InteractiveFly: GeneBrief

Ajuba LIM protein: Biological Overview | References

BIOLOGICAL OVERVIEW

The Hippo signaling network controls organ growth through YAP family transcription factors, including the Drosophila Yorkie protein. YAP activity is responsive to both biochemical and biomechanical cues, with one key input being tension within the F-actin cytoskeleton. Several potential mechanisms for biomechanical regulation of YAP proteins have been described, including tension-dependent recruitment of Ajuba family proteins, which inhibit kinases that inactivate YAP proteins, to adherens junctions (AJ). This study investigated the mechanism by which the Drosophila Ajuba family protein, Ajuba LIM protein (Jub) is recruited to adherens junctions, and the contribution of this recruitment to the regulation of Yorkie. Alpha-catenin is identifed as the mechanotransducer responsible for tension-dependent recruitment of Jub by identifying a region of α-catenin that associates with Jub, and by identifying a region, which when deleted, allows constitutive, tension-independent recruitment of Jub. Increased Jub recruitment to alpha-catenin is associated with increased Yorkie activity and wing growth, even in the absence of increased cytoskeletal tension. These observations establish alpha-catenin as a multi-functional mechanotransducer and confirm Jub recruitment to alpha-catenin as a key contributor to biomechanical regulation of Hippo signaling (Alegot, 2019).

To evaluate the role of α-catenin in the recruitment of Jub to AJ, Jub localization was examined in wing discs isolated from animals expressing truncated forms of α-catenin. This was achieved by using RNAi to knock-down expression of endogenous α-catenin and expressing RNAi-insensitive Venus- or V5-tagged α-catenin transgenes under UAS-Gal4 control. Transgenes were expressed in posterior cells using an en-Gal4 driver, so that anterior cells could serve as a control for Jub localization. Full-length α-catenin expressed under UAS-Gal4 control rescued both the lethality associated with knockdown of α-catenin, and Jub localization. α-catenin has similarity to Vinculin in N-terminal, middle, and C-terminal regions termed VH1, VH2, and VH3. An initial series of constructs deleted regions of α-catenin centered around these Vinculin homology regions, as well as separate deletions of either the N-terminal or C- terminal half of VH2. Constructs lacking either the N- or C-terminus of α-catenin (ΔVH1 or ΔVH3) could not rescue the lethality associated with α-catenin knockdown. Thus, to enable visualization of the consequences of these deletions on Jub, conditional knockdown and expression of α-catenin was achieved using a temperature sensitive-allele of Gal80 (Gal80ts) that represses Gal4-driven expression at 18°C but not at 29°C. After a 30 hour shift to higher temperature, Jub was mostly lost from apical cell junctions. However, E-cadherin was also mostly lost, indicating that these regions of α-catenin are required to maintain AJ. This is consistent with the roles of F-actin and α-catenin in stabilizing AJ, as the VH1 region is required for association with β-catenin and thus localization to AJ, and the VH3 region is required for association with F-actin. The ΔVH1 and ΔVH3 constructs also did not themselves localize to junctions. The loss of Jub from cell junctions under these conditions is consistent with studies indicating its association with AJ, but does not provide specific information about its interactions with α-catenin (Alegot, 2019).

Conversely, deletions within the central region of α-catenin increased Jub recruitment to AJ. Deletion of the entire VH2 region increased Jub recruitment but was also associated with abnormal cell morphology and E-cadherin localization. However, two smaller deletions comprising the N- and C-terminal halves of the VH2 region (ΔVH2-N and ΔVH2-C) rescued knock-down of α-catenin, resulting in cells that appear to have normal localization of E-cadherin and α-catenin. The ΔVH2-C deletion also appears to have normal Jub localization. In contrast, the ΔVH2-N deletion is associated with increased Jub localization at AJ. Moreover, in cells with the ΔVH2-N deletion, Jub is distributed relatively uniformly around cell junctions, whereas in cells with wild-type α-catenin, Jub localization to junctions is often punctate. The ΔVH2-N deletion was not only associated with increased Jub recruitment when expressed in place of endogenous α-catenin; it could also promote Jub recruitment even when expressed in the presence of endogenous α-catenin. Thus, expression of this isoform dominantly increases Jub recruitment to AJ (Alegot, 2019).

To further investigate the influence of α-catenin on Jub, smaller deletion constructs were created. To aid in the design of these additional constructs, Phyre2 was used to predict the structure of Drosophila α-catenin, based on its sequence similarity to mammalian α-catenin proteins with experimentally-determined structures. Mammalian α-catenin consists largely of a series of α-helical bundles: two N-terminal 4-helix bundles (N1 and N2) that share one long α-helix; three central 4-helix bundles (M1, M2, and M3), and a C-terminal 5-helix bundle. These structural features are also predicted for Drosophila α-catenin (Alegot, 2019).

The ΔVH2-N deletion that increases Jub recruitment to AJ deletes both the M1 and M2 helical bundles. Thus, separate deletions were created of either the M1 or M2 bundles. Deletion of the M2 bundle (ΔM2) slightly increased Jub localization to AJ, but a much stronger increase in Jub binding was detected when the M1 bundle was deleted (ΔM1a or ΔM1b). Recruitment of Jub to AJ is normally promoted by cytoskeletal tension. To examine the possibility that M1 deletions increase Jub recruitment by increasing tension, levels were examined of junctional myosin (using a GFP-tagged myosin light chain) that correlate with junctional tension, but no difference was observed. F-actin levels were similarly unaffected. Staining was carried out specifically for the phosphorylated (activated) form of myosin regulatory light chain (pMLC) in discs expressing Jub:GFP. This revealed similar levels of pMLC in control cells and cells expressing α-catenin ΔM1b, whereas expression of rok RNAi or an activated form of Rok visibly decreased or increased, respectively, junctional levels of both pMLC and Jub. Since Jub associates with α-catenin (Rauskolb, 2014), the observation that deletion of the M1 bundle increases Jub localization to AJ without increasing tension suggests that deletion of this region alters the structure of α-catenin in a way that makes a Jub-binding region more accessible. The M1 bundle might thus act as an 'inhibitory' region that prevents Jub binding, with this inhibition normally alleviated in wild-type α-catenin by a conformational change induced by high tension. Alternatively, deletion of the M1 bundle might destabilize a non-Jub binding conformation of α-catenin and thereby indirectly increase accessibility of a Jub-binding region (Alegot, 2019).

While both Jub and Vinculin exhibit tension-dependent association with α-catenin, the results suggest that their interactions are distinct. A region initially identified as inhibitory for Vinculin binding, corresponds to the M3 helical bundle and the linker between VH2 and VH3. Moreover, the M1 helical bundle includes the Vinculin binding interface. To directly compare the influence of α-catenin deletions on Jub and Vinculin binding, advantage was taken of a Drosophila genomic GFP-tagged Vinculin (Vinc:GFP). Both of the M1 deletions substantially reduced, but did not eliminate, Vinculin localization to AJ, which fits with observations that Vinculin interacts with sequences in the M1 bundle, but implies that additional mechanisms also contribute to Vinculin localization. Deletion of the M2 bundle increased Vinculin localization to AJ, consistent with observations that M2 participates in interactions that stabilize a conformation of α-catenin that is 'closed' with respect to Vinculin binding (Alegot, 2019).

If M1 deletions mimic the influence of cytoskeletal tension on the ability of α-catenin to bind to Jub, the increased recruitment of Jub in M1 deletion isoforms could occur even in the absence of tension. To test this, Jub localization was examined in flies expressing α-catenin constructs and with cytoskeletal tension decreased by RNAi of Rho-associated protein kinase (Rok), a promoter of myosin activity. In the presence of full-length α-catenin, rok RNAi decreases Jub recruitment to junctions. Conversely, in the presence of deletions that include the M1 bundle, Jub recruitment to junctions remains elevated. Quantitation of Jub at junctions, normalized to E-cadherin, revealed a decrease in Jub recruitment when tension is lowered even in cells expressing M1 deletions, but Jub recruitment nonetheless remains above that in control cells. Thus, these deletions recruit Jub even under low-tension conditions, which implicates α-catenin as the key mechanotransducer responsible for tension-dependent recruitment of Jub (Alegot, 2019).

Cytoskeletal tension promotes Yki activity, and Yki activity is suppressed by knockdown of Jub. However, other mechanisms by which tension might increase Yki activity have also been suggested, such as an influence on spectrins. The observation that deletions of the M1 bundle lead to increased Jub at AJ without increasing myosin identifies a condition under which it was possible to determine whether recruitment of Jub to junctions is sufficient to increase Yki activity, and thus distinguish the contribution of Jub from other potential influences of cytoskeletal tension (Alegot, 2019).

Yki activity was evaluated by examining ex-lacZ expression, which is a reporter for the Yki target gene expanded (ex). Expression of α-catenin with deletion of the M1 bundle increased ex-lacZ, whereas expression of full length α-catenin did not. Increased Yki activity is associated with increased accumulation of Yki in the nucleus, and nuclear levels of Yki were slightly increased in wing disc cells expressing M1 region deletions. To further examine the influence of the M1 deletion, adult wing size was examined, as wing growth is promoted by Yki activity. Expression of M1 deletion isoforms throughout the developing wing under nub-Gal4 control increased wing size as compared to wings expressing wild-type α-catenin. The increased Yki activity and wing growth in M1 deletion mutants are Jub-dependent, because they are reversed by RNAi of jub. As mutation or knock-down of Vinculin has only minor phenotypic consequences in Drosophila, the effects of M1 deletion on Yki activity and wing growth cannot be attributed to loss of Vinculin. Altogether, these observations establish that increased Jub recruitment to junctions can be sufficient to elevate Yki activity. This strongly supports the conclusion that Jub recruitment to AJ is a key component of the biomechanical response linking cytoskeletal tension to Yki activity (Alegot, 2019).

However, analysis of the larger ΔVH2N deletion suggests that the role of α-catenin in promoting Yki activity is more complex. Although expression of ΔVH2N α-catenin increased Jub recruitment at AJ, it did not detectably increase ex-lacZ expression or wing size. Similarly, even though deletion of the M2 helical bundle modestly increased Jub recruitment to AJ, it did not detectably increase ex-lacZ expression or wing size. Thus, it is inferred that additional features of α-catenin may contribute to Yki regulation (Alegot, 2019).

Jub requires α-catenin to localize to junctions (Rauskolb, 2014). To map regions of α-catenin that mediate association with Jub, co-immunoprecipitation experiments were performed in Drosophila S2 cells expressing transfected constructs. Initially, co- immunoprecipitation of Jub with α-catenin full length, VH1, VH2, and VH3 region constructs was compared. Significant association of Jub and a VH1 region construct was detected. Conversely, association of Jub with full length, VH2, or VH3 constructs was weaker, and close to the non- specific background. The association with the VH1 region of α-catenin was not as strong as binding of Jub to Warts, which was included as a positive control. Association of Jub with the VH1 region is consistent with reports that the mammalian Ajuba protein can associate with an N- terminal fragment of α-catenin, corresponding roughly to VH1 (Marie, 2003). The stronger association of Jub with a VH1 fragment as compared to full length α-catenin is consistent with normal binding depending upon junctional tension, as S2 cells are not epithelial, so it would be expected that the transfected α-catenin would be in a low-tension conformation. Moreover, co-immunoprecipitation experiments comparing M1 bundle deletion constructs to full length α-catenin revealed that M1 deletions significantly increased association of Jub with α-catenin (Alegot, 2019).

To further refine the Jub-binding region, the predicted Drosophila α-catenin structure was used to design constructs corresponding to the N1 or N2 helical bundles. No significant co-immunoprecipitation with the N1 bundle was detected, whereas co- immunoprecipitation with N2 was comparable to that for the VH1 region construct. Thus, it is inferred that the primary site of association of Jub with α-catenin is within N2. While the simplest model would be that Jub directly binds to this region of α-catenin, it remains possible that their association is mediated through other proteins. The N2 bundle is near the M1 bundle, but they do not directly contact each other in the predicted structure. Thus, the effect of M1 deletion on Jub binding may be an indirect consequence of a conformational change in α-catenin when this region is deleted, or reduced stability of a more closed conformation, rather than the M1 bundle directly obscuring a binding site within N2. Attempts were also mad to examine the consequences of loss of Jub recruitment to junctions by expressing α-catenin with N2 deleted in vivo. However, this protein failed to rescue the viability of wing disc cells expressing α-catenin RNAi, so this could not be examined. Moreover, when expressed in wild-type cells, the ΔN2 construct failed to localize to adherens junctions (Alegot, 2019).

These results establish key features of α-catenin mechanotransduction and tension- dependent regulation of Yki activity. They implicate α-catenin as a multi-functional mechanotransducer that associates with both Vinculin and Jub upon a tension-induced conformational change in α-catenin, but through distinct sites. The complete structure of the open conformation of α-catenin has not been determined, but it is inferred that it makes Jub- associating regions accessible. M1 deletions also make the Jub-associating region more accessible, but it's not clear whether they do so in a manner similar to, or distinct from, the effect of tension. The observation that M1 deletions increase Yki activity also supports the crucial role of Jub recruitment to AJ in promoting Yki activity. This does not exclude the possibility that of other consequences of cytoskeletal tension contribute to Yki regulation, but clearly implicates recruitment of Jub as a key factor (Alegot, 2019).

Role of alpha-Catenin and its mechanosensing properties in regulating Hippo/YAP-dependent tissue growth

alpha-Catenin is a key protein of adherens junctions (AJs) with mechanosensory properties. It also acts as a tumor suppressor that limits tissue growth. This study analyzed the function of Drosophila alpha-Catenin (alpha-Cat) in growth regulation of the wing epithelium. Different alpha-Cat levels led to a differential activation of Hippo/Yorkie or JNK signaling causing tissue overgrowth or degeneration, respectively. alpha-Cat can modulate Yorkie-dependent tissue growth through recruitment of Ajuba, a negative regulator of Hippo signaling to AJs but also through a mechanism independent of Ajuba recruitment to AJs. Both mechanosensory regions of alpha-Cat, the M region and the actin-binding domain (ABD), contribute to growth regulation. Whereas M is dispensable for alpha-Cat function in the wing, individual M domains (M1, M2, M3) have opposing effects on growth regulation. In particular, M limits Ajuba recruitment. Loss of M causes Ajuba hyper-recruitment to AJs, promoting tissue-tension independent overgrowth. Although M binds Vinculin, Vinculin is not responsible for this effect. Moreover, disruption of mechanosensing of the alpha-Cat ABD affects tissue growth, with enhanced actin interactions stabilizing junctions and leading to tissue overgrowth. Together, these findings indicate that alpha-Cat acts through multiple mechanisms to control tissue growth, including regulation of AJ stability, mechanosensitive Ajuba recruitment, and dynamic direct F-actin interactions (Sarpal, 2019).

One key factor that determines the impact of α-Cat on tissue growth, leading either to overgrowth or tissue degeneration, is the amount of α-Cat at AJs. Analysis of phenotypic defects resulting from the differential reduction of gene function revealed that Drosophila α-Cat is a negative regulator of tissue growth. Moderate α-Cat overexpression or moderate depletion of α-Cat or DEcad caused Yki activation and overproliferation. Moreover, consistent with these findings, a previous analysis of strong loss-of-function conditions for α-Cat, DEcad, or Arm led to the conclusion that a loss of the cadherin-catenin complex (CCC) reduces Yki activity and causes JNK-meditated tissue degeneration. This conclusion was at odds with some mammalian studies where the loss of E-cadherin or αE-catenin causes YAP or TAZ-dependent overgrowth in a number of different tissues and cell lines. The current data showing that α-Cat and DEcad limit Yki activity and tissue growth in Drosophila similar to mammalian tissues suggest a conserved functional relationship between AJs and the regulation of Yki/YAP/TAZ activity. Only when the Drosophila CCC is strongly depleted does a substantive activation of JNK signaling override Yki activation, causing tissue degeneration. This could potentially involve a direct inhibition of Yki by JNK (Sarpal, 2019).

α-Cat loss-of-function conditions in conjunction with a block of cell death defined three distinct phenotypic classes that broadly align with the progression sequence of epithelial cancer from adenoma (epithelial overgrowth), to adenocarcinoma (overgrowth associated with a partial loss of epithelial integrity), to carcinoma (loss of epithelial integrity with cells showing protrusive activity). Similarly, in mammalian cancer, such as human colon cancer is the down-regulation of αE-catenin associated with an increased propensity for cells to become invasive. On the other hand, loss of αE-catenin was reported to suppress colorectal adenomas induced by APC loss-of-function, an effect that could be mediated by the Rho-Rho kinase signalling-dependent cell death elicited by the loss of the CCC in an APC mutant background. Also in Drosophila, it was found that Rho1 is required for cell death resulting from the loss of α-Cat, likely mediated by the Rho1 signalling dependent activation of the JNK pathway (Sarpal, 2019).

Loss of α-Cat leads to a corresponding decline of DEcad and Arm at AJs. Quantification of CCC proteins in the late stage embryonic epidermis of zygotic α-Cat null mutants suggested that AJs can be retained and support normal tissue architecture when CCC levels are reduced to less than 10% of normal. As heterozygous α-Cat animals are normal, it is anticipated that reduction of α-Cat to somewhere between 50% and 10% of wild-type levels will cross a threshold that will increase Yki activity without activating JNK signaling sufficiently to cause cell death. How strongly α-Cat and DEcad have to be reduced to cause an activation of Yki remains to be explored. For example, reducing α-Cat could compromise the interactions between the AJ protein Echinoid and Salvadore, a Hippo binding partner important for normal Hippo activity, or compromise the interactions between Crb and Ex that could deregulate Hippo signaling. Loss of α-Cat could also affect actin polymerization as mature AJs suppress actin polymerization, whereas enhanced actin polymerization is a known activator of Yki. Reducing α-Cat could therefore directly promote actin polymerization prior to an overt defect in cell adhesion, and consequently stimulate Yki activity and tissue growth. Finally, loss of α-Cat may affect Yki not through the Hippo pathway as was documented for αE-catenin in mammalian keratinocytes (Sarpal, 2019).

Mammalian studies raised the possibility that αE-catenin could act independently of AJs to regulate tissue growth. In contrast, several observations argue that Drosophila α-Cat acts as an AJ component to limit tumorigenesis: (1) The mechanosensory properties of α-Cat regulate the recruitment of Jub to AJs, which stimulates Yki activity and tissue growth and implies that α-Cat is suspended between the E-cadherin/β-catenin complex and actomyosin. In particular, α-Cat mutants that disrupt mechanosensing and cause overgrowth (α-CatR-ΔM1 and α-CatR-H1) are effectively recruited to AJs where they support normal epithelial organization, suggesting that a specific junctional defect disrupts growth regulation. (2) A reduction of DEcad is also associated with hyperplastic tissue overgrowth similar to the partial loss of α-Cat. (3) The expression of a DEcad::αCat fusion protein restores both the epithelial organization and normal growth of α-Cat compromised wing discs, and rescues α-Cat null mutant clones which normally fail to develop in wing discs. Together, these data suggest that α-Cat acts as a component of the CCC at AJs to regulate tissue growth (Sarpal, 2019).

The mechanosensitive interactions between Jub and α-Cat are thought to transmit tissue tension into growth regulatory signals. Cytoskeletal tension enhances recruitment of Jub to junctional α-Cat where Jub forms a complex with Wts, preventing it from phosphorylating and hence deactivating Yki. Supporting this model, Jub and Yki-dependent tissue overgrowth was observed that correlates with an enhanced recruitment of Jub to AJs. In particular, the data suggest that the M1 domain acts as a gatekeeper for Jub recruitment. In the absence of M1, junctional Jub levels become strikingly high, suggesting that M region mechanosensing has become ineffective in limiting Jub recruitment, causing Yki activation and overgrowth. Tension is thought to cause a conformational change in the M region and an unfurling of the M1 domain exposing a Vinc binding site. However, Vinc is not required for limiting Jub recruitment and deletion of M1 causes a reduction of junctional Vinc but not a complete loss. This leaves the mechanism of how M1 moderates Jub recruitment unresolved. A second unknown binding partner of M1 may be involved or an intramolecular interaction between M1 and the Jub binding site in the N domain could control Jub binding (Sarpal, 2019).

This Jub recruitment-dependent model of how mechanotransduction by the α-Cat M region regulates tissue growth does not explain all the observations and therefore needs to be extended to incorporate additional mechanisms of how AJs can control tissue growth. First, this model cannot explain the Yki-dependent overgrowth precipitated by low α-Cat levels, and corresponding low Jub levels at AJs. Second, it was observed that two fusion proteins between DEcad and α-Cat, one containing both full-length proteins (DEcad::αCat) and one lacking the Jub binding domain in α-Cat (DEcadΔβ::αCatΔN), can both support normal growth in α-Cat KD tissue but only DEcad::αCat restored normal junctional Jub levels whereas DEcadΔβ::αCatΔN did not. Third, expression of α-CatR in α-Cat KD tissue restores α-Cat to approximately 63% of normal levels. However, Jub increases to 119% without a noticeable increase in tissue size. One possibility is that mechanical force distributed over fewer α-Cat molecules enhances the mechanosensory response of individual α-Cat molecules in a non-linear manner resulting in higher Jub recruitment. Evidence for such a mechanism was recently reported for the recruitment of Vinc to AJs in the Drosophila germband, and may be similar for Jub recruitment to AJ in that tissue. Fourth, removing the entire M region results in an α-Cat protein that can support normal wing development, implying that M region mechanosensing is not an essential aspect of regulating tissue growth. In light of the results with α-CatR-ΔM1, this can only be explained by assuming that removing M2 and M3 in addition to M1 has a compensatory effect. Whereas α-CatR and α-CatR-ΔM expression in α-Cat depleted PC tissue enhances Jub recruitment above AC control levels, loss of M2 or M3 causes Jub levels to remain below AC levels suggesting that these two domains somehow normally support the ability of α-Cat to recruit Jub, possibly by supporting the stability of the CCC at AJs. Collectively, these findings suggest that M region mechanosensing contributes to Jub recruitment and Hippo/Yki pathway regulation but that the actual mechanisms involved have considerable complexity and require further analysis to be resolved (Sarpal, 2019).

Increased levels of Jub were also observed in response to disrupting the mechanosensory properties of the α-Cat ABD. Disruption of the α1-helix of ABD did not only enhance F-actin binding in vitro but also stabilized the CCC at AJs in the wing epithelium as suggested by the current observations. A corresponding increase in Jub levels at AJs could account for the persistent overgrowth observed in α-Cat KD tissue expressing α1-helix compromised α-Cat (α-CatR-H1). Thus, the mechanosensory properties of the M region and the α-Cat ABD are both important for regulating Jub recruitment to AJs and growth regulation through the Hippo/Yki pathway. As changes in tissue tension are thought to modulate the Hippo/Yki pathway through junctional recruitment of Jub it was asked whether α-Cat mutants that compromise mechanosensing cause changes in tissue tension or viscoelasticity that could indirectly affect Jub recruitment to AJs. We did not observe such changes after laser ablation of cell-cell junction. This is consistent with the finding that replacing α-Cat with α-CatR-ΔM1 did not change junctional myosin II levels, the major tension generator in the wing disc epithelium. Taken together, these data suggest a direct molecular role of α-Cat in Jub recruitment and strongly argue that α-Cat operates as a crucial mechanosensor to regulate tissue growth (Sarpal, 2019).

In summary, it is concluded that α-Cat uses multiple mechanisms to act as an important regulator of tissue growth in the Drosophila wing disc epithelium. It is doing so at least in part by operating as a mechanotransducer, engaging both M region and ABD mechanosensing, to relay cytoskeletal tension into growth regulatory signals. One of these mechanisms involves the recruitment of Jub to the N domain of α-Cat that can be modulated by both mechanosensing mechanisms. However, α-Cat also engages mechanisms that are independent of the junctional recruitment of Jub to control tissue growth, which remain to be explored further (Sarpal, 2019).

Differential growth triggers mechanical feedback that elevates Hippo signaling

Mechanical stress can influence cell proliferation in vitro, but whether it makes a significant contribution to growth control in vivo, and how it is modulated and experienced by cells within developing tissues, has remained unclear. This study reports that differential growth reduces cytoskeletal tension along cell junctions within faster-growing cells. A theoretical model is proposed to explain the observed reduction of tension within faster-growing clones, supporting it by computer simulations based on a generalized vertex model. This reduced tension modulates a biomechanical Hippo pathway, decreasing recruitment of Ajuba LIM protein and the Hippo pathway kinase Warts, and decreasing the activity of the growth-promoting transcription factor Yorkie. These observations provide a specific mechanism for a mechanical feedback that contributes to evenly distributed growth, and it is shown genetically that suppressing mechanical feedback alters patterns of cell proliferation in the developing Drosophila wing. By providing experimental support for the induction of mechanical stress by differential growth, and a molecular mechanism linking this stress to the regulation of growth in developing organs, these results confirm and extend the mechanical feedback hypothesis (Pan, 2016).

Scalloped and Yorkie are required in Drosophila for cell cycle re-entry of quiescent cells after tissue damage

Regeneration of damaged tissues typically requires a population of active stem cells. How damaged tissue is regenerated in quiescent tissues lacking a stem cell population is less well understood. This study used a genetic screen in the developing Drosophila melanogaster eye to investigate the mechanisms that trigger quiescent cells to re-enter the cell cycle and proliferate in response to tissue damage. Hippo signaling was found to regulate compensatory proliferation after extensive cell death in the developing eye. Scalloped and Yorkie, transcriptional effectors of the Hippo pathway, drive Cyclin E expression to induce cell cycle re-entry in cells that normally remain quiescent in the absence of damage. Ajuba, an upstream regulator of Hippo signaling that functions as a sensor of epithelial integrity, is also required for cell cycle re-entry. Thus, in addition to its well-established role in modulating proliferation during periods of tissue growth, Hippo signaling maintains homeostasis by regulating quiescent cell populations affected by tissue damage (Meserve, 2015).

Localization of Hippo signalling complexes and Warts activation in vivo

Hippo signalling controls organ growth and cell fate by regulating the activity of the kinase Warts. Multiple Hippo pathway components localize to apical junctions in epithelial cells, but the spatial and functional relationships among components have not been clarified, nor is it known where Warts activation occurs. This study reports that Hippo pathway components in Drosophila wing imaginal discs are organized into distinct junctional complexes, including separate distributions for Salvador, Expanded, Warts and Hippo. These complexes are reorganized on Hippo pathway activation, when Warts shifts from associating with its inhibitor Ajuba LIM protein (Jub) to its activator Expanded, and Hippo concentrates at Salvador sites. This study identify mechanisms promoting Warts relocalization, and using a phospho-specific antisera and genetic manipulations, where Warts activation occurs was identified: at apical junctions where Expanded, Salvador, Hippo and Warts overlap. These observations define spatial relationships among Hippo signalling components and establish the functional importance of their localization to Warts activation (Sun, 2015).

Wts is a key control point within the Hippo pathway, where multiple upstream regulatory processes converge. A fundamental gap in understanding of Hippo signal transduction has been the cellular location of Wts activation. This study established that Wts activation in wing disc epithelial cells occurs at sub-apical junctions where Hpo, Sav, Ex and Wts overlap. Co-recruitment of Hpo and Wts kinases to a common scaffold is implicated as a central feature of Hippo pathway activation, and this helps to explain why genes required for apical junctions and apical-basal polarity promote Hippo signalling and can act as tumour suppressors (Sun, 2015).

These studies indicate that a key step in Wts activation in disc epithelia is its relocalization from Jub to Ex. No special mechanism is needed to transport Wts from Jub to Ex, as Wts localization could simply be governed by equilibrium binding with a limited cytoplasmic pool. That is, if Wts normally binds relatively strongly to Jub, and relatively weakly to Ex, it could, depending on its concentration, accumulate at Jub sites but not at Ex sites. Expression of activated Yki induced a robust relocalization of Wts from Jub to Ex, and these studies identify three factors that contribute to the visible accumulation of Wts at Ex sites under these conditions. First, Yki activation appears to increase Hpo activity. It was also found that hpo RNAi suppresses the relocalization of Wts from Jub to Ex, and that increased Hpo activity promotes Ex-Wts binding, as assayed by co-immunoprecipitation experiments. These observations are consistent with the hypothesis that Wts shifts from Jub sites towards Ex sites due to an increased Ex-Wts binding affinity induced by Hpo. Second, Yki activation increases levels of Ex, which under equilibrium binding would also increase the recruitment of Wts to Ex sites. The relocalization of Wts back to adherens junctions in the absence of Ex indicates that the shift in Wts localization is Ex dependent, and implies that Jub and Ex can compete for association with Wts. A third factor that contributes to detection of Wts-Ex co-localization is the increase in Wts protein levels induced by activated Yki, which could lead to Wts concentrations high enough to bind even lower-affinity Ex sites, and indeed it was observed that simply overexpressing Wts was sufficient to induce Wts-Ex overlap, without removing Wts from adherens junctions where it co-localizes with Jub. It is suggested that an additional consequence of increased Wts levels that enables detection of Wts and pWts overlapping Ex could be a saturation of pWts removal. While at present this remains speculative, all signal transduction pathways require mechanisms to turn off after they have been activated, so there should exist mechanisms that either degrade or dephosphorylate pWts. Relatively low levels of pWts due to rapid turnover could also help explain why pWts was undetectable in wild-type wing discs (Sun, 2015).

The discovery of Ex-Wts binding, together with earlier studies that identified Ex-Hpo binding, implicate Ex as a scaffold that could promote Wts activation by co-localizing it with Hpo, and thus define a role for Ex distinct from previous suggestions that it functions as an activator of Hpo. Similarly, recent studies in cultured cell models showed that activated forms of Mer could bind Wts, and suggested a model in which Mer promotes Wts activation by recruiting it to membranes where it could be activated by Hpo. This suggests that in tissues where Mer, rather than Ex, plays key roles in Wts activation, such as glia, Mer, which can also associate with Hpo, through Sav, could play an analogous role in assembling a Wts activation complex. It is thus noteworthy that the best characterized upstream branches of Hippo signalling characterized in Drosophila (Fat, Ex and Mer) can all now be said to act principally at the levels of Wts regulation rather than Hpo regulation. Moreover, it is noted that Kibra, which has been suggested to act at a similar point in the Hippo pathway as Mer and Ex, has also been reported to be able to physically interact with both Hpo and Wts, and thus might also act principally as a scaffold that links them together rather than as a promoter of Hpo activation (Sun, 2015).

Indeed, external signals that impinge directly on Hpo activity have not yet been identified. The current discovery that Hpo localization to Sav is greatly increased by Yki activation reveals that regulators of Hpo localization exist, and implies that they are subject to negative feedback regulation downstream of Yki. As Hpo kinase activity can be promoted by Hpo dimerization, it is proposed that the increased recruitment of Hpo to Sav could elevate Hpo activity by increasing its local concentration, and thereby its dimerization. Relocalization of Hpo might also affect its interactions with kinases that can modulate Hpo activity. Recruitment of Hpo to Sav also concentrates Hpo near Ex, where it would more efficiently phosphorylate Ex-bound Wts. However, since most junctional Wts in disc epithelia is normally complexed with Jub rather than Ex, a mechanism-based solely on Hpo recruitment to apical junctions would not be expected to induce robust Wts activation. Importantly, then, these studies revealed that Hpo can increase Ex-Wts binding, possibly by phosphorylating Ex. Increased Ex-Wts binding would help recruit Wts to Ex, where it could then be phosphorylated by Hpo. Thus, it is now possible to suggest a sequential model for Hippo pathway activation in which Hpo is first recruited to membranes and activated, activated Hpo then phosphorylates Ex to recruit Wts and finally Hpo phosphorylates and activates Wts complexed with Ex. While further studies will be required to validate this model, it provides a framework that could guide future investigations, and these current studies clearly emphasize the importance of determining the in vivo localization of endogenous pathway components (Sun, 2015).

Cytoskeletal tension inhibits Hippo signaling through an Ajuba-Warts complex

Mechanical forces have been proposed to modulate organ growth, but a molecular mechanism that links them to growth regulation in vivo has been lacking. This study reports that increasing tension within the cytoskeleton increases Drosophila wing growth, whereas decreasing cytoskeletal tension decreases wing growth. These changes in growth can be accounted for by changes in the activity of Yorkie, a transcription factor regulated by the Hippo pathway. The influence of myosin activity on Yorkie depends genetically on the Ajuba LIM protein (jub), a negative regulator of Warts within the Hippo pathway. This study further shows that Jub associates with alpha-catenin and that its localization to adherens junctions and association with alpha-catenin are promoted by cytoskeletal tension. Jub recruits Warts to junctions in a tension-dependent manner. These observations delineate a mechanism that links cytoskeletal tension to regulation of Hippo pathway activity, providing a molecular understanding of how mechanical forces can modulate organ growth (Rauskolb, 2014).

Mechanical forces have long been known to modulate the proliferation of cultured cells in vitro and have received attention as an attractive mechanism for modulating organ growth in vivo. Moreover, increased stiffness is well known to correlate with tumor progression. Although progress has been reported in identifying components of growth regulatory pathways that could respond to mechanical force, understanding of how mechanical signals are integrated into growth regulatory pathways has remained poor. This study has documented an influence of cytoskeletal tension on wing growth in Drosophila and delineated a molecular pathway linking this cytoskeletal tension to the regulation of growth through inhibition of the Hippo pathway (Rauskolb, 2014).

Jub is identified as a protein regulated by mechanical tension, as its localization to foci at adherens junctions is elevated during normal development along compartment boundaries, which are sites of increased tension, and its localization to junctions can be increased or decreased by increasing or decreasing, respectively, Myo II activity. Because this localization requires α-catenin, which associates with Jub, and α-catenin has been identified as a mechanotransducer (Yonemura, 2010), it is proposed that this modulation of Jub localization occurs as a consequence of a tension-induced conformational change of α-catenin that increases its binding to Jub. α-catenin is well positioned to act as a mechanotransducer: it interacts with the β-catenin:E-cad complex, which is effectively anchored through binding E-cad in neighboring cells, and it also associates with the F-actin cytoskeleton, which could pull on α-catenin through myosin-mediated contraction (Yonemura, 2010). It is further proposed that association with α-catenin increases the binding of Jub to Wts, leading to an inhibitory recruitment of Wts to apical junctions and consequently increased Yki activity. This proposal is supported by the observations that Wts colocalizes with and physically associates with Jub in vivo, the promotion of this physical association by Myo II activity, and genetic and biochemical evidence that wts and jub are required for tension-dependent modulation of Yki activity (Rauskolb, 2014).

Cytoskeletal tension has previously been reported to increase YAP activity in cultured mammalian cells. The molecular mechanism that mediates this mechanoregulation of YAP is unknown but was determined to be independent of Hippo signaling, because it did not involve LATS (the mammalian homolog of Wts). This clearly distinguishes it from the Hippo-pathway-dependent mechanoregulation of Yki in wing discs that ia describe in this study. Among many differences in experimental conditions, it is note dthat experiments on cultured cells frequently involve manipulating cytoskeletal tension through cell-extracellular matrix attachment, whereas cytoskeletal tension at cell-cell attachments of epithelial cells was manipulated in these experiments. Whether this accounts for the distinct mechanisms involved remains to be determined, but just as there are multiple ligand-regulated biochemical signal transduction pathways that influence organ growth, there are likely also multiple mechanically-regulated pathways that influence organ growth. Indeed, other experimental regimes of altered attachment of cultured cells to matrix in vitro have been associated with LATS-dependent effects on YAP activity. Moreover, in Drosophila, increased accumulation of F-actin elevates Yki activity, also potentially consistent with mechanical regulation of Hippo signaling. However, it has recently been reported that F-actin levels modulate interaction between Wts and the Wts activator Merlin, and further studies are needed to address the mechanisms by which changes in F-actin levels associated with genetic or pharmacological manipulations impinge on Hippo signaling (Rauskolb, 2014).

Despite the crucial role of Wts as the key direct regulator of Yki within the Hippo pathway, the endogenous localization of Wts protein in vivo has not previously been characterized. The observations that Wts associates with Jub at cell junctions, and that this association is increased under conditions leading to elevated Yki activity, indicate that recruitment of Wts to junctions is associated with inhibition of Wts. Several positive upstream regulators of Wts also localize near cell junctions, and it was recently proposed that recruitment of Wts to the membrane is associated with Wts activation. However, as is shown in this study, at least some membrane-associated activators exhibit distinct localization profiles from Wts. These observations suggest that there could be distinct sites for Wts activation and Wts inhibition at apical junctions, with most junctional Wts localized to an inhibitory complex. It could be that only a small fraction of endogenous Wts is normally active or that Wts activation involves transient association of Wts with activators, as opposed to the stable association of Wts with Jub revealed by these studies (Rauskolb, 2014).

Whereas the results described in this study identify Jub as a key player in mechanoregulation of Hippo signaling, other recent studies have identified Jub, and two its mammalian homologs, LIMD1 and WTIP, as targets for cross-regulation of Hippo signaling by the EGFR and JNK signaling pathways (Reddy, 2013, Sun, 2013). These observations emphasize the importance of Ajuba family proteins as a key regulatory node within the Hippo signaling pathway and as a point of convergence between mechanical and biochemical signaling pathways. Indeed, organ growth in vivo must integrate multiple inputs. For example, the dorsal-ventral compartment boundary is not only a region of elevated cytoskeletal tension but is also, in late third-instar discs, a region of low cell proliferation, due to repression of cell proliferation downstream of Notch signaling. Thus, at late third instar, Notch activation might override or bypass the influence of tension on growth along the compartment boundary (Rauskolb, 2014).

The observation of Wts complexing with Jub at apical junctions has implications for how growth is controlled in developing tissues. These complexes are promoted by cytoskeletal tension, which provides a mechanism for tension-dependent regulation of growth. However, even under conditions of constant tension, an increase in the apical perimeter of cells at junctions could result in more of these Wts-inhibitory Jub complexes, whereas a decrease in apical perimeter could lead to fewer of these Wts-inhibitory complexes. This suggests the potential of shape-dependent regulation of Hippo-signaling, with cells of small apical perimeter having higher Hippo signaling and consequently reduced growth. Reduced apical area (and consequently perimeter) correlates with reduced cell proliferation, both in vivo and in cell culture models. Apical area is also expected to be affected by relative mechanical forces (e.g., how much is a cell stretched by its neighbors), and thus it is speculated that it could be used by cells to assess not only their own tension but also the mechanical environment in which they reside. Effects of tension on shape and the formation of junctional Wts-inhibitory complexes could thus provide a mechanism for cell density-dependent regulation of organ size (Rauskolb, 2014).

Zyxin links fat signaling to the hippo pathway

The Hippo signaling pathway has a conserved role in growth control and is of fundamental importance during both normal development and oncogenesis. Despite rapid progress in recent years, key steps in the pathway remain poorly understood, in part due to the incomplete identification of components. Through a genetic screen, this study identified the Drosophila Zyxin family gene, Zyx102 (Zyx), as a component of the Hippo pathway. Zyx positively regulates the Hippo pathway transcriptional co-activator Yorkie, as its loss reduces Yorkie activity and organ growth. Through epistasis tests, the requirement for Zyx was positioned within the Fat branch of Hippo signaling, downstream of Fat and Dco, and upstream of the Yorkie kinase Warts, and Zyx was found to be required for the influence of Fat on Warts protein levels. Zyx localizes to the sub-apical membrane, with distinctive peaks of accumulation at intercellular vertices. This partially overlaps the membrane localization of the myosin Dachs, which has similar effects on Fat-Hippo signaling. Co-immunoprecipitation experiments show that Zyx can bind to Dachs and that Dachs stimulates binding of Zyx to Warts. This study also extended characterization of the Ajuba LIM protein Jub and determined that although Jub and Zyx share C-terminal LIM domains, they regulate Hippo signaling in distinct ways. The results identify a role for Zyx in the Hippo pathway and suggest a mechanism for the role of Dachs: because Fat regulates the localization of Dachs to the membrane, where it can overlap with Zyx, it is proposed that the regulated localization of Dachs influences downstream signaling by modulating Zyx-Warts binding. Mammalian Zyxin proteins have been implicated in linking effects of mechanical strain to cell behavior. This identification of Zyx as a regulator of Hippo signaling thus also raises the possibility that mechanical strain could be linked to the regulation of gene expression and growth through Hippo signaling (Rauskolb, 2011).

This characterization of Zyx identifies a role for it as a novel and integral component of the Hippo pathway, which is required for the Fat branch, but not the Ex branch, of Hippo signaling. Unlike most previously identified components, loss of Zyx reduces the activity of the key transcriptional effector of the pathway, Yki, and consequently its loss reduces organ growth. Genetic epistasis experiments position the requirement for Zyx in between fat and wts, and concordant protein binding experiments identify a Dachs-stimulated ability of Zyx to bind Wts protein. In is inferred that this association of Zyx with Wts then downregulates Wts, at least in part, by targeting it for degradation (Rauskolb, 2011).

Zyx localizes to the sub-apical membrane independently of Fat or Dachs. Since Fat regulates the localization of Dachs, this regulated localization provides a mechanism by which Fat could modulate the interaction of Dachs with Zyx (although it is noted that Fat might affect the activity of Dachs in addition to affecting its localization). Since Dachs stimulates Zyx-Wts binding, this regulated localization provides a means for Fat signaling to modulate Zyx-Wts binding. It is inferred that Dachs effects a conformational change in Zyx, as in the absence of Dachs a Zyx LIM-domains polypeptide binds efficiently to Wts, whereas full-length Zyx binds poorly. Intriguingly, the association of vertebrate homologues of Zyx and Warts can also be post-translationally regulated, as the ability of the LIM domains of human LATS1 to bind Zyxin is masked within full-length Zyxin, but uncovered by Cdc2-mediated phosphorylation, presumably due to conformational change (Hirota, 2000). It is hypothesized that the ability of Dachs to bind to both the N-terminus and the LIM domains of Zyx enables it to effect a conformational change in Zyx, resulting in an open configuration that can bind to Wts. It is also possible that Dachs binding stimulates a post-translational modification of Zyx to induce a conformational change (Rauskolb, 2011).

Prior studies identified two mechanisms by which Fat signaling could influence Yki activity, as fat mutation reduces both the levels of Wts protein and the amount of Ex at the sub-apical membrane. It has not been possible to completely uncouple these two pathways for Fat-Hippo signaling, although the observation that over-expression of Wts can efficiently suppress fat overgrowth phenotypes, but only partially suppresses ex overgrowth phenotypes, suggested that the influence of Fat on Wts levels might be more critical. Analysis of the influence of Zyx on Ex is complicated by its influence on ex transcription, but the current observation that reduction of Zyx does not appear to suppress the influence of fat on Ex staining, even though it does suppress the influence of fat on Wts levels, also suggests that the influence of Fat on Wts levels might be more critical than its effects on Ex. Intriguingly, mutation of dachs did suppress the influence of fat on Ex levels. Although it is possible that this difference between dachs and Zyx results from technical differences in the experimental paradigms (e.g., mutant clones versus RNAi), it is also possible that dachs can influence Ex levels independently from its association with Zyx (Rauskolb, 2011).

The discovery of the Fat-specific effect on Wts levels, by contrast to the Hippo-pathway-mediated effect on Wts kinase activity, established the concept of distinct mechanisms for regulating Wts -- one mechanism that affects Wts levels and another that affects Wts activity. This study's identification of distinct genetic requirements for Zyx and Jub provide further support for this concept. As Jub is equally required for both Fat-Hippo and Ex-Hippo signaling and acts genetically between hippo and wts, Jub appears to inhibit Wts activation. In the current working model, the epistasis of Jub to fat could be explained by an increased activity of residual Wts, which then acts catalytically to repress Yki activity. Zyx is required for the influence of fat on Wts levels. It is noted that when measured within a whole tissue lysate, Wts levels are only reduced to approximately half their normal levels. However, as Wts appears to function within multi-protein complexes, including some components that can localize preferentially to the sub-apical membrane, it is hypothesized that Fat signaling affects a discrete pool of Wts within a complex at the membrane that is crucial for Hippo signaling, whereas there might be additional pools of Wts within the cell that are unaffected. It is also noted that while effects on Wts protein levels are clearly seen, the results do not exclude the possibility that Fat signaling also influences Wts activity (Rauskolb, 2011).

The characterization of Zyx and Jub also provides new tools for analyzing critical steps in Hippo signaling. For example, in addition to influencing Hpo and Wts kinase activity, it has been observed that Ex can bind directly to Yki and that when Ex is over-expressed it can repress Yki through a mechanism that involves direct sequestration of Yki, rather than regulation of Yki phosphorylation. Because this direct repression mechanism was based on over-expression experiments, the extent to which it contributes to normal Yki regulation in vivo remained uncertain. The observations that Jub acts genetically upstream of wts, yet is required for ex phenotypes, suggests that Ex regulates Yki principally through its effects on Wts activity, rather than through direct interaction with Yki (Rauskolb, 2011).

The ability of Zyx LIM domains to interact with Wts is conserved in their human homologues. Although the functional significance of this interaction in vertebrates has not yet been established, the observations raise the possibility that the oncogenic effects of human LPP mutations could be due to an ability of these aberrant LPP fusion proteins to negatively regulate LATS proteins, resulting in inappropriate activation of YAP or TAZ (Rauskolb, 2011).

One of the most intriguing aspects of Zyxin family proteins is their role in mediating effects of mechanical force on cell behavior (Hirata, 2008). Zyxin family proteins can localize to focal adhesions of cultured fibroblasts, and this localization is modulated by mechanical tension. The observation that increasing tension on stress fibers stimulates Zyxin accumulation at focal adhesions is intriguing in light of the observation that Zyx tends to accumulate at higher levels at intercellular vertices in imaginal discs, as these could be points of increased tension. As the association of unconventional myosins with F-actin can also be influenced by external force, this study's discovery of binding between a myosin protein (Dachs) and Zyx raises the possibility that other myosins might also interact with Zyxin family proteins, which could potentially influence either their tension-based recruitment or their activity (Rauskolb, 2011).

Finally, it is noted that theoretical models of growth control in developing tissues have proposed that growth should be controlled by mechanical tension, and direct evidence for mechanical effects on growth has been obtained in cultured cell models. However, a mechanism for how this might be achieved has been lacking. The discovery that Zyx, a member of a family of proteins implicated in responding to and transducing the effects of mechanical tension, is also a component of the Hippo signaling pathway, a crucial regulator of growth from Drosophila to humans, raises the intriguing possibility that Zyxin family proteins might form part of a molecular link between mechanical tension and the control of growth (Rauskolb, 2011).

Drosophila Ajuba is not an Aurora-A activator but is required to maintain Aurora-A at the centrosome

The LIM-domain protein Ajuba localizes at sites of epithelial cell-cell adhesion and has also been implicated in the activation of Aurora-A (Aur-A). Despite the expected importance of Ajuba, Ajuba-deficient mice are viable, which has been attributed to functional redundancy with the related LIM-domain protein LIMD1. To gain insights into the function of Ajuba, this study investigated its role in Drosophila, where a single gene (jub) encodes a protein closely related to Ajuba and LIMD1. A key function were identified in neural stem cells, where Jub localizes to the centrosome. In these cells, mutation in jub leads to centrosome separation defects and aberrant mitotic spindles, which is a phenotype similar to that of aur-A mutants. In jub mutants Aur-A activity is not perturbed, but that recruitment and maintenance at the centrosome is affected. As a consequence the active kinase is displaced from the centrosome. On the basis of studies in Drosophila neuroblasts, it is proposed that a key function of Ajuba, in these cells, is to maintain active Aur-A at the centrosome during mitosis (Sabino, 2010).

The regulation of kinase activity in time and space is crucial for the coordination of cellular events. Aurora-A (Aur-A), one of the three members of the Aurora family of kinases in mammals, is a serine/threonine kinase that functions as a key regulator of several events. The kinase Aur-A was first identified in Drosophila as a mitotic kinase. In flies, mutations in aur-a cause severe developmental defects and pleiotropic phenotypes, which include abnormal centrosome and spindle behavior, lack of astral microtubules (MTs), defects in chromosome segregation, spindle positioning, cortical targeting of cell fate determinants and neural stem-cell self-renewal (Sabino, 2010).

In vertebrate cells, Aur-A also plays a major role in mitosis, and recently an unexpected role for this kinase has been described in non-mitotic cells. Aur-A phosphorylates and activates the tubulin deacetylase HDAC-6 to promote disassembly of cilia and cell cycle re-entry. The large spectrum of functions attributed to the kinase Aur-A is thought to be, at least in part, regulated by different cofactors or activators. TPX2, a MT-associated protein (MAP), binds Aur-A, thereby promoting Aur-A autophosphorylation and targeting it to the mitotic spindle. Hef-1 (also known as Nedd9) binding and activation of Aur-A is required for HDAC-6 phosphorylation. In Drosophila, a single Aur-A activator, Bora, has been described so far. In bora mutants, defects in centrosome behavior and spindle assembly, together with defects in the asymmetric cell division of sensory organ precursors (SOPs), have been identified (Sabino, 2010).

Ajuba (Jub) is a LIM-domain protein that localizes at the sites of cell-cell adhesion in epithelial cells and has also been implicated in the activation of Aur-A. Surprisingly, however, Jub-deficient mice are viable; this has been attributed to functional redundancy with the related LIM-domain protein LIMD1. To gain insight into the function of Jub, its role was investigated in Drosophila, where a single gene encodes a protein closely related to mouse Jub and LIMD1. A mutation was generated in ajuba (jub) and jub mutants were found to die at the larval-pupal transition. No defects were detected in cell adhesion or epithelial polarity. However, a key function was detected in neural stem cells, where Jub localized to the centrosome. In these cells, mutation of jub led to centrosome separation defects and abnormal mitotic spindles. Surprisingly, It was found that in jub mutants Aur-A activity was not perturbed, but that Aur-A recruitment and maintenance at the centrosome was affected. As a consequence the active kinase was ectopically displaced into the cytoplasm, which resulted in abnormalities of the mitotic spindle. On the basis of these studies, it is proposed that a major function of Jub in Drosophila neuroblasts is to restrict active Aur-A to the centrosome during mitosis, but that Jub does not function as an Aur-A activator (Sabino, 2010).

This study generated mutations in the jub gene in Drosophila in order to examine its functions within an intact animal without the complications of potential redundancy with closely related genes. Unexpectedly, it was discovered that Jub had an essential role in just a subset of cells within the animal, namely the neural stem cells. Although not all cell types were exhaustively examined, cell cycles that are normally very sensitive to centrosome or MT perturbation, such as the nuclear divisions of the early embryo and the meiotic divisions of the male germline, occurred normally in the absence of Jub. Thus, Nbs are especially dependent on Jub to generate normal centrosomes and spindles, consistent with the clearly detectable levels of Jub-GFP on the centrosomes in these cells, but not in other cell types (Sabino, 2010).

Within the Nbs lacking Jub, three related, but distinct, phenotypes were detected: defects in the separation of centrosomes following mitosis, defects in spindle assembly, and defects in cortical targeting of determinants and orientation of the mitotic spindle. These phenotypes are shared by Nbs lacking Aur-A, consistent with previous work demonstrating that Jub and Aur-A proteins bind to each other and function together (Hirota, 2003). However, it was not possible to detect a biochemical interaction between these two proteins in Drosophila brains. In addition, the loss of Aur-A causes a number of additional defects that were not observed in jub mutant Nbs, such as defects in centrosome maturation and increased levels of genomic instability, demonstrating that Jub is not required for the majority of Aur-A functions (Sabino, 2010).

It is worth mentioning that no Nbs were obserged with supernumerary centrosomes in jub mutants. Defects in centrosome separation should result in the generation of daughter cells without centrosomes (which was see in 10% of the cells in jub mutant brain cells) and in those with two centrosomes, which should undergo duplication during the following cell cycle to produce extra centrosomes. Future work will be required to explain the absence of Nbs with supernumerary centrosomes (Sabino, 2010).

The results show that, in the absence of Jub, Aur-A is not as concentrated at the centrosome, and hence Tacc recruitment is affected. However, even in the absence of Jub, Tacc (transforming acidic coiled-coil protein), a MT-associated protein, can be phosphorylated by Aur-A, which further supports the idea that Jub is not an Aur-A activator, at least in Drosophila. Furthermore, it appears that loss of Jub results in a displacement of Aur-A from the centrosome. Thus, the key question is whether the defects caused by loss of Jub are due to diminished Aur-A activity on the centrosome, elevation of activity in the cytoplasm or both. The defects in centrosome separation, just after cell division, might be due to diminished levels of Aur-A on the centrosome, whereas the loss of astral MTs might be explained by diminished levels of P-Tacc or other MAPs. The manipulation of Aur-A levels in the presence or absence of Jub suggests that it is the elevated cytoplasmic Aur-A activity that is causing the defects in spindle assembly. Elevated cytoplasmic Aur-A is also likely to account for the defects in spindle positioning during asymmetric cell division (Sabino, 2010).

One possible explanation for the Nb-specific requirement for Jub is that the centrosome cycle is significantly different in these cells. In Nbs, just after centrosome duplication and migration to the apical cortex, one of the centrosomes moves away from the other. This dynamic centrosome continues to move throughout the S and G2 phases, which means that centrosome separation in Nbs takes place substantially before mitosis, in contrast with the timing in other cell types. It is therefore possible that Jub is only required for centrosome separation in cells where centrosomes separate earlier in the cell cycle, substantially before the following mitosis, when Aur-A activity is still present (Sabino, 2010).

Finally, no evidence has bee obtained to support a role for Jub as an Aur-A activator, since no reduction was seen in the phosphorylation of the Aur-A substrate Tacc. Many cell types require Aur-A function, including embryos and male spermatocytes. No Jub-GFP was observed at the centrosome in these cells and no jub mutant phenotypes were seen in the early embryo or male germline. In addition, no co-immunoprecipitate was seem of Jub and Aur-A in brain extracts. It is therefore proposed that the main function of Jub is to bind Aur-A at the centrosome, not to activate the kinase, but rather to restrict its activity in time and space. Too much active Aur-A in the cytoplasm during mitosis seems to perturb astral MT nucleation and centrosomal spindle assembly. Alternatively, Jub might also help to recruit and/or maintain Aur-A at the centrosome, so that it can be activated by another protein concentrated there, and, most crucially in Nbs, ‘hold’ the active Aur-A away from the cytoplasm. Unfortunately, such a candidate protein has not yet been identified in flies. Flies do not have an obvious TPX2 orthologue and the only Aur-A activator identified so far lacks a function in the fly brain. The failure of Jub to regulate Aur-A in Drosophila could also just reflect differences in the way Aur-A is regulated between vertebrates and invertebrates. In human cells, Jub is also associated with kinetochores and spindle MTs, and it has been shown that Jub, together with BubR1 and Aurora B, plays a role in the regulation of the metaphase-to-anaphase transition. However, the lack of a mitotic phenotype in Jub-knockout mice also strongly suggests that it might not play an essential role in Aur-A activation (Sabino, 2010).

The force-sensitive protein Ajuba regulates cell adhesion during epithelial morphogenesis

The reorganization of cells in response to mechanical forces converts simple epithelial sheets into complex tissues of various shapes and dimensions. Epithelial integrity is maintained throughout tissue remodeling, but the mechanisms that regulate dynamic changes in cell adhesion under tension are not well understood. In Drosophila melanogaster, planar polarized actomyosin forces direct spatially organized cell rearrangements that elongate the body axis. The LIM-domain protein Ajuba is recruited to adherens junctions in a tension-dependent fashion during axis elongation. Ajuba localizes to sites of myosin accumulation at adherens junctions within seconds, and the force-sensitive localization of Ajuba requires its N-terminal domain and two of its three LIM domains. This study demonstrates that Ajuba stabilizes adherens junctions in regions of high tension during axis elongation, and that Ajuba activity is required to maintain cell adhesion during cell rearrangement and epithelial closure. These results demonstrate that Ajuba plays an essential role in regulating cell adhesion in response to mechanical forces generated by epithelial morphogenesis (Razzell, 2018).

LIM Protein Ajuba associates with the RPA complex through direct cell cycle-dependent interaction with the RPA70 subunit

DNA damage response pathways are essential for genome stability and cell survival. Specifically, the ATR kinase is activated by DNA replication stress. An early event in this activation is the recruitment and phosphorylation of RPA, a single stranded DNA binding complex composed of three subunits, RPA70, RPA32 and RPA14. Previous work has shown that the LIM protein Ajuba associates with RPA and that depletion of Ajuba leads to potent activation of ATR. This study, provides evidence that the Ajuba-RPA interaction occurs through direct protein contact with RPA70 and that their association is cell cycle-regulated and is reduced upon DNA replication stress. A model in which Ajuba negatively regulates the ATR pathway by directly interacting with RPA70, thereby preventing inappropriate ATR activation. These results provide a framework to further understanding of the mechanism of ATR regulation in human cells in the context of cellular transformation (Fowler, 2018).

The Ajuba family protein Wtip regulates actomyosin contractility during vertebrate neural tube closure

Ajuba family proteins are implicated in the assembly of cell junctions and have been reported to antagonize Hippo signaling in response to cytoskeletal tension. To assess the role of these proteins in actomyosin contractility, the localization and function of Wtip, a member of the Ajuba family, was examined in Xenopus early embryos. Targeted in vivo depletion of Wtip inhibited apical constriction in neuroepithelial cells and elicited neural tube defects. Fluorescent protein-tagged Wtip showed predominant punctate localization along the cell junctions in the epidermis and a linear junctional pattern in the neuroectoderm. In cells undergoing Shroom3-induced apical constriction, the punctate distribution was reorganized into a linear pattern. Conversely, the linear junctional pattern of Wtip in neuroectoderm changed to a more punctate distribution in cells with reduced myosin II activity. The C-terminal fragment of Wtip physically associated with Shroom3 and interfered with Shroom3 activity and neural fold formation. It is therefore proposed that Wtip is a tension-sensitive cytoskeletal adaptor that regulates apical constriction during vertebrate neurulation (Chu, 2018).

The scaffold protein Ajuba suppresses CdGAP activity in epithelia to maintain stable cell-cell contacts

Levels of active Rac1 at epithelial junctions are partially modulated via interaction with Ajuba, an actin binding and scaffolding protein. This study demonstratest hat Ajuba interacts with the Cdc42 GTPase activating protein CdGAP, a GAP for Rac1 and Cdc42, at cell-cell contacts. CdGAP recruitment to junctions does not require Ajuba; rather Ajuba seems to control CdGAP residence at sites of cell-cell adhesion. CdGAP expression potently perturbs junctions and Ajuba binding inhibits CdGAP activity. Ajuba interacts with Rac1 and CdGAP via distinct domains and can potentially bring them in close proximity at junctions to facilitate activity regulation. Functionally, CdGAP-Ajuba interaction maintains junctional integrity in homeostasis and diseases: (1) gain-of-function CdGAP mutants found in Adams-Oliver Syndrome patients strongly destabilize cell-cell contacts and (2) CdGAP mRNA levels are inversely correlated with E-cadherin protein expression in different cancers. This study presenta conceptual insights on how Ajuba can integrate CdGAP binding and inactivation with the spatio-temporal regulation of Rac1 activity at junctions. Ajuba provides a novel mechanism due to its ability to bind to CdGAP and Rac1 via distinct domains and influence the activation status of both proteins. This functional interplay may contribute towards conserving the epithelial tissue architecture at steady-state and in different pathologies (McCormack, 2017).

AJUBA LIM proteins limit Hippo activity in proliferating cells by sequestering the Hippo core kinase complex in the cytosol

The Hippo pathway controls organ growth and is implicated in cancer development. Whether and how Hippo pathway activity is limited to sustain or initiate cell growth when needed is not understood. The members of the AJUBA family of LIM proteins are negative regulators of the Hippo pathway. In mammalian epithelial cells, this study found that AJUBA LIM proteins limit Hippo regulation of YAP, in proliferating cells only, by sequestering a cytosolic Hippo kinase complex in which LATS kinase is inhibited. At the plasma membranes of growth-arrested cells, AJUBA LIM proteins do not inhibit or associate with the Hippo kinase complex. The ability of AJUBA LIM proteins to inhibit YAP regulation by Hippo and to associate with the kinase complex directly correlate with their capacity to limit Hippo signaling during Drosophila wing development. AJUBA LIM proteins did not influence YAP activity in response to cell-extrinsic or cell-intrinsic mechanical signals. Thus, AJUBA LIM proteins limit Hippo pathway activity in contexts where cell proliferation is needed (Jagannathan, 2016).

The results indicate that the AJUBA LIM proteins limit Hippo pathway-mediated YAP inactivation in proliferating cells. In growth-arrested cells, CIP-mediated Hippo activation is not inhibited by the presence of AJUBA LIM proteins. The data suggest a model whereby AJUBA LIM proteins inhibit Hippo core kinase complex activation of YAP in proliferating cells by sequestering the Hippo core kinase complex, including LATS kinase, in the cytosol and inhibiting activation of LATS kinase. At the plasma membrane, where LATS kinases are thought to be activated by the Hippo core kinase complex, AJUBA LIM proteins do not associate with the Hippo core kinase complex or LATS kinases and do not inhibit Hippo pathway-mediated YAP regulation. If a primary function of AJUBA LIM proteins is to limit Hippo pathway inactivation of YAP/TAZ in proliferating cells, then this could explain why Jub is required for Drosophila embryo development, a state of high cell proliferation and organ growth when YAP transcriptional activity should be high and Hippo pathway inhibition of YAP low. In the absence of Jub in Drosophila, Hippo activity would be unrestrained, Yki would be inhibited, and cells would cease to proliferate and undergo apoptosis. In support of this, in mammalian cells, it was not possible to RNAi deplete all three AJUBA LIM proteins, as they would undergo apoptosis whenever the third family member was depleted (Jagannathan, 2016).

In contrast to other studies, this study found that in mammalian cells, the AJUBA LIM proteins did not affect YAP regulation in response to mechanical signals. Since AJUBA LIM proteins regulate YAP by inhibiting activation of LATS kinases by the Hippo core kinase complex, this could be a reflection of LATS-independent regulation of YAP in response to mechanical signals, but in other studies, morphological manipulation of single cells affected YAP regulation in a LATS-dependent manner. When this study likewise manipulated single cells (i.e., intracellular tension), AJUBA LIM proteins did not affect YAP nuclear/cytoplasmic distribution or transcriptional activity in response to changes in intracellular tension. This suggests that LATS activation in response to mechanical signals in mammalian cells, a process that is not fully understood, is not influenced by the AJUBA LIM proteins. Furthermore, if AJUBA LIM proteins affect only Hippo-dependent activation of LATS, then this result would be consistent with the model of mechanical activation of LATS independent of the Hippo core kinase complex (Jagannathan, 2016).

During Drosophila wing development, genetic experiments have shown that Jub influences tension-dependent Yki-mediated wing growth. There, it is argued that Jub does so by recruiting Wts (LATS) to the cell junction in a tension-dependent manner. This study also observed that the presence of LIMD1 and AJUBA influenced the recruitment of LATS1 to cell-cell junctions in a confluent mammalian epithelium, but in confluent epithelia undergoing CIP, no inhibition of LATS kinases by AJUBA LIM proteins was observed, nor did AJUBA LIM proteins associate with LATS kinases or the Hippo core kinase complex despite their presence at cell-cell junctions. Moreover, forced recruitment of LATS2 to the plasma membrane by the mp-MOB1A mutant did not recruit LIMD1 to the plasma membrane and LATS activation was not inhibited, nor did LIMD1 associate with LATS2 or the Hippo core kinase components (Jagannathan, 2016).

In MCF10A cells, cyclic or static stretch was found to activate YAP as a result of Hippo pathway inhibition. There, JNK activation, downstream of a stretch signal, phosphorylated LIMD1, which enhanced its interaction with and inhibition of LATS kinase. In this work, RNAi depletion of LIMD1 alone was sufficient to produce an effect. In these experiments, no effects were seen when any single AJUBA LIM protein was depleted in MCF10A cells. Since AJUBA, LIMD1, and trace amounts of WTIP (the AJUBA family LIM proteins) are present in MCF10A cells, depletion of at least two (AJUBA and LIMD1) was required to observe any effect of this family of proteins upon Hippo pathway activation of LATS. Furthermore, AJUBA-/- and LIMD1-/- mice have minimal developmental or adult phenotypes unless stressed. This study did not assess stretch as a mechanical stimulus in these studies, however. Thus, it is possible that stretch signals versus exposure to a stiff ECM activate distinct mechanotransduction pathways and that stretch-activated pathways are more sensitive to AJUBA LIM protein levels (Jagannathan, 2016).

AJUBA LIM proteins and the Hippo core kinase components are both recruited to sites of cell contact, yet no association was detected between them at this site. Possibly, they are recruited to different cell contact components. AJUBA LIM proteins interact with α-catenin bound to E-cadherin at AJ. The ERM protein NF2 (Mer), which associates with the apical membrane protein Crumbs, recruits LATS (Wts) to the plasma membrane. AJUBA LIM proteins do not associate with Mer, Ex, or Kibra. Therefore, physical separation of AJUBA LIM proteins and Hippo core kinase components at cell-cell contacts possibly preclude an association and inhibition. Interestingly, in the absence of AJUBA and LIMD1, endogenous LATS1 was not recruited to the cell surface despite the presumed presence of NF2. Perhaps AJUBA LIM proteins serve to facilitate delivery of LATS to NF2 at the cell surface. This observation also suggests the possibility that LATS could be activated without plasma membrane recruitment. The possibility cannot be excluded that transient recruitment of LATS to the cell surface still occurs, through NF2 for example, and that this may be enough for its activation and phosphorylation of YAP. Despite the significant change in membrane recruitment of LATS1 in confluent epithelia lacking AJUBA and LIMD1, YAP was still inactivated. Perhaps this reflects Hippo-independent or actin-mediated regulation of YAP in this setting (Jagannathan, 2016).

Other signals could regulate the association of AJUBA LIM proteins with LATS and the Hippo core kinase complex. MAPK can phosphorylate Drosophila Jub and increase its association with Wts in S2 cells, for example, and in mammalian cells, overexpression of AJUBA increases MAPK activity. In the context of tissue repair, JNK can also phosphorylate AJUBA, and this increases its association with LATS. In light of these findings, AJUBA LIM proteins are possibly phosphorylated in the cytosol and not at adherens junctions. In the absence of phosphorylation, they do not interact with the Hippo core kinase complex (Jagannathan, 2016).

In Drosophila, AJUBA LIM protein-mediated regulation of the Hippo pathway is critical for wing development. The determination of organ size, such as the fly wing and fly eye and the mammalian liver, is a cell intrinsic property involving the Hippo pathway, as shown by organ-specific transgenic models. The current work suggests that the AJUBA LIM proteins limit Hippo pathway activity only in proliferating cells. If so, then they may be critical to tune the proliferative response of cells during organ development, repair of injured tissue, and early cancer development (Jagannathan, 2016).

Regulation of YAP by mechanical strain through Jnk and Hippo signaling

Experiments conducted mainly on cultured cells have established that altering mechanical forces influences cell behaviors, including migration, differentiation, apoptosis, and proliferation. The transcriptional coactivator YAP has been identified as a nuclear relay of mechanical signals, but the molecular mechanisms that lead to YAP activation were not identified. YAP is the main transcriptional effector of the Hippo signaling pathway, a major growth regulatory pathway within metazoa, but at least in some instances, the influence of mechanical strain on YAP was reported to be independent of Hippo signaling. This study identified a molecular pathway that can promote the proliferation of cultured mammary epithelial cells in response to cyclic or static stretch. These mechanical stimuli are associated with increased activity of the transcriptional coactivator YAP, which is due at least in part to inhibition of Hippo pathway activity. Much of this influence on Hippo signaling can be accounted for by the activation of c-Jun N-terminal kinase activity by mechanical strain and subsequent inhibition of Hippo signaling by JNK. LATS1 (see Drosophila Warts) is a key negative regulator of YAP within the Hippo pathway, and this study further shows that cyclic stretch is associated with a JNK-dependent increase in binding of a LATS inhibitor, LIMD1 (Drosophila homolog: Ajuba LIM protein), to the LATS1 kinase and that reduction of LIMD1 expression suppresses the activation of YAP by cyclic stretch. Together, these observations establish a pathway for mechanical regulation of cell proliferation via JNK-mediated inhibition of Hippo signaling (Codelia, 2014).

The LIM protein Ajuba is recruited to cadherin-dependent cell junctions through an association with alpha-catenin

Cell-cell adhesive events affect cell growth and fate decisions and provide spatial clues for cell polarity within tissues. The complete molecular determinants required for adhesive junction formation and their function are not completely understood. LIM domain-containing proteins have been shown to be present at cell-cell contact sites and are known to shuttle into the nucleus where they can affect cell fate and growth; however, their precise localization at cell-cell contacts, how they localize to these sites, and what their functions are at these sites is unknown. This study shows that, in primary keratinocytes, the LIM domain protein Ajuba is recruited to cadherin-dependent cell-cell adhesive complexes in a regulated manner. At cadherin adhesive complexes Ajuba interacts with alpha-catenin, and alpha-catenin is required for efficient recruitment of Ajuba to cell junctions. Ajuba also interacts directly with F-actin. Keratinocytes from Ajuba null mice exhibit abnormal cell-cell junction formation and/or stability and function. These data reveal Ajuba as a new component at cadherin-mediated cell-cell junctions and suggest that Ajuba may contribute to the bridging of the cadherin adhesive complexes to the actin cytoskeleton and as such contribute to the formation or strengthening of cadherin-mediated cell-cell adhesion (Marie, 2003).

Search PubMed for articles about Drosophila Ajuba

Alegot, H., Markosian, C., Rauskolb, C., Yang, J., Kirichenko, E., Wang, Y. C. and Irvine, K. D. (2019). Recruitment of Jub by alpha-catenin promotes Yki activity and Drosophila wing growth. J Cell Sci. PubMed ID: 30659113

Chu, C. W., Xiang, B., Ossipova, O., Ioannou, A. and Sokol, S. Y. (2018). The Ajuba family protein Wtip regulates actomyosin contractility during vertebrate neural tube closure. J Cell Sci 131(10). PubMed ID: 29661847

Codelia, V. A., Sun, G. and Irvine, K. D. (2014). Regulation of YAP by mechanical strain through Jnk and Hippo signaling. Curr Biol 24: 2012-2017. PubMed ID: 25127217

Fowler, S., Maguin, P., Kalan, S. and Loayza, D. (2018). LIM Protein Ajuba associates with the RPA complex through direct cell cycle-dependent interaction with the RPA70 subunit. Sci Rep 8(1): 9536. PubMed ID: 29934626

Jagannathan, R., Schimizzi, G. V., Zhang, K., Loza, A. J., Yabuta, N., Nojima, H. and Longmore, G. D. (2016). AJUBA LIM proteins limit Hippo activity in proliferating cells by sequestering the Hippo core kinase complex in the cytosol. Mol Cell Biol 36(20): 2526-2542. PubMed ID: 27457617

Marie, H., Pratt, S. J., Betson, M., Epple, H., Kittler, J. T., Meek, L., Moss, S. J., Troyanovsky, S., Attwell, D., Longmore, G. D. and Braga, V. M. (2003). The LIM protein Ajuba is recruited to cadherin-dependent cell junctions through an association with alpha-catenin. J Biol Chem 278(2): 1220-1228. PubMed ID: 12417594

McCormack, J. J., Bruche, S., Ouadda, A. B. D., Ishii, H., Lu, H., Garcia-Cattaneo, A., Chavez-Olortegui, C., Lamarche-Vane, N. and Braga, V. M. M. (2017). The scaffold protein Ajuba suppresses CdGAP activity in epithelia to maintain stable cell-cell contacts. Sci Rep 7(1): 9249. PubMed ID: 28835688

Meserve, J. H. and Duronio, R. J. (2015). Scalloped and Yorkie are required in Drosophila for cell cycle re-entry of quiescent cells after tissue damage. Development [Epub ahead of print]. PubMed ID: 26160905

Pan, Y., Heemskerk, I., Ibar, C., Shraiman, B. I. and Irvine, K. D. (2016). Differential growth triggers mechanical feedback that elevates Hippo signaling. Proc Natl Acad Sci U S A 113(45): E6974-E6983. PubMed ID: 27791172

Rauskolb, C., Pan, G., Reddy, B. V., Oh, H. and Irvine, K. D. (2011). Zyxin links fat signaling to the hippo pathway. PLoS Biol 9: e1000624. PubMed ID: 21666802

Rauskolb, C., Sun, S., Sun, G., Pan, Y. and Irvine, K. D. (2014). Cytoskeletal tension inhibits Hippo signaling through an Ajuba-Warts complex. Cell 158: 143-156. PubMed ID: 24995985

Razzell, W., Bustillo, M. E. and Zallen, J. A. (2018). The force-sensitive protein Ajuba regulates cell adhesion during epithelial morphogenesis. J Cell Biol 217(10): 3715-3730. PubMed ID: 30006462

Reddy, B. V. and Irvine, K. D. (2013). Regulation of Hippo signaling by EGFR-MAPK signaling through Ajuba family proteins. Dev Cell 24: 459-471. PubMed ID: 23484853

Sabino, D., Brown, N. H. and Basto, R. (2010). Drosophila Ajuba is not an Aurora-A activator but is required to maintain Aurora-A at the centrosome. J. Cell Science 124: 1156-1166. PubMed Citation: 21402878

Sarpal, R., Yan, V., Kazakova, L., Sheppard, L., Yu, J. C., Fernandez-Gonzalez, R. and Tepass, U. (2019). Role of alpha-Catenin and its mechanosensing properties in regulating Hippo/YAP-dependent tissue growth. PLoS Genet 15(11): e1008454. PubMed ID: 31697683

Sun, G. and Irvine, K. D. (2013). Ajuba family proteins link JNK to Hippo signaling. Sci Signal 6(292): ra81. PubMed ID: 24023255

Sun, S., Reddy, B. V. and Irvine, K. D. (2015). Localization of Hippo signalling complexes and Warts activation in vivo. Nat Commun 6: 8402. PubMed ID: 26420589

Yonemura, S., Wada, Y., Watanabe, T., Nagafuchi, A. and Shibata, M. (2010). alpha-Catenin as a tension transducer that induces adherens junction development. Nat Cell Biol 12(6): 533-542. PubMed ID: 20453849

date revised: 12 August 2023

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