those from the embryo suggests that Talin's role may be to promote integrin clustering, which, in turn, allows the establishment of a strong connection with the cytoskeleton (Brown, 2002).
The involvement of Talin in the transmission of integrin signals regulating gene expression was also examined. In Drosophila one signaling assay uses the enhancer trap 258, which is expressed in the midgut and fails to be repressed in the absence of PS1 integrin. The expression of this integrin target gene was examined in the absence of Talin. In embryos lacking maternal and zygotic Talin, midgut development was too disrupted to assay 258 expression. In the absence of the zygotic Talin, the midgut shows the characteristic phenotype of an integrin mutation: the gastric caeca fail to split from two initial evaginations into four slender tubes, the midgut does not elongate into a slender tube, and the proventriculus does not form properly. Despite these morphological defects, the 258 gene was repressed. The same result was obtained in more than 30 mutant midguts. While the possiblility cannot be ruled out that the small amount of maternal Talin left is sufficient for integrin signaling, but not for integrin adhesion, these results suggest that Talin is not required for integrin signaling to the nucleus (Brown, 2002).
Integrins are evolutionarily conserved transmembrane alpha,beta heterodimeric receptors involved in cell-to-matrix and cell-to-cell adhesions. In Drosophila the position-specific (PS) integrins mediate the formation and maintenance of junctions between muscle and epidermis and between the two epidermal wing surfaces. Besides integrins, other proteins are implicated in integrin-dependent adhesion. In Drosophila, somatic clones of mutations in PS integrin genes disrupt adhesion between wing surfaces to produce wing blisters. To identify other genes whose products function in adhesion between wing surfaces, a screen was conducted for autosomal mutations that produce blisters in somatic wing clones. Seventy-six independent mutations in 25 complementation groups were isolated, 15 of which contain more than one allele. Chromosomal sites were determined by deficiency mapping, and genetic interactions with mutations in the beta PS integrin gene myospheroid were investigated. Mutations in four known genes (blistered, Delta, dumpy and mastermind) were isolated. Mutations were isolated in three new genes (Piopio, rhea and steamer duck) that affect myo-epidermal junctions or muscle function in embryos. Mutations in three other genes (kakapo, kiwi and moa) may also affect cell adhesion or muscle function at hatching. These new mutants provide valuable material for the study of integrin-dependent cell-to-cell adhesion (Prout, 1997).
Epithelial tubes that compose many organs are typically long lasting, except under specific developmental and physiological conditions when network remodeling occurs. Although there has been progress elucidating mechanisms of tube formation, little is known of the mechanisms that maintain tubes and destabilize them during network remodeling. This study describes Drosophila tendrils mutations that compromise maintenance of tracheal terminal branches, fine gauge tubes formed by tracheal terminal cells that ramify on and adhere tightly to tissues in order to supply them with oxygen. Homozygous tendrils terminal cell clones have fewer terminal branches than normal but individual branches contain multiple convoluted lumens. The phenotype arises late in development: terminal branches bud and form lumens normally early in development, but during larval life lumens become convoluted and mature branches degenerate. Their lumens, however, are retained in the remaining branches, resulting in the distinctive multi-lumen phenotype. Mapping and molecular studies demonstrate that tendrils is allelic to rhea, which encodes Drosophila talin, a large cytoskeletal protein that links integrins to the cytoskeleton. Terminal cells mutant for myospheroid, the major Drosophila ß-integrin, or doubly mutant for multiple edematous wings and inflated α-integrins, also show the tendrils phenotype, and localization of myospheroid ß-integrin protein is disrupted in tendrils mutant terminal cells. The results provide evidence that integrin-talin adhesion complexes are necessary to maintain tracheal terminal branches and luminal organization. Similar complexes may stabilize other tubular networks and may be targeted for inactivation during network remodeling events (Levi, 2006; full text of article).
The establishment of a multicellular body plan requires coordinating changes in cell adhesion and the cytoskeleton to ensure proper cell shape and position within a tissue. Cell adhesion to the extracellular matrix (ECM) via integrins plays diverse, essential roles during animal embryogenesis and therefore must be precisely regulated. Talin, a FERM-domain containing protein, forms a direct link between integrin adhesion receptors and the actin cytoskeleton and is an important regulator of integrin function. Similar to other FERM proteins, talin makes an intramolecular interaction that could autoinhibit its activity. However, the functional consequence of such an interaction has not been previously explored in vivo. This study demonstrates that targeted disruption of talin autoinhibition gives rise to morphogenetic defects during fly development and specifically that dorsal closure (DC), a process that resembles wound healing, is delayed. Impairment of autoinhibition leads to reduced talin turnover at and increased talin and integrin recruitment to sites of integrin-ECM attachment. Finally, evidence is presented that talin autoinhibition is regulated by Rap1-dependent signaling. Based on these data, it is proposed that talin autoinhibition provides a switch for modulating adhesion turnover and adhesion stability that is essential for morphogenesis (Ellis, 2013).
Overall, this study identifies an important role for the regulation of talin function through autoinhibition. Failure to autoinhibit talin impairs morphogenetic processes, but this is not due to defects in integrin-mediated attachment to the ECM or in the assembly of the adhesion complex. Thus, it is unlikely that the FERM domain mutation E1777A, which completely blocks autoinhibition, blocks integrin-mediated cell-ECM attachment in a dominant-negative fashion. An alternative explanation for the phenotype is that the E1777A mutant behaves like a gain-of-function allele of talin and that the morphogenetic defects that were observe are due to too much rather than too little adhesion. This would not be the first time such a phenomenon has been observed; for example, overexpression of integrins in either the wing or the muscle gives rise to phenotypes identical to those found in integrin-null mutants. How could the E1777A mutation give rise to stronger adhesion? It was shown that this mutation enhances the recruitment and colocalization of talin and integrin at sites of adhesion. Importantly, it was shown that the E1777A mutation effectively reduces talin turnover at sites of adhesion. Indeed, the data fit with a gain-of-function model: blocking talin autoinhibition leads to increased integrin-mediated adhesion, and this impairs morphogenetic processes that require cyclic adhesion assembly and disassembly. Further consistent with this model is the observation that adhesion at myotendinous junctions (MTJs), a non-morphogenetic context, is not perturbed upon blocking autoinhibition of talin. The possibility cannot be excluded that E1777A may confer its effect on talin function through a means other than disruption of autoinhibition. Encouragingly, however, homology modeling and NMR analyses strongly suggest that the fly protein behaves much as the mammalian homolog does (Ellis, 2013).
How does prevention of autoinhibition stabilize integrin-mediated adhesion? This study shows that autoinhibition regulates talin recruitment to adhesions through a RIAM-Rap1-dependent mechanism. Interestingly, the E1777A autoinhibition mutant talin is more strongly recruited to adhesions than WT talin; this enhanced recruitment occurs independent of RIAM-Rap1 activity. Thus, it is possible that constitutive relief of autoinhibition works to stabilize and promote adhesion by enhancing recruitment of the talin molecule to adhesions, thus bypassing the need of the RIAM-Rap1 pathway for recruitment. At the membrane, adhesion strengthening may occur via talin's scaffolding function, as talin can interact with multiple components of the integrin adhesion complex IAC, and these interactions may increase and/or change when talin assumes a more extended conformation. Another possibility, consistent with structural studies, is that relief of autoinhibition frees up the FERM/IBS-1 domain of talin such that it can activate integrins. It is predicted that mutations in talin that block IBS-1-mediated integrin activation would lead to more dynamic adhesions, and this is indeed what was observed. According to the model, talin recruitment is determined by the sum of interactions that a single molecule can make with other IAC components at any one time. For example, the autoinhibited form of talin relies on Rap1/RIAM for efficient recruitment, even though it may still bind integrin through its free IBS-2 domain; both mechanisms may contribute to targetting of talin to adhesions. It is speculated that relief of autoinhibition makes the IBS-1 available, as well as the many other binding sites for IAC components that are found in the talin rod domain (e.g., vinculin binding sites), thereby substantially increasing the number of possible interactions that can lead to talin recruitment to the IAC (Ellis, 2013).
There are likely to be multiple avenues leading to relief of talin autoinhibition. Recent superresolution studies provided elegant evidence that autoinhibition is primarily relieved within adhesion complexes, implicating the need for a mechanism to specifically recruit autoinhibited talin to adhesions. This study has showm that forcing talin to remain in an open, nonautoinhibited conformation gives rise to very similar phenotypes as activating the RIAM-Rap1 pathway (RIAM is Rap1 interacting adaptor protein). Based on the results, it is proposed that RIAM-Rap1 brings autoinhibited talin to the membrane where autoinhibition can subsequently be relieved, possibly through electrostatic interactions with the membrane/ PIP2. RIAM-Rap1 has a previously established role in mediating the recruitment of talin to sites of adhesion, but it has recently been demonstrated that the requirement for RIAM-Rap1 is context dependent. Structural and biochemical studies have revealed that the binding of talin to either RIAM or vinculin is mutually exclusive and likely dependent on force. Moreover, in cell culture, vinculin-stimulated integrin activation is RIAM-Rap1 independent, raising the possibility that more mature adhesions might not need RIAM-Rap1 to promote talin activation in this case. Along similar lines, this study demonstrated that RIAM-Rap1 activity is dispensable for recruitment of a nonautoinhibited talin molecule (Ellis, 2013).
In summary, the results suggest that talin autoinhibition confers a switch through which fine control of integrin-mediated adhesion can be exerted in vivo. The findings also reveal RIAM-Rap1-mediated regulation of integrin adhesion is an important modulator of morphogenesis, and evidence is provided for an autoinhibition-based pathway for control of talin function through RIAM-Rap1. Furthermore, this study exemplifies how subtle tuning of adhesion complex composition and stability elicits different adhesive functions and cellular behaviors during development (Ellis, 2013).
Many aspects of glial development are regulated by extracellular signals, including those from the extracellular matrix (ECM). Signals from the ECM are received by cell surface receptors, including the integrin family. Previous studies have shown that Drosophila integrins form adhesion complexes with Integrin-linked kinase and talin in the peripheral nerve glia and have conserved roles in glial sheath formation. However, integrin function in other aspects of glial development is unclear. The Drosophila eye imaginal disc (ED) and optic stalk (OS) complex is an excellent model with which to study glial migration, differentiation and glia-neuron interactions. The roles of the integrin complexes was studied in these glial developmental processes during OS/eye development. The common β subunit βPS and two α subunits, αPS2 and αPS3, are located in puncta at both glia-glia and glia-ECM interfaces. Depletion of βPS integrin and talin by RNAi impaired the migration and distribution of glia within the OS resulting in morphological defects. Reduction of integrin or talin in the glia also disrupted photoreceptor axon outgrowth leading to axon stalling in the OS and ED. The neuronal defects were correlated with a disruption of the carpet glia tube paired with invasion of glia into the core of the OS and the formation of a glial cap. These results suggest that integrin-mediated extracellular signals are important for multiple aspects of glial development and non-autonomously affect axonal migration during Drosophila eye development (Xie, 2014).
This study found that OS glia express integrin complexes that play a role in the development of the glia and axons of the ED and OS. βPS integrin is located in puncta at the glial membrane and associates with Talin and ILK. These focal adhesion markers are found between the perineural glia (PG) and ECM, plus at the interfaces of the PG-CG (carpet glia) and CG-CG layers. A different distribution was found for the αPS2 and αPS3 integrins, which were concentrated at the periphery and interior of the OS, respectively. The results from RNAi-mediated knockdown revealed that these complexes play important roles in OS glial development, as knockdown led to disruption of PG and CG morphology. Specifically, the loss of βPS integrin or talin caused PG to aggregate in the distal half of the OS, resulting in an accumulation of glia in the OS. The PG formed clusters instead of a surrounding monolayer, suggesting that PG make integrin-mediated associations that maintain their distribution (Xie, 2014).
The PG migrate between the CG and the basal ECM, and loss of focal adhesions led to a disruption in PG migration, suggesting that integrin complexes on one or both surfaces play a role in mediating glia migration. The αPS2/βPS heterodimer binds ligands containing the tripeptide RGD sequence and αPS3/βPS binds laminins, so either or both could mediate adhesion of the PG to the ECM. However, it appears that depletion of the integrin complex in apposing glial layers is necessary to disrupt glial migration into the OS, as MARCM clones within the PG alone did not disrupt migration. Integrin function is conserved in mediating glial cell migration either on ECM or neighboring glial surfaces. For example, vertebrate glial studies found that integrins are involved in astrocyte, oligodendrocyte precursor and Schwann cell migration on various ECM molecules. Loss of β1-integrin in Bergmann glia leads to mislocalization, ectopic migration and disruption of process growth within the vertebrate cerebellum. ILK and CDC42 within Bergmann glia are required for the β1-integrin-dependent control of process outgrowth (Xie, 2014).
Reduction of focal adhesions disrupted the CG sheath and the integrity of the blood-nerve barrier, suggesting that maintenance of the CG tube also requires integrin-mediated adhesion. The disruption of the CG tube is similar to observations made in vertebrates, in which glial tubes are necessary for chain migration of neuroblasts along the rostral migratory stream; β1-integrin plays a role in both chain migration and maintenance of the glial tubes. However, the link between the integrin complex and the formation or stabilization of the CG tube is currently unknown (Xie, 2014).
The integrin complex appears to play a limited role in the migration of the WG into the OS. After knockdown of βPS or talin, WG with normal bipolar membrane processes were observed and WG mys1 MARCM clones had morphologies similar to control clones, suggesting that integrin signaling is not required for migration in differentiated WG. TEM analysis suggests that the WG failed to properly ensheath and segregate the bundles of photoreceptor axons, a phenotype consistent with that observed in vertebrate glia, although it is also possible that the lack of WG ensheathment is a secondary effect of axon stalling (Xie, 2014).
Loss of integrin complexes resulted in a failure of photoreceptor axons to exit the ED, navigate the OS or correctly target the optic lobe. Previously, blocking glial migration from the OS using dominant-negative Ras1 (Ras85D -- FlyBase) resulted in photoreceptor axons stalling in the ED but not the OS. The phenotype suggested that photoreceptor axons require physical contact with retinal glia to exit the ED. However, this mechanism does not seem to apply to the stalling phenotype observed with knockdown of the integrin complex. In the majority of samples, glia were still present in the ED and around the axonal stalling region, suggesting that the axon stalling phenotypes are likely to be due to a different mechanism (Xie, 2014).
It is possible that axon stalling results from a combination of glial changes affecting the multiple subtypes of OS glia and disruption of both the βPS/αPS2 and βPS/αPS3 adhesion complexes. Only the simultaneous loss of both αPS2 and αPS3 triggered the axonal phenotypes. Similarly, knockdown of βPS or talin within individual glial subtypes does not trigger axon stalling, whereas disruption of adhesion complexes in all glial subtypes does. It might be that only the repo-GAL4 driver is sufficiently strong or expressed early enough for the effective knockdown of integrin or talin to trigger the axon stalling phenotypes. However, the axon stalling phenotype can be effectively produced by delaying the expression of the RNAi with the repo-GAL4 driver until the second instar, suggesting that early expression is not key. Overall, the results suggest that it is the combined loss of the focal adhesion complex in multiple glial layers that led to the axon stalling phenotype, although the underlying mechanism is not known. It is possible that the simultaneous aggregation of the stalled PG and disruption of the CG sheath triggers axon stalling by allowing ectopic PG to enter the center of the OS or form the glial cap. The ectopic PG within the axon stalling area and in the glial cap were likely to be PG given their expression of LanB2, Apt and the lack of the WG Gli-lacZ marker. Normally, the PG migrate into the ED and differentiate into WG in the presence of photoreceptor axons. However, in the RNAi-treated OS many of the Apt-positive glia also expressed Gli-lacZ, suggesting a change in the normal differentiation pathway, perhaps owing to the premature and ectopic contact of the PG with the photoreceptors within the OS. Loss of integrins throughout the entire glial population could also lead to global changes to the ECM, as loss of integrins can alter the deposition of ECM components during epithelial morphogenesis. Although loss of integrins in the PNS does not lead to changes in the neural lamella of the peripheral nerve, it is possible that ECM changes in terms of structural integrity or the ability to recruit protein components could result in the multiple glial morphological changes (Xie, 2014).
In summary, this study has shown that glia in the OS and ED express integrins and Talin, through which they receive external signals important for PG migration, organization and CG barrier formation. The combined impact of integrin complexes on the morphology and development of both glial layers is crucial for proper axonal outgrowth through the OS and targeting in the brain (Xie, 2014).
Mechanotransduction of tension can govern the remodeling of cardiomyocytes during growth or cardiomyopathy. Tension is signaled through the integrin adhesion complexes found at muscle insertions and costameres but the relative importance of signalling during cardiomyocyte growth versus remodelling has not been assessed. Employing the Drosophila cardiomyocyte as a genetically amenable model, this study depleted the levels of Talin, a central component of the integrin adhesion complex, at different stages of heart growth and remodeling. A continuous requirement for Talin was demonstrated during heart growth to maintain the one-to-one apposition of myofibril ends between cardiomyocytes. Retracted myofibrils cannot regenerate appositions to adjacent cells after restoration of normal Talin expression, and the resulting deficit reduces heart contraction and lifespan. Reduction of Talin during heart remodeling after hatching or during metamorphosis results in pervasive degeneration of cell contacts, myofibril length and number, for which restored Talin expression is insufficient for regeneration. Resultant dilated cardiomyopathy results in a fibrillating heart with poor rhythmicity. Cardiomyocytes have poor capacity to regenerate deficits in myofibril orientation and insertion, despite an ongoing capacity to remodel integrin based adhesions (Bogatan, 2015).
In late embryogenesis, the Drosophila heart is a 4 micron diameter tube enclosed by 2 cardiomyocytes, attached at the dorsal and ventral midline with cadherin based cell junctions, and an integrin rich lumen. The larval cardiomyocytes are dominated by myofibrils that terminate in integrin rich insertions at the dorsal and ventral midline, without a cadherin rich domain. Therefore early heart development is marked by dramatic reorganisation of cell adhesion and polarity. For the remainder of a fly’s life, cardiomyocyte differentiation is remarkable for increase in cell size but not cell number, and for the pupal remodelling of posterior aorta myocytes into heart myocytes (Bogatan, 2015).
This study examined the role of Talin production in the differentiation, growth and remodelling of cardiomyocytes. The requirement of integrin function for cardiomyocyte adhesion was verified, and it was noted that, like body wall muscle, the insertions are integrin-rich, and that the muscle costameres coincide with myocyte surface integrin adhesions. Normally, myofibrils of each cell are aligned end-to-end with myofibrils of the contralateral cardiomyocyte, suggesting that extracellular matrix (ECM) linkages at the end of myofibrils are different from the rest of the cell surface, reminiscent of the mammalian intercalated disc. If levels of Talin production are reduced, cardiomyocyte insertion, particularly at points of myofibril termination are vulnerable to degeneration (Bogatan, 2015).
During first instar heart differentiation and pupal remodelling, cardiomyocytes are most susceptible to depletion of Talin, resulting in significant cell shrinkage. At less susceptible stages of heart growth, less cell shrinkage, but loss of myofibril apposition between cells results. The resulting degeneration of heart structure is likely due to the loss of adhesion caused by the depletion of Talin. This reflects the ongoing turnover of Talin and Integrin at adhesions, shown to be modulated by tension in Drosophila muscle. Remarkably, restoration of normal Talin expression does not enable regeneration of myofibril length, inter-cardiomyocyte cell junctions or apposition of myofibril ends between myocytes at any larval or adult stage. Instead, the cardiomyocyte perimeter is marked by a broader band of integrin, suggestive of expanded adhesion to the heart ECM, and hence less direct transmission of tension between cardiomyocytes. Nevertheless, affected cardiomyocytes continue to grow as the larva grows, without restoring cell to cell apposition and alignment of myofibrils (Bogatan, 2015).
Heart contraction is reduced subsequent to Talin reduction at each larval stage, including during the second instar, when myocyte degeneration is minimal, but midline apposition of myofibrils is disrupted. Nevertheless, this disruption does not reduce the rhythmicity of second instar treated hearts. Heart dilation, rhythmicity and contraction are most affected by transient depletion of Talin during cardiomyocyte remodelling in the first instar, suggesting that synchronicity of cardiomyocyte contraction requires cell to cell contact, possibly along the ipsilateral domains of cardiomyocytes. This cell surface domain contains the costameres, where components of the IAC are implicated in tension signalling (Bogatan, 2015).
Myofibril stability may depend upon linkage to integrin adhesion at insertions, or at the costameres, as Talin depleted cardiomyocytes have fewer myofibrils. However muscle insertion structure is far more sensitive to the level of Talin than the structure of the costamere. Weakened costameres, observed in Drosophila mutants of muscle Trim32, are depleted of Integrin Adhesion complex (IAC) proteins, including Talin, resulting in unbundling of myofibrils and muscle “wasting”. Similarly, increased or decreased Integrin function in vertebrate heart muscle alters intercalated disc structure and cardiomyocyte contractility. In Drosophila and vertebrates, integrin adhesion signalling is required for homeostasis of the contractile apparatus (Bogatan, 2015).
ECM is visible on the luminal and abluminal surfaces of cardiomyocytes. As heart diameter grows normally, new matrix must be deposited on both surfaces. Similarly, when cardiomyocytes retract, the remaining ECM likely stretches and expands as the heart vessel becomes dilated. In the Drosophila model, this dilation results in the deposition of a more elaborate network of Pericardin containing ECM fibrils. This process is analogous to mammalian Dilated Cardiomyopathy (DCM). DCM can be triggered by mutations in proteins that link the sarcomere to the ECM, such as IAC proteins vinculin and tintin. Expression of IAC proteins is elevated in cardiac hypertrophy. Analysis of IAC gene function in genetic models such as Drosophila reveals the temporal dimension of the stability and remodeling of myofibrils. This study indicates myofibril stability requires ongoing Talin renewal, and that regeneration after perturbation is very limited. Further study of IAC function subsequent to changes in cardiac load in Drosophila cardiomyocytes should be instructive in revealing the signalling pathways activated in DCM (Bogatan, 2015).
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