Transcriptional Regulation

The proventriculus is a multiply folded muscular organ of the foregut formed from a simple epithelial tube, whose function is grinding and masticating food. Coordinated cell movements are critical for tissue and organ morphogenesis in animal development. Drosophila genes hedgehog and wingless, which encode signaling molecules, and the gene myospheroid, which encodes a beta subunit of the integrins, are required for epithelial morphogenesis during proventriculus development. In contrast, this morphogenetic process is suppressed by the decapentaplegic gene (Pankratz, 1995).

Apterous plays a role controlling patterns of gene expression in the developing wing disc. The PS1 and PS2 integrins are normally expressed in primarily dorsal-specific and ventral-specific patterns, respectively. Ectopic expression of apterous induces ectopic ventral expression of alphaPS1 mRNA and PS1 integrin while loss of apterous can induce the ectopic dorsal expression of PS2 integrin. Thus, apterous plays a selector-like role both in terms of the control of lineage restrictions and the regulation of downstream gene expression (Blair, 1994).

There is a genetic interaction between blistered/BSRF and integrin genes inflated and myospheroid suggesting that blistered/DSRF might regulate integrin expression. There is an increased frequency and severity of blisters in progeny when mutant blistered males are crossed to females carrying if or mys. For the most part blistered/integrin combinations do not affect venation even when the blisters are very large (Fristrom, 1994).

Spatial regulation of cell adhesion in the Drosophila wing is mediated by Delilah, a potent activator of βPS integrin expression

In spite of conceptual views of how differential gene expression is used to define different cell identities, it is still not understood how different cell identities are translated into actual cell properties. The fly wing is composed of two main cell types, vein and intervein. These two types differ in many features, including their adhesive properties. One of the major differences is that intervein cells express integrins, which are required for the attachment of the two wing layers to each other, whereas vein cells are devoid of integrin expression. The major signaling pathways that divide the wing to vein and intervein domains have been characterized. However, the genetic programs that execute these alternative differentiation programs are still very roughly drawn. This study identifies the bHLH protein Delilah (Dei) as a mediator between signaling pathways that specify intervein cell-fate and one of the most significant realizators of this fate, βPS integrin. Dei's expression is restricted to intervein territories where it acts as a potent activator of βPS integrin expression. In the absence of normal Dei activity the level of βPS integrin is reduced, leading to a failure of adhesion between the dorsal and ventral wing layers and a consequent formation of wing blisters. The effect of Dei on βPS expression is not restricted to the wing, suggesting that Dei functions as a general genetic switch, which is turned on wherever a sticky cell-identity is determined and integrin-based adhesion is required (Egoz-Matia, 2011).

This study has identified the bHLH transcription factor Dei as an important positive regulator of the expression of βPS, the major β subunit in Drosophila. During embryonic development Dei's expression is confined mainly to cells that adhere strongly to other cells and are able to withstand mechanical strain, for instance, tendon cells that attach body wall muscle to the cuticle. Moreover, when different types of cells arise from within a uniform cell population, or through asymmetric cell division, Dei's expression is restricted to the ‘stickier’ types of cells. For example, in the chordotonal organ lineage, Dei is expressed in the four types of support cells (cap, ligament, cap-attachment and ligament-attachment), but is excluded from the neuron and glia. Similar phenomenon is seen in the developing wing where Dei is expressed only in intervein territories, where the ventral and dorsal layers adhere to each other, and is not expressed in vein cells that do not adhere to cells of the opposite layer. In all these systems, Dei does not function as a selector of cell identity, but it is required to realize the selected fate by activating a developmental program that specifies adhesive properties of cells (Egoz-Matia, 2011).

Although dei's expression has not been characterized in all developmental stages and tissues, published data of various microarray analyses suggest that dei is expressed in other developmental and physiological contexts where up-regulation of βPS integrin is required. For example, dei was up-regulated when larvae were exposed to immune challenge, or when mutant larvae exhibited an increase in lamellocyte cell population. Lamellocytes represent a subset of hemocytes in Drosophila, which differentiate in response to specific immune challenge. The lamellocytes aggregate around large pathogens to form a rigid laminated capsule that confines the pathogen and enables its elimination. This encapsulation process requires members of the integrin family that presumably mediate the lamellocyte's attachment (Egoz-Matia, 2011).

This work focused mainly on the role of dei in intervein cells and showed that dei provides a missing link between the genetic specification of these epithelial cells and their differentiation. The data place dei downstream to the major signaling pathways that divide the wing to regions of veins and interveins and downstream to Bs, which works as a selector of intervein identity. It remains to be determined whether dei is a direct target of Bs, and whether it is a direct regulator of βPS, however the results of the rescue experiment suggest that the effect of Bs on integrin expression is mediated, at least in part, by the activity of Dei (Egoz-Matia, 2011).

The venation phenotypes caused by weak dei alleles could be also attributed to the effects of Dei on βPS expression. Even though vein and intervein territories are established during early stages of wing development, the decision remains plastic for at least 24-h APF. Maintenance of the right fates depends on both vein-specific and intervein-specific genes. Appropriate levels of integrin expression are required for the maintenance of intervein fate, as suggested by the ectopic vein phenotype of certain mys alleles, which is very similar to the venation phenotype of weak dei alleles (Egoz-Matia, 2011).

It is reasonable to assume that Dei regulates multiple target genes in different cells and tissues. However, as for integrins, Dei regulates specifically βPS integrin. No evidence was found for regulation of αPS1 or αPS2, which are expressed differentially in the two wing layers, by Dei. Since βPS is the dimerization partner of both αPS1 and αPS2, by regulating its expression Dei practically affects all integrin-based adhesion processes at both the dorsal and ventral wing layers. The data also suggest that the effects of Dei on integrin-dependent adhesion are not restricted to the wing. Ectopic expression of Dei led to up-regulation of βPS expression in embryonic tissues, whereas loss of Dei's activity caused a reduction in the level of βPS expression in the cone cells of the eye (Egoz-Matia, 2011).

In summary, Dei is thought of as a general switch that turns on βPS integrin expression wherever a sticky cell has to develop. Since such a switch needs to be turned on in different tissues and different developmental and physiological contexts, it is predicted that the dei gene can respond to various signaling pathways and transcription factors. Indeed, analysis of the regulatory region of the dei locus demonstrated that it harbors multiple regulatory modules that respond to different transcription factors working in different developmental contexts (Egoz-Matia, 2011).

Analysis of integrin turnover in fly myotendinous junctions

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).

Phosphoinositide regulation of integrin trafficking required for muscle attachment and maintenance

Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. This study showa that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. These results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease (Ribeiro, 2011).

mtm regulates integrin adhesions in muscle and in the developing wing, and integrin localization was found to be disrupted in human XLMTM, pointing to a central role for Mtm/MTM1 in a trafficking pathway important for localization of β-integrin at the plasma membrane. It is well-established that integrin turnover contributes to cell motility, whereby targeted integrin recycling and reassembly of localized adhesions mediate polarized matrix attachments and signaling responses. The current results reveal that regulated integrin turnover is also important for integrin adhesions in non-motile myofibers, after the establishment of attachments. Importantly, mtm disruption uncovered a demand for βPS-integrin trafficking in the maintenance of adhesions both at MTJs as well as at costameres, a less-understood adhesion site with putative roles in muscle integrity, mechanotransduction, and myofibril assembly. Costameres are associated with repeating sarcomeric Z-lines attach peripheral myofibrils to the extracellular matrix. Although integrin was destabilized at larval MTJs in mtm mutants, the most severe consequences occurred later with specific loss of pupal or adult mtm function during developmental myofiber remodeling or adult muscle use, respectively. This is consistent with costamere sensitivity to integrin depletion in adult muscle and the possibility that mtm similarly regulates integrin turnover with myofiber remodeling that occurs both in development and with demands in adult muscle growth, repair and aging (Ribeiro, 2011).

In fly macrophages, Class II Pi3K68D and mtm co-depletion could revert both an imbalance in PI(3)P and defects in cortical remodeling that impaired macrophage shape and in vivo immune cell distribution. This study found Pi3K68D disruption is also a specific and potent suppressor of integrin adhesion defects in mtm-depleted muscle. Despite distinct macrophage and myofiber morphology and function, a shared requirement for a PI3KC2/Mtm pathway highlights common functions during cellular remodeling. Loss of Mtm phosphatase activity could be considered a gain of function condition, analogous to ectopic kinase activity, leading to inappropriate phosphoinositide accumulation. In line with this, either mtm depletion or Pi3K68D overexpression disrupted integrin adhesion in the fly wing, presumably through imbalanced responses to an accumulation of the same phosphoinositide pool. PI3KC2 and Mtm family members in vertebrates have been associated with antagonistic functions related to regulation of traffic to the plasma membrane. PI3KC2 isoforms are required to promote while overexpression of MTM1 impairs GLUT4 trafficking and integrin-mediated cell motility. Together, the observations point to a broad and conserved relationship for PI3KC2/Mtm co-regulation at the plasma membrane (Ribeiro, 2011).

How might PI3KC2 and Mtm co-regulate integrin trafficking? One possibility is that the cycle of phosphoinositides co-regulated by PI3KC2/Mtm tunes the balance between endocytic-exocytic flux. The strong genetic interaction between mtm and Pi3K68D, in conjunction with Pi3KC2 ability to create PI(3)P in vivo, supports the possibility that Pi3K68D could generate a PI(3)P substrate pool acted on by Mtm phosphatase. Alternatively, Pi3K68D could act more distantly on an interrelated phosphoinositide pool. It is envisioned that Pi3K68D mediates early endocytic trafficking, tethering or sorting of integrin-containing vesicles. The integrin detected on large inclusions in mtm-depleted and XLMTM muscles in flies and humans, respectively, and evidence that mtm promotes membrane tubulation from PI(3)P compartments, point to an Mtm/MTM1 role in membrane efflux for delivery of integrin to the plasma membrane. Mtm phosphatase could act to promote recycling or to negatively regulate retention, for example, through a PI(3)P-mediated fusion of integrin-containing vesicles with endosomes-lysosomes. An accumulation of β1-integrin on enlarged, perinuclear compartments has been observed with certain genetic manipulations in non-muscle cells. These results raise the possibility that normal Mtm phosphatase activity functions antagonistically to Rab21 GTPase or in concert with PKCepsilon kinase, Rab11 and/or Arf6 GTPase, respectively, to control redelivery of β-integrin to the plasma membrane. It was found that class III PI3K, Vps34, also contributes to integrin localization upon myofiber remodeling, but with no effect on integrin-containing inclusions. A requirement for class III Pi3K could be at a shared step with the early endosomal Rab5 GTPase shown to be involved in integrin turnover at larval MTJs. Thus, regulation of distinct PI(3)P pools is important for differential regulation of integrin endosomal trafficking, whereby Pi3KC2 and Mtm are dedicated to specific paired antagonistic functions (Ribeiro, 2011).

mtm was found to be required in muscle for both integrin-mediated adhesions and T-tubule organization. The T-tubule requirement for mtm was similar to but not as severe as that for amph, the sole homolog of human AMPH2 that is also associated with centronuclear myopathy. However, unlike mtm, null alleles of amph did not share a defect of myofiber detachment. Despite localization of betaPS-integrin at T-tubules, and the dual requirements for mtm, it was found that normal integrin adhesions and abnormal betaPS-integrin localization on inclusions are independent of T-tubule organization. This suggests that mtm may serve a common function for integrin turnover and T-tubule formation at a shared precursor compartment, for example, at recycling endosomes, or alternatively, act independently at two distinct sites. β-integrin, Dlg and Amph are known to functionally interact at postsynaptic junctions, and MTMR2 has been shown to interact with Dlg1/SAP-97 and Dlg4/PSD-95 to promote postsynaptic function. Thus, the shared accumulation of betaPS-integrin, Dlg and Amph on central membrane inclusions in mtm-depleted myofibers, and their elimination with Pi3K68D co-depletion, points to a possible role for a PI3KC2/Mtm pathway in endocytic recycling at neuromuscular junctions, as well as at MTJs (Ribeiro, 2011).

Many of the defects observed in mtm mutant muscle parallel those associated with the human disease, XLMTM, demonstrating that the fly offers a tractable model for the cellular basis of centronuclear myopathy. Importantly, the discovery that mtm broadly regulates betaPS-integrin turnover through endocytic trafficking led to the uncovering of a previously untested defect in beta1-integrin localization in human XLMTM myofibers. Normal myofiber organization and function rely on integrin adhesions in vertebrate muscle. Thus, disruption of integrin regulation provides a basis for aspects of the severity of myofiber disorganization and dysfunction observed in XLMTM. The conservation between fly mtm and human MTM1 functions brings further significance to the potent interaction demonstrated between mtm and class II Pi3K68D for integrin regulation in flies. Whereas Class I and III PI3-kinases have been the focus of intense study as potential therapeutic targets of specific inhibitory compounds, the Class II PI3-kinases have received little attention. The knowledge of PI3KC2 contributions to specific MTM pathways is significant towards motivating similar studies for potential strategies addressing MTM-related disease (Ribeiro, 2011).

Mtm is the single fly homolog related to both human MTM1 and MTMR2, and human MTMR2 expression was able to rescue integrin-related defects in mtm-depleted fly myofibers. An mtm pathway function in endocytic trafficking is therefore relevant to a more general understanding of the cell biological functions employed by MTM subfamily members. Mutations in MTMR2 associated with CMT4B neuropathy affect the morphology and function of myelinating Schwann cells, which like myofibers, share features of having an extensive plasma membrane and a reliance on integrin adhesions. The regulation of integrin trafficking under the control of a conserved PI3KC2/Mtm pathway may be an important mechanism for controlling cell compartmentalization more broadly in different contexts, and relevant to different MTM-related human disease (Ribeiro, 2011).

Membrane protein location-dependent regulation by PI3K (III) and rabenosyn-5 in Drosophila wing cells: A berrant localization phenotypes of beta-integrin

The class III phosphatidylinositol-3 kinase [PI3K (III)] regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate [PI(3)P]. While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. This study, in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) were examined in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein beta-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of beta-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes (Abe, 2009).

Cell polarity along the apical-basal axis is essential for the function of epithelial cells. This polarity is formed and maintained by distinct localization of membrane spanning and associated proteins, to apical, lateral or basal membrane domains. Membrane proteins localized to the apical or basolateral plasma membrane are endocytosed into early and apical or basolateral endosomes. For example, horseradish peroxidase (HRP) administered to the apical cell surface is incorporated into the apical early endosome. By contrast, HRP or dimeric IgA administered to the basolateral cell surface or transferring receptor (TfR) in the basolateral domain are internalized into the basolateral early endosome, which remain distinct. Sorting of proteins for transcytosis, recycling and degradation takes place in these early endosomes. The proteins, incorporated into apical and basolateral early endosomes, meet in common endosomes, a process that can be observed within 15 min after the onset of internalization in MDCK cells. The significance of keeping the apical and basolateral early endosomes distinct is thought to ensure that proteins from the apical and basolateral plasma membrane remain apart before the sorting processes proceeds. Although it is plausible that the trafficking of proteins in distinct membrane domains is regulated differently, the factors involved in such a differential regulation remain elusive (Abe, 2009).

One of the key molecules regulating membrane trafficking is PI3K (III), a heterodimer of Vps34p and Vps15p/p150, which produces phosphatidylinositol-3-phosphate (PI(3)P). PI(3)P is found to localize with early endosome and internal vesicles of multivesicular bodies (MVBs) in mammalian cells in culture. Genetic and pharmacological analysis, using yeast and mammalian cells in culture, suggests that PI3K (III) is required for five distinct processes. These are: (1) the fusion of clathrin-coated vesicles and early endosomes as well as the fusion between early endosomes; (2) the recycling from early endosomes back to the Golgi complex or other destinations; (3) the entry of proteins into the lysosomal degradation pathway; (4) the formation of internal vesicles of MVBs and (5) autophagy. Moreover, inactivation of PI3K (III) by Vps34 mutation leads to an expansion of the outer nuclear membrane and an abnormal reduction of the LDL receptor at the apical membrane in C. elegans. In Drosophila, dVps34 mutation results in defective endocytosis of the apical membrane protein Notch and a defective onset of autophagy. It has been suggested that PI3K (III) utilizes different effectors at apical and basolateral endosomes. However, the role of PI3K (III) in the regulation of protein localization at different membrane domains has remained unclear (Abe, 2009 and references therein).

To understand the various functions of PI3K (III), it is crucial to clarify which downstream effectors are involved in each of the processes it regulates. PI3K (III) is thought to exert its function through the recruitment of proteins that contain PI(3)P-binding motifs such as FYVE or PX domains. Among such proteins, Rabenosyn-5 (Rbsn-5) has been shown to contribute to endosome fusion and recycling processes in mammalian cells. Genetic studies on C. elegans and Drosophila also show that Rbsn-5 is essential for receptor-mediated endocytosis and endosome fusion, although it is not clear whether or not Rbsn-5 is involved in other PI3K (III)-related phenomena (Abe, 2009).

To determine how the proteins in distinct membrane domains are regulated by PI3K (III) and its effector Rbsn-5 this study analyzed Drosophila wing development. This provides a good model since wing primordial cells have a clear polarity along the apical-basal axis. In addition a number of membrane proteins are known to be transported in an organized manner along the apical-basal axis. For example DE-cadherin, a cell adhesion protein and Fmi, a planar cell polarity (PCP) core protein, are localized in the apical junctions or zonula adherens (ZA), whereas the cell adhesion molecules FasIII and β-integrin are localized in lateral and basal membranes, respectively. This study found that inactivation of PI3K (III) in the wing primordial cells by knockdown of dVps15 affects the localization of these membrane proteins differently. In particular, it was found that dVps15 knockdown results in the accumulation of FasIII at the lateral membrane, whereas it results in intracellular accumulation of DE-cadherin, Fmi and β-integrin. Importantly, inactivation of Rbsn-5 shows accumulation of FasIII and β-integrin at the lateral membrane and intracellular vesicles, respectively, but no effects of DE-cadherin and Fmi localization (see in contrast Mottola, 2010). These results provide evidence for a differential regulation of protein localization by PI3K (III) and Rbsn-5 at distinct membrane domains (Abe, 2009).

This study demonstrated that PI3K (III) differentially regulates the localization of proteins at distinct membrane domains. The intracellular accumulation of Fmi, DE-cadherin and β-integrin induced by the dVps15 knockdown might be due to defects in the degradation pathway, since the maturation of MVBs and the lysosomal trafficking were defective in these cells. However, unlike these proteins, Fas III did not accumulate in the intracellular compartments, but rather accumulated at the surface of the lateral plasma membrane. It is possible that PI3K (III) regulates proteins at the lateral membrane differently from those localized at other membrane domains. It is also possible that PI3K (III) regulates Fas III in a different way, irrespective of the membrane domain to which it is localized. Whichever is the case it will be important to elucidate the mechanism underlying this difference in a future study (Abe, 2009).

Rbsn-5, a FYVE domain-containing protein, shares a part of the functions of PI3K (III), in that it is necessary for the regulation of Fas III and β-integrin localization, but not that of DE-cadherin and Fmi localization. Although the Rbsn-5C241 null mutant clones may not completely lack Rbsn-5 activity, the requirement of Rbsn-5, or at least the requirement of an appropriate amount, differs between these proteins with respect to normal trafficking. It appears that Rbsn-5 preferentially controls the events at the basolateral regions, given that Rbsn-5 is necessary for the formation of large endosomes at the basal region, whereas it is indispensable for the formation of actin bundles at the apical surface (Abe, 2009).

PI3K (III) has been implicated in the differential regulation of vesicle trafficking at apical and basolateral regions. For instance, a reduction of PI(3)P dissociates EEA1, a FYVE-domain containing protein essential for early endosome fusion, selectively from basolateral endosomes. However, which proteins, including EEA1, regulate the different trafficking pathways downstream of PI3K (III) has remained unknown. Rbsn-5 has been proposed to be a PI3K (III) effector, since Rbsn-5 harbors a FYVE domain. The current results provide further evidence supporting a possible functional interaction between these two molecules, based on their genetic interaction on the wing morphogenesis and the PI3K (III)-dependent Rbsn-5 immunostaining. Importantly, the different requirement of Rbsn-5 for trafficking at apical junction and basolateral membrane domains suggests that Rbsn-5 may a selective regulator under the control of PI3K (III) (Abe, 2009).

The Bro1-domain-containing protein Myopic/HDPTP coordinates with Rab4 to regulate cell adhesion and migration

Protein tyrosine phosphatases (PTPs) are a group of tightly regulated enzymes that coordinate with protein tyrosine kinases to control protein phosphorylation during various cellular processes. Using genetic analysis in Drosophila non-transmembrane PTPs, one role was identified that Myopic (Mop), the Drosophila homolog of the human His domain phosphotyrosine phosphatase (HDPTP), plays in cell adhesion. Depletion of Mop results in aberrant integrin distribution and border cell dissociation during Drosophila oogenesis. Interestingly, Mop phosphatase activity is not required for its role in maintaining border cell cluster integrity. Rab4 GTPase was further identified as a Mop interactor in a yeast two-hybrid screen. Expression of the Rab4 dominant-negative mutant leads to border cell dissociation and suppression of Mop-induced wing-blade adhesion defects, suggesting a critical role of Rab4 in Mop-mediated signaling. In mammals, it has been shown that Rab4-dependent recycling of integrins is necessary for cell adhesion and migration. This study found that human HDPTP regulates the spatial distribution of Rab4 and integrin trafficking. Depletion of HDPTP resulted in actin reorganization and increased cell motility. Together, these findings suggest an evolutionarily conserved function of HDPTP-Rab4 in the regulation of endocytic trafficking, cell adhesion and migration (Chen, 2012).

Cell adhesion and cell migration are essential for the development and coordinated function of multicellular organisms. Aberrant regulation of these processes often results in the progression of many diseases, including cancer invasion and metastasis. Accumulating evidence has indicated that dynamic and reversible protein tyrosine phosphorylation is essential for the regulation of cell migration and cell adhesion. While many studies have been devoted to the role of protein tyrosine kinases in these processes, the function of protein tyrosine phosphatases (PTPs) in cell adhesion and migration remains unclear (Chen, 2012).

The dynamic change of integrin-mediated focal adhesions plays a critical role in cell adhesion and migration. Many focal adhesion regulators such as focal adhesion kinase (FAK), Src, p130Cas, and paxillin are tyrosine phosphorylated. The tyrosine phosphorylation of these proteins affects focal adhesion dynamics. Phosphorylation of tyrosine 397 in FAK promotes its association with Src, and the activated FAK-Src complex subsequently regulates focal adhesion dynamics by signaling downstream targets. Several PTPs have been implicated in integrin signaling, cell adhesion and motility. One study has shown that SHP-2 phosphatase influences FAK activity. SHP-2 also promotes Src kinase activation by inhibiting Csk. Depletion of PTP-PEST has been found to lead to the hyperphosphorylation of p130Cas, FAK and paxillin, and a marked increase in focal adhesions. Moreover, PTP1B and PTPα, have also been found to regulate Src phosphorylation and integrin-mediated adhesion (Chen, 2012).

In Drosophila, a total of sixteen putative classical PTPs have been identified. Compared to mammalian PTPs, Drosophila PTP family members are relatively simple, most containing only one gene corresponding to each subtype (except for DPTP10D and DPTP4E, which share similar domain structures). Therefore, Drosophila can serve as an excellent model system for the study of the physiological and developmental function of PTPs. While much research has been devoted to the function of receptor PTPs, the role of non-transmembrane PTPs (NT-PTPs) in Drosophila development remains unknown. One of the most well studied Drosophila NT-PTPs is Corkscrew (Csw). Csw is the ortholog of human SHP-2 which has two SH2 domains at the N-terminus and a PTP domain at the C-terminus. Csw functions as a downstream effecter of Sevenless PTK and is essential for the development of the R7 photoreceptor. Phenotypic analysis showed that Csw can also act downstream of many receptor tyrosine kinases, such as the Drosophila epidermal growth factor receptor (DER) and the fibroblast growth factor (Breathless). PTP-ER has been shown to function as a negative regulator downstream of Ras1 and to be involved in RAS1/MAPK-mediated R7 photoreceptor differentiation. PTP61F, the Drosophila ortholog of human PTP1B and TCPTP, has been reported to interact with Dock, an adapter protein required for axon guidance. PTP61F has recent been shown to coordinate with dAbl in regulating actin cytoskeleton organization via reversible tyrosine phosphorylation of Abi and Kette ). Moreover, dPtpmeg, a FERM and PDZ domain-containing NT-PTP, is reported to be involved in the formation of neuronal circuits in the Drosophila brain, though its molecular function in this process is not known (Chen, 2012).

To explore the functional role of Drosophila NT-PTPs in cell adhesion and migration, genetic analyses was performed to identify NT-PTPs that could modulate border cell migration during oogenesis. This study found that Myopic (Mop), the Drosophila homolog of the human His domain phosphotyrosine phosphatase (HDPTP), plays an important role in maintaining border cell cluster integrity. Depletion of Mop altered the normal distribution of integrin receptor. While Mop has recently been reported to regulate EGFR and Toll receptor signaling, its molecular mechanism has remained elusive. This study found that Mop interacts with Rab4 GTPase in controlling integrin distribution and cell adhesion. It was further demonstrated that human HDPTP is essential for the intracellular positioning of Rab4, integrin trafficking, and cell migration. These findings provide some insight into the mechanisms underlying HDPTP in the regulation of cell adhesion and migration (Chen, 2012).

Accumulating evidence has indicated that vesicular trafficking regulates the distribution of plasma membrane content as well as the localization of cytoskeletal proteins during cell adhesion and migration. Drosophila border cells migrate as a cluster of strongly adherent cells during the development of the egg chamber. During this process, JNK signaling and endocytosis-mediated spatial distribution of receptor tyrosine kinases play a critical role, though mechanisms involved in this process have remained elusive. This identified Mop, the Drosophila homolog of human HDPTP, as a regulator of integrin trafficking. Mop is essential for proper integrin localization and for maintaining border cell integrity during oogenesis. It was further demonstrated that Mop and HDPTP interacts with Rab4 GTPase in both Drosophila and mammals. Rab4 has been shown to regulate integrin recycling and cell migration. The current findings indicate that Mop/HDPTP-mediated endocytic trafficking plays an essential role in integrin-mediated cell adhesion and migration. Mop has been predicted as a nontransmembrane-PTP. However, amino acid sequence analysis revealed that Mop displays several differences from conserved PTP motifs within the phosphatase domain. For example, the catalytic essential aspartic acid (D) within motif 8 (WPDXGXP) is replaced by a lysine residue (K). Although the active site cysteine (C) in the catalytic motif 9 (VHCSAGXGR[T/S]G) could be found, the overall signature motif of Mop was much more divergent compared to other PTPs. Moreover, no Mop tyrosine phosphatase activity could be detected using in vitro phosphatase assays. These results suggest that Drosophila Mop may not be enzymatic active. Alternatively, Mop may exhibit weak phosphatase activity which can not be detected using either pNPP or in gel phosphatase assay. A recent study by Lin (2011) indicated that human PTPN23/HDPTP exhibits relatively low activity that is comparable with the specific activity of PTP1B D181E mutant. This study also found that expression of Mop-C/S mutant, in which the catalytic cysteine in the active site is replaced by serine, or Mop phosphatase domain deletion mutant rescued the Mop-RNAi induced border cell dissociation defects as effectively as the wild-type Mop, indicating that the putative tyrosine phosphatase activity is not essential for maintaining border cell cluster integrity (Chen, 2012).

In addition to having a C-terminus phosphatase domain, Mop has a sequence similar to that of yeast Bro1 at the N-terminus. The Bro1 domain consists of a folded core of about 370 residues and has been found in many proteins, including Bro1, Alix, and Rim20, known to regulate endosome trafficking. The Bro1 domain has been shown to bind with multivesicular body components (ESCRT-III proteins) such as yeast Snf7 and mammalian CHMP4B for targeting Bro1 domain-containing proteins to endosomes. Interestingly, mutational analysis revealed that CHMP4B binding is not required for HDPTP function, suggesting that the Bro1 domain may have other functions This study identified Rab4 as an interactor with Mop and HDPTP through the Bro1 domain. The Rab GTPases cycle between the GTP-bound active and the GDP-bound inactive forms and are necessary for efficient membrane vesicle transport between different subcellular compartments. Among them, Rab4 and Rab11 have been implicated in controlling membrane trafficking through the endocytic recycling pathways. Several lines of evidence suggest that Rab4 is involved in Mop-mediated integrin distribution. First, it was found that expression of dominant-negative Rab4S22N or depletion of Rab4 in migrating border cells resulted in integrin redistribution and border cell dissociation. Second, genetic analysis revealed that Rab4S22N and Rab4- RNAi suppressed Mop-induced wing blistering phenotypes. Third, in mammalian cells, Rab4 regulated integrin recycling from early endosomes and was required for cell adhesion and spreading. This study found that downregulation of HDPTP disrupted integrin distribution, focal adhesion formation, actin organization, and enhanced cancer cell molility. Moreover, since Mop interacts with Rab4 in a nulcleotide-independent manner, Mop may act as an adaptor rather than an effector of Rab4 during endosomal trafficking processes. In mammals, Rab4 has been shown to regulate fast integrin recycling and persistent cell migration. This study also noticed that dominant-negative Rab4 caused integrin redistribution to the perinuclear region. However, it was found that expression of HDPTP could not rescue the dominant-negative Rab4-induced integrin redistribution. This is consistent with the genetic data that Rab4 acts downstream of Mop. It is proposed that Mop/HDPTP may act as an adaptor to keep Rab4 in proper endosomal domains. Depletion of HDPTP resulted in Rab4 mislocalization, changes in the integrin dynamics and increases in cell migration. Indeed, it was found that misexpression of βPS-integrin resulted in a marked increase in border cell cluster dissociation and βPS-integrin knockdown suppressed MopRNAi-induced border cell dissociation phenotype. Mop/HDPTP is likely to function via its association with Rab4 on early endosomes to regulate integrin sorting and recycling (Chen, 2012).

Integrins activate multiple signaling pathways involved in regulating cell proliferation, survival and migration. One major signaling event stimulated by integrins is mediated by the FAK and Src tyrosine kinases. FAK and Src are crucial regulators of cell adhesion and motility which they regulate by controlling the formation and turnover of focal adhesions. Several reports have shown interactions between HDPTP and FAK-Src complex. HDPTP has been proposed to act as a molecular bridge between FAK and Src in regulating endothelial and bladder carcinoma cell motility. Moreover, Lin identified PTPN23/HDPTP as a negative regulator of cell invasion in mammary epithelial cells. In those studies, FAK and Src were found to be substrates of HDPTP, suggesting that loss of HDPTP may increase FAK-Src activity to promote cell motility. Intriguingly, in Drosophila, this study showed that Mop regulates the border cell association in a PTP domain-independent manner. Recent studies in Drosophila and mammalian cells also found that the phosphatase activity of Mop and HDPTP is not required for its biological function in receptor signaling and tumor suppression. The current findings on Mop/HDPTPRab4 interaction and the regulation of integrin recycling provide new insights into the role of Mop/HDPTP in the regulation of cell adhesion and migration, and they are not mutually exclusive from previous findings (Chen, 2012).

Signaling downstream of integrins

The Drosophila PS1 and PS2 integrins are required to maintain the connection between the dorsal and ventral wing epithelia. αPS subunits are inappropriately expressed during early pupariation via the Blistermaker chromosome (containing a PS2 gene driven by the wing pouch enhancer trap, 684). Inappropriate expression of αPS2 results in the separation of epithelia, causing a wing blister. Two lines of evidence indicate that this apparent loss-of-function phenotype is not a dominant negative effect, but is due to inappropriate expression of functional integrins: (1) wing blisters are not generated efficiently by misexpression of loss-of-function αPS2 subunits with mutations that inhibit ligand binding, and (2) gain-of-function, hyperactivated mutant αPS2 proteins cause blistering at expression levels well below those required by wild-type proteins. A genetic screen was carried out for dominant suppressors of Blistermaker induced wing blisters. Suppression was induced by null alleles of a gene named moleskin, which encodes the protein DIM-7. DIM-7, a Drosophila homolog of vertebrate importin-7, has been shown to bind the SHP-2 tyrosine phosphatase homolog Corkscrew and to be important in the nuclear translocation of activated D-ERK (Rolled). Consistent with this latter finding, homozygous mutant clones of moleskin fail to grow in the wing. Genetic tests suggest that the moleskin suppression of wing blisters is not directly related to inhibition of D-ERK nuclear import (Baker, 2002).

The ß-importin family of proteins is principally linked with nuclear import of protein cargos. However, recently other functions have been associated with members of the importin superfamily. For example, importin-ß, in some cases with importin-α, functions in vertebrates to sequester microtubule polymerization factors early in mitosis. Mitotic microtubule formation can be triggered by the release of the polymerizaion regulators by RanGTP, just as RanGTP binding to importin-ß leads to release of cargos inside the nucleus. DIM-7 protein can be detected immunologically at the cell cortex, both in early Drosophila embryos and in S2 cells in culture. It thus seems reasonable to consider a more direct connection between the peripheral DIM-7 and integrin regulation. Additionally, it appears that a mutation in corkscrew, the Drosophila SHP-2 homolog, can also suppress Blistermaker and that Corkscrew protein binds directly to DIM-7. Although Corkscrew has been implicated primarily in signaling events downstream of receptor tyrosine kinases, vertebrate SHP-2 has been implicated in signaling via a host of growth factor receptors, cytokines, hormones, and antigens. Most relevant to this study, SHP-2, often in association with the membrane glycoproteins PECAM-1 or SHPS-1, has been shown to be involved in many integrin-dependent signaling events and also to be important in regulating integrin-mediated cell adhesion, spreading, or migration. While SHP-2 is a cytoplasmic tyrosine phosphatase, some experiments suggest that it can serve as a scaffolding protein at or near the plasma membrane. For example, a Corkscrew protein mutated in the phosphatase domain retains significant wild-type activity in situ, and this activity is increased if the protein is targeted to the plasma membrane (Baker, 2002).

It is likely therefore that cell surface receptors mediate a localized Corkscrew/SHP-2 activation of cortical DIM-7. This active DIM-7, in combination with associated factors such as D-ERK, could then function more directly in integrin regulation. A more direct connection between DIM-7 and integrin function is also consistent with the fact that moleskin mutations were especially common among the suppressors isolated in the screen. A key question for future work, therefore, will be defining the subcellular location at which DIM-7 functions with respect to integrin-related phenotypes (Baker, 2002).

Recently, evidence has begun to appear that integrin engagement with the ECM can regulate nuclear import of regulatory molecules. For example, there is an association between αLß2 and the c-Jun coactivator JAB1; this connection is suggested to regulate the nuclear localization of JAB1. More directly relevant to these results, ERK nuclear translocation in fibroblasts is dependent on an integrin-mediated event, also involving the actin cytoskeleton. Also, primary mouse embryo fibroblasts with a ß1 integrin cytoplasmic mutant show reduced nuclear translocation of phosphorylated ERK. Regardless of the importance of nuclear transport in Blistermaker suppression, the genetic data indicate a functional connection between integrins and a specific importin-ß that can transport activated ERK and suggest another potential molecular mechanism whereby integrin and growth factor signals can be integrated by the cell (Baker, 2002).

Tensin is an actin-binding protein that is localized in focal adhesions. At focal adhesion sites, tensin participates in the protein complex that establishes transmembrane linkage between the extracellular matrix and cytoskeletal actin filaments. Even though there have been many studies on tensin as an adaptor protein, the role of tensin during development has not yet been clearly elucidated. Thus, this study was designed to dissect the developmental role of tensin by isolating Drosophila tensin mutants and characterizing its role in wing development. The Drosophila tensin loss-of-function mutations results in the formation of blisters in the wings, that are due to a defective wing unfolding process. Interestingly, by1 -- the mutant allele of the gene blistery (by) -- also shows a blistered wing phenotype, but fails to complement the wing blister phenotype of the Drosophila tensin mutants, and contains Y62N/T163R point mutations in Drosophila tensin coding sequences. These results demonstrate that by encodes Drosophila Tensin protein and that the Drosophila tensin mutants are alleles of by. Using a genetic approach, it has been demonstrated that Tensin interacts with integrin and also with the components of the JNK signaling pathway during wing development; overexpression of by in wing imaginal discs significantly increases JNK activity and induces apoptotic cell death. Besides the defects in wing cell adhesion process, another distinct mutant phenotype was observed in the by mutants; they lay rounded eggs due to defective oocyte elongation during oogenesis. Collectively, these data suggest that Tensin relays signals from the extracellular matrix to the cytoskeleton through interaction with integrin, and through the modulation of the JNK signal transduction pathway during Drosophila wing development (Lee, 2003).

Drosophila wing development after pupariation (AP) consists of two distinct stages: prepupal and pupal wing morphogenesis. Pupal wing morphogenesis is further divided into three stages: separation (11-12 hours AP) of the ventral cell layer from the dorsal layer, re-apposition of the inter-vein cells (21-36 hours AP) and re-separation (60 hours AP) of the two cell layers. Shortly after eclosion, wings expand and unfold by an influx of hemolymph. PS integrins are required for the attachment of the two wing surfaces during pupal wing re-apposition and for the maintenance of the wing bilayer (Lee, 2003).

To determine the detailed roles of tensin during wing morphogenesis, the pupal wings of the by2 flies were examined. No differences were observed in the attachment of two wing surfaces and in the integrin localization between wild type and by2 wings during both prepupal apposition (4-6 hours AP) and pupal reapposition stages (30-36 hours AP) (Lee, 2003).

Because the pupal wing development was not disturbed in the by2 mutants, the expansion and unfolding processes of adult wings in the by2 mutants was investigated. After eclosion, the by2 flies display folded wings similar to the controls. Then, a sudden and rapid influx of hemolymph induces the unfolding of folded wings in the by2 mutants. However, as soon as the wings of by2 flies unfold, fluid-filled blisters began to appear at the distal part of the wings, and the boundary of the blisters expand to a certain extent. After the fluid dries, the wing blisters are fixed in place. Taken together, although the dorsal and ventral layers of a wing can be brought into close association during apposition and re-apposition processes in the by2 flies, the link between them may not be strong enough to resist the hydrostatic pressures during the wing unfolding process (Lee, 2003).

The functional significance of each domain of tensin in normal wing development was examined. UAS lines overexpressing either full-length tensin protein or various deletion mutant forms of tensin were generated. Unlike DeltaN and DeltaC, overexpression of DeltaPTB by MS1096-GAL4 driver completely rescues the blistered wing phenotype of by2. These data suggest that both the N-terminal region and the SH2 domain of tensin are required for proper attachment of two wing surfaces (Lee, 2003).

Since mammalian tensin is known to participate in the integrin signaling, whether tensin genetically interacts with integrin was examined. As expected, the blistered wing phenotype become more severe in the if3; by2/+ mutants and extremely severe in the double homozygotic mutants for if3 and by2, compared with if3 homozygotes or by2 heterozygotes. In addition, the rate of flies showing blistered wings in the total population greatly increases in the double mutants (Lee, 2003).

In mammalian cells, tensin has been implicated in signal transduction related to cell adhesion such as Src, JNK and PI3K. To examine the role of tensin in the signaling processes related to wing development, the in vivo interaction between tensin and signaling molecules including rl/Erk, Src, JNK and PI3K was investigated. Interestingly, it was found that the JNK signaling pathway is tightly correlated with tensin in the wing development, while other signaling molecules including rl/Erk do not show any interactions with tensin. Homozygous by2 mutants with heterozygotic mutations of the JNK signaling components bsk1 or hep1 (the loss-of-function mutants for Drosophila JNK and MKK7, respectively) display a highly severe blistered wing phenotype, compared with either homozygous by2, heterozygous bsk1 or heterozygous hep1 mutants. Notably, the rate of flies, which show Class II blistered wings, increases from 46.5% to 70% for these double mutants compared with homozygous by2 mutants, and about 15% of these flies had multiple blisters in their wings. Furthermore, the double homozygotic mutants for by2 and hep1 die at pharate adult stage. The lethality of these double mutants may be due to an impairment of essential in vivo interactions between tensin and the JNK signaling pathway in Drosophila (Lee, 2003).

Next, whether overexpressed by also interacts with the components of the JNK signaling pathway was tested. Overexpression of by using MS1096-GAL4 driver turns the adult wings into a convex shape with a smaller overall size, and this phenotype becomes more severe when two copies of the by gene are overexpressed. Simultaneous overexpression of bsk or hep with by results in a severely curled wing phenotype, which is fully penetrant, whereas overexpression of bsk or hep alone by MS1096-GAL4 driver did not induce any detectable phenotypes in the wing. Collectively, these data suggest that tensin activity is highly related to the JNK signaling pathway during wing development in Drosophila (Lee, 2003).

To further confirm the genetic interaction between tensin and the JNK pathway, the effect of tensin on JNK activity in vivo was tested. The extent of JNK phosphorylation was tested using anti-phosphospecific JNK antibody in the by overexpression line and the by2 mutants. As expected, JNK phosphorylation is dramatically increased in the wing imaginal discs overexpressing by; this directly demonstrates increased JNK activity by by. On the contrary, JNK phosphorylation in the imaginal discs of the by2 mutants is reduced compared with the control (Lee, 2003).

Moreover, the reduced size and the convex wing phenotype observed in the wings overexpressing by can be most easily explained by apoptosis in the wings. Since the induction of apoptosis by the JNK signaling is well established, it was expected that the wing phenotype induced by by overexpression might be due to apoptosis. To confirm the by-induced apoptosis in vivo, Acridine Orange staining of the relevant wing imaginal discs was carried out. As expected, the overexpression of by dramatically increases apoptotic cell death compared with the control (Lee, 2003).

Thus, tensin genetically interacts with the components of the JNK signaling pathway, and regulates JNK activity during wing development. The supporting evidence for the engagement of tensin in the JNK signaling pathway comes from a recent report that transfected mammalian tensin activates JNK signaling in HEK 293T cells (Katz, 2000). Interestingly, in mammalian cells, JNK is also activated via adaptor proteins p130 CAS and Crk which receive a signal from the FAK/Src tyrosine kinase complex in the cell adhesion sites when cells attach to the ECM. Since tensin is a possible substrate for FAK, and p130 CAS is able to interact with the C terminus of tensin, it is highly possible that tensin is involved in this signaling cascade and mediates signals from integrin and FAK to the JNK signaling pathway (Lee, 2003).

Integrin-ECM interactions regulate the changes in cell shape driving the morphogenesis of the Drosophila wing epithelium

During development, morphogenesis involves migration and changes in the shape of epithelial sheets, both of which require coordination of cell adhesion. Thus, while modulation of integrin-mediated adhesion to the ECM regulates epithelial motility, cell-cell adhesion via cadherins controls the remodelling of epithelial sheets. The Drosophila wing epithelium was used to demonstrate that cell-ECM interactions mediated by integrins also regulate the changes in cell shape that underly epithelial morphogenesis. Integrins control the transitions from columnar to cuboidal cell shape underlying wing formation, and eliminating the ECM has the same effect on cell shape as inhibiting integrin function. Furthermore, lack of integrin activity also induces detachment of the basal lamina and failure to assemble the basal matrix. Hence, it is proposed that integrins control epithelial cell shape by mediating adherence of these cells to the ECM. Finally, it was shown that the ECM has an instructive rather than a structural role, because inhibition of Raf reverses the cell shape changes caused by perturbing integrins (Dominguez-Gimenez, 2007).

The generation of form in early animal development involves key cellular process such as epithelial morphogenesis. The reorganisation of cell shape is commonly associated with epithelial morphogenesis, which requires a precise and coordinated remodelling of the cytoskeleton and the adhesive properties of cells. In view of the two predominant cell adhesion systems, there is considerable evidence indicating that interactions between cells and the ECM modulate the shape of cells in culture. This study used the Drosophila wing to show that the regulation of cell shape by integrins also plays an important role during epithelial organ morphogenesis. Furthermore, this integrin function is shown to rely on interactions with a matrix whose assembly also depends on integrin activity. Finally, evidence is provided that the Raf kinase may act as a putative intracellular regulator of this integrin activity (Dominguez-Gimenez, 2007).

A chimera was formed in which the integrin PSβ subunit cytoplasmic domain was fused to the extracellular and transmembrane domains of mutant forms of the Torso receptor tyrosine kinase. These chimeras localise to the sites of endogenous integrins. There, they can act both as activated integrins and as dominant negatives. Whereas on one hand, they can substitute for the endogenous integrin and regulate integrin target genes in the Drosophila midgut they can, on the other hand, also inhibit cell adhesion, matrix assembly and cell migration mediated by the endogenous integrins. Since the key feature of this chimera appears to be the dimerisation of the PSβ cytoplasmic tail, it is henceforth referred to as diβ (Dominguez-Gimenez, 2007).

Integrins are thought to perform two distinct functions in the wing: an early regulatory one in which integrins signal to make cells competent for re-apposition and a later, more traditional one, where integrins mediate adhesion. This study has unravelled a new early function for integrins in maintaining the columnar cell shape of wing epithelial cells. It is proposed that maintenance of this columnar shape is necessary to achieve proper contact and recognition of cells on opposing surfaces during folding. Thus, interfering with this activity results in cells adopting a cuboidal shape, which prevents them from establishing appropriate dorso-ventral connections. These early connections are probably necessary for re-apposition and final adhesion between the dorsal and ventral epithelia. Indeed, disruption of integrin function by diβ overexpression during the initial apposition period results in the formation of wing blisters in the adult (Dominguez-Gimenez, 2007).

The simplest hypothesis as to how integrins maintain the columnar shape of cells is that they keep the wing disc cells firmly attached to the basal matrix. However, recent evidence supports the idea that integrins also play a role in mediating adhesion between the lateral surfaces of cells during the process of dorsal closure in the embryo. In the wing, integrins seem to be distributed basolaterally when cells are in close contact, such as during apposition and adhesion. By contrast, integrins are absent form the lateral cell surfaces and become restricted to basal junctions when cells diminish their basal contact, i.e. during the expansion period. It therefore seems reasonable to propose that, integrins can maintain the columnar shape by mediating basolateral contact between adjacent cells (Dominguez-Gimenez, 2007).

Cell culture studies have shown that integrins and members of the Rho family of GTPases function in a coordinated manner to regulate the morphological changes that accompany cell spreading and migration upon binding to the ECM. In the Drosophila wing, the Rho GTPase Dcdc42 localises predominantly to the basal and apical regions of epithelial columnar cells. Furthermore, expression of a dominant-negative form of Dcdc42 results in a shortening of epithelial cells in the third instar larvae and produces a wing blister in the adult. Therefore, it is possible that integrins and Rho GTPases also interact to regulate the changes in cell shape underlying epithelial morphogenesis during development (Dominguez-Gimenez, 2007).

Maintenance of the columnar state through integrins is also required for folding along the wing margin. During normal wing morphogenesis this is mainly accomplished by local changes in cell shape of the wing margin cells, involving a reduction in height. This ensures that folding only occurs at the middle of the wing disc, thereby allowing an alignment match between dorsal and ventral cells. This study shows that, when integrin function is disrupted in most of the wing pouch, folding does not always occur along the wing margin, probably because all cells now adopt a similar shape. In fact, wing margin cells cannot be distinguished morphologically from the rest, although they do maintain their identity. This results in a mismatch between dorsal and ventral cells that might also be important for later differentiation processes. In summary, this study has show that cell-ECM interactions mediated by integrins are required for the temporal and, most likely, the spatial regulation of the changes in cell shape that accompany the folding of epithelial sheets during organogenesis (Dominguez-Gimenez, 2007).

It still remains unclear to what extent signalling contributes to the activity of integrins during development. One of the main problems is how to distinguish between direct integrin signalling and the indirect effects caused by a lack of integrin adhesion. In Drosophila, the differentiation of some but not all cell types depends directly on integrin signalling downstream of diβ. The results presented here support the idea that the regulation of cell morphology by integrins depends more on integrin adhesion to the ECM than on signalling. Indeed, the diβ chimera does not prevent the changes in cell shape by interfering with integrin activity. However, the possibility that the signal pathway activated by diβ does not fully mimic integrin signalling cannot be ruled out. In fact, it has been demonstrated in muscle that the chimeric diβ integrin is not capable of recruiting certain proteins associated with sites of integrin activity, such as ILK, paxillin, PINCH and tensin (Tanentzapf, 2006a). Hence, it remains possible that the regulation of cell shape requires integrin signals that are only triggered when a complete integrin complex is assembled (Dominguez-Gimenez, 2007).

Additional evidence has been generated to support the idea that the interactions between integrins and the matrix affect cell shape. The elimination of ECM components by overexpressing metalloproteinases provoked changes in cell morphology that strongly resemble those observed when integrin activity is disrupted. Moreover, overexpression of metalloproteinases does not affect the normal distribution of endogenous integrins, which can still cluster in focal-adhesion like structures. Hence, integrins alone are insufficient to regulate changes in cell shape but, rather, they must interact with ECM components. This is in agreement with findings that, in most cases, a threshold of both clustering and binding to integrins must be reached before fully functional focal adhesion complexes are formed (Dominguez-Gimenez, 2007).

The interactions of cells with the ECM have long been proposed to involve 'dynamic reciprocity', whereby a cell response to its ECM affects the composition of the new matrix it secretes, which in turn alters the ensuing response of the cell. This study shows that disrupting integrin function leads to changes in the basal matrix containing laminin. As such, it seems reasonable to consider a model by which the main function of integrins in regulating cell shape during wing development is the correct assembly and/or attachment to the ECM. An organised ECM can then in turn modulate the activity of the integrins themselves and/or other receptors to regulate cell morphology (Dominguez-Gimenez, 2007).

Overexpression of chimeras containing the cytoplasmic domains of the β1 or β3 subunits reduces integrin affinity. By contrast, chimeras containing a mutated β3 cytoplasmic domain with defective inside-out signalling, reduce the ability of the β3 cytoplasmic domain to block activation. These results suggest that there are limiting factors that bind to the cytoplasmic domains of integrins and which regulate ligand binding affinity. Modulation of these factors could be a way of regulating integrin activity. This study shows that diβ recruits the cytoplasmic protein Talin, opening the possibility that diβ exerts its dominant-negative effect by competing for Talin. However, this does not seem to be the case because overexpression of Talin does not rescue the diβ phenotype, contradicting data from CHO cells showing that competition for Talin underlies the trans-dominant inhibition exerted by isolated β tails. Nevertheless, complementation has been demonstrated between mutations in different motifs of the βPS cytoplasmic domain that eliminate the dominant-negative activity of diβ. This suggests that, if the dominant negative activity of diβ were due to competition for cytoplasmic components, this would involve the recruitment of at least two cytoplasmic proteins (Dominguez-Gimenez, 2007).

Alternatively, diβ could initiate a signalling cascade leading to the activation of a Raf-dependent integrin-suppressing pathway. Integrin-mediated adhesion to the ECM can trigger clustering and increase tyrosine phosphorylation of a number of intracellular proteins, including focal adhesion kinase (FAK), Raf, Ras, and MAPKs. However, it has been shown that activation of MAPK suppresses high-affinity ligand binding in integrins. Thus, a model has been proposed in which MAPK regulates integrin function through a negative feedback loop. This study shows that a dominant-negative form of Raf suppresses the capacity of diβ to inhibit integrin function. Furthermore, it is demonstrated that overexpression of diβ enhances Raf activity. Therefore, it is proposed that the trans-dominant inhibition exerted by diβ could result from the activation of a Raf-dependent signal transduction pathway that inhibits or modifies integrin-ECM interactions (Dominguez-Gimenez, 2007).

The negative regulation exerted by the Raf pathway could be part of a negative feedback loop that regulates integrin function during normal development. If this were the case, the expression of RafDN would be expected to constitutively activate integrin signalling and, therefore. provoke changes in cell morphology. But, none of these effects have been demonstrated upon expression of RafDN in the wing disc. However, since integrins can activate other pathways that are Raf independent, affecting one of these pathways might not produce a dramatic effect because the other pathways may compensate this deficiency. In this context, the results suggest that the chimeric diβ integrin is not able to activate intracellular signals other than those associated to the Raf pathway - probably be due to the failure of diβ to assemble a complete integrin complex (Dominguez-Gimenez, 2007).

The regulation of cell shape through cell-ECM interactions has been shown to have a dramatic influence on cell proliferation, patterning, differentiation, cell migration, cell branching and matrix production during development. This study shows that these interactions also play a crucial role in regulating the changes in cell shape that drive epithelial morphogenesis underlying the formation of organs and tissues. The molecular mechanisms by which these cell-ECM interactions influence the cytoskeleton and regulate cell shape during morphogenesis must now be identified. The easily detectable wing blister phenotype caused by expression of diβ in the wing provides a foundation to screen for mutations in genes required to modulate these integrin-ECM interactions (Dominguez-Gimenez, 2007).

Nuclear localization of the ERK MAP kinase mediated by Drosophila alphaPS2betaPS integrin and importin-7

The control of gene expression by the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK) requires its translocation into the nucleus. In Drosophila S2 cells nuclear accumulation of diphospho-ERK (dpERK) is greatly reduced by interfering double-stranded RNA against Drosophila importin-7 (DIM-7) or by the expression of integrin mutants (see Myospheroid), either during active cell spreading or after stimulation by insulin. In both cases, total ERK phosphorylation is not significantly affected, and ERK accumulates in a perinuclear ring. Tyrosine phosphorylation of DIM-7 is reduced in cells expressing integrin mutants, indicating a mechanistic link between these components. DIM-7 and integrins localize to the same actin-containing peripheral regions in spreading cells, but DIM-7 is not concentrated in paxillin-positive focal contacts or stable focal adhesions. The Corkscrew (SHP-2) tyrosine phosphatase binds DIM-7, and Corkscrew is required for the cortical localization of DIM-7. These data suggest a model in which ERK phosphorylation must be spatially coupled to integrin-mediated DIM-7 activation to make a complex that can be imported efficiently. Moreover, dpERK nuclear import can be restored in DIM-7-deficient cells by Xenopus Importin-7, demonstrating that ERK import is an evolutionarily conserved function of this protein (James, 2007).

The integrin cell surface receptors regulate numerous cellular processes, including growth, differentiation, apoptosis and migration. Integrins are heterodimers made up of α and β subunits, each with short cytoplasmic tails and large extracellular domains. Integrins function as adhesion molecules and frequently form a physical connection between the extracellular matrix (ECM) and the actin cytoskeleton (James, 2007).

In addition to their function in cell adhesion, integrins are critical to many of the signaling pathways of cells. Of particular relevance to these studies, numerous examples have been documented in which integrins regulate the activity of mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase (ERK), or in turn are regulated by these enzymes. Integrins may directly mediate ERK activation, or in other cases, they may function to modulate the activities of growth factor receptors on ERK signaling (James, 2007).

ERK-induced gene expression requires the transport of ERK into the nucleus. In the absence of stimulation, ERK is maintained in the cytoplasm through an interaction with its upstream activator mitogen-activated protein kinase kinase (MEK). MEK phosphorylates both tyrosine and threonine residues in the activation loop of ERK. After phosphorylation by MEK, diphospho-ERK (dpERK) probably dimerizes and enters the nucleus via an active transport mechanism. The subcellular localization of dpERK after activation offers an additional level of regulation of ERK signaling (James, 2007 and references therein).

In general, cells in suspension respond weakly to growth factor stimulation compared with cells adhering to the ECM, and regulation of ERK nuclear import is one potential step where integrin and receptor tyrosine kinase (RTK) signals may be integrated. For example, after activation by MEK in NIH 3T3 cells maintained in suspension the majority of ERK remains in the cytoplasm, and ERK activation of the transcription factor Elk-1 is reduced compared with adherent cells. A β4 integrin signaling domain has been shown to affect the nuclear translocation of MAP kinases and NF-kappaB although the large cytoplasmic domain of β4 is not homologous to that of other integrin β subunits. The nuclear localization of other transcriptional regulators also has been shown to be altered by integrin function in mammalian cells, including the c-Abl tyrosine kinase in mouse fibroblasts and the transcriptional coactivator JAB1 in a variety of cell types. Additional connections between integrins and nuclear import are suggested by studies on proteins that are typically considered to be downstream of integrins. For example, integrin-linked kinase (ILK) has been shown to regulate the nuclear import of a c-Jun coactivator protein (James, 2007).

A potential link between integrins and nuclear import has been further suggested by studies of wing development in Drosophila. In Drosophila, integrins are required to maintain the attachment of the dorsal and ventral wing epithelia during adult morphogenesis, and this process depends on the differential expression αPS1 and αPS2 integrin subunits on the dorsal and ventral cells, respectively. Loss of integrin function leads to wing blisters, where the two surfaces separate after eclosion of the adult from the pupal case. Surprisingly, wing blisters can also occur when an α subunit is inappropriately expressed on the wrong side of the wing, and experiments with various mutants have demonstrated that this is a gain-of-function phenotype. That is, the activity of an integrin in the wrong place during a specific morphogenetic event causes a subsequent loss of epithelial attachment. A genetic screen for dominant suppressors of this gain-of-function wing blister phenotype (Baker, 2002) identified null mutations in a gene named moleskin (msk) (James, 2007).

The moleskin gene encodes Drosophila Importin-7 (DIM-7), which is a close homologue of vertebrate Importin-7, also known as Ran Binding Protein-7 (RanBP-7). Importin-7 is a member of the importin β superfamily of nuclear importers, which can bind directly to the nuclear pore complex. Vertebrate Importin-7 has been shown to mediate nuclear import of ribosomal proteins, histone H1, the HIV-1 reverse transcription complex and the glucocorticoid receptor. In Drosophila, DIM-7 is tyrosine phosphorylated in response to growth factor stimulation of RTKs, and it physically binds Drosophila ERK. Additionally, DIM-7 binds the tyrosine phosphatase Corkscrew (CSW), the Drosophila homologue of SHP-2. Corkscrew is generally required for ERK signaling via RTKs, and in vertebrate cells SHP-2 has been associated with integrin signaling and regulation of integrin activity, although the molecular bases of these interactions remain unclear (James, 2007).

Until recently, it has not been clear how dpERK gains entry to the nucleus after activation. In addition to regulated nuclear import, the cellular localization of phosphorylated ERK dimers can be influenced by release from cytoplasmic anchors and regulated nuclear retention or export, and in at least one case it has been suggested that ERK2 may not require any additional import proteins. Genetic experiments with Drosophila embryos demonstrate that DIM-7 is largely responsible for the nuclear import of activated ERK in this system. The suppression of integrin-related phenotypes in fly wings by moleskin mutations led to an examination a potential connection between integrins and the regulation of ERK import in a Drosophila cell culture system, and the results suggest that DIM-7 may represent a novel nexus of integrin and RTK signaling (James, 2007).

This study shows that a vertebrate homologue of DIM-7 can rescue the ERK localization phenotype of DIM-7 dsRNA treated cells. Thus, ERK nuclear translocation is a property of members of the Importin-7 family of proteins generally. This function cannot necessarily be extended to other MAP kinases; for example, no change is seen in nuclear localization of the p38 MAP kinase in S2 cells grown in DIM-7 dsRNA, although c-Jun NH2-terminal kinase transport does seem to involve DIM-7 (James, 2007).

The ability of growth factors to activate ERK signaling is often linked to integrins; however, specific integrin functions typically have not been examined. The reduced levels of nuclear dpERK in cells expressing βPS-G1 (which contains a frameshift mutation in the cytoplasmic domain that eliminates the two NPXY motifs that are critical for interaction with a number of cytoplasmic proteins, including talin) or βPS-G4 (which has a mutation in the second serine of the MIDAS domain (DXSXS), which would be expected to inhibit extracellular ligand binding) show that ERK signaling seen in Drosophila S2 cells is dependent on functional integrins and that it is not due solely to changes in cell adhesion or shape. Specifically, both the extracellular and cytoplasmic integrin domains must be able to interact properly with ECM or intracellular components for the integrins to support high levels of nuclear dpERK (James, 2007).

A simple model in which soluble dpERK (activated by integrins or growth factors) finds DIM-7 ('activated' by integrins) for import cannot explain all of the data. The experiments that examine total ERK distribution in the integrin mutants suggest that most of the activated ERK cannot enter the nucleus even after translocation into the DIM-7-rich perinuclear region. One would expect that the importins here are actively working with various other cargos and that a significant fraction of the DIM-7 is generally capable of import. This result suggests a model in which ERK phosphorylation must be spatially coupled to DIM-7 activation to make a complex that can be imported efficiently (see Model for the role of integrins in ERK nuclear import in S2 cells). Interestingly, a specific membrane localization of DIM-7 has been suggested as a regulatory mechanism in developing Drosophila eyes (Vrailas, 2006); however, in this case the targeting to apical epithelia has been seen as an inhibitory mechanism (James, 2007).

moleskin (DIM-7) function is required for normal cell proliferation in animals, where patches of mutant cells disappear in developing epithelia. Examples in which a 50% reduction in DIM-7 function has produced phenotypes in developing flies have involved circumstances in which a signaling pathway has been stimulated to high levels by gene overexpression. In the current study DIM-7 levels are reduced significantly, but they are not eliminated, and the cells show no obvious phenotype during normal growth. However, clear effects are seen after acute stimulation of the ERK pathway. The integrin-mediated activation of DIM-7 may be especially important as a regulatory component in such cases of acute, high-level signaling (James, 2007).

Mouse embryo fibroblasts expressing a β1 mutant similar to βPS-G1 transiently display elevated phospho-ERK after stimulation with growth factor, but subsequently the same groups reported that ERK does not necessarily enter the nucleus after plating on fibronectin. Interestingly, the import defect seen in the integrin mutants can be rescued by adding constitutively active Rac. Although multiple pathways may couple integrins to ERK activation and transport in different cell types, Importin-7 family members are likely to be a common feature of dpERK nuclear translocation, and it will be interesting to see whether various pathways leading from integrins to import converge at this protein (James, 2007).

Perhaps most intriguing with respect to the current studies is the work on a natural human β1 variant. β1C is an alternatively spliced form that, like βPS-G1, replaces the cytoplasmic NPXY motifs with other sequence. Cells that express β1C show reduced proliferation and reduced activation of the Ras-ERK pathway, relative to cells expressing the more common β1A. β1A-containing integrins seem to form a complex that includes insulin-like growth factor-I receptor and the insulin receptor substrate-1 (IRS-1), and the addition of insulin leads to cell proliferation and inhibition of adhesion to laminin. In contrast, β1C expression leads to decreased proliferation and increased adhesion, and these effects seem to be mediated by a complex that includes Gab1 and SHP-2, but not IRS-1. There is no β1C variant naturally in Drosophila, but the βPS-G1 mutant does show some dominant-negative effects in flies, and the work reported in this study suggests that this might result at least in part from disruptive effects on intracellular signaling (James, 2007 and references therein).

Significant amounts of cortical DIM-7 are found where integrins are located at the spreading edges of cells. Consistent with a role of integrins in peripheral DIM-7 localization, cells that are protease treated, heat shocked to induce integrin expression, and spread in serum-free media (where spreading is integrin independent), DIM-7 is not found at the periphery in fully spread cells at early times, but it appears when integrin expression is detected after a few hours. One striking feature of the peripheral DIM-7 is that it does not colocalize with integrins in more organized cell-substratum adhesion sites. Thus, peripheral integrin-DIM-7 associations seem to depend on the functional state of the integrins (James, 2007).

Vertebrate SHP-2 is necessary in many contexts for growth factor activation of ERK, and SHP-2 has also been shown to be involved in integrin-dependent signaling. However, the data from different cell types fail to paint a simple, cohesive picture of SHP-2 molecular function, especially with respect to signaling downstream of integrins. Previous biochemical data indicate that DIM-7 binds Drosophila Corkscrew (SHP-2) (Lorenzen, 2001). Corkscrew is generally an essential component in signaling via receptor tyrosine kinases; however, Corkscrew lacking tyrosine phosphatase activity can rescue some phenotypes when reintroduced into corkscrew mutants, suggesting that in some contexts Corkscrew functions primarily as a scaffolding protein. The role of Corkscrew in DIM-7 activation may be largely as a scaffold, because DIM-7 disappears from the periphery when Corkscrew is depleted. This does not seem to be an indirect result of a defect in DIM-7 activation, because cortical DIM-7 remains in other situations that affect its ability to import dpERK, for example, in both of the integrin mutants tested (James, 2007 and references therein).

Interestingly, the screen that identified moleskin (DIM-7) as a suppressor of Blistermaker assayed only ~40% of the Drosophila genome (the third chromosome). Further elucidation of the molecular mechanisms underlying the DIM-7/integrin connection is likely to be facilitated by the identification of additional Blistermaker suppressors on other chromosomes. Screens for such loci are in progress (James, 2007).

dGRASP-mediated noncanonical integrin secretion is required for Drosophila epithelial remodeling

Integral plasma membrane proteins are typically transported in the secretory pathway from the endoplasmic reticulum and the Golgi complex. This study shows that at specific stages of Drosophila development corresponding to morphological changes in epithelia, apposed basolateral membranes separate slightly, allowing new plasma membrane contacts with basal extracellular matrix. At these sites, newly synthesized integrin α subunits are deposited via a mechanism that appears to bypass the Golgi. The Drosophila Golgi resident protein dGRASP localizes to these membrane domains, and in the absence of dGRASP, the integrin subunit is retained intracellularly in both follicular and wing epithelia that are found disrupted. It is proposed that this dGRASP-mediated noncanonical secretion route allows for developmental regulation of integrin function upon epithelial remodeling. It is speculated that this mechanism might be used during development as a means of targeting a specific subset of transmembrane proteins to the plasma membrane (Schotman, 2008).

This study has identified a developmentally regulated noncanonical dGRASP-dependent and dSyntaxin5-independent secretion route that displays several characteristics. (1) It is specifically built in epithelia that undergo rearrangement, such as the elongating discs and the flattening follicle cells. There, it is used by the transmembrane integrin subunit αPS1 for its transport and deposition at the open zone of contact (ZOC), the basolateral portion of the plasma membrane that was engaged in cell-cell contact and, after the morphological changes, is now facing the extracellular matrix. This deposition elicits the building of a focal adhesion that helps maintain epithelium integrity at stage 11 onward. In the absence of dGRASP, the specific deposition of αPS1 is dramatically impaired and the resulting epithelium is severely disrupted in a similar fashion as in a hypomorphic mew. (2) This pathway is insensitive to BFA and the absence of the SNARE dSyntaxin5, suggesting that it bypasses the Golgi (Schotman, 2008).

It is proposed that the building of this pathway starts with the upregulation of a subset of mRNAs encoding proteins of the Golgi, dGRASP and dGos28. These mRNAs are targeted to the open ZOC, where they elicit the de novo synthesis of the corresponding proteins that are found anchored at the plasma membrane lining the open ZOC in the follicular epithelium. This RNA pattern was also observed with a handful of other transcripts. Remaining to be answered is what triggers the upregulation and localization of the dgrasp mRNA and the other transcripts to the open ZOC in response to epithelial morphological changes and how they are moved and anchored there. As mechanical tension and integrin binding have already been shown to induce the recruitment of mRNAs to focal adhesions, integrins themselves could be the sensor for the mechanical stretching during disc elongation and the centripetal movement of the follicle cells (Schotman, 2008).

Concomitant with the targeting of dgrasp transcripts, αPS1 mRNA is also upregulated and basally concentrated. It is proposed that at stage 10B, the ER cisternae that reside near the open ZOC are actively involved in the local synthesis of αPS1. After synthesis in the ER membrane, a yet-unknown cargo receptor likely provides a very efficient exit for the newly synthesized αPS1 and prevents its diffusion through the entire ER membrane, similar to Gurken in the oocyte . From these αPS1-enriched ER cisternae, carriers would form, although their nature remains elusive. Although Sar1 localization has not been addressed, none of the COPII subunits were concentrated near the open ZOC (Schotman, 2008).

These ER-derived carriers bypass the Golgi and specifically fuse with the plasma membrane outlining the open ZOC. This membrane domain has become, at stage 10B, an acceptor compartment of an unexpectedly mixed nature, comprising plasma membrane resident proteins as well as cis-Golgi proteins dGRASP, dGM130, and dGos28 that are specifically localized there at this stage. These proteins could form a platform to which the αPS1-enriched ER-derived carriers fuse through the formation of a SNARE complex involving dGos28, and other SNAREs that have yet to be identified. dGRASP/dGM130 is likely to be involved in their tethering through oligomerization of dGRASP and promote the formation of this unusual complex. The fusion would involve the activity of the ATPase dNSF1 and its cofactor dSNAP, meaning that this system is clearly different from the Golgi-independent deposition of the transmembrane protein Ist2 from Saccharomyces cerevisiae that is Sec18/NSF independent (Schotman, 2008).

In addition to Ist2, a few proteins have already been shown to traffic directly from the ER to the plasma membrane, such as the cystic fibrosis transductance regulator in BTK cells and the simian rotavirus RRV in Caco-2 cells. Further research might reveal whether a pathway equivalent to the dGRASP-mediated pathway is involved in Golgi bypass in these cases (Schotman, 2008).

A number of mammalian factors, including Galectin, Interleukin, and Fibroblast growth factor 2, have been shown to be secreted in an unconventional manner that completely bypasses the exocytic pathway. Very recently, the Dictyostelium GRASP homolog GrhA has been shown to be involved in the unconventional secretion of the polypeptide AcbA that is predicted to harbor no signal peptide in its coding sequence (Schotman, 2008).

It is striking that both dGRASP and GrhA mediate unconventional secretion routes yet the pathways appear to be different. Unlike AcbA in Dictyostelium, αPS1 is translocated into the ER lumen using its signal peptide. In follicle cells earlier than stage 10B, αPS1 transport requires dSyntaxin5, suggesting that it travels via the typical ER-Golgi-plasma membrane transport route. There is no evidence suggesting that the signal peptide might be omitted at stage 10B and, importantly, no evidence of transmembrane proteins transported to the plasma membrane by the unconventional secretion pathway that AcbA is proposed to use (Schotman, 2008).

AcbA has been postulated to be captured from the cytosol and stored in endosomes prior to release from this compartment to the extracellular medium. In contrast, αPS1 could be made de novo and stored in an endosomal compartment (perhaps similar to Glut4 in adipocytes) localized near the open ZOC before being specifically recycled to the adjacent plasma membrane. Alternatively, the integrin could be stored in endosomes upon internalization from the plasma membrane, although the inhibition of integrin deposition to the open ZOC by protein synthesis inhibitors argues against this (Schotman, 2008).

Both trafficking events could be insensitive to BFA or to the loss of dSyntaxin5 function, and the tethering and fusion of the recycling vesicles, or even a whole endosome, could require dGRASP and the other proteins found near the open ZOC, in a similar fashion as the exocytic carriers proposed above. GrhA could have an equivalent role. However, the recycling to the plasma membrane of the mammalian integrins, like Glut4, has been shown to depend to a great extent on Rab11, and if this small GTPase is involved in αPS1 recycling, it would be expected to be fond concentrated near the open ZOC. This is not the case. The identification of αPS1-positive carriers in follicle cells will shed light on the mechanism involved in its deposition (Schotman, 2008).

In the Drosophila wing epithelia, αPS1 and αPS2 are substrates of the dGRASP-mediated pathway and the dgrasp wings exhibit blisters. However, when compared to the mew and inflated phenotype (not shown), dgrasp wings are smaller and rounder. This could be a result of the additive effect of stopping the transport of both αPS1 and αPS2. dGRASP itself could be involved in wing development, perhaps with a role in cell-cycle control. Other unidentified proteins involved in growth could use the same dGRASP-dependent pathway. It is also possible that the α subunits of integrin are involved in disc elongation, as they have been proposed to be in follicle cells (Schotman, 2008).

The intriguing question is why in the follicular epithelium, αPS1 uses an alternative pathway at stage 10B. The Golgi houses glycosylases and glycosyltransferases allowing the processing and building of complex oligosaccharides that are often required for the biological activity of glycoproteins. The Golgi bypass of αPS1 suggests that the oligosaccharide modifications carried out in this compartment are not necessary for αPS1 function at the open ZOC. Because the lack of a series of Golgi glycosylases enhances the adhesion activity of integrins, the Golgi bypass might indeed enhance or modulate integrin adhesion properties at this specific time of oocyte development. βPS is not a substrate of this noncanonical pathway. This is surprising, because α and β integrin subunits have been shown to oligomerize early in the secretory pathway, probably leading to their increased stability and efficient transport. These results suggest that the subunits are also able to travel on their own, perhaps by binding to other proteins (Schotman, 2008).

This study has shown that the integrin subunits αPS1 and αPS2 are not properly deposited in two different dgrasp mutant epithelia. The mechanism unraveled in this study could therefore also be used in other tissue remodeling events throughout Drosophila development involving adhesion. In this context, the basal adhesion of follicle cells shares many similarities with dorsal closure in embryos. The secretory process described could also apply here, and perhaps more generally in embryogenesis (Schotman, 2008).

This also gives an additional molecular handle to adhesion at the basal site that is crucially involved in the maintenance of epithelium integrity. Adhesion can be modulated by the phosphorylation of focal adhesion components leading to a change in integrin adhesive properties. In the follicular epithelium, the receptor tyrosine phosphatase Dlar genetically interacts with βPS with which it colocalizes in basal tricellular junctions in stage 7-8. There, it is involved in F-actin organization that ultimately stabilizes the epithelium. This study shows that adhesion can also be modulated at a pretranslational level by the transport (albeit noncanonical) and targeting of newly synthesized integrins to future adhesion sites (Schotman, 2008).

Taken together, it is proposed that the GRASP-mediated secretory route might be used during development as a means of targeting a specific subset of transmembrane proteins crucial for development to the plasma membrane (Schotman, 2008).

Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila

Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. This study demonstrates that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. This study investigated the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function (Bharadwaj, 2013).

Interactions between cells and the extracellular matrix (ECM) are crucial for many biological processes. These include cell migration, directed process outgrowth, basement membrane-mediated support of tissues and maintenance of cell shape. Communication between cells and ECM proteins often occurs through the action of α/β-integrin heterodimers, a receptor complex that forms adhesive contacts, including focal adhesions, hemiadherens junctions, costameres and myotendinous junctions. In response to extracellular forces, focal adhesions undergo structural changes and initiate signaling events that allow adaptation to tensile stress. Vinculin is thought to be the primary force sensor in the integrin complex, mediating homeostatic adaptation to external forces (Bharadwaj, 2013 and references therein).

Vinculin-binding partners include proteins belonging to the CAP (Cbl-associated protein) protein family. However, the physiological significance of this association is unknown. Mammalian CAP proteins are components of focal adhesions in cell culture. In myocytes, CAP localizes to integrin-containing complexes called costameres that anchor sarcomeres to muscle cell membranes. There are three mammalian CAP protein family members: CAP, Vinexin and ArgBP2. CAP associates in vitro with many proteins, including the cytoskeletal regulators Paxillin, Afadin and Filamin, vesicle trafficking regulators such as Dynamin and Cbl, and the lipid raft protein Flotillin. In vitro studies demonstrate that CAP regulates the reassembly of focal adhesions following nocodazole dissolution. However, despite extensive studies on CAP, little is known about its functions in vivo. Cap (Sorbs1) mutant mice are defective in fat metabolism, and targeted deletion of the vinexin gene results in wound-healing defects. Drosophila CAP binds to axin and is implicated in glucose metabolism . Analysis of CAP function in mammals is complicated by potential functional redundancy of the three related CAP proteins. Therefore, the function of Drosophila CAP, the single CAP family member in Drosophila, was examined in vivo (Bharadwaj, 2013 and references therein).

The Drosophila muscle attachment site (MAS) is an excellent system for studying integrin signaling. Somatic muscles in each segment of the fly embryo and larva are connected to the body wall through integrin-mediated hemiadherens junctions. Somatic muscles in flies lacking integrins lose their connection to the body wall. Surprisingly, flies lacking Vinculin, a major component of cytosolic integrin signaling complexes, are viable and show no muscle defects. Thus, unlike its mammalian counterpart, Drosophila Vinculin is apparently dispensable for the initial assembly of integrin-mediated adhesion complexes at somatic MASs (Bharadwaj, 2013).

The fly MAS is structurally analogous to the fly chordotonal organ. These organs transduce sensations from various stimuli, including vibration, sound, gravity, airflow and body wall movements. The chordotonal organ is composed of individual subunits called scolopidia, each containing six cell types: neuron, scolopale, cap, ligament, cap attachment and ligament attachment cells. Chordotonal neurons are monodendritic, and their dendrites are located in the scolopale space, a lymph-filled extracellular space completely enveloped by the scolopale cell. Within the scolopale cell, a cage composed of actin bars, called scolopale rods, facilitates scolopale cell envelopment of the scolopale space. Thus, like the MAS, the actin cytoskeleton plays a specialized role in defining chordotonal organ morphology. Similarities between MASs and chordotonal organs include the requirement during development in both tendon and cap cells for the transcription factor Stripe. Furthermore, both of these cell types maintain structural integrity under force and so are likely to share common molecular components dedicated to this function (Bharadwaj, 2013 and references therein).

This study shows that the Drosophila CAP protein is selectively localized to both muscle attachment sites and chordotonal organs. In Drosophila CAP mutants morphological defects are observed that are indicative of actin disorganization in both larval MASs and the scolopale cells of Johnston's organ in the adult. The morphological defects in scolopale cells result in vibration sensation defects in larvae and hearing deficits in adults. It was also found that, like its mammalian homologues, Drosophila CAP interacts with Vinculin both in vitro and in vivo. These results reveal novel CAP functions required for actin-mediated organization of cellular morphology, lending insight into how CAP mediates muscle and sensory organ development and function (Bharadwaj, 2013).

Integrin-based adhesion complexes are crucial for cell attachment to the extracellular matrix. These complexes change their composition and architecture in response to extracellular forces, initiating downstream signaling events that regulate cytoskeletal organization. This study has investigated the role played by the CAP protein in two stretch-sensitive structures in Drosophila: the MAS and the chordotonal organ. CAP mutants exhibit aberrant junctional invaginations at the MAS and collapse of scolopale cells in chordotonal organs. This study highlights a crucial integrin signaling function during development: the maintenance of membrane morphology in stretch-sensitive structures (Bharadwaj, 2013).

The morphological defects observed in CAP mutants could result from an excessive integrin signaling, or possibly accumulation of additional membranous components related to integrin signaling, in CAP mutants, owing to defects in endocytosis at the MAS. This is consistent with known interactions between CAP family members and vesicle trafficking regulators, including Dynamin and Synaptojanin, which are required for internalization of transmembrane proteins. Alternatively, CAP may be required for proper organization of the actin cytoskeleton at MASs, and the aberrant membrane invaginations that were observe are a secondary consequence of these cytoskeletal defects. This idea garners support from known interactions between CAP and various actin-binding proteins, including Vinculin, Paxillin, Actinin, Filamin and WAVE2. A third possibility is that CAP and Vinculin are regulators of membrane stiffness at the MAS, and aberrant junctional infoldings observed in CAP and vinculin mutants derive from diminished membrane rigidity in the presence of persistent myofilament contractile forces. Biophysical studies demonstrate that Vinculin-deficient mammalian cells in vitro show reduced membrane stiffness. Interestingly, the CAP protein ArgBP2 interacts with Spectrin, a protein important for cell membrane rigidity maintenance. These models for CAP function at MASs, however, are not mutually exclusive. Interestingly, disruption of the ECM protein Tiggrin leads to MAS phenotypes similar to CAP. Future studies on CAP interaction with Tiggrin and other CAP-interacting proteins will shed light on mechanisms underlying CAP function. Nevertheless, this study demonstrates in vivo the importance of CAP in stretch-sensitive organ morphogenesis, and it will be interesting to determine whether this function is phylogenetically conserved (Bharadwaj, 2013).

Apart from the MAS, CAP is also expressed at high levels in chordotonal organ scolopale cells, and this study has found that CAP mutants are defective in vibration sensation, a hallmark of chordotonal organ dysfunction. However, only the initial fast hunching response to vibration is disrupted in CAP mutant larvae. This may result from a partial loss of chordotonal function in these organs in the absence of CAP. A functional defect was also observed in the adult Johnston's organ; CAP mutant flies show diminished sound-evoked potentials. Importantly, the scolopale cells in CAP mutants appear partially collapsed. The extracellular space within the scolopale cell is lined by an actin cage, and CAP may influence the proper assembly of this actin cage or its association with the scolopale cell membrane. Ch organs are mechanosensory detectors and are constantly exposed to tensile forces. Thus, CAP apparently influences cytoskeletal integrity in two actin-rich structures: the MAS and the chordotonal organ, both of which are involved in force transduction (Bharadwaj, 2013).

Mammalian and Drosophila CAP bind to Vinculin. Vinculin is required for the recruitment of the mammalian CAP protein vinexin to focal adhesions in NIH3T3 cells in vitro. Consistent with this observation, a dramatic decrease was seen in CAP levels at MASs in vinculin mutants, but residual levels of CAP protein remain. Furthermore, CAP localization at the muscle fiber Z-lines is completely unaltered in vinculin mutants. These observations indicate that Vinculin is not the sole upstream regulator of CAP localization. vinculin mutants show some of the phenotypic defects observed in CAP mutants; however, these defects are less pronounced. Therefore, the residual CAP pool that is recruited to MASs in a Vinculin-independent manner is apparently sufficient for partial CAP function. Assessment of CAP and Vinculin function at the larval MAS shows that these proteins are required for maintaining the integrity of junctional membranes in the face of tensile forces. CAP proteins may serve as scaffolding proteins at membrane-cytoskeleton interfaces and facilitate the assembly of protein complexes involved in cytoskeletal regulation and membrane turnover (Bharadwaj, 2013).

Mutations in the CAP-binding protein filamin cause myofibrillar myopathy. This, in combination with data showing a crucial role for CAP in regulation of muscle morphology, sets the stage for investigating how loss of CAP protein function might influence the etiology of myopathies (Bharadwaj, 2013).

Protein Interactions

scab was initially described in a study of mutations that affect the pattern of the larval cuticle. The defect in dorsal closure that describes the effects of scab mutation is similar to that seen in myospheroid mutant embryos. Myospheroid (also known as beta PS) is the dimerization partner of two previously characterized alpha integrins: alphaPS1 (Multiple edematous wings) and alphaPS2 (Inflated). Dorsal closure defect is not seen in null mutations of these two alpha integrins, indicating that some other alpha integrin must team up with betaPS during dorsal closure.

In a search for the presumed missing integrin, attention was focussed on a 90kDa band associated with immunoprecipitates of Myospheroid, resulting from the application of an anti-betaPS antiserum. This 90 kDa protein forms a non-covalent, divalent cation-dependent complex with Myospheroid. The 90 kDa protein binds well to both lentil lectin and Concanavilin A beads, suggesting that it is a glycoprotein. The protein was purified by immunoprecipitation, lecitin binding, elution and SDS gel electrophoresis and subjected to tryptic digestion; the resulting peptides were then sequenced. Degenerate primers based on the amino acid sequences were used to identify the cDNA coding for the 90 kDa protein. The sequence revealed an alpha integrin subunit that has been designated alphaPS3. Thus, the cloning of scab, revealing as it does the gene coding for the missing integrin, completes a picture of integrin activity with the discovery of a third alpha integrin partnering Myospheroid (Stark, 1997).

Scab RNA is localized to tissues undergoing invagination, tissue movement and morphogenesis: for example, salivary gland, trachea, midgut, dorsal vessel, midline of the ventral nerve cord, amnioserosa and the amnioproctodeal invagination. AlphaPS3 DNA localizes to the chromosomal vicinity of scab (scb), previously identified by a failure of dorsal closure. Embryos homozygous for the 119 allele of scb have no detectable alphaPS3 RNA. The 1035 allele of scb contains a P element inserted just 5' of the coding region for the shorter of the gene's two transcripts. Mutations in the scb locus exhibit additional defects corresponding to sites of alphaPS3 transcription, including abnormal salivary glands, mislocalization of the pericardial cells and interrupted trachea. Removal of both maternal and zygotic betaPS produces similar defects, indicating that these two integrin subunits associate in vivo and function in the movement and morphogenesis of tissues during development in Drosophila. Phenotypic similarities suggest that laminin A is a potential ligand for this integrin, at least in some tissues (Stark, 1997).

Tiggrin, a novel Drosophila extracellular matrix protein contains the potential integrin recognition sequence Arg-Gly-Asp (RGD) and 16 repeats of a novel 73-77 amino acid motif. The tiggrin gene is expressed by embryonic hemocytes and fat body cells. Tiggrin protein is detected in matrices, especially at muscle attachment sites that also strongly express integrins. Tiggrin may help to mediate integrin-dependent adhesion at embryonic muscle insertions. Tiggrin-coated surfaces support primary embryo cell culture and provide excellent substrates for alphaPS2 betaPS integrin-mediated cell spreading. Soluble RGD-peptides, able to act as ligands for integrins, inhibit this cell spreading (Fogerty, 1994).

PS1 (alphaPS1 betaPS) integrin is a laminin receptor. Both PS1 and PS2 integrins promote cell spreading on two different Drosophila extracellular matrix molecules, Laminin and Tiggrin, respectively. The differing ligand specificities of these two integrins, combined with data on the in vivo expression patterns of the integrins and their ligands, leads to a model for the structure of integrin-dependent attachments in the pupal wings and embryonic muscles of Drosophila (Gotwals, 1994a).

Two new potential ligands of the Drosophila PS2 integrins have been characterized by functional interaction in cell culture. These potential ligands are a new Drosophila laminin alpha2 chain encoded by the wing blister locus and Tenascin-major, an extracellular protein known to be involved in embryonic pattern formation. As with previously identified PS2 ligands, both contain RGD sequences, and RGD-containing fragments of these two proteins (DLAM-RGD and TENM-RGD) can support PS2 integrin-mediated cell spreading. In all cases, this spreading is inhibited specifically by short RGD-containing peptides. As previously found for the PS2 ligand Tiggrin (and the Tiggrin fragment TIG-RGD), TENM-RGD induces maximal spreading of cells expressing integrin containing the alphaPS2C splice variant. This is in contrast to DLAM-RGD, which is the first Drosophila polypeptide shown to interact preferentially with cells expressing the alphaPS2 m8 splice variant. The betaPS integrin subunit also varies in the presumed ligand binding region as a result of alternative splicing. For TIG-RGD and TENM-RGD, the beta splice variant has little effect, but for DLAM-RGD, maximal cell spreading is supported only by the betaPS4A form of the protein. Thus, the diversity in PS2 integrins due to splicing variations, in combination with diversity of matrix ligands, can greatly enhance the functional complexity of PS2-ligand interactions in the developing animal. The data also suggest that the splice variants may alter regions of the subunits that are directly involved in ligand interactions, and this is discussed with respect to models of integrin structure (Graner, 1998).

Curiously, the ten-m gene is expressed in an embryonic pair-rule pattern, and ten-m mutants display pair-rule patterning defects. Since the protein influences expression of downstream genes, it must communicate its presence to the cell nucleus. However, it does not appear that integrin signal transduction is important in early embryonic segmentation. PS integrins are not strongly expressed at this time, and, more importantly, mutations in integrin subunit genes do not cause segmentation phenotypes (Graner, 1998 and references).

Ten-m is later localized (among other places) at muscle attachment sites, where integrins are known to accumulate. This localization of Ten-m in vivo, as well as the demonstration of TENM-RGD interactions with PS2 integrins in vitro, suggests that Ten-m may function with PS2 integrins in muscle attachment. Interestingly, the heparan sulfate-containing protein D-syndecan also localizes to muscle attachments, and Ten-m contains a consensus heparin-binding sequence near the RGD, suggesting the potential of a Ten-m-syndecan-integrin complex. Syndecan proteoglycans recently have been shown to be important in signal transduction in focal adhesions in vertebrate cells (Graner, 1998 and references).

The available data, although very suggestive, do not demonstrate unequivocally that Ten-m serves as an integrin ligand at muscle attachment sites. However, other potential PS2 ligands, such as Tiggrin, also accumulate at muscle attachment sites, and genetic studies of tiggrin suggest considerable functional redundancy among the extracellular matrix components there. Because of this redundancy, a direct genetic demonstration of a role for Ten-m in muscle attachment may require simultaneous disruption of multiple genes encoding matrix proteins, and the early embryonic phenotype of ten-m mutants will further complicate such an analysis. One potential approach might be to demonstrate a dominant genetic effect of ten-m mutations in a background that has been sensitized for loss of function phenotypes by viable mutations in other genes that encode proteins important for muscle attachment or other integrin-dependent processes (Graner, 1998).

The integrin family of cell surface receptors mediates cell-substrate and cell-to-cell adhesion and transmits intracellular signals. In Drosophila there is good evidence for an adhesive role for integrins, but evidence for integrin signaling has remained elusive. Each integrin is an alphabeta heterodimer; the Drosophila betaPS subunit forms at least two integrins by association with different alpha subunits: alphaPS1betaPS (PS1) and alphaPS2betaPS (PS2). The complex pattern of PS2 integrin expression includes, but is more extensive than, the sites where PS2 has a known requirement. a comprehensive genetic analysis was carried out on the gene inflated, which encodes alphaPS2. Thirty-five new inflated alleles were isolated; 10 additional alleles were obtained from other investigators. The majority of alleles are amorphs (36/45) or hypomorphs (4/45), but five alleles that affect specific developmental processes were identified. Interallelic complementation between these alleles suggests that some alleles may affect distinct functional domains of the alphaPS2 protein, which specify particular interactions that promote adhesion or signaling. One new allele reveals that the PS2 integrin is required for the development of the adult halteres and legs as well as the wing (Bloor, 1998).

An examination of the phenotypes of the new classes of inflated alleles demonstrates that the inflated gene has separate functions in the somatic musculature vs. the gut and nerve cord. The class II allele, if SEF, is an embryonic lethal yet the phenotype is surprisingly mild: the nerve cord is fully condensed, midgut morphogenesis occurs normally, and the vast majority of muscles remain attached to the epidermis. The somatic muscles in if SEF mutant embryos, however, do exhibit a defect in the contractile ultrastructure. Examination by polarized light shows little evidence of sarcomeric structure in these muscles; staining for filamentous actin with rhodamine-phalloidin reveals that the f-actin fails to become properly organized. Embryos were examined at earlier times during stage 17 with polarized light. The appearance of striations in if SEF mutant embryos was not observed, suggesting that the PS2 integrin is required for the formation of muscle sarcomeric structure rather than for its maintainance. The strong waves of muscle contraction that normally accompany hatching from the vitelline membrane and chorion are not observed in these mutants, although some residual muscle function is present, since weak muscle contractions occur if the mutant animal is poked with a needle. Therefore, the if SEF mutant appears to be unable to form normal contractile somatic muscles. When if SEF mutant embryos were stained with the PS2hc/2 monoclonal antibody, staining was detected; however, wild-type staining could be detected with a polyclonal antisera directed against the C-terminal 15 amino acids of the PS2 subunit. This suggests that this mutant alters the conformation of the PS2 integrin (and the PS2hc/2 epitope) rather than its expression (Bloor, 1998).

A significant fraction of embryos mutant for the class III inflated alleles hatch to first instar larvae. Approximately one-fifth of the mutant individuals carrying the class III if C2B allele hatch: these larvae slowly become less motile and die over the next 48 hr. if C2B mutant embryos that fail to hatch have been examined by polarized light and rhodamine-phalloidin staining. The muscles remain attached and have normal sarcomeric structure. In contrast, the midgut fails to elongate and only two fat gastric caecae are formed. Staining of the visceral muscles in the mutant midguts shows that there is some detachment of the visceral muscle layer, but that the sarcomeric structure is not perturbed. Hatched if C2B larvae possess the same phenotypic characteristics as their unhatched counterparts, that is, wild-type muscles and abnormal midguts. It seems likely that the larval lethality is a result of their inability to feed, altough it is unknown why some of the mutant embryos fail to hatch. The lethal class III embryos also have defective nerve cord condensation. When these alleles are placed over Df(1)rif, the gut and nerve cord phenotypes are enhanced, and rare muscle detachments are observed (Bloor, 1998).

The if V2 allele causes a substantial reduction in the expression of the PS2 subunit in the third instar wing imaginal disc and is semilethal with a very strong adult phenotype. There is some larval lethality (but no embryonic lethality), to judge by the reduced numbers of if V2 pupae and adults relative to their siblings. The majority of the mutant individuals die while eclosing: they get their head and legs out but then become stuck. This is likely due to the inflated wings sticking to the pupal case. A few mutant individuals do successfully eclose and have severe adult abnormalities, although they are viable and fertile. The two layers of the wing blade are completely separated and the wings appear as hemolymph filled balloons. The hemolymph often becomes dried and blackened within the wing. In addition to this extreme version of the wing blister phenotype previously observed for inflated, two novel phenotypes are observed in this mutant. The halteres are distorted, appearing longer and less rounded than in wild type and have a rougher surface. The legs are also misshapen, with a kink in the femur, particularly in the second and third legs (Bloor, 1998).

Using a Drosophila cell line, a monoclonal antibody that inhibits not only cell clumping but also cell spreading has been generated. This antibody immunoprecipitates a complex of proteins identical to PS beta and other proteins. The antibody preferentially recognizes the PS beta associated with particular alpha chains in situ. The cells spread very well on dishes coated with vitronectin and, to a lesser extent, on those with fibronectin. The cells also can attach to dishes coated with laminin but without spreading; this attachment was not inhibited by antibody (Hirano, 1991).

The closest mammalian equivalents of Drosophila alphaPS1 are alpha3, alpha6 and alpha7, while the closest mammalian equivalents of alphaPS2 are alpha11b, alpha8, alphaV and alpha5. The ability of different Drosophila integrin alpha subunits to substitute for one another during embryonic development was tested. Two alpha subunits, which form heterodimers with the same betaPS subunit, are expressed in complementary tissues in the Drosophila embryo, with alphaPS1 expressed in the epidermis and endoderm, and alphaPS2 expressed in the mesoderm. As a result the two integrin heterodimers are present on opposite surfaces at sites of interaction between the mesoderm and the other cell layers, where they are required for normal development. Using the GAL4 system, the embryonic lethality of an alphaPS2 null mutation was rescued with a UAS-alphaPS2 transgene, but only partially with a UAS-alphaPS1 gene, as evidenced by the partial rescue of both muscle and midgut phenotypes. Similarly the embryonic/first instar larval lethality of an alphaPS1 null mutation gene was rescued with UAS-alphaPS1, but only partially with UAS-alphaPS2. Each UAS-alpha gene, when it contains the cytoplasmic domain from the other alpha subunit, maintains an equivalent ability to rescue its own mutation and cannot fully rescue a mutation in the other alpha. It is concluded that the two alpha subunits are not equivalent and have distinct functions that reside in the extracellular domains (Martin-Bermudo, 1997).

Integrin cell surface receptors are ideally suited to coordinate cellular differentiation and tissue assembly during embryogenesis because they can mediate both signaling and adhesion. The identification of two genes, Mt and 258, that require integrin function for their normal expression in Drosophila midgut endodermal cells has shown that integrins regulate gene expression in the intact developing embryo. The relative roles of integrin adhesion versus signaling in the regulation of these integrin target genes was determined. Integrin-mediated adhesion is not required between the endodermal cells and the surrounding visceral mesoderm for integrin target gene expression. In addition, a chimeric protein that lacks integrin-adhesive function, but maintains the ability to signal (TorsoD), can substitute for the endogenous integrin and regulate integrin target genes. This chimera consists of an oligomeric extracellular domain fused to the integrin betaPS subunit cytoplasmic domain; a control monomeric extracellular domain fusion does not alter integrin target gene expression. Therefore, oligomerization of the 47-amino-acid betaPS intracellular domain is sufficient to initiate a signaling pathway that regulates gene expression in the developing embryo (Martin-Bermudo, 1999a).

Integrin regulation of gene expression does not require specific alpha subunit function. Whereas the betaPS cytoplasmic domain alone can mimic PS1 integrin signaling when fused to TorsoD, in the intact integrin the alpha subunit will be required for interaction with the extracellular ligands to promote clustering and may also play a role inside the cell in the signaling pathway. To test whether specific alpha subunits are required for signaling by PS integrin heterodimers, the consequences of switching alpha subunits were examined in the endodermal cells. alphaPS2 is not able to substitute for alphaPS1 function in the midgut when assayed by larval lethality. Expression of UAS-alphaPS1 with the GAL4 driver can substitute for endogenous alphaPS1 function to repress the target gene 258 . Next to be tested were two chimeric alpha subunits, in which the cytoplasmic domains were swapped between alphaPS1 and alphaPS2, were tested, as well at the normal alphaPS2 subunit. It was found that all three can substitute for alphaPS1 and repress 258 expression. This shows that the alpha subunits do not provide specificity to this signaling event. In addition, it shows that the PS2 integrin is able to interact with enough ligands to become clustered and initiate a signaling pathway, even when it is expressed in an ectopic location. These results suggest that dimerization of the PS subunit intracellular domain is sufficient to initiate a signaling pathway that can upregulate and downregulate gene expression. This shows that whereas integrin ligand binding is used for adhesion to the extracellular matrix, as signaling receptors, the integrins are formally equivalent to growth factor receptors, in that their ability to mediate adhesion is not required for integrins to regulate gene expression. Thus, these results have confirmed the importance of integrins in providing a vital link between cell adhesion during morphogenesis and cellular differentiation (Martin-Bermudo, 1999a).

The localized assembly of extracellular matrix integrin ligand Tiggrin requires cell-cell contact

The assembly of an organism requires the interaction between different layers of cells, in many cases via an extracellular matrix. In the developing Drosophila larva, muscles attach in an integrin-dependent manner to the epidermis, via a specialized extracellular matrix called tendon matrix. Tiggrin, a tendon matrix integrin ligand, is primarily synthesized by cells distant to the muscle attachment sites, yet it accumulates specifically at these sites. Previous work has shown that the PS integrins are not required for tiggrin localization, suggesting that there is redundancy among tiggrin receptors. This was examined by testing whether the PS2 integrin can recruit Tiggrin to ectopic locations within the Drosophila embryo. It was found that neither the wild type nor modified forms of the PS2 integrin, which have higher affinity for Tiggrin, can recruit Tiggrin to new cellular contexts. Next, the fate was genetically manipulated of the muscles and the epidermal muscle attachment cells; this demonstrated that muscles have the primary role in recruiting Tiggrin to the tendon matrix and that cell-cell contact is necessary for this recruitment. Thus it is proposed that the inherent polarity of the muscle cells leads to a molecular specialization of their ends, and interactions between the ends produces an integrin-independent Tiggrin receptor. Thus, interaction between cells generates an extracellular environment capable of nucleating extracellular matrix assembly (Martin-Bermudo, 2000b).

This paper uses the muscle attachment sites and the integrin ligand Tiggrin as a model system to study the mechanisms that regulate the spatial and temporal assembly of ECM during embryogenesis. How the extracellular matrix protein Tiggrin comes to be tightly localized at the interface between the specialized epidermal tendon cells and the ends of the muscles at the muscle attachment sites was examined. Whether one Tiggrin cell surface receptor, the PS2 integrin, is able to localize Tiggrin to new sites within the embryo was tested -- it is not. Then an examination was performed of what cells are required for the localization of Tiggrin; muscles are required, while the tendon cells are not. Unexpectedly it was found that the localization of Tiggrin to the end of a muscle requires contact between the muscle and another cell (Martin-Bermudo, 2000b).

The requirement for integrins in the assembly of ECM in vivo is clearly variable. In amphibian embryos, the accumulation of fibronectin fibrils is blocked by antibodies against the integrins. However, genetic elimination of integrin function has more modest effects. The initial assembly of extracellular matrices appears normal in mouse embryos lacking a variety of integrin subunits, although the matrices formed may be less stable. Ultrastructural analysis shows that in the absence of PS integrin function the tendon matrix still accumulates at the muscle attachment sites, although it is separated from the cell surfaces. This is further supported by light microscopic findings showing that the tendon matrix protein Tiggrin accumulates correctly in embryos lacking PS integrins. In addition, the PS2 integrin is not only not necessary for Tiggrin localization but is also not sufficient. Therefore, a mechanism for the assembly of extracellular matrix at the muscle attachment sites has to be integrin-independent (Martin-Bermudo, 2000b).

This work has posed two key questions that will have to be resolved in order to understand the mechanism of Tiggrin localization: why does it require cell-cell contact, and why do some sites of cell-cell contact recruit Tiggrin, while others do not? The latter point implies that something must be special about the ends of the muscles; when they make cell-cell contacts they recruit Tiggrin, while other cell-cell contacts, for example between the lateral surfaces of the muscles, do not. This difference reflects the inherent polarity present within the developing muscles, which has been revealed by two separate experiments. When the rat transmembrane protein CD2 is expressed in Drosophila muscles it is uniformly distributed on membrane, but when its cytoplasmic tail is replaced with that from the ßPS subunit, then the chimeric protein is localized to the ends of the muscles. This demonstrates that the ßPS cytoplasmic tail is recognized inside the cell and localized to the ends of the muscles. By examination of the localization of kinesin-ß-galactosidase fusion proteins, it has been shown that the muscles contain a bipolar arrangement of microtubules, with the plus ends at the termini of the muscles. Thus the muscles clearly have an internal polarity that is able to localize molecules specifically to the ends of the muscles, and it is proposed that this is the first step leading to the localization of Tiggrin (Martin-Bermudo, 2000b).

There are a variety of possible models to explain why cell-cell contact is required for Tiggrin localization and three will be outlined here. In the first model, the inherent polarity of the muscles leads to the localization of a transmembrane Tiggrin receptor at the ends of the muscle. However, this receptor is not active until the muscle cells have made contact with another cell, such as the end of the equivalent muscle in the next segment. Following this cell contact-dependent interaction, Tiggrin can bind to the receptor, and later in development binds more strongly to other proteins in the extracellular matrix, possibly by becoming crosslinked to them, so that when the muscle detaches in the PS2 integrin mutant, Tiggrin remains with the extracellular matrix. In a second model, polarization of the cells results in the specific transport of vesicles containing transmembrane receptors and or extracellular matrix components to the ends of the muscles. Then, the fusion of these vesicles with the plasma membrane, which releases the contents, requires cell-cell contact. This could be achieved by the interaction of a transmembrane receptor with a ligand on the apposing cell, which triggers an intracellular pathway leading to vesicle fusion. In this model, since the vesicles are localized, the receptor that triggers fusion does not have to be. One of the proteins in the vesicle is not freely diffusible (for example by being tethered to the membrane) and contains a binding site for Tiggrin, thus recruiting Tiggrin to the tendon matrix. Such polarized discharge of matrix components has been described in diverse vertebrate cells, although it has not been shown to require cell-cell contact (Martin-Bermudo, 2000b).

In these models, focus was placed on the muscles, since the localization of Tiggrin requires only the muscles. However, both models may also be applicable to the localization by tendon cells of ECM proteins involved in tendon cell attachment to the tendon matrix, such as the proteins affected by the rhea mutation. The main difference between the cells on the two sides of muscle attachments is that the muscles make cell-cell contacts with each other and with the tendon cells, while the tendon cells only make contact with the muscles. One of the attractive aspects to the vectorial discharge model is that the epidermal cells and muscles could secrete components that crosslink together when they interact in the extracellular space, forming a stable matrix. This would be similar to basement membrane assembly at the interface between the epidermis and the dermis, where laminin and its interacting partner nidogen are expressed in different layers. Such interaction between components secreted by the two layers may be important for the formation of a functional tendon matrix, as suggested by the rhea phenotype, but is not required for Tiggrin localization (Martin-Bermudo, 2000b).

The two models described above involve cell contact-dependent activation of Tiggrin receptors, but it is difficult to rule out the third model, where cell-cell contact has a more mechanical role and is only required to reduce Tiggrin diffusion. For example the inherent polarity could produce a Tiggrin receptor at the ends of the muscles that is fully active prior to cell-cell contact. The interaction of Tiggrin with this receptor could be short-lived, so that it comes off again and diffuses away. The cell-cell interaction would serve to make an enclosed space or 'basket', where the resulting concentration of Tiggrin would reach a high enough concentration to assemble into an insoluble matrix. However, the assembly of the tendon matrix clearly differs from the assembly of basement membranes and fibronectin fibrils, which can form on a cell surface that faces the extracellular fluid. Of course the actual mechanism of tendon matrix localization could easily involve all of these possible mechanisms (Martin-Bermudo, 2000b).

In summary, a combination of cell polarity and cellular interaction result in the assembly of the tendon matrix in the right place. These results have shown that in the developing embryo cell-cell contact is necessary and may be sufficient for the formation of a localized matrix, and have allowed the formulation of models that are consistent with diverse experimental results. Further characterization of the components of the tendon matrix and the transmembrane receptors on the cells should allow a determination of the mechanism of cell contact dependent localization (Martin-Bermudo, 2000b).

Evidence for an interaction between integrins and Talin

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).

Amino acid changes in Drosophila alphaPS2ßPS integrins that affect ligand affinity

A ligand-mimetic antibody Fab fragment specific for Drosophila alphaPS2ßPS integrins was developed to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the alphavß3 ligand-mimetic antibody WOW-1. The specificity of alphaPS2ßPS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the ßPS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the alphaPS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn2+. Although Mn2+ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn2+ could be increased further by the alphaPS2 GFFNR --> GFANA mutation. A mutation in the ßPS I domain (ßPS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by alphaPS2ßPS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin ß subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. Complete deletion of the ßPS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because ßPS-DeltaPSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system (Bunch, 2006).

Integrins are the primary family of receptors that connects cells to the extracellular matrix (ECM). The cytoplasmic tails of integrin subunits associate with multiple intracellular components, which mediate both signaling functions and ECM-cytoskeleton connections. Cells can regulate the functions of integrins at least in part by inducing conformational changes in integrin structure that alter the affinity of the integrin heterodimer for ECM ligands. Similarly integrin binding to ligands can lead to outside-in signal propagation, which can regulate cellular behaviors such as growth, differentiation, and survival (Bunch, 2006)

The integrin heterodimer is composed of alpha and ß subunits that are nonhomologous to one another but strongly conserved structurally across the animal kingdom. A long history of studies with conformation-sensitive antibodies indicates that integrins undergo large and concerted conformational changes as a result of cellular activation, and recent studies have begun to provide details of this structural switching. Inactive integrins are likely to be bent in the middle so that the headpiece faces in toward the membrane-proximal part of the extracellular stalks. As a result of cellular activation, the integrin adopts an extended conformation with the headpiece facing away from the cell in optimal position to engage ECM proteins. The headpiece also can adopt two states, termed the open or closed conformations, corresponding to high and low affinity states for ligand binding. Binding of substrate ligands traps the integrin in the open conformation, and this shift in the equilibrium may then trigger outside-in signaling in many cases involving integrin clustering. This view of integrin dynamics is almost certainly simplified with other intermediate states probable (for example, bent integrins can also bind ligand, but overall it can provide a consistent explanation of the structure-function relationships between cellular regulation and signaling (Bunch, 2006).

Invertebrates such as Drosophila provide a system for sophisticated genetic studies of integrin function that are not practical with vertebrates. Genetic studies with flies can also be complimented with cell biology experiments in cell culture, and this system has become especially attractive with the simplicity of RNA interference methods in Drosophila. However, one persistent drawback to studies of integrin structure and function in invertebrates has been a lack of probes for measuring affinity states of integrins directly. Even in cell culture, Drosophila experiments have relied on indirect assays such as cell adhesion or spreading to quantitate integrin activity. Although these methods have generally provided a consistent set of data, a tool for measuring ligand affinity directly could permit much more reliable and rapid assays that can complement genetic analyses of integrin function in Drosophila (Bunch, 2006).

This study describes the generation of TWOW-1, a novel monovalent antibody Fab fragment that functions as a ligand-mimetic affinity sensor for alphaPS2ßPS. Using this probe, it is shown that previously described mutants in both the alphaPS2 and ßPS subunits lead to changes in affinity of the fly integrins. Finally it is demonstrated that complete removal of the ß subunit N-terminal PSI domain causes an increase in ligand affinity (Bunch, 2006).

The integrin ligand Thrombospondin mediates adhesion that is essential for the formation of the myotendinous junction in Drosophila

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 development of functional musculature depends on the correct encounter and adhesion of muscles with their corresponding tendon cells. In Drosophila the hemiadherens junctions, formed on both sides of the myotendinous junction, mediate the adhesion between muscles and their corresponding tendon cells. The muscle-specific integrin heterodimer αPS2ßPS accumulates at the muscle counterpart of this junction, and binds to its specific extracellular matrix (ECM) ligand Tiggrin. Correspondingly, the tendon-specific integrin heterodimer αPS1ßPS accumulates at the tendon counterpart of the myotendinous junction, and is thought to associate with the laminin ligand. Both hemiadherens junctions on each cell type exhibit a symmetrical distribution, raising the possibility that, although each cell utilizes a distinct integrin heterodimer, the formation of the myotendinous junction is coordinated between the two cell types. In the absence of the common ßPS subunit Myospheroid, muscles initially interact with tendon cells; however, following muscle contraction the muscles detach from the tendon cells and round up (the myospheroid phenotype). Notably, lack of the muscle-specific αPS2 subunit similarly leads to muscle detachment; by contrast, however, lack of the tendon-specific αPS1 (e.g., in the mew mutant embryos) does not lead to muscle detachment. mew mutant embryos hatch, suggesting that occupation of the muscle-specific αPS2ßPS by its ligand may be sufficient for the formation of embryonic myotendinous junctions. The αPS1 belongs to the laminin-binding type α family of receptors and binds to laminin. Drosophila laminin may consist of ß1 and ß2 subunits and either of two laminin α subunits. The αPS1 is thought to associate with laminin containing the LanA subunit (also known as α3,5), which when deleted does not exhibit significant muscle-tendon attachment defects. By contrast, lack of the laminin α1,2 (wing blister), which associates with the αPS2ßPS (Graner, 1998), results in a mild muscle-detachment phenotype (e.g., wing blister mutants) , pointing to the crucial function of the muscle-specific PS2 in the formation of the myotendinous junction (Subramanian, 2007).

Tiggrin, a Drosophila-specific ECM component, has been shown to associate with the muscle-specific αPS2ßPS integrin. However, homozygous tiggrin mutant embryos do form muscle-tendon junctions and the adult flies are only semilethal (Subramanian, 2007).

In addition to its role in the establishment of myotendinous junctions, integrin-mediated adhesion is essential for several biological processes, including dorsal closure, visceral mesoderm development and the development of the adult fly wing. Wing epithelial cells from the dorsal and ventral aspects of the wing form specialized integrin-mediated adherens junctions required for the development of the adult fly wing. At morphogenesis dorsal wing epithelial cells expressing αPS1ßPS are brought together with ventral cells that express αPS2ßPS. Adhesion between these two epithelial sheets of cells is presumably mediated by specific ECM ligands. Although the involvement of the laminin α1,2 (wing blister) has been described, ligand specificity of each of the PS integrin receptors in this context has yet to be elucidated (Subramanian, 2007).

Tendon cells are specified in the Drosophila ectoderm as a result of the activity of the tendon-specific transcription factor Stripe. Embryos mutant for stripe do not develop normal tendon cells. Conversely, ectopic expression of Stripe leads to ectopic development of tendon cells. Thrombospondin was recovered in a search for genes that are regulated by the tendon-specific transcription factor Stripe (Subramanian, 2007).

Thrombospondins (Tsps) are a family of extracellular matrix proteins that mediate cell-cell and cell-matrix interactions by binding membrane receptors, extracellular matrix proteins and cytokines (Adams, 2001; Lawler, 2000). In vertebrates there are five tsp genes expressed in various tissues, including the brain (TSP1 and Tsp2), bones (Tsp5) and tendons (Tsp4). Tsp1 and Tsp2 are closely related trimeric proteins that share the same set of structural and functional domains. Tsp4 and Tsp5 are pentameric and differ from Tsp1 and Tsp2 in their domain arrangement. All Tsps share a typical C-terminal domain (CTD) that contains epidermal growth factor (EGF)-like repeats, and a Ca-binding domain. The N-terminal domain contains additional conserved regions including the laminin G-like domain (which is not present in Tsp5). Drosophila tsp is encoded by a single gene that is spliced into four variants, among which only one (TspA) contains the conserved CTD, which in addition to the EGF repeats and Ca-binding domains also includes a putative integrin-binding KGD motif. The N-terminal domain contains a conserved heparin-binding domain and putative integrin-binding motifs RGD and KGD. Drosophila Tsp is closest in structure to vertebrate Tsp-5/COMP, which is expressed mainly in cartilage and certain other connective tissues and has a role in chondrocyte attachment, differentiation and cartilage ECM assembly (Subramanian, 2007).

A wide range of functions has been attributed to the different Tsps, including a role in platelet aggregation, inflammatory response, regulation of angiogenesis during wound healing, and tumor growth. Tsp1 and Tsp2 have been described as astrocyte-secreted components that promote synapse formation in the CNS (Subramanian, 2007).

The large isoform of Drosophila Tsp has been shown to form pentamers and exhibits heparin-binding activity. Its major sites of expression in the embryo are the muscle attachment sites, and also the precursors of the longitudinal visceral muscles. In larval stages it is expressed in wing imaginal discs (Subramanian, 2007).

This study reports that Drosophila Tsp is a key ECM component that is required for muscle-specific adhesion to tendon cells. In tsp mutant embryos muscles fail to attach to tendon cells, and often aggregate and form ectopic integrin-mediated junctions with neighboring muscles. This leads to nonfunctional somatic musculature and embryonic lethality. In the embryo, Tsp is required for integrin-mediated adhesion as measured by Talin-specific accumulation. Furthermore, Tsp can functionally bind to αPS2ßPS-integrins; the purified CTD of Tsp mediates PS2 integrin-dependent cell spreading in a KGD- and PS2-dependent manner (Subramanian, 2007).

Taken together, these results suggest a model whereby Tsp produced by tendon cells is required for muscle-specific adhesion to tendons by binding the muscle-specific αPS2ßPS integrin receptors, and a subsequent consolidation of the junction by enhanced tendon-specific production of Tsp secreted into the junctional space (Subramanian, 2007).

It is suggested that the dynamics of myotendinous junction formation involve the following sequential steps. (1) When the myotube is very close to the tendon cell, Tsp secreted continuously from the tendon cell associates with the muscle leading edge and binds to the muscle-specific αPS2ßPS integrin receptors. Because Drosophila Tsp forms pentamers, each pentamer potentially associates with several PS2 receptors, leading to accumulation of αPS2ßPS receptors at the myotube leading edge. This association triggers integrin-mediated adhesion and Talin accumulation at the cytoplasmic tail of the PS2 integrin receptors. (2) Tsp may bind to the tendon surfaces through an unknown ligand. (3) Stripe levels in the tendon cell are elevated following the establishment of the muscle-tendon junction, because of Vein-EGF receptor (EGFR) signaling. Stripe induces the elevation of Tsp levels, creating a positive feedback loop that encourages further secretion and accumulation of Tsp at the junction site, strengthening the myotendinous junction (Subramanian, 2007).

The KGD site in the CTD of Tsp was shown to trigger PS2 integrin-dependent cell spreading. This sequence had been shown to bind certain types of vertebrate integrin receptors (Scarborough, 1993). The N-terminal domain of Tsp contains an additional KGD site, and an RGD site, both implicated in integrin-binding activity. These sites may also contribute to the binding of the PS2 muscle-specific integrins. Therefore, each Tsp pentamer contains multiple binding sites for PS2 integrin receptors, and thus may induce receptor aggregation at the muscle leading edge. It remains to be determined whether Tsp is capable of binding to PS1 integrins or other receptors expressed by the tendon cell (Subramanian, 2007).

Whether Tsp functions as an integrin ligand in other tissues (e.g., midgut, salivary gland, dorsal closure and the wing epithelium) is yet to be elucidated. Phenotypic analysis of the tsp8R mutant embryos did not reveal a major phenotype in the gut, CNS or dorsal closure. Similarly, tsp8R mutant clones induced at the larvae stage did not result with wing blisters as in integrin-induced clones. Although mutants for the tsp8R allele did not show staining with the anti-Tsp antibody, it is still possible that residual Tsp activity is retained in the mutants because of the activity of the other TSP isoforms (which were not affected by the deletion of the EP excision at the CTD). In addition, maternal tsp transcripts were detected that may partially rescue the zygotic tsp phenotype in the early developmental stages (Subramanian, 2007).

An additional relevant ECM component at the myotendinous junction is laminin. Laminin α1,2 (encoded by wing blister) is required for the formation of the myotendinous junction (Martin, 1999). Laminin α1,2 contains an RGD sequence and also binds to the PS2 integrins (Graner, 1998), demonstrating the crucial role of these receptors in the formation of the myotendinous junctions. It is possible that laminin containing the laminin α1,2 subunit associates with Tsp in the myotendinous junctional space. Both laminin and Tsp carry a heparin-binding domain and it is possible that they interact indirectly through a putative heparin-containing proteoglycan. Because no changes in laminin distribution was detected following overexpression of Tsp (using anti-laminin antibody), it is not thought that there is any direct Tsp-laminin interaction. The heparan sulfate glycoprotein Syndecan is produced by the muscle cells. In syndecan mutant embryos the somatic muscle pattern is defective, a phenotype that is attributed to an effect of Syndecan on Slit distribution and function. However, Syndecan at the muscle cell membrane may contribute to a putative indirect interaction between Tsp and laminin through its heparin-containing domain. Such interaction may enhance the accumulation of ECM components such as Tsp and laminin at the myotendinous junction. In support of this hypothesis, vertebrate Tsp has been shown to bind Syndecan at its CTD (Adams, 2004). However, syndecan homozygous mutant embryos do not exhibit alterations in Tsp distribution, arguing against a central role for Syndecan in Tsp distribution. Nevertheless, it remains possible that another heparin domain-containing protein functions to promote Tsp and laminin deposition at the myotendinous junction (Subramanian, 2007).

It is considered that the Stripe-dependent positive feedback that upregulates tsp transcription contributes significantly to the establishment of the myotendinous junction. Previous studies have shown that muscle-tendon interactions form a signaling center, which is initiated by muscle-dependent Vein secretion and accumulation at the myotendinous junction. Vein activates the EGFR pathway in the tendon cell, leading to a significant elevation of the transcription factor Stripe. This study shows that Stripe induces upregulation of Tsp. Taking these results together, it is suggested that the initial formation of the hemiadherens junction creates a self-auto-regulatory nucleation center, which leads to additional deposition of Tsp and possibly other ECM components. These, in turn, gradually strengthen the hemiadherence junction formed between the muscle and the tendon cell (Subramanian, 2007).

Vertebrate Thrombospondins are essential for a variety of biological activities, including cell adhesion, migration, angiogenesis, etc. This work reveals an intriguing similarity between the role of Tsp in the formation of the myotendinous junction and the role of vertebrate Tsp1 and Tsp2 in the induction of synapses. It was shown that Tsp provided by oligodendrocytes is a potent inducer of synapse formation on the dendrites of cultured neurons (Christopherson, 2005). Although these synapses are not electrically active, the Tsp-induced synapses exhibit typical synaptic ultra-structures. The biogenesis of the myotendinous junction carries several similarities to the biogenesis of synapses, including the mutual crosstalk between the two cell types involved and the gradual formation of the junction at both cell membranes involved (Subramanian, 2007).

In summary, this analysis of Tsp function reveals the molecular dynamics and biogenesis of the myotendinous junction. A similar scenario may unfold during Tsp-dependent synapse formation in the development of vertebrate embryos (Subramanian, 2007).

αPS2 integrin-mediated muscle attachment in Drosophila requires the ECM protein Thrombospondin

During Drosophila embryogenesis, the attachment of somatic muscles to epidermal tendon cells requires heterodimeric PS-integrin proteins (α- and β-subunits). The α-subunits are expressed complementarily, either tendon cell- or muscle-specific, whereas the β-integrin subunit is expressed in both tissues. Mutations of β-integrin cause a severe muscle detachment phenotype, whereas α-subunit mutations have weaker or only larval muscle detachment phenotypes. Furthermore, mutations of extracellular matrix (ECM) proteins known to act as integrin binding partners have comparatively weak effects only, suggesting the presence of additional integrin binding ECM proteins required for proper muscle attachment. This study reports that mutations in the Drosophila gene thrombospondin (tsp) cause embryonic muscle detachment. tsp is specifically expressed in both developing and mature epidermal tendon cells. Its initial expression in segment border cells, the tendon precursors, is under the control of hedgehog-dependent signaling, whereas tsp expression in differentiated tendon cells depends on the transcription factor encoded by stripe. In the absence of tsp activity, no aspect of muscle pattern is affected, nor is formation of the initial contact between muscle and tendon cells or muscle-to-muscle attachments. However, when muscle contractions occur during late embryogenesis, muscles detach from the tendon cells. The Tsp protein is localized to the tendon cell ECM where muscles attach. Genetic interaction studies indicate that Tsp specifically interacts with the αPS2 integrin and that this interaction is needed to withstand the forces of muscle contractions at the tendon cells (Chanana, 2007).

Attachment of muscles to tendon cells critically depends on integrin activity. The integrin-like muscle detachment phenotype of the tsp mutants as well as the co-localization of Tsp and integrin proteins strongly suggest that Tsp plays a decisive role in the integrin-mediated cell adhesion process of muscles and tendon cells. In fact, Tsp was shown to encode a pentameric glycosylated protein that is part of the ECM (Adams, 2003). Thus, it could indeed function as a direct binding partner of the PS-integrins (Chanana, 2007).

In order to establish such a functional link between tsp and PS-integrins by genetic means, double mutant embryos were generated that carry either a strong loss of function allele for the αPS1 subunit of integrin due to the mewM6 mutation or a hypomorphic allele for the αPS2 subunit due to the ifB2 mutation in combination with only one wild-type copy of tsp (tspΔ6 and tspΔ79) (Chanana, 2007).

In comparison with wild-type embryos mewM6 mutants bearing two copies of tsp develop a normal muscle pattern with muscle detachment in only few segments. mewM6 mutant embryos that have only one wild-type copy of tsp develop a weak muscle detachment phenotype affecting a small number of longitudinal muscles. In contrast, ifB2 mutants bearing two copies of tsp developed a mild detachment phenotype in several segments, which was strongly enhanced, both with respect to the extent of detachment and penetrance in embryos with only one remaining wild-type copy of tsp. In these embryos, longitudinal, ventral as well as dorsal muscles were found to be detached, a phenomenon not observed in tsp mutant embryos. Furthermore, in mewM6 mutants lacking both wild-type copies of tsp, only a mild enhancement of the tsp mutant phenotype was observed. In contrast, ifB2 lacking both wild-type copies of tsp develop dramatic muscle pattern defects beyond an additive effect of the two individual mutant phenotypes. The enhancement of the muscle detachment phenotype of if mutants by the removal of one copy of tsp and the dramatic enhancement that affects even the set of muscles that are not affected in each of the single if or tsp mutants establishes an essential role of Tsp in the αPS2-dependent muscle attachment process. In contrast, the weak effects of the reduction of the tsp dose in hemizygous mewM6 mutants makes a prominent role of Tsp in αPS1-dependent muscle attachment rather unlikely (Chanana, 2007).

Vertebrate Tsp is a glycosylated protein that forms oligomers and is capable of interacting with both calcium and heparin (Adams, 2001). Furthermore, it has been shown to directly interact with the extracellular part of integrin proteins (Lawler, 1988). This interaction depends on a highly conserved RGB motif, which is characteristic of integrin binding proteins of the ECM (Chanana, 2007).Vertebrate genomes code for up to five Tsps (Adams, 2001), whereas the Drosophila genome contains only a single tsp-coding sequence (Adams, 2003), which, however, encodes two Tsp variants which differ in their carboxyterminal regions. Previous biochemical studies on Drosophila Tsp showed that the protein is secreted and able to form a pentameric structure as suggested by the molecular weight of the secreted native complex (Adams, 2003). At the sequence level, the conserved Drosophila Tsp contains all functionally characterized domains including the critical RGD motif required for integrin binding. In contrast to vertebrate Tsp, the RGD motif in Drosophila Tsp is positioned in the aminoterminal region, close to two BBXB sequence motifs known to bind to heparin, instead of the third Tsp/COMP domain (Adams, 2003). In addition, a KGD motif is observed in the third Tsp/COMP domain of Drosophila Tsp which was shown to serve also as an interaction motif for integrins (Ruoslahti, 1996). Drosophila Tsp contains therefore two RGD/KGD motifs that would allow direct binding of PS2, the integrin heterodimer that was previously found to associate with Tig, an interaction that was shown to be dependent on the presence of the RGD motif. Furthermore, an RGD motif is also required to mediate the interaction of PS2 with the laminin α-chain Wb (Chanana, 2007).

Drosophila tsp is expressed in all ectodermal tendon precursor cells, strongly enriched in those positioned at the segment border of the embryo. Furthermore, tsp is expressed in all differentiated tendon cells after muscle contact. Therefore, tsp is expressed in all cells that have previously been identified by the expression of stripe. stripe encodes an EGR-type Zn-finger transcription factor that is required for tendon cell differentiation. Like stripe, the initial expression of tsp is controlled by Hedgehog signaling at the segment borders and requires stripe activity only during the later stages when the tendon cells are already differentiated. These results suggest that the genes stripe and tsp are activated in parallel by Hh-dependent Ci activity, and that stripe activity maintains the expression of tsp during the later stages when Ci activity has ceased (Chanana, 2007).

Tsp is secreted from epidermal tendon cells and accumulates at the tendon cell matrix, a specific ECM to which the muscles attach. The functional characterisation of the newly generated tsp alleles, which fail to express detectable amounts of tsp transcript, showed that Tsp is necessary for the proper anchoring of muscles at the tendons cells. As observed with mutants affecting the β subunit and the αPS2 subunit of integrin, mys and if, respectively, the muscles were found to detach from their epidermal attachment sites once muscle contraction occurs. Thus, tsp activity is not required for any aspect of muscle pattern formation and/or muscle guidance as well as proper adherence to tendon cells but plays an essential role in maintaining the interconnection between muscles and tendons cells once contraction occurs. Although the muscle detachments are less pronounced than in mys or if mutants, the detachment phenotype of tsp mutants is by far stronger than the corresponding phenotypes that are caused by the loss of other ECM proteins, such as Tig, Wb and LanA, known to be integrin interaction partners. The strong and specific enhancement of the detachment phenotype of mutants that carry a weak if allele, in response to the loss of one or both tsp wild-type alleles strongly suggests that Tsp functions as an essential ECM binding partner of αPS2 encoded by if. The specificity of the genetic interaction shown in this study is consistent with the finding that binding to αPS2 requires an RGD motif as has been found in Tig and Wb as well. Mutations of either tig or wb cause weak muscle detachment phenotypes as observed with tsp mutants, suggesting a redundant αPS2 integrin interaction system in which ECM binding is provided by different partners and that each of them is required for the proper anchoring of the muscles. This conclusion is consistent with the finding that tig, wb and tsp mutants display a weaker phenotype than the if loss of function mutants. Based on the specific expression of tsp in both tendon cell precursors and differentiated tendon cells, which differs from the multiple expression sites of tig and wb, together with the strong enhancement of the muscle detachment phenotype in if and tsp double mutants, it appears likely that tsp is the crucial interaction partner of the αPS2 integrin subunit to provide proper anchoring of muscles to tendon cells. This proposal, and the relative contribution of each of the by now three different αPS2 integrin subunit binding proteins, can be tested once double and triple mutant combinations for all the genes involved and biochemical test systems become available (Chanana, 2007).

Slowdown promotes muscle integrity by modulating integrin-mediated adhesion at the myotendinous junction

The correct assembly of the myotendinous junction (MTJ) is crucial for proper muscle function. In Drosophila, this junction comprises hemi-adherens junctions that are formed upon arrival of muscles at their corresponding tendon cells. The MTJ mainly comprises muscle-specific alphaPS2betaPS integrin receptors and their tendon-derived extracellular matrix ligand Thrombospondin (Tsp). Reported here is the identification and functional analysis of a novel tendon-derived secreted protein named Slowdown (Slow). Homozygous slow mutant larvae exhibit muscle or tendon rupture, sluggish larval movement, partial lethality, and the surviving adult flies are unable to fly. These defects result from improper assembly of the embryonic MTJ. In slow mutants, Tsp prematurely accumulates at muscle ends, the morphology of the muscle leading edge changes and the MTJ architecture is aberrant. Slow was found to form a protein complex with Tsp. This complex is biologically active and capable of altering the morphology and directionality of muscle ends. This analysis implicates Slow as an essential component of the MTJ, crucial for ensuring muscle and tendon integrity during larval locomotion (Gilsohn, 2010).

When migrating muscles reach their target tendons they reorganize their leading edge in order to arrest migration and form the integrin-mediated MTJ. The possible molecular link between the arrest of muscle migration and the formation of the MTJ has not yet been characterized. The present study analyzes these processes, revealing the function of the novel gene product Slow, a Drosophila ortholog of vertebrate Egfl7, as an important modulator of integrin-mediated adhesion (Gilsohn, 2010).

The phenotype of muscle/tendon rupture observed in slow mutant larvae is unique and does not resemble that of mutants for the muscle- or tendon-specific integrins, or for their specific ligands Tsp, Laminin or Tiggrin, in which the typical phenotype is muscle detachment from tendon cells and muscle cell rounding following initial muscle contraction in the embryo. Despite the localization of Slow at the MTJ, its deletion leads to phenotypes similar to those caused by mutations affecting either the cytoskeletal arrangement of mature muscles. These unique phenotypes enabled identification of a novel and crucial aspect of MTJ construction, namely the correct assembly of the integrin receptors and their ECM ligand Tsp at the surfaces of the muscle ends (Gilsohn, 2010).

It is suggested that the defect in slow mutants, manifested by the rupture of both muscles and tendons, might be explained by two mechanisms, which are not mutually exclusive. First, the lack of Slow may result in an aberrant arrangement of the ECM material deposited between the muscle and the tendon, which becomes too rigid and compact; therefore, the mechanical stress imposed by muscle contraction would lead to muscle or tendon rupture. Second, the fine architecture of the muscle-tendon hemi-adherens junction is aberrant, leading to an unequal distribution of mechanical forces upon muscle contraction, resulting in sporadic muscle or tendon rupture. Ultrastructure electron microscopy analysis of the larval muscle-tendon junction did not reveal significant changes in the arrangement of the electron-dense material deposited between the two cell types. Thus, the possiblity if favored that muscle/tendon rupture occurs due to aberrant formation of the MTJ and the unequal distribution of mechanical forces occurring following muscle contraction (Gilsohn, 2010).

When the muscle leading edge reaches the tendon cell, it must undergo morphological changes prior to the establishment of the hemi-adherens junction with the ECM ligand(s) provided by the tendon cell. Should junction formation precede the smoothening and widening of the muscle leading edge, it might lead to the formation of an MTJ with aberrant morphology, in which the integrin receptors are not homogenously distributed and the muscle surfaces are rough. Such a scenario is consistent with observation that in slow mutant embryos, Tsp and muscle-specific integrins prematurely accumulate at the muscle leading edge prior to its arrival at the tendon cell. It was also shown, both in live embryos and fixed material, that at a later developmental stage the muscle leading edge in slow mutant embryos does not spread, resulting in a narrow contact area. These two processes may be interconnected so that the premature accumulation of Tsp and integrin leads to the abnormal pointed morphology of the muscle leading edge. These changes then lead to abnormal MTJ architecture and eventually to defective muscle function, resulting in muscle/tendon rupture and lethality. Additional support for this model comes from the observation that the sole overexpression of αPS2βPS integrin at an early stage of muscle migration leads to a muscle phenotype that is reminiscent of slow, i.e., an altered morphology of the muscle ends and torn muscles at the larval stage (Gilsohn, 2010).

It is therefore suggested that Slow activity allows the muscle leading edges to correctly spread along the surfaces of the tendon cells in order to maximize the contact surface area and to enable the gradual and homogenous distribution of integrins along the entire surfaces of the contact area (Gilsohn, 2010).

Whether the formation of the Slow-Tsp complex (observed in S2 cells) is directly linked to the premature accumulation of Tsp and integrin at the muscle leading edge is not clear at this stage. It is possible that the KGD domain in Tsp, which is required both for integrin binding and for association with Slow, is masked by Slow, attenuating the Tsp-integrin association with this site; thus, in the absence of Slow, Tsp-integrin interaction occurs prematurely by the association of the muscle integrin with this site. Alternatively, Slow-Tsp association at the KGD site may facilitate the association of the integrin receptors with the alternative RGD site located at the N-terminal region of Tsp, and this might be important for proper Tsp-integrin interaction. Experiments could not distinguish between these possibilities. In vitro spreading assays of αPS2βPS-expressing S2 cells showed that Slow reduces integrin-dependent cell spreading on a Tsp matrix. This favors the possibility that Slow attenuates integrin-Tsp binding (Gilsohn, 2010).

Importantly, the genetic interaction found between slow and inflated indicates that in the absence of Slow, the muscle-specific integrin functions less efficiently in mediating proper muscle function. This supports the possibility that Slow regulates integrin-dependent MTJ formation by allowing gradual accumulation of αPS2βPS integrin at the MTJ, and that reducing integrin levels further worsens MTJ construction (Gilsohn, 2010).

The ectopic expression of Tsp and Slow led to clear changes in the somatic muscle pattern, as well as to altered morphology of the muscle leading edge. Upon arrival at the ectopic expression site of Tsp and Slow, the leading edge of several muscles displayed a pointed morphology directed towards the ectopic expression site. In other cases, muscles arrived at their respective tendons, although their leading edge conformation was also altered to a narrower edge. This result further suggests a role for Slow in regulating the shape of muscle ends and emphasizes the biological significance of Tsp-Slow complex formation. Ectopic integrin accumulation was barely detected at the muscle edges, which terminated at the ectopic Tsp-Slow sites, supporting the notion that Slow attenuates integrin-Tsp association and providing an explanation for the premature integrin and Tsp accumulation in the absence of Slow (Gilsohn, 2010).

Taken together, these results demonstrate that Slow modulates the interaction between Tsp and integrin to impose the correct MTJ architecture, although the exact mechanism of Slow action at the molecular level requires further analysis (Gilsohn, 2010).

Vertebrate Egfl7 is highly expressed in endothelial cells during embryonic stages and after injury, and is downregulated in most fully differentiated blood vessels of adult tissue. Egfl7 knockout in zebrafish and mice leads to aberrant blood vessel formation, resulting in severe hemorrhages throughout the body and potential lethality. Recently, this phenotype was attributed to the deletion of the Mir126 regulatory microRNA, which resides within intronic sequences of Egfl7. Therefore, the unique role of Egfl7 in blood vessel formation remains to be further clarified. Significantly, Mir126 is not included within intronic sequences of Drosophila slow (Gilsohn, 2010).

In summary, the results demonstrate a unique and novel function for the Drosophila Egfl7 ortholog Slow in coordinating the morphological changes that occur at the muscle leading edge following its arrival at the tendon cell and in the establishment of the MTJ. The link between Slow and Tsp might be highly relevant to blood vessel development in vertebrates, which, in addition to Egfl7, is also characterized by expression of Tsp1 and Tsp2, representing a potential general molecular paradigm for Slow/Egfl7 activity (Gilsohn, 2010).

Presynaptic secretion of Mind-the-gap organizes the synaptic extracellular matrix-integrin interface and postsynaptic environments

Mind the gap (MTG) is required during synaptogenesis of the Drosophila glutamatergic neuromuscular junction (NMJ) to organize the postsynaptic domain. In this study MTG:GFP transgenic animals were generated to demonstrate MTG is synaptically targeted, secreted, and localized to punctate domains in the synaptic extracellular matrix (ECM). Drosophila NMJs form specialized ECM carbohydrate domains, with carbohydrate moieties and integrin ECM receptors occupying overlapping territories. Presynaptically secreted MTG recruits and reorganizes secreted carbohydrates, and acts to recruit synaptic integrins and ECM glycans. Transgenic MTG::GFP expression rescues hatching, movement, and synaptogenic defects in embryonic-lethal mtg null mutants. Targeted neuronal MTG expression rescues mutant synaptogenesis defects, and increases rescue of adult viability, supporting an essential neuronal function. These results indicate that presynaptically secreted MTG regulates the ECM-integrin interface, and drives an inductive mechanism for the functional differentiation of the postsynaptic domain of glutamatergic synapses. It is suggested that MTG pioneers a novel protein family involved in ECM-dependent synaptic differentiation (Rushton, 2009).

It is hypothesized that presynaptically secreted Mind-the-Gap (MTG) binds within the synaptic cleft extra-cellular matrix (ECM) to establish the synaptic cleft environment required for inductive signaling pathways driving postsynaptic assembly, including glutamate receptor (GluR) localization/maintenance. It has been shown previously that the MTG protein contains a predicted secretion signal peptide, and a 6-cysteine domain related to “cysteine-knot” domains, with high homology to the carbohydrate-binding domain (CBD) in ECM-binding protein families. Endogenous MTG expression peaks sharply during late embryonic stages (16 – 8 hr), corresponding to the period of functional synapse differentiation. The key features of the mtg null mutant phenotype include loss of the electron-dense ECM material characterizing the synaptic cleft, a matrix of unknown composition and function; and the reduction/mislocalization of multiple postsynaptic density (PSD) proteins, including dPix, dPak, DLG, and Dock/Dreadlocks, which function in the pathway(s) regulating GluR localization and abundance. Loss of MTG prevents GluR accumulation at the synapse, resulting in a severe (~80%) loss of the functional postsynaptic glutamate response. Neuronally-targeted mtg RNAi knockdown partially phenocopies these defects, supporting the hypothesis of an inductive, presynaptic requirement for MTG in postsynaptic assembly (Rushton, 2009).

Several key questions that were not adequately answered in earlier studies have been thoroughly addressed in this study. mtg gene identity was conclusively confirmws by demonstrating rescue of mtg null mutant viability with a single copy of a CG7549 cDNA GFP fusion construct. It was further shown that mtg overexpression is deleterious, demonstrating the need for precise regulation of MTG function, consistent with the sharp temporal regulation of endogenous mtg developmental expression levels. In mtg null animals, it was first shown that the MTG::GFP transgene rescues MTG function. It was then shown that neuronally expressed MTG is subcellularly trafficked to synapses both in the CNS and at the NMJ. Cell-specific targeting of the MTG::GFP transgene using the GAL4-UAS approach was critical for demonstrating specific neuronal requirements, including presynaptic targeting and secretion of MTG, as well as non-neuronal requirements (Rushton, 2009).

The embryonic and larval central synaptic neuropil contains multiple classes of chemical synapses, including predominantly excitatory cholinergic connections driving glutamatergic motor output, as well as GABAergic and glutamatergic connections and neurosecretory terminals. The entire neuropil contains concentrated synaptic connections, as evident from the dense presentation of presynaptic active-zone proteins (BRP), synaptic vesicle-associated proteins and vesicular neurotransmitter transporters, and postsynaptic proteins (e.g., DLG). MTG::GFP is prominently localized in similar punctae distributed primarily along longitudinal axon tracts in central and medial regions of the ventral neuropil. When viewed in thin transverse or longitudinal neuropil sections, most MTG punctae appear closely adjacent to or surrounded by BRP punctae, rather than obviously co-localized with BRP. This finding is consistent with MTG functional synaptic localization both presynaptically near active zones and in secreted extracellular aggregates closely opposed to presynaptic boutons. Since MTG is targeted to and secreted at glutamatergic NMJ terminals, an attractive possibility is that MTG also has a specific parallel function at central glutamatergic synapses. A recent study suggested glutamatergic central synapses are primarily concentrated in dorsal neuropil regions in the larval ventral nerve cord. Future localization studies, in combination with glutamatergic- and cholinergic-specific synaptic markers, are needed to identify whether MTG is localized to a particular subclass of chemical synapse (Rushton, 2009).

Utilizing the salivary gland (SG) as an accessible, specialized secretory tissue, this study showed that transgenically expressed MTG is prominently targeted to SG vesicles, subsequently secreted, and strongly accumulated in the lumen, providing in vivo demonstration of predicted MTG secretory function and validating earlier in vitro studies. Secreted MTG remains closely associated with the external secretory cell membrane, revealing the profiles of fused vesicles, suggesting that the secreted protein remains bound to ECM. Neuronally expressed MTG::GFP is prominently contained within punctate aggregates both in the central neuropil and at NMJ synaptic boutons. Using detergent-free conditions to directly test secretion at NMJ synapses, this study showed that MTG aggregates are extracellularly localized immediately surrounding presynaptic boutons. The source of targeted MTG::GFP is entirely presynaptic, showing this externally localized MTG to be secreted from presynaptic terminals (Rushton, 2009).

The results indicate that secreted MTG occupies subregions of synaptic ECM. Using detergent-free lectin-labeling and immunostaining assays to isolate the extracellular domain, it was shown that the Drosophila NMJ synaptic ECM represents a specialized carbohydrate- and receptor-containing matrix domain, bearing many similarities, but also differences, compared to the vertebrate cholinergic NMJ. A punctate βPS integrin distribution defines a synaptic ECM subdomain surrounding type I synaptic boutons. ECM glycans, revealed by VVA and WGA lectins, occupy characteristic but overlapping subdomains with integrin receptors broadly surrounding the synaptic terminal. Other lectin probes (e.g., DBA, PNA) show no synaptic localization, underscoring the important fact that the synaptic ECM is defined by the exclusion of certain extracellular components, as well as the inclusion of synapse-specific molecules. Importantly, it was shown that secreted MTG::GFP aggregates localize within this broader ECM environment, overlapping with βPS integrins and VVA distribution, showing that MTG is positioned to interact with integrins and other ECM molecules. One potential interpretation is that MTG has a signaling function via integrins and/or specific glycans, and that these interactions occur in discrete extracellular synaptic signaling domains (Rushton, 2009).

A critical finding is that presynaptic MTG is necessary for normal synaptic integrin localization. βPS is a required subunit in all Drosophila synaptic integrin receptor subclasses; thus, the MTG-dependent reduction of βPS predicts a concurrent loss of α-integrin proteins and functional synaptic integrin receptors. The importance of bidirectional integrin-ligand interactions and patterning in other tissues suggests that synaptic PS integrins may have a major role in shaping synaptic cleft ECM organization and composition. Since secreted MTG occupies a subset of the βPS synaptic domain, another possibility is that MTG regulates an ECM integrin ligand, such as laminin-A, which in turn regulates integrin localization or maintenance within the synapse. It is difficult to assess the absolute requirement for synaptic integrins in ECM regulation because Drosophila βPS null mutants (myospheroid; mys) are 100% early embryonic lethal, with severely abnormal muscle patterning and altered NMJ morphological differentiation. A residual (~20% of normal) level of NMJ-localized βPS in mysxg43/mysts1 hypomorphs is sufficient for relatively normal synaptic composition, although mutant NMJ morphology and synaptic function are significantly perturbed. In these same mutants, nonsynaptic VVA labeling is abnormally elevated, showing an altered synapse-specificity in ECM glycans with reduced levels of the βPS synaptic integrin receptors (Rushton, 2009).

A second series of critical findings is that the MTG level regulates the lectin-defined carbohydrate distribution in the salivary gland, and that presynaptically targeted MTG modifies NMJ synaptic carbohydrate ECM domains. MTG::GFP strongly overlaps with VVA and WGA fluorescence within SG cells and at the synapse, suggesting that MTG and lectin probes recognize accumulations of similar carbohydrates. Lectins are not primarily recognizing MTG itself, as MTG::GFP and lectin signals can be spatially separated, and where they tightly overlap, their fluorescence intensities are not proportional. Thus, MTG overexpression results in increased recruitment or accumulation of carbohydrates, even in regions where MTG itself is only weakly localized. It is of particular interest to consider the in vivo synaptic glycoproteins and/or glycolipids that are recognized by VVA and WGA, and modulated by MTG expression level. In the synaptic ECM, candidate targets for VVA and WGA include the integrin receptors, which are glycosylated and have a distribution overlapping that of both lectins. However, synaptic VVA labeling persists in βPS mys mutants with greatly reduced synaptic integrin levels, suggesting that integrins do not carry the glycans recognized by VVA. An alternative possibility is that integrin glycans are lectin targets, but in mys mutants the ECM is remodeled in such a way as to restore these glycan moieties (Rushton, 2009).

One potential synaptic target of Vicia villosa agglutinin (VVA) lectins is O-linked glycans on dystroglycan (DG), an important postsynaptic ECM receptor linked to the muscle cytoskeleton. DG has roles in regulating quantal content and vesicle release probability in the presynaptic bouton, and also in recruiting GluRIIB to the postsynaptic domain. It has been reported that NMJ VVA staining is increased by DG overexpression, but not by overexpression of DG lacking the extracellular mucin domain. If VVA labeling at the NMJ is primarily recognizing DG, alterations in VVA resulting from changes in MTG or βPS integrin expression may be mediated in part through changes in the synaptic localization or modification of the DG receptor. Alternatively, DG may itself be recruiting VVA targets to the NMJ ECM. These possibilities will be addressed in future studies. It is stressed that no assumptions can be made about which specific glycoproteins or glycolipids are recognized in vivo by a given lectin. Indeed, it is recognized that lectins may bind other glycans at lower affinity than their preferred substrate target. However, the inhibition of WGA and VVA in vivo labeling by preincubation with their preferred sugars strongly suggests that these lectins recognize these same carbohydrates at the synapse. Given the possible range of synaptic lectin targets, it is of great interest that MTG is able to significantly regulate the entire lectin-labeled glycan pool (Rushton, 2009).

Finally, this study has demonstrated that transgenic MTG expression confers rescue of the GluR, dPak, and DLG punctate postsynaptic domains that are severely disrupted in mtg null synapses. This restoration of postsynaptic differentiation occurs in parallel with the demonstrated central neuropil and NMJ MTG synaptic targeting, localized punctate presynaptic expression, and secreted external localization in the ECM, and with restored mutant movement and viability. Together, these results indicate a synaptic requirement for functional rescue. Evidence for clear postsynaptic rescue with neuronal-specific presynaptic MTG expression supports this conclusion, and is consistent with previous and present results showing a specific presynaptic requirement for MTG in PSD/GluR and βPS localization. The mechanism for the MTG inductive requirement in postsynaptic assembly remains unknown. If MTG acts through integrins in this pathway, then null βPS mutants would be predicted to show loss or mislocalization phenotypes for at least a subset of PSD and GluR. This hypothesis cannot be rigorously tested owing to the essential, pleiotropic requirements for mys during embryogenesis. Alternatively, MTG may act through unidentified matrix or postsynaptic signaling proteins, perhaps including the dystroglycan complex. Several important questions remain to be addressed in future work, including determining the in vivo binding target(s) of MTG, which additional matrix proteins may interact with or be regulated by secreted MTG, and whether MTG directly or indirectly governs the composition/function of the specialized synaptic cleft microdomain (Rushton, 2009).

The Rho-family GTPase Rac1 regulates integrin localization in Drosophila immunosurveillance cells

When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys) is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and it has been shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. This study teste the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response. Rac1 was shown to be required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results it is concluded that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization. This study shows that Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila larvae are often raise at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes (Xavier, 2011; full text of article).

Zasp regulates integrin activation

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)

The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion

Neurons develop highly stereotyped receptive fields by coordinated growth of their dendrites. Although cell surface cues play a major role in this process, few dendrite specific signals have been identified to date. An in vivo RNAi screen in Drosophila class IV dendritic arborization (C4da) neurons identified the conserved Ret receptor (Ret oncogene), known to play a role in axon guidance, as an important regulator of dendrite development. The loss of Ret results in severe dendrite defects due to loss of extracellular matrix adhesion, thus impairing growth within a 2D plane. Evidence is provided that Ret interacts with integrins to regulate dendrite adhesion via rac1. In addition, Ret is required for dendrite stability and normal F-actin distribution suggesting it has an essential role in dendrite maintenance. Novel functions are proposed for Ret as a regulator in dendrite patterning and adhesion distinct from its role in axon guidance (Soba, 2015).

Accurate functional connectivity and sensory perception require proper development of the neuronal dendritic field, which ultimately determines the (sensory) input a specific neuron can receive and detect. Thus, coordinated dendrite growth and patterning is important for establishing the often complex, but highly stereotyped organization of receptive fields. Two of the organizing principles in dendrite development are self-avoidance and tiling. While self-avoidance describes the phenomenon of recognition and repulsion of isoneuronal dendritic branches, tiling refers to the complete yet non-redundant coverage of a receptive field by neighboring neurons of the same type. Both phenomena have been described in different systems across species including the mouse, zebrafish, medicinal leech, Caenorhabditis elegans, and Drosophila melanogaster (Soba, 2015).

Dendritic patterning by self-avoidance, tiling, and other mechanisms is thought to be mediated by cell surface receptors and cell adhesion molecules (CAMs), which play a pivotal role in integrating environmental and cellular cues into appropriate growth and adhesion responses. Many such receptors, prominently Robo and Ephrin receptors, have well understood roles in axon guidance. Although some of these axonal cues including Robo/Slit play a role in dendrite development as well, dendritic surface receptors and their functions are not fully characterized to date. Recent efforts have yielded some progress in this area. Down's syndrome cell adhesion molecule (Dscam) has been shown to regulate dendrite self-avoidance in Drosophila. Studies on protocadherins have revealed that they play an important role in dendrite self-avoidance in mammals. In C. elegans, sax-7/L1-CAM and menorin (mnr-1) form a defined pattern in the surrounding hypodermal tissue to guide PVD sensory neuron dendrite growth via the neuronal receptor dma-1. However, given the complexity and stereotypy of dendritic arbors within individual neuronal subtypes, it is important to search for additional signals for directing dendrite growth (Soba, 2015).

The Drosophila peripheral nervous system (PNS) has served as an excellent model which has helped to elucidate several molecular mechanisms regulating dendrite development. The larval PNS contains segmentally repeated dendritic arborization (da) neurons which have been classified as class I-IV according to their increasing dendritic complexity. All da neuron classes feature highly stereotyped sensory dendrite projections. Moreover, all da neurons exhibit self-avoidance behavior allowing them to develop their individual receptive fields without overlap. It has been demonstrated that all da neuron classes require Dscam for dendrite self-avoidance. In addition, the atypical cadherin flamingo and immunoglobulin super family (IgSF) member turtle might play a more restricted role in C4da neuron self-avoidance. Netrin and its receptor frazzled have also been shown to act in parallel to Dscam in class III da neurons ensuring their proper dendritic field size and location by providing an attractive growth cue which is counterbalanced by self-avoidance. For tiling, no surface receptor has been identified to date. However, the conserved hippo and tricornered kinases, and more recently the torc2 complex, have been implicated in C4da neuron tiling, as the loss of function of these genes results in iso- and hetero-neuronal crossing of dendrites (Soba, 2015).

Recent work has further shown that dendrite substrate adhesion plays an essential role in patterning. Da neuron dendrites are normally confined to a 2D space through interaction with the epithelial cell layer and the extracellular matrix (ECM) on the basal side of the epidermis. 2D growth of da neuron dendrites requires integrins, as loss of the α-integrin mew (multiple edomatous wing) or ß-integrin mys (myospheroid) results in dendrites being freed from the 2D confinement due to detachment from the ECM. Thus, they can avoid dendrites by growing into the epidermis leading to 3D crossing of iso- and hetero-neuronal branches . Integrins are therefore essential to ensure repulsion-mediated self-avoidance and tiling mechanisms, which restrict growth of dendrites competing for the same territory. How integrins are recruited to dendrite adhesion sites and whether they cooperate with other cell surface receptors is unknown (Soba, 2015).

To identify novel receptors required for generating complex, stereotypical dendritic fields, an in vivo RNAi screen was performed for cell surface molecules in C4da neurons. The Drosophila homolog of Ret (rearranged during transfection) was identified as a patterning receptor of C4da dendrites. Loss of Ret function in C4da neurons severely affects dendrite coverage, dynamics, growth, and adhesion. In particular, dendrite stability and 2D growth are impaired resulting in reduced dendritic field coverage and abnormal 3D dendrite crossing, respectively. These defects can be completely rescued by Ret expression in C4da neurons. It was further shown that Ret interaction with integrins is needed to mediate C4da dendrite-ECM adhesion, but not dendrite growth. These data suggest that Ret together with integrins acts through the small GTPase rac1, which is required for dendrite adhesion and 2D growth of C4da neuron dendrites as well. This study thus describes a novel role for the Ret receptor in dendrite development and adhesion by direct receptor crosstalk with integrins and its downstream signals (Soba, 2015).

This study provides evidence that Ret is a regulator of dendrite growth and patterning of C4da neurons. Ret is a conserved receptor tyrosine kinase (RTK) expressed in the nervous system of vertebrates and D. melanogaster , and has been shown to have a number of important functions in nervous system development and maintenance: it regulates motor neuron axon guidance (Kramer, 2006), dopaminergic neuron maintenance and regeneration, and mechanoreceptor differentiation and projection to the spinal cord and medulla. Ret signaling is activated by binding to glial cell line derived neurotrophic factor (GDNF) family ligands and their high affinity co-receptors, the GDNF family receptors (GFRα). Ret also plays an important role in human development and disease as loss of function mutations of Ret lead to Hirschprung's disease displaying colonic aganglionosis due to defective enteric nervous system development. Conversely, Ret gain of function mutations are causal for autosomal dominant MEN2 (multiple endocrine neoplasia type 2) type medullary thyroid carcinoma (Soba, 2015 and references).

Prior to this study, Ret has not been implicated in dendrite development. This study shows that Ret is required specifically for 2D growth of C4da neurons by regulating integrin dependent dendrite-ECM adhesion. Normally, C4da neuron dendrites are virtually always in contact with the ECM and the basal surface of the epithelium lining the larval cuticle, and thus tightly sandwiched between the two compartments. In both integrin and Ret mutants, dendrite-ECM adhesion is impaired. Ret and integrins can co-localize in dendrites and thus likely form a functional complex that could induce and maintain adhesion of dendrites to the ECM. Since Ret loss of function primarily leads to detached terminal dendrite branches, it is tempting to speculate that Ret might be required to recruit integrins to sites of growing dendrites to promote ECM interaction. This is supported by the colocalization of Ret and integrins on the dendrite surface. Their cooperative interaction could thus ensure proper adhesion of growing branches and, conversely, the fidelity of self-avoidance and tiling (Soba, 2015).

These results also highlight the importance of integrating different guidance and adhesion cues to achieve precise neuronal patterning. This has so far only been studied in axon guidance in vivo. Interestingly, vertebrate Ret has been shown to cooperate with Ephrins to ensure high fidelity axon guidance in motor neurons by mediating attractive EphrinA reverse signaling (Kramer, 2006; Bonanomi, 2012). Similar mechanisms may conceivably be employed for growing dendrites, which also encounter a multitude of attractive, repulsive, and adhesive cues that have to be properly integrated. Besides pathways acting independently or in a parallel fashion, an emerging view is that receptors exhibit direct crosstalk to integrate incoming signals. So far, only parallel receptor pathways like Dscam and Netrin-Frazzled signaling in class III da neurons or Dscam/integrins have been identified co-regulating dendrite morphogenesis. The current data show that the Ret receptor and integrins integrate dendrite adhesion and growth by collaborative interaction of the two cell surface receptors. The molecular and genetic link between Ret and integrins suggests that in this case direct receptor crosstalk plays a major role in their function. How exactly these cell surface receptors cooperate and interact remains to be elucidated. Integrins have been shown to display extensive crosstalk with other signaling receptors, including RTKs. Although integrins are involved in adhesion of virtually all cell types, the underlying signaling and recruitment of integrins to sites of adhesion in vivo is complex and not completely understood. It has been suggested that integrin and growth factor receptor crosstalk can occur by concomitant signaling, collaborative activation, or direct activation of associated signaling pathways. For example, matrix-bound VEGF can induce complex formation between VEGFR2 and β1-integrin with concomitant targeting of β1-integrin to focal adhesions in endothelial cells. The current findings of biochemical interaction and colocalization of Ret with the α/β-integrins mys and mew in C4da neuron dendrites argue in favor of direct receptor interaction and subsequent activation of a common signaling pathway (Soba, 2015).

Integrins and RTKs like Ret do share some of the same intracellular signaling components. These comprise, among others, the MAPK (mitogen-activated protein kinase) pathway, Pi3-Kinase (Pi3K), and Rho family GTPases including Rac1. Previous studies provide evidence for Ret-integrin-Rac1 interplay in vitro showing that Ret can enhance integrin mediated adhesion and induce Rac1 dependent lamellipodia formation in cell culture models. In primary chick motor neurons, Rac1 is involved in neurite outgrowth on the integrin substrates laminin and fibronectin. Interestingly, Rac1 has previously been shown to regulate dendrite branching in C4da neurons, however a role in dendrite adhesion in vivo has not been described before. This study shows that Rac1 is required for dendrite-ECM adhesion similarly to what has been described for integrins, and Ret and integrin dependent adhesion was genetically linked with Rac1 function. In Drosophila, MAPK, Src and PI3K can be activated by constitutively active Ret overexpression in the compound eye. Moreover, novel inhibitors of Ret signaling targeting Raf, Src, and S6-Kinase (S6K) prevent lethality induced by Ret over-activation in a Drosophila multiple endocrine neoplasia (MEN2) model. Interestingly, S6K has been shown to be involved in dendrite growth but not tiling in C4da neurons. It remains to be shown if these pathways play a direct role in Ret function in dendrite adhesion and growth (Soba, 2015).

Notwithstanding important commonalities, Ret function in C4da neurons cannot be fully explained by crosstalk with integrins and rac1. Reduced dendritic field coverage, likely due to the observed increase in dendrite turnover, is only evident in Ret but not in integrin or rac1 mutant C4da neurons. Moreover, increasing integrin expression in a Ret mutant background did not rescue dendrite coverage defects, albeit it prevented dendrite crossing. These findings indicate that Ret has additional functions in dendritic branch growth and stability that require as yet unknown extracellular and intracellular mediators. This is also supported by the aberrant F-actin localization in neurons lacking Ret. In this study, Ret dependent intracellular effectors are likely important for F-actin assembly to support directed dendrite growth and stabilization, and their localization and activity might be deregulated in the absence of Ret (Soba, 2015).

Drosophila Ret is a highly conserved molecule, its cognate vertebrate ligand GDNF, however is not (Airaksinen, 2006). In addition, Drosophila Ret can neither bind GDNF nor transduce GDNF signaling, although it has been shown to contain a functional tyrosine kinase domain. In mammals, the GFRα co-receptors are essential components of GDNF/Ret signaling. A Drosophila GFR-like homolog (dGFRL) has recently been characterized and was found to function and interact with the NCAM homolog FasII. Therefore, it appears that Ret's functional interaction partners in dendrite development differ significantly from the previously described co-factors in other systems. It is interesting to speculate that a yet undiscovered Ret ligand is involved in Ret mediated dendrite growth and branch stabilization, which might have implications for mammalian Ret function as well: due to its role in the maintenance of dopaminergic neurons and motor axon growth in mouse, adhesion related signaling via integrins could well be important during these processes. Moreover, the formation of a dorsal root ganglia derived mechanosensory neurons and their afferent and efferent fiber growth and innervation depends on Ret expression. It will be interesting to investigate the functional interplay of Ret and integrins in central and peripheral target innervation and neurite maintenance in these systems, given the interdependent function of Ret and integrins in sensory dendrite growth as shown in this study (Soba, 2015).

In summary, this study describes a novel role for the Ret receptor in dendrite branch growth and stability in Drosophila C4da neurons. This role involves cell-autonomous effects of Ret on ECM adhesion, and F-actin localization in these neurons. Moreover, dendritic adhesion defects attributable to Ret have been linked to integrin and rac1 function featuring a novel and possibly conserved mode of action for Ret in dendrite development (Soba, 2015).

myospheroid: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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