In wild-type embryos, PS antigens are found at the interface between mesoderm and ectoderm, and later mainly at the attachment sites of muscles to the epidermis and gut. Together these results indicate that during embryogenesis, Drosophila integrins are used to attach mesoderm to ectoderm, and are required for the proper assembly of the extracellular matrix and for muscle attachment (Leptin, 1989).

Time-lapse videomicroscopy was used to examine living embryos integrins. Roles for these molecules have been demonstrated as early as gastrulation [Images]. Abnormalities in mutant embryos include: separation and twisting of the embryonic germband, abnormal shape and migration of midgut primordia, irregular visceral mesoderm, detachment of amnioserosa cells, rupture of the cuticle along the dorsal midline, lack of midgut constriction, and detachment of somatic muscles. These observations suggest multiple roles for integrins in the adhesion of cells and in the formation, organization, and migration of embryonic tissues. The complete loss of both alpha subunits does not produce all of the phenotypes observed in embryos lacking betaPS. This suggests that alphaPS1 betaPS and alphaPS2 betaPS are not required in all embryonic processes utilizing integrins (Roote, 1995).

Integrins help to coordinate the differentiation of the internal, sarcomeric cytoarchitecture of a muscle fiber with its immediate environment and are essential for correct integration of muscle cells into tissue. Integrin alphaPS2 betaPS accumulates at contact regions of Drosophila cultured embryo cells. Myotubes form, but subsequent addition of serum or fibronectin is needed for sarcomere formation: Integrin and Actin become concentrated at Z-bands; Myosin and Actin are positioned between the Z-bands. This change fails to occur myotubes derived from myospheroid null myospheroid mutants (Volk, 1990).

The role of integrins was examined in the formation of the cell junctions that connect muscles to epidermis (muscle attachments) and muscles to neurons (neuromuscular junctions). At the ultrastructural level two types of muscle attachments can be distinguished: direct and indirect. At the direct muscle attachments, single muscles (such as the transverse muscles) attach to epidermal cells directly such that the hemiadherens junctions (HAJs) in opposing cells are separated by only 30-40nm, with a thin line of extracellular electron-dense material in between. These closed paired HAJs are referred to as connecting HAJs. Indirect muscle attachments occur at the segmental border, where the ends of multiple muscles attach at the same epidermal site, and contain extensive extracellular matrix consisting of fuzzy electron dense fibers, separated by up to several micrometers. This is referred to as tendon matrix because, like the vertebrate tendons, it is an extracellular matrix used to attach the muscles. Since HAJs at indirect muscle attachments are not closely paired but connected to the tendon matrix, they are referred to as tendon HAJs. Both types of muscle attachments have a common molecular basis: both contain PS integrins; both are sites were large secreted proteins Tiggrin and Masquerade accumulate; the intracellular appearance of connecting HAJs and tendon HAJs looks similar; connecting HAJs and tendon HAJs can appear together at the same site; they both appear to arise from short connecting HAJs; and both HAJs are separated from the extracellular electron dense matrix by a translucent gap of a few nanometers (Prokop, 1998).

Muscle attachments and neuromuscular junctions were examined ultrastructurally in single or double mutant Drosophila embryos lacking PS1 integrin (alphaPS1betaPS), PS2 integrin (alphaPS2betaPS), and/or their potential extracellular ligand Laminin A. At the muscle attachments PS integrins are essential for the adhesion of hemiadherens junctions to extracellular matrix, but not for their intracellular link to the cytoskeleton. The intracellular electron-dense material of connecting HAJs and tendon HAJs connects to microfilaments in the muscles, and to microtubules in the epidermis. The epidermal microtubules are anchored at the other end to apical focal HAJs that connect to the cuticle (Prokop, 1998).

The PS2 integrin is only expressed in the muscles, but it is essential for the adhesion of muscle and epidermal HAJs to electron dense extracellular matrix. PS2 integrin is also required for adhesion of muscle HAJs to a less electron dense form of extracellular matrix, the basement membrane. The PS1 integrin is expressed in epidermal cells and can mediate adhesion of the epidermal HAJs to the basement membrane. The ligands involved in adhesion mediated by both PS integrins seem distinct because adhesion mediated by PS1 appears to require the extracellular matrix component Laminin A, while adhesion mediated by PS2 integrin does not (Prokop, 1998).

At neuromuscular junctions (NMJs) the formation of functional synapses occurs normally in embryos lacking PS integrins and/or Laminin A, but the extent of contact between neuronal and muscle surfaces is altered significantly in embryos lacking laminin A. It is suggested that neuromuscular contact does not require laminin A directly at its point of contact, but requires basement membrane adhesion to the general muscle surface, and this form of adhesion is completely abolished in the absence of Laminin A. In contrast, loss of PS integrin function causes the boutons to make a more extensive contact with the muscle surface. Since no PS integrins are found at neuromuscular contacts it seems likely that the boutons can adhere to more muscle area because the muscle surfaces are more relaxed (allowing them to bend around the bouton) in the severely detached muscles of embryos lacking both PS integrins functions. Adhesion molecules expressed at Drosophila NMJs, like Fasciclin II, Fasciclin III or Connectin, are unlikely to mediate adhesion at the mature embryonic NMJ because they either fade during stage 16 or show no phenotype when mutated. Instead, mutant analysis reveals the existence of yet unknown embryonic adhesion factors downstream of mef2 regulation. Such factors might include laminin receptors that promote adhesion, or other receptors that displace the basement synaptic cell junction. Identification of mef2-dependent receptors might be aided by the use of lamA mutation as a sensitized background (Prokop, 1998).

Heterodimeric cell surface receptor integrin is widely expressed in the nervous system, but its specific role during axon development has not been directly tested in vivo. The Drosophila nervous system expresses low levels of positron-specific (PS) integrin subunits alphaPS1, alphaPS2, and betaPS during embryonic axogenesis. Furthermore, certain subsets of neurons express higher levels of integrin mRNAs than do the rest (Hoang, 1998).

The expression pattern of alphaPS1, alphaPS2, and betaPS subunits were examined using in situ hybridization and immunocytochemistry in the embryonic nervous system. All three PS subunit mRNAs are expressed widely in the nervous system. Their expression levels during hours 9-18 of embryogenesis are notably low compared with that in the other tissues that have been studied previously, such as muscles and apodemes. alphaPS1 mRNA is detected widely in the CNS at a steady level during hours 13-18. During this period, axogenesis occurs within the CNS as well as in the periphery. At the ventral midline, cells appear to accumulate slightly higher levels of alphaPS1, as compared with most other cells in the CNS. The alphaPS2 mRNA expression pattern differs somewhat from that of alphaPS1. Specific clusters of cells, one near the midline and a bilateral pair at mediolateral sites, express alphaPS2 at relatively high levels in each segment of the CNS. The alphaPS2 expression in the CNS peaks during hours 9-15 of embryogenesis. The betaPS mRNA expression pattern partially overlaps with those of alphaPS1 and alphaPS2 mRNA in the CNS, with one prominent cluster of cells expressing relatively high levels at the ventral midline. betaPS mRNA expression in the CNS persists through hours 9-18. These in situ hybridization data suggest that the embryonic CNS expresses both PS1 (alphaPS1/betaPS heterodimer) and PS2 (alphaPS2/betaPS heterodimer) integrins during the period of axogenesis. Consistent with the mRNA data, immunocytochemistry shows that the betaPS protein subunit is unambiguously revealed on neuronal cell surfaces during hours 16-18. The major axon fascicles within the CNS, including the longitudinal connectives and the anterior and posterior nerve tracts as well as the cell surfaces of at least some identified neurons, are labeled with the betaPS antibodies. Unfortunately, both alphaPS1 and alphaPS2 subunits are below the threshold of detection with available antibodies. However, the detection of the betaPS protein subunit on the neuronal cell surfaces suggests that the alphaPS subunits are likely forming functional heterodimers with the betaPS subunit in those cells. These observations with in situ hybridization and immunocytochemistry have led to the conclusion that, similar to vertebrate neurons, many Drosophila neurons express relatively low levels of integrin on the neuronal surface during axogenesis (Hoang, 1998).

Null mutations in either the alphaPS1 or alphaPS2 subunit gene cause widespread axon pathfinding errors that can be rescued by supplying the wild-type integrin subunit to the mutant nervous system. In contrast, misexpressing either the alphaPS1 or alphaPS2 integrin subunit in all neurons leads to no obvious axon pathfinding errors. In the CNS, the longitudinal connective normally contains three prominent axon fascicles that are easily visualized by mAb 1D4. Axons in the alphaPS1 or alphaPS2 mutants appear somewhat wiggly or partially disconnected (Hoang, 1998).

In the PNS, one can visualize specific groups of motoneuron axons with higher cellular resolution than is possible in the CNS. Null mutations in alphaPS1 and alphaPS2 subunit genes both result in similarly widespread axon pathfinding defects for all five known motoneuron groups, despite apparently normal muscle development. The axon defects observed can be interpreted as a consequence of failing to turn at choice points and/or invading into neighboring muscle fields. These defects are most frequently detected in the SNb group. It is important to note that the axons in these loss-of-function mutants can extend as far as in wild type and sometimes beyond their normal stopping points. This suggests that integrin is unlikely to serve simply as a clutch-constituting molecule, on which growth cones depend for the adequate traction needed to extend forward. Another point is that each axon group selects a range of alternative pathways without obvious preferences. It is therefore likely that the loss of integrin leads to losses in the responsiveness of an axon to a large array, rather than a small specific set, of guidance cues. Finally, in general, loss of PS2 integrin (alphaPS2 null mutation) leads to higher axon guidance errors than does loss of PS1 integrin (alphaPS1 null mutation). Future analysis is needed to determine specific contributions of these two forms of integrin. On the basis of the current data, it is suggested that both the laminin-binding PS1 and RGD-dependent PS2 integrins are necessary for accurate axon guidance. It is proposed that integrin serves as part of a molecular network that cooperatively guarantees accurate axon guidance (Hoang, 1998).

Analysis of Talin protein distribution during embryogenesis shows that it is maternally deposited and is evenly distributed in the cytoplasm following cellularization. Talin becomes progressively concentrated at the membrane, first detected in the migrating primordial midgut cells and then at muscle attachment sites, where the muscles and epidermal cells are linked via integrins. Talin protein shows only a hint of the strong pattern of mRNA expressed in the nervous system. Consistent with the low level of protein in the nervous system, zygotic mutant rhea embryos do not have any defects in the structure of the nervous system, as assayed with glial and neuronal cell markers. The subcellular distribution of talin at the muscle attachment sites was examined by immunoelectron microscopy (IEM). Talin is found within submembranous electron-dense material associated with the hemiadherens junctions at muscle attachment sites. Most sites of Talin concentration at membranes correspond to sites of integrin concentration, and colocalization of the two proteins is seen at the edge of the epidermis, during dorsal closure, and at muscle attachments. No PS integrin staining lacking colocalized Talin was observed. However, Talin is concentrated at the membrane of some cells lacking integrin. This is clearest in the gonadal mesoderm, where recruitment of Talin to the cortex of the gonadal mesoderm cells occurs as the gonad condenses. To test whether recruitment of Talin to sites of integrin expression requires integrins, Talin distribution was examined in embryos lacking PS integrins. Embryos lacking the ßPS subunit show a loss of Talin concentration at muscle attachment sites. In embryos that lack the mesodermally expressed alphaPS2 integrin subunit, but that still contain epidermal PS1 (alphaPS1ßPS) integrin, Talin is lost from muscle ends but still concentrated at the basal ends of the attaching epidermal cells, the tendon cells. These results show that talin is recruited from the cytoplasm by integrins in both cell layers of the muscle attachment site (Brown, 2002).

The final overall shape of the salivary gland and its position within the developing embryo arise as a consequence of both its intrinsic properties and its interactions with surrounding tissues. This study focuses on the role of directed cell migration in shaping and positioning the Drosophila salivary gland. The salivary gland turns and migrates along the visceral mesoderm (VM) to become properly oriented with respect to the overall embryo. Salivary gland posterior migration requires the activities of genes that position the visceral mesoderm precursors, such as heartless, thickveins, and tinman, but does not require a differentiated visceral mesoderm. A role for integrin function in salivary gland migration is demonstrated. Although the mutations affecting salivary gland motility and directional migration cause defects in the final positioning of the salivary gland, most do not affect the length or diameter of the salivary gland tube. These findings suggest that salivary tube dimensions may be an intrinsic property of salivary gland cells (Bradley, 2003).

In htl, tkv, and tinman, the residual fragments of VM express Fas3, have a VM-like structure, and are able to direct salivary gland migration if present along its migratory path. Thus, the residual structures appeared to be differentiated VM with wild-type properties. To determine whether salivary gland migration requires a differentiated VM, embryos with mutations in the VM-specific gene biniou (bin) were examined. In bin mutant embryos, VM precursors segregate from dorsal mesoderm and move internally where they coalesce into the typical VM band; however, all tested VM-specific genes, including Fas3, fail to be expressed in bin mutants. Thus, an intact structure formed from VM precursors is present in bin mutants, but the VM precursor cells fail to express markers indicative of differentiation from a general mesodermal cell into a VM-specific cell. The salivary glands in bin mutants had no defects in turning or posterior migration, suggesting that guidance of salivary gland posterior migration by the VM requires neither the terminal differentiation of the precursors nor the function of any VM gene whose expression is bin-dependent (Bradley, 2003).

The VM forms a contiguous structure that may physically block salivary cells from further dorsal movement, thereby causing the cells to move posteriorly, in the path of least resistance. Alternatively or additionally, there may be a bin-independent factor (or factors) that guides salivary gland migration in a more instructive way, perhaps via a secreted signal or a transmembrane guidance molecule. If the mesodermal cue were informational, a signaling pathway functioning within salivary gland cells would have to be involved. A screen of several candidate pathways revealed that mutations disrupting the FGFR1-, FGFR2-, EGF-, DPP-, JNK-, or Wg-signaling pathway did not have phenotypes consistent with a role in the salivary cells for their migration. Thus, focus was placed on molecules known to have a more direct role in migration, specifically the integrin family of cell adhesion molecules, which are heterodimers of two transmembrane proteins, an alpha and a ß subunit. In Drosophila, each of the five identified alpha subunits (alphaPS1-5) is thought to dimerize with the ßPS subunit encoded by the myospheroid (mys) gene. The alpha subunit of alphaPS2ßPS (PS2) integrin is expressed in all mesodermal cells beginning at a very early stage, suggesting that PS2 integrin is likely to be present in the VM precursor cells prior to bin-dependent differentiation. Indeed, alphaPS2 RNA expression was observed in the mesoderm of binR22 homozygotes. In embryos mutant for inflated (if), the gene encoding the alphaPS2 subunit, migration of two tissues along the VM is affected, the endoderm and the tracheal visceral branch. Thus, the PS2 integrin is required to make the VM a suitable substrate for the migration of at least two distinct cell populations (Bradley, 2003).

Whether PS2 integrin is required for salivary gland migration was examined by staining if mutant embryos for several salivary gland proteins. In if homozygotes, salivary cells appear to invaginate normally. The first group of salivary cells to be internalized reaches the approximate level of the wild-type turning point but fails to migrate. During subsequent stages, the remaining if salivary cells continue to internalize, but the distal tip remains at the approximate VM turning point, and the tube is often slightly bent. By late stages, if salivary tubes are frequently folded in half with the distal tips oriented anteriorly. The apparent lack of salivary gland migration in if mutants is distinct from the mismigration phenotypes in htl, hbr, tkv, and tin mutants (Bradley, 2003).

Analysis of PINCH function in Drosophila demonstrates its requirement in integrin-dependent cellular processes

Integrins play a crucial role in cell motility, cell proliferation and cell survival. The evolutionarily conserved LIM protein PINCH is postulated to act as part of an integrin-dependent signaling complex. The molecular architecture of PINCH (Particularly Interesting New Cysteine-Histidine rich protein), which consists exclusively of multiple LIM domains suggests that it may function as a platform for the docking and/or productive juxtaposition of proteins involved in integrin signaling. In order to evaluate the role of PINCH in integrin-mediated cellular events, function of PINCH in Drosophila melanogaster was directly tested in vivo. The steamer duck (stck) alleles, that were first identified in a screen for potential integrin effectors, represent mutations in Drosophila pinch. stck mutants die during embryogenesis, revealing a key role for PINCH in development. Muscle cells within embryos that have compromised PINCH function display disturbed actin organization and cell-substratum adhesion. Mutation of stck also causes failure of integrin-dependent epithelial cell adhesion in the wing. Consistent with the idea that PINCH could contribute to integrin function, PINCH protein colocalizes with ßPS integrin at sites of actin filament anchorage in both muscle and wing epithelial cells. Furthermore, it is shown that integrins are required for proper localization of PINCH at the myotendinous junction. Integrin-linked kinase (Ilk), is also essential for integrin function. Drosophila PINCH and Ilk are complexed in vivo and are coincident at the integrin-rich muscle-attachment sites in embryonic muscle. Interestingly, Ilk localizes appropriately in stck mutant embryos, therefore the phenotypes exhibited by the stck mutants are not attributable to mislocalization of Ilk. These results provide direct genetic evidence that PINCH is essential for Drosophila development and is required for integrin-dependent cell adhesion (Clark, 2003).

The genetic analysis of PINCH function has led to four main conclusions: (1) Drosophila PINCH is encoded by the stck locus and is essential for embryonic development and maintenance of tissue architecture; (2) PINCH is necessary for stable actin-membrane anchorage in muscle and contributes to integrin-dependent adhesion in muscle cells and epithelial cells; (3) integrins are required for the stable association of PINCH with muscle-attachment sites; and (4) the lethal stck mutant phenotype cannot be attributed to mislocalization of the PINCH-binding partner, Ilk, whose recruitment to muscle-attachment sites appears normal in stck mutant embryos (Clark, 2003).

Genetic analyses of the roles of integrins in Drosophila have clearly highlighted the importance of integrins for adhesion and signaling in vivo. Drosophila PINCH is colocalized with integrins in both muscle and epithelial cells. Integrins retain the capacity to accumulate at muscle-attachment sites in stck mutants, illustrating that PINCH does not have an obligatory role in the proper processing and membrane targeting of integrins in vivo. The integrin staining in stck mutants does lack the high degree of order and lateral registration observed in wild-type embryos. In the Drosophila system, it is difficult to distinguish whether this modest disorganization simply reflects the underlying disturbance of the musculature or if it is revealing some contribution of PINCH to maintenance of spatially restricted integrin localization. In C. elegans embryos in which PINCH function is compromised by unc-97 mutation, both integrin and vinculin spread laterally beyond their normal zones of accumulation in dense plaques, suggesting a role for PINCH in clustering of adhesive junction components in this system (Clark, 2003).

Interestingly, PINCH depends on the presence of integrins for its stable accumulation at muscle-attachment sites. Several other proteins, including Talin, Ilk, Myosin II and Short stop colocalize with ßPS integrin at Drosophila muscle-attachment sites. These proteins display variable levels of dependence on integrins for their localization. Like Talin, a well-established integrin effector, PINCH depends on the presence of integrins for its concentration at muscle-attachment sites. The reliance of PINCH and Talin on integrins for their spatially restricted accumulation in muscle emphasizes their connection to the integrin receptors (Clark, 2003).

Integrins must establish links to both extracellular determinants and to intracellular cytoskeletal elements in order to support strong adhesion. Examination of the cellular defects in stck mutant muscle suggests that PINCH contributes to the stabilization of actin-membrane linkages at integrin-rich adhesion sites. In a stck mutant muscle cell, the actin filaments lose their linear organization and eventually accumulate in clumps at one end of the cell. These defects are interpreted to mean that a primary consequence of disturbed PINCH function is a destabilization of the linkage between the actin cytoskeleton and the muscle membrane; it appears that the actin-membrane attachments in stck mutants lack the mechanical strength to remain intact during cyclic muscle contraction. Because integrin functionality relies on the ability of the receptors to establish a transmembrane link between the cytoskeletal elements and the extracellular matrix, reduced substratum attachment strength and/or stability might also be expected to occur if membrane cytoskeletal linkages were compromised. Consistent with this prediction, loss of adhesion is evident in the stck17-/- wing cell clones and, to some extent, in muscles of stck mutant embryos (Clark, 2003).

The molecular architecture of PINCH suggests that it may function as a platform for the docking and/or productive juxtaposition of protein partners. Ilk, a binding partner of PINCH, is thus a candidate to collaborate with PINCH in the stabilization of integrin-cytoskeletal linkages. Consistent with the view that PINCH and Ilk cooperate to promote stable actin anchorage at sites of integrin-mediated adhesion, the phenotypes that result from compromised function of either protein in Drosophila are very similar (Zervas, 2001; Clark, 2003). Moreover, PINCH and Ilk are colocalized in Drosophila embryos and are recovered in a protein complex isolated from embryos by immunoprecipitation. Drosophila PINCH also interacts directly with Ilk using two-hybrid methods. These results are consistent with findings for vertebrate PINCH and Ilk. PINCH and Ilk also colocalize at actin-membrane anchorage sites in C. elegans muscle, and elimination of either gene product was shown to produce a paralyzed at twofold stage (PAT) phenotype similar to that seen for ß-integrin mutants. Collectively, results in both invertebrate and vertebrate systems illustrate that the capacity to form a PINCH/Ilk complex has been conserved through evolution (Clark, 2003 and references therein).

Given the fact that Ilk and PINCH colocalize, co-precipitate and have similar loss of function phenotypes, it is possible that disturbed PINCH function could adversely affect Ilk localization and that such mislocalization might account for the stck mutant phenotype. To explore this possibility the localization of Ilk was examined in stck mutant embryos; Ilk was found to be unperturbed in its ability to accumulate at muscle-attachment sites, even when a dramatic lethal phenotype is evident in stck mutant embryos. As noted above, ßPS integrin also accumulates at muscle-attachment sites in stck mutant embryos. These findings illustrate that the proper localization of integrin and Ilk is not sufficient to stabilize actin membrane linkages at sites of integrin-dependent adhesion, and define PINCH as a critical component of the molecular machinery necessary for the tethering of actin to the integrin-rich membranes (Clark, 2003).

The demonstration that single ilk and stck mutants both display deficiencies in integrin-dependent processes illustrates that neither PINCH nor Ilk is sufficient on its own to support full integrin function. It is possible that PINCH acts as a positive regulator of Ilk function, either by modulating Ilk function by direct binding or by recruitment of an Ilk-modifying factor. Alternatively, Ilk may activate some PINCH function that is crucial for stabilization of actin-membrane linkages. Finally, a PINCH-Ilk protein complex may be a key component of the platform necessary for the recruitment of other proteins required to achieve stable actin-membrane associations. In this regard, it is of interest that PINCH and Ilk can be recovered in a complex with the Ilk-binding partner, CH-IlkBP, a calponin domain-containing protein related to Affixin and Actopaxin that could provide the link to actin filaments. Because the localization of Drosophila PINCH is dependent on integrins, the establishment of PINCH-Ilk complexes at muscle-attachment sites is not be supported in the absence of integrin function. This dependence of PINCH localization on integrins could provide a means to couple integrin adhesive function to its role in cytoskeletal anchorage (Clark, 2003).

In vertebrate cells, PINCH and Ilk appear to be mutually dependent on each other for their localization to integrin-rich focal adhesions (Zhang, 2002b). However, as noted above, despite their ability to interact with each other, PINCH and Ilk show distinct requirements for their recruitment to specific subcellular domains in Drosophila. In particular, it is shown that PINCH requires functional integrins for its localization to muscle-attachment sites, whereas it has previously been demonstrated that Drosophila Ilk fails to bind integrins directly and localizes normally in an integrin mutant. Rather than employing an association with integrins, Ilk may rely on a protein such as Paxillin for its targeting to integrin-rich sites. Although Drosophila PINCH requires integrins for its stable accumulation at muscle-attachment sites, there is no evidence that PINCH can associate directly with integrin cytoplasmic domains, therefore additional proteins probably act as a bridge (Clark, 2003 and references therein).

Crk-associated substrate (Cas) signaling protein functions with integrins to specify axon guidance during development

Members of the Cas family of Src homology 3 (SH3)-domain-containing cytosolic signaling proteins are crucial regulators of actin cytoskeletal dynamics in non-neuronal cells; however, their neuronal functions are poorly understood. This study identified a Drosophila Cas (DCas, CG1212, p130CAS; not to be confused with the Drosophila genes CAS/CSE1 segregation protein and castor), found that Cas proteins are highly expressed in neurons and showed that DCas is required for correct axon guidance during development. Functional analyses reveal that Cas specifies axon guidance by regulating the degree of fasciculation among axons. These guidance defects are similar to those observed in integrin mutants, and genetic analysis shows that integrins function together with Cas to facilitate axonal defasciculation. These results strongly support Cas proteins working together with integrins in vivo to direct axon guidance events (Huang, 2007).

In mammals, Cas proteins function downstream of several different receptors in non-neuronal cells, including growth factor receptors, G-protein-coupled receptors, T-cell receptors, B-cell receptors and integrins. Interestingly, integrin receptor subunit mutations in Drosophila give rise to CNS and motor axon guidance defects that are strikingly similar to those observed in DCas mutants, suggesting that Cas might function together with integrin receptors to guide axons (Huang, 2007).

Drosophila integrins, like vertebrate integrins, are composed of an α-subunit and a ß-subunit. In Drosophila, there is one gene encoding a typical laminin-binding-type α-subunit (α1, called mew), one encoding an RGD-binding-type α-subunit (α2, called if), and a single ß-subunit gene (ß1, called mys) very similar to the prototype vertebrate ß1 receptor. To investigate the connection between integrin and Cas signaling, the role of integrin receptor function in embryonic motor axon pathfinding was revisited and it was found that integrin-null mutant embryos exhibit defects that are qualitatively and quantitatively similar to DCas mutants. Embryos harboring null alleles for either α1 (mewM6) or α2 (ifK27E) integrin genes exhibited ISNb and SNa axon guidance defects very similar to those observed in DCas mutants, including increased fasciculation resulting in the absence of muscle innervation. CNS axon guidance defects in were also observed both α1 and α2 integrin mutants that were similar to those observed in DCas mutants (Huang, 2007).

To further address DCas involvement in integrin-mediated axon guidance, dominant genetic interactions were sought between DCas and integrin subunit LOF mutations. Such transheterozygous interactions provide genetic support for two proteins functioning together in the same signaling pathway. It was asked whether removal of a single copy of DCas dominantly enhances heterozygosity at the α1, α2 or ß1 integrin loci. It was found that in α1, α2, ß1 or DCas heterozygotes, motor axon trajectories were not significantly different from wild type. However, removal of a single copy of DCas together with a single copy of α1, α2 or ß1 integrin resulted in highly penetrant axon guidance defects, suggesting that these three genes function in the same signaling pathway. Importantly, the phenotypes resulting from dominant enhancement by DCas are indicative of increased fasciculation, similar to those observed in DCas, α1 or α2 integrin LOF embryos (Huang, 2007).

To further assess the role integrins play in DCas-mediated axon guidance, it was asked whether ß1integrin LOF mutants dominantly suppress DCas GOF motor axon guidance phenotypes. The Drosophila ß1 integrin (encoded by mys1) is the predominant neuronal ß1 integrin and therefore likely to mediate most, if not all, nervous system integrin signaling. If integrins are indeed necessary for activating DCas signaling, removing a single copy of ß1 integrin should suppress DCas GOF phenotypes. When low levels of DCas were expressed in all neurons in an otherwise wild-type background, moderate guidance defects were observed involving axons of the ISNb, SNa and CNS third longitudinal. Removing a single copy of the ß1 integrin gene in this same neuronal DCas GOF genetic background significantly rescued axon guidance phenotypes resulting from DCas GOF. Importantly, these results also show that neuronal overexpression of DCas does not simply function in a dominant-negative fashion to block integrin, or other, signaling pathways (Huang, 2007).

Supporting a model for how integrins and Cas regulate axonal fasciculation and pathfinding is extensive work on neuronal integrin functions in vitro. Growing axons and migrating cells preferentially elongate on surfaces to which they adhere most strongly, including integrin ligands. How might adhesive interactions influence axonal guidance decisions in vivo? Neuronal growth cones tend to form extensive lamellae, which are indicative of strong adhesive interactions, when cultured on highly adhesive substrata containing integrin ligands. These adhesive interactions stabilize elongating nerve fibers by promoting filopodial extension and expansion of growth cone surfaces. Disruption of axon-substrate attachment in vitro with integrin function-blocking antibodies encourages axon-axon adhesive interactions (fasciculation) in place of axon-substrate adhesion. Furthermore, contact with integrin ligands can slow axon elongation, as axons encountering an increasing gradient of laminin peptide exhibit reduced velocity, but growth cone velocity returns to previous rates when axons turn down the gradient. This in vitro observation resembles in vivo situations in which growth cones slow at a choice point, exhibit increased morphological complexity and then extend along distinct pathways. Drosophila motor axon growth cones also exhibit similar changes in morphological complexity upon contacting different substrates in vivo, suggesting that similar processes function to generate motor axon trajectories. Different combinations of integrin ligands might be responsible for these effects. When vertebrate growth cones in vitro contact either the α1 or α2 integrin ligands, laminin and fibronectin respectively, they decelerate, pause and exhibit short-term growth arrest. Interestingly, in vivo observations show that DCas functions with both α1 (laminin-binding) and α2 [RGD (e.g., Tiggrin)-binding] integrins to mediate correct axon navigation by regulating motor axon fasciculation at choice points, suggesting that integrin/Cas-mediated spatial regulation of growth cone extension underlies correct navigation at these choice points (Huang, 2007 and references therein).

The molecular mechanisms underlying integrin-mediated axon guidance remain to be completely defined. However, results derived from analysis of integrin/Cas signaling on cell migration shed light on how Cas and integrins might specify axonal defasciculation events in vivo. During cell migration, Cas proteins serve to establish linkage between migrating cells and the ECM. Cas plays an important role in regulating cytoskeletal organization, cell adhesion and force sensing, and fibroblasts isolated from p130Cas-null mutant mouse embryos exhibit disorganized and short actin filaments and decreased cell migration. In non-neuronal cells, Cas becomes phosphorylated in response to integrin engagement by many ECM components, including fibronectin and laminin (Defilippi, 2006). FAK and Src family kinases have been implicated in integrin-dependent phosphorylation of Cas. Interestingly, recent in vitro observations reveal that FAK signaling at sites of integrin-mediated adhesion controls axon pathfinding. Furthermore, pharmacological inhibitors of Src family kinases decrease the level of neuronal phosphorylated Cas in vitro, supporting a role for Src kinases in regulating Cas proteins in neurons. Finally, the activity of Rho-family small GTPases is also regulated by Cas interactions with the guanine nucleotide exchange factor Dock180 (Dock1) (Defilippi, 2006). Taken together, these links between Cas signaling components and cytoskeletal reorganization suggest that some of these signaling proteins might also influence axon guidance in vivo during development (Huang, 2007 and references therein).

The results demonstrate that integrin/Cas-mediated signaling is necessary but not sufficient for axonal defasciculation, revealing that integrin/Cas-mediated axon guidance must be integrated with other axon guidance signaling cascades to regulate axon defasciculation events during development. The identity of these other axon guidance pathways is not known. The attractive/permissive guidance cue Netrin binds to integrins, and functions with integrins in non-neuronal cells. The Netrin receptor Deleted in colorectal cancer (DCC) has been found to utilize the integrin effector FAK and recently p130Cas, to mediate Netrin-dependent attractive growth cone steering. Ephrins, best known for their role as repulsive axon guidance cues, also induce cell adhesion and actin cytoskeletal changes in fibroblasts in a p130Cas-dependent manner. Repulsive axon guidance cues may also regulate integrin/Cas-dependent axon guidance during development. The axonal repellent Slit genetically interacts with integrins and their ligands to guide commissural axons in Drosophila. Semaphorin and Ephrin-mediated repulsive effects on non-neuronal cells also appear to involve inhibition of integrin signaling events. Interestingly, a crucial component of semaphorin-dependent repulsive axon guidance, a member of the molecule interacting with Cas-L (MICAL) family, physically associates with Cas-L and preliminary data suggest that these interactions are functionally important for axon guidance. The observation that Cas functions with integrins to mediate axon guidance during development suggests new directions to better understand how integrin/Cas signaling modulates neuronal guidance through interactions with distinct axon guidance signaling pathways (Huang, 2007).

Drosophila Importin-7 functions upstream of the Elmo signaling module to mediate the formation and stability of muscle attachments

Establishment and maintenance of stable muscle attachments is essential for coordinated body movement. Studies in Drosophila have pioneered a molecular understanding of the morphological events in the conserved process of muscle attachment formation, including myofiber migration, muscle-tendon signaling, and stable junctional adhesion between muscle cells and their corresponding target insertion sites. In both Drosophila and vertebrate models, integrin complexes play a key role in the biogenesis and stability of muscle attachments through the interactions of integrins with extracellular matrix (ECM) ligands. This study shows that Drosophila Importin7 (Dim7) is an upstream regulator of the conserved Elmo-Mbc-->Rac signaling pathway in the formation of embryonic muscle attachment sites (MASs). Dim7 is encoded by the moleskin (msk) locus and was identified as an Elmo-interacting protein. Both Dim7 and Elmo localize to the ends of myofibers coincident with the timing of muscle-tendon attachment in late myogenesis. Phenotypic analysis of elmo mutants reveal muscle attachment defects similar to that previously described for integrin mutants. Furthermore, Elmo and Dim7 interact both biochemically and genetically in the developing musculature. The muscle detachment phenotype resulting from mutations in the msk locus can be rescued by components in the Elmo-signaling pathway, including the Elmo-Mbc complex, an activated Elmo variant, or a constitutively active form of Rac. In larval muscles, the localization of Dim7 and activated Elmo to the sites of muscle attachment is attenuated upon RNAi knockdown of integrin heterodimer complex components. These results show that integrins function as upstream signals to mediate Dim7-Elmo enrichment to the MASs (Liu, 2013).

Previous studies have shown that Dim7 localizes to developing muscle-tendon insertion sites and removal of Dim7 has severe consequences in muscle attachment maintenance (Liu, 2011). The current studies extend these observations to elucidate the functional contribution of Dim7-Elmo in regulating Drosophila muscle attachment. The results show that Dim7 is an upstream adaptor protein that recruits Elmo in response to integrin adhesion and/or signaling. Thus, it is proposed that the spatial and temporal regulation Elmo-Mbc activity results in regulation of the Rac-mediated actin cytoskeleton changes at the MASs (Liu, 2013).

The 'myospheroid' phenotype in elmo or msk mutants resemble attachment defects first characterized in mutated genes that encode for integrins, ILK and Talin, and is not due to earlier developmental defects in myogenesis. A similar number of cells expressing the muscle differentiation factor DMef2 was present in elmo or msk mutants, indicating that muscle specification was not affected (Geisbrecht, 2008; Liu, 2011). Genes essential for muscle migration and targeting also lead to detached muscles. For example, in kon/perd or grip mutants, the early arrest of migrating myotubes resulting from defective migration eventually leads to a linkage failure between the muscle and tendon cells. In mutant embryos with reduced levels of Elmo or Dim7, the muscle detachment phenotype did not appear to result from muscle migration defects. First, the spatial-temporal accumulation of Elmo and Dim7 is developmentally regulated. Both proteins are not detected at the leading edges of migrating muscles, but begin to accumulate at MASs after stage 15. Second, failure of muscle ends contacting their corresponding attachment sites was not observed in elmo or msk mutants at late stage 15, when muscle migration was almost complete (Liu and Geisbrecht, 2011) (Liu, 2013).

Both membrane localization and Rac-dependent cell spreading of the uninhibited, active version of Elmo is enhanced compared to native WT Elmo in cultured mammalian cells (Patel, 2010). These in vitro results are in agreement with the current in vivo analysis, where ElmoEDE (a mutation that prevents the autoinhibitory interaction of Elmo) is enriched at larval muscle ends compared to the poor accumulation of full-length Elmo-YFP. This may reflect a potential regulatory mechanism controlling the subcellular localization of Elmo from the cytoplasm to the muscle ends upon the release of Elmo autoinhibition. Within different cells or tissues, various proteins may regulate Elmo localization to the cell periphery, or other sites where active Elmo is needed. In cultured mammalian epithelial cells, membrane recruitment of the Elmo-Dock180 complex is dependent on active RhoG for cell spreading. Consistent with a functional role for membrane-targeted Elmo, active Elmo promotes cell elongation in Hela cells, when co-expressed with RhoG (Patel, 2010; Liu, 2013 and references therein).

The data argues that adaptor proteins may be required in muscle cells for activated Elmo membrane recruitment. Decreased levels of ElmoEDE are observed at the polarized ends of muscle insertion sites when Dim7 levels are decreased. It is still not clear if Dim7 binding is required for the conformational change that results in Elmo activation or if an activated Dim7-Elmo complex already exists within the cell and gets recruited as a complex upon integrin activation. Furthermore, complete loss of Elmo-EDE protein levels is not observed, suggesting that either Dim7 protein levels are not depleted enough or other proteins in addition to Dim7 play a role in Elmo membrane recruitment. Alternatively, post-translational modification(s), such as phosphorylation, could be an additional mechanism for the relief of Elmo autoinhibition. Thus, it is concluded that in muscle, Dim 7 is an essential adaptor protein for the polarized membrane localization of active Elmo or the active Elmo-Mbc complex downstream of integrin signaling pathway (Liu, 2013).

What is the relationship between the integrin adhesome and the Dim7-Elmo complex? Two explanations are proposed that are not mutually exclusive. One possibility is that the Dim7-Elmo-Mbc complex assembles at MASs via integrin-mediated 'outside-in' signaling. Upon ligand binding to ECM molecules, integrin activation results in Dim7-Elmo-Mbc complex localization for the spatial-temporal regulation of Rac activity to maintain dynamic actin filament adhesion at the MASs. It is predicted that localization of activated Elmo to the MASs is a prerequisite regulatory mechanism for actin cytoskeleton remodeling via Rac to maintain stable attachments. This hypothesis is supported by three lines of evidence: (1) muscle attachment defects upon loss of Dim7 or Elmo are only observed after the establishment of the integrin adhesion complex and onset of muscle contraction; (2) muscle detachment in msk mutants can be rescued by expressing low levels of activated Rac; and (3) the enrichment of Dim7 and Elmo-EDE proteins at the ends of muscle fibers is greatly reduced in integrin-deficient larvae (Liu, 2013).

It is also possible that accumulation of the Dim7-Elmo complex to the ends of muscles regulates 'inside-out' signaling to dynamically regulate integrin affinity for strong ligand binding and stable muscle attachments. Previously, it was reported that Dim7 acts upstream of the Vein-Egfr signaling pathway in muscle to tendon cell signaling (Liu, 2011). Combined with previous results that muscle-specific Vein secretion is dependent on the adhesive role of βPS integrin, the Dim7-Elmo complex may be internally required for integrins to regulate Vein secretion. A decrease in Vein-Egfr signaling and loss of tendon cell terminal fate results in a reduction in ECM secretion and weakened integrin-ECM attachment. This is consistent with the observation that msk or elmo mutants phenocopy embryos with reduced or excessive amounts of the αPSβPS integrin complex, where pointed muscle ends result in smaller muscle attachments. Future studies analyzing Dim7-Elmo-Mbc complex localization and function in the background of integrin deletion constructs which separate the 'inside-out' and 'outside-in' signaling pathways will be essential to uncover more detailed molecular mechanisms (Liu, 2013).

What is the relationship between the Dim7-Elmo-Mbc-->Rac signaling pathway and the integrin mediated adhesome complex assembly (including the Talin, IPP [integrin linked kinase (ILK)-PINCH-Parvin-α) complex]? It is proposed that actin filaments within the muscle cell are anchored to the muscle cell membrane via the IPP complex, while regulation of MAS-actin remodeling iscontrolled by the Dim7-Elmo-Mbc-->Rac pathway. The data suggests that these two complexes assemble independently at the muscle ends. In msk mutant embryos, both ILK and Talin properly accumulate at the MASs, suggesting that Dim7 is not responsible for their localization (Liu, 2011). Similarly, both MAS-enriched Dim7 and active Elmo can be detected at two ends of the muscles in ILK-deficient larva, even in fully detached muscles. In a vertebrate cell culture model, Elmo2 was found to physically interact with ILK for the establishment for cell polarity (Ho and Dagnino, 2012; Ho, 2009). Thus, it is possible that the current approaches have not fully knocked down Ilk levels or that the Dim7-Elmo recruitment by Ilk is redundant with another attachment site protein. Alternatively, an upstream scaffold protein may function to recruit both the IPP and Dim7-Elmo complex to the MASs. It is likely that these two complexes are temporally regulated in embryogenesis, where the actin remodeling complex is not needed until initial muscle-tendon initiation has been established (Liu, 2013).

Talin autoinhibition is required for morphogenesis

The establishment of a multicellular body plan requires coordinating changes in cell adhesion and the cytoskeleton to ensure proper cell shape and position within a tissue. Cell adhesion to the extracellular matrix (ECM) via integrins plays diverse, essential roles during animal embryogenesis and therefore must be precisely regulated. Talin, a FERM-domain containing protein, forms a direct link between integrin adhesion receptors and the actin cytoskeleton and is an important regulator of integrin function. Similar to other FERM proteins, talin makes an intramolecular interaction that could autoinhibit its activity. However, the functional consequence of such an interaction has not been previously explored in vivo. This study demonstrates that targeted disruption of talin autoinhibition gives rise to morphogenetic defects during fly development and specifically that dorsal closure (DC), a process that resembles wound healing, is delayed. Impairment of autoinhibition leads to reduced talin turnover at and increased talin and integrin recruitment to sites of integrin-ECM attachment. Finally, evidence is presented that talin autoinhibition is regulated by Rap1-dependent signaling. Based on these data, it is proposed that talin autoinhibition provides a switch for modulating adhesion turnover and adhesion stability that is essential for morphogenesis (Ellis, 2013).

Overall, this study identifies an important role for the regulation of talin function through autoinhibition. Failure to autoinhibit talin impairs morphogenetic processes, but this is not due to defects in integrin-mediated attachment to the ECM or in the assembly of the adhesion complex. Thus, it is unlikely that the FERM domain mutation E1777A, which completely blocks autoinhibition, blocks integrin-mediated cell-ECM attachment in a dominant-negative fashion. An alternative explanation for the phenotype is that the E1777A mutant behaves like a gain-of-function allele of talin and that the morphogenetic defects that were observe are due to too much rather than too little adhesion. This would not be the first time such a phenomenon has been observed; for example, overexpression of integrins in either the wing or the muscle gives rise to phenotypes identical to those found in integrin-null mutants. How could the E1777A mutation give rise to stronger adhesion? It was shown that this mutation enhances the recruitment and colocalization of talin and integrin at sites of adhesion. Importantly, it was shown that the E1777A mutation effectively reduces talin turnover at sites of adhesion. Indeed, the data fit with a gain-of-function model: blocking talin autoinhibition leads to increased integrin-mediated adhesion, and this impairs morphogenetic processes that require cyclic adhesion assembly and disassembly. Further consistent with this model is the observation that adhesion at myotendinous junctions (MTJs), a non-morphogenetic context, is not perturbed upon blocking autoinhibition of talin. The possibility cannot be excluded that E1777A may confer its effect on talin function through a means other than disruption of autoinhibition. Encouragingly, however, homology modeling and NMR analyses strongly suggest that the fly protein behaves much as the mammalian homolog does (Ellis, 2013).

How does prevention of autoinhibition stabilize integrin-mediated adhesion? This study shows that autoinhibition regulates talin recruitment to adhesions through a RIAM-Rap1-dependent mechanism. Interestingly, the E1777A autoinhibition mutant talin is more strongly recruited to adhesions than WT talin; this enhanced recruitment occurs independent of RIAM-Rap1 activity. Thus, it is possible that constitutive relief of autoinhibition works to stabilize and promote adhesion by enhancing recruitment of the talin molecule to adhesions, thus bypassing the need of the RIAM-Rap1 pathway for recruitment. At the membrane, adhesion strengthening may occur via talin's scaffolding function, as talin can interact with multiple components of the integrin adhesion complex IAC, and these interactions may increase and/or change when talin assumes a more extended conformation. Another possibility, consistent with structural studies, is that relief of autoinhibition frees up the FERM/IBS-1 domain of talin such that it can activate integrins. It is predicted that mutations in talin that block IBS-1-mediated integrin activation would lead to more dynamic adhesions, and this is indeed what was observed. According to the model, talin recruitment is determined by the sum of interactions that a single molecule can make with other IAC components at any one time. For example, the autoinhibited form of talin relies on Rap1/RIAM for efficient recruitment, even though it may still bind integrin through its free IBS-2 domain; both mechanisms may contribute to targetting of talin to adhesions. It is speculated that relief of autoinhibition makes the IBS-1 available, as well as the many other binding sites for IAC components that are found in the talin rod domain (e.g., vinculin binding sites), thereby substantially increasing the number of possible interactions that can lead to talin recruitment to the IAC (Ellis, 2013).

There are likely to be multiple avenues leading to relief of talin autoinhibition. Recent superresolution studies provided elegant evidence that autoinhibition is primarily relieved within adhesion complexes, implicating the need for a mechanism to specifically recruit autoinhibited talin to adhesions. This study has showm that forcing talin to remain in an open, nonautoinhibited conformation gives rise to very similar phenotypes as activating the RIAM-Rap1 pathway (RIAM is Rap1 interacting adaptor protein). Based on the results, it is proposed that RIAM-Rap1 brings autoinhibited talin to the membrane where autoinhibition can subsequently be relieved, possibly through electrostatic interactions with the membrane/ PIP2. RIAM-Rap1 has a previously established role in mediating the recruitment of talin to sites of adhesion, but it has recently been demonstrated that the requirement for RIAM-Rap1 is context dependent. Structural and biochemical studies have revealed that the binding of talin to either RIAM or vinculin is mutually exclusive and likely dependent on force. Moreover, in cell culture, vinculin-stimulated integrin activation is RIAM-Rap1 independent, raising the possibility that more mature adhesions might not need RIAM-Rap1 to promote talin activation in this case. Along similar lines, this study demonstrated that RIAM-Rap1 activity is dispensable for recruitment of a nonautoinhibited talin molecule (Ellis, 2013).

In summary, the results suggest that talin autoinhibition confers a switch through which fine control of integrin-mediated adhesion can be exerted in vivo. The findings also reveal RIAM-Rap1-mediated regulation of integrin adhesion is an important modulator of morphogenesis, and evidence is provided for an autoinhibition-based pathway for control of talin function through RIAM-Rap1. Furthermore, this study exemplifies how subtle tuning of adhesion complex composition and stability elicits different adhesive functions and cellular behaviors during development (Ellis, 2013).

Talin is required to position and expand the luminal domain of the Drosophila heart tube

Fluid- and gas-transporting tubular organs are critical to metazoan development and homeostasis. Tubulogenesis involves cell polarization and morphogenesis to specify the luminal, adhesive, and basal cell domains and to establish an open lumen. This study explores a requirement for Talin, a cytoplasmic integrin adaptor, during Drosophila embryonic heart tube development. Talin marked the presumptive luminal domain and was required to orient and develop an open luminal space within the heart. Genetic analysis demonstrated that loss of zygotic or maternal-and-zygotic Talin disrupted heart cell migratory dynamics, morphogenesis, and polarity. Talin is essential for subsequent polarization of luminal determinants Slit, Robo, and Dystroglycan as well as stabilization of extracellular and intracellular integrin adhesion factors. In the absence of Talin function, mini-lumens enriched in luminal factors form in ectopic locations. Rescue experiments performed with mutant Talin transgenes suggested actin-binding was required for normal lumen formation, but not for initial heart cell polarization. The study proposes that Talin provides instructive cues to position the luminal domain and coordinate the actin cytoskeleton during Drosophila heart lumen development (Vanderploeg, 2015).

These experiments establish an essential function for the integrin adapter Talin in the assembly of the Drosophila embryonic heart. During the cardioblast (CB) migratory phase preceding tubulogenesis, Talin localizes along the CB apical surface, immediately ventral to the leading edge which extends towards the dorsal midline. As this Talin rich domain persists throughout embryonic heart assembly, eventually surrounding the lumen of the open cardiac tube, this surface is termed the pre-luminal domain. Talin is essential for the dynamic cell morphology and the leading edge features that characterise collective cardial cell migration. Furthermore, following migration, Talin is required to enclose a continuous lumen between the bilateral CB rows (Vanderploeg, 2015).

Analysis of late stage hearts in rhea zygotic mutants reveals that Talin is essential to correctly orient the CB polarity such that a continuous lumen is enclosed along the midline. In wildtype, many membrane receptors including Robo, Dg, Unc5, and Syndecan accumulate along the luminal domain. E-cadherin, Dlg, and other cell-cell adhesion factors are restricted to cell contact points immediately dorsal and ventral to the lumen and to the lateral cell domains between ipsilateral CBs. As evidenced by Robo and Dg immunolabeling experiments, the midline luminal domain is absent or, at best, is discontinuous along the midline in rhea mutant embryos. However, the Robo and Dg enriched luminal domains are not completely absent in null rhea homozygotes, but are found ectopically along lateral membranes between ipsilateral CBs. Robo's ligand, Slit, is also detected within these ectopic lumina. Similar ipsilateral Slit and Robo accumulations were observed in embryos mutant for the integrin subunit genes scab (αPS3) or mys (βPS1). Thus, the expanded Dlg-rich adhesive contact observed in rhea null embryonic hearts is consistent with a model in which integrins and Talin instruct the localization of Slit and Robo. These cues are essential to orient the lumen and to restrict the adhesive regions. In the absence of Talin, other components of the luminal structure, including Dg and the Slit-Robo complex, can self-assemble and create non-adherent luminal domains. However, proper midline positioning of the lumen requires Talin function (Vanderploeg, 2015).

Using an array of Talin transgenes previously shown to modify integrin adhesion strength and actin recruitment, this study assessed and compared the importance of these Talin-dependent processes. Binding of Talin's integrin binding site 1 (IBS1) to a membrane proximal NPxY motif on the β-integrin tail induces conformational changes within the integrin dimer, activating it and increasing the affinity for ECM ligands. Integrin activation is likely required prior to Talin IBS2 binding, an interaction which promotes a strong and stable integrin-cytoplasmic adhesome linkage. The current data indicates that either of Talin's two integrin binding sites are sufficient to promote CB morphogenesis and heart tube assembly. The ability of the heart to form in the presence of only IBS1 or IBS2 suggests that strong, long-lasting integrin-mediated adhesions are unnecessary. This idea is reinforced by the late accumulation of CAP, a protein recruited to more mature muscle adhesions. It is likely that transient adhesions are sufficient for lumenogenesis. It remains possible that an essential role for either IBS1 or IBS2 is masked by the perdurant maternal Talin in zygotic mutants. However, the functional redundancy of these domains is consistent with in vitro and in vivo studies suggesting that a subset of Talin functions can be fulfilled by either IBS domain (Vanderploeg, 2015).

Talin links integrins to the actin cytoskeleton both directly through an actin binding domain, or indirectly through recruitment of actin regulators such as Vinculin. Bond force studies of the C-terminal ABD suggest that although the ABD-actin linkage is direct, it is a weak bond which likely relies on additional direct or indirect Talin-actin linkages to form a strong and stable connection. Supporting this, TalinABD is essential for morphogenetic processes which rely on transient and dynamic integrin-actin linkages, but it is at least partially dispensable for longer-lasting adhesions which are likely stabilized by indirect Talin-actin interactions through Vinculin. The current studies demonstrate that Drosophila heart development is sensitive to disruptions in Talin's C-terminal ABD, which implicates cytoskeletal reorganization as a key process downstream of integrins during tubulogenesis. Supporting this, expression of constitutively active Diaphanous or dDAAM, formin proteins which promote actin polymerization, induced ectopic lumina similar to those that have been characterized in rhea mutants. These data are consistent with Talin promoting CB morphogenesis and lumen formation through direct, but dynamic actin linkages and suggest that formins may act downstream of Talin in apicalizing lumen formation (Vanderploeg, 2015).

To date, most studies on the Drosophila embryonic heart have focused on cell surface factors including receptors and their respective ligands; few studies have moved into the cell to establish the downstream signaling pathways involved. Insights into in vitro models suggest that polarity pathways and vesicle trafficking will be informative areas of study. For example, in the MDCK cyst model, the small GTPases Rab8a and Rab11a coordinate with the exocyst complex to deliver luminal factors to the pre-luminal initiation site. It remains to be determined whether similar exocytosis or secretion mechanisms are required for Drosophila heart lumen initiation or expansion. Furthermore, although it is unclear which classical apical polarity proteins are conserved in the Drosophila heart, epithelial and endothelial models suggest that the Cdc42-Par6-aPKC complex is a conserved master regulator of tube formation in both vertebrates and flies. Indeed, Drosophila heart tubulogenesis fails in embryos with heart specific inhibition of Cdc42 and expression of activated Cdc42 results in lateral lumina reminiscent of those characterized in rhea homozygotes. A mechanism is envisioned of heart tubulogenesis in which Talin provides instructive cues to the vesicle trafficking and polarity networks that target luminal factors and inhibit the assembly of cell-cell adhesion structures within the pre-luminal domain (Vanderploeg, 2015).

Basal cell-extracellular matrix adhesion regulates force transmission during tissue morphogenesis

Tissue morphogenesis requires force-generating mechanisms to organize cells into complex structures. Although many such mechanisms have been characterized, little is known about how forces are integrated across developing tissues. This study provides evidence that integrin-mediated cell-extracellular matrix (ECM) adhesion modulates the transmission of apically generated tension during dorsal closure (DC) in Drosophila. Integrin-containing adhesive structures (see Myospheroid) resembling focal adhesions were identified on the basal surface of the amnioserosa (AS), an extraembryonic epithelium essential for DC. Genetic modulation of integrin-mediated adhesion results in defective DC. Quantitative image analysis and laser ablation experiments reveal that basal cell-ECM adhesions provide resistance to apical cell displacements and force transmission between neighboring cells in the AS. Finally, the study provides evidence for integrin-dependent force transmission to the AS substrate. Overall, these data indicate that integrins regulate force transmission within and between cells, thereby playing an essential role in transmitting tension in developing tissues (Goodwin, 2016).

During morphogenesis, cells undergo complex rearrangements to generate tissues and organs. Forces generated through the actin cytoskeleton and transmitted through cell-cell adhesion receptors govern the changes in cell shape and position that drive morphogenetic events. Interactions between cells and their extracellular environment, mediated by cell to extracellular matrix (ECM) adhesions, also play an important role in establishing appropriate tissue mechanics during development (Goodwin, 2016).

Integrin heterodimers are the main family of cell-ECM adhesion receptors in metazoans. They are composed of an α and a β subunit and connect to the actin cytoskeleton through an intracellular adhesion complex comprising many adapter and signaling proteins that modulate integrin receptor function. Understanding of integrin biology has largely been obtained from study of focal adhesions (FAs), prominent integrin-mediated adhesion structures observed in 2D cell culture. These studies have demonstrated that integrins can be regulated not only by biochemical signals but also by extracellular and/or intracellular mechanical cues. In FAs, integrins are key regulatory hubs for both sensing and responding to changes in the mechanical properties of the cellular microenvironment; this function impinges greatly on cell behavior. Translating the insights generated from the study of FAs in 2D culture into 3D models has proved to be contentious because FAs do not form as readily in 3D, and by some accounts do not form at all. Therefore, identification of contexts in intact living organisms in which FAs form and affect cell behavior is of great interest for corroborating and building upon mechanisms delineated in cell culture (Goodwin, 2016).

Studies of morphogenetic movements that occur during Drosophila development have provided many insights into the role of mechanical forces in shaping developing tissues, as well as elucidation of the molecular mechanisms that underlie tissue biomechanics. One such process, dorsal closure (DC), is a well-studied integrin-dependent morphogenetic event that occurs midway through fly embryogenesis to create a continuous epidermal sheet over the dorsal surface of the embryo. An extraembryonic epithelium called the amnioserosa (AS) contracts and ingresses, allowing the lateral epidermis from opposing sides of the embryo to migrate toward the dorsal midline. Integrin adhesion complexes form in both tissues, and are thought to carry out a variety of functions. In particular, integrins mediate attachment between the AS and the underlying yolk cell through adhesion to a layer of laminin-rich ECM; disruption of laminin phenocopies loss of integrin function, suggesting that integrin-dependent cell-ECM adhesion is required for this process. However, the specific role of integrins in the regulation of tissue biomechanics during DC has not been addressed. Since integrins are known to transduce traction forces that are generated by actomyosin networks in spreading and migrating cells, it is possible that they may play a similar role in force transmission during DC (Goodwin, 2016).

The role of mechanical forces driving DC is well established. Forces originating within the AS alone are sufficient to drive tissue closure (Wells, 2014). AS contractile forces are generated through cell death and extrusion, which reduces AS surface area, as well as by apical ratcheting, generated by coordinated pulses of medial actomyosin networks. Ratcheting drives apical oscillations and constriction of individual AS cells and generates tension in the apical plane of the tissue. Mechanical forces generated in the leading-edge epidermis also contribute to closure. A contractile actin cable assembles at the leading edge of the epidermis and forms a purse-string-like structure that generates tension to help contract the hole. Cells in the leading-edge epidermis also elongate to provide a counteractive pulling force away from the dorsal midline. Later in closure, projections formed at the leading edge participate in zippering, a process hypothesized to provide additional pulling forces for closure. The essential requirement for forces contributed from the AS versus the leading edge remains a somewhat contentious issue; it is likely an optimized balance of forces originating from both tissues that leads to efficient DC. However, in neither tissue has the question of how forces are transmitted and coordinated between cells been addressed. Cell-ECM adhesions have been shown to regulate the balance between force transmission across cells and to the substrate in culture models. Given the precise spatiotemporal patterning of forces required for DC and the known requirement for integrins in the AS, it was hypothesized that integrins may act in a similar capacity during DC to facilitate controlled propagation of forces generated across the apical plane of the tissue between neighboring cells (Goodwin, 2016).

By focusing on subcellular regulation of integrins in the AS, and through a combination of quantitative live imaging, genetics, and biophysical approaches, this study identified adhesive structures and describes a role for cell-ECM adhesion in regulating local cell displacements in the AS. Perturbation of integrin function was found to lead to changes in force transmission across the apical plane of cells. Furthermore, evidence is provided for integrin-dependent mechanical coupling between the AS and its substrate. Based on these results, it is proposed that the apical pole of AS cells is tethered to the substrate via apical-basal mechanical coupling and cell-ECM adhesions; thus, apically generated forces that drive cell oscillations are subject to passive resistance from basal adhesions to the ECM. Finally, evidence is provided that tethering at cell-ECM adhesions may resist large-scale cell displacements during tissue contraction in DC. These findings highlight the importance of integrin-mediated coordination of mechanical forces during collective cell behaviors that drive tissue morphogenesis (Goodwin, 2016).

This work uncovers an essential role for integrin-dependent cell-ECM adhesion in the coordination of forces during tissue morphogenesis. It significantly enhances understanding of how integrin function in the AS contributes to DC. Specifically, deviations in either direction from wild-type levels of cell-ECM adhesion are sufficient to disrupt DC. Through identification of focal adhesion-like structures (FALS) and characterization of their morphology and behavior, subcellular insights were gained into how cell-ECM adhesions regulate displacement of AS cells. Furthermore, evidence is provided that FALS morphology dictates tissue response to ablation, implicating cell-ECM adhesion in the efficiency of apical force transmission across AS cells. Genetic perturbation of cell-ECM adhesion leads to altered force transmission and, consequently, tissue response to ablation and cell displacement are misregulated. Finally, evidence is provided for integrin-dependent force transmission to the ECM by demonstrating that cell-substrate coupling is lost in the absence of FALS. Therefore, it is concluded that cell-ECM adhesion plays a critical role in modulating the transmission of cell-generated forces between neighboring cells and to the substrate (Goodwin, 2016).

This study identifies FALS, integrin-dependent adhesive structures that in many ways resemble FAs. In particular, like FAs, FALS are force responsive. Previous studies have demonstrated that integrins play a conserved role in force sensing in Drosophila ( Pines, 2012). Since the AS is a morphogenetically active epithelial tissue, FALS represent a particularly attractive system in which to study FAs in the context of an intact, developing organism. Links between FAs and cell behavior are well studied in cell culture models. In the AS, cells exhibit non-directional mobility as a result of neighboring cell deformation, and the extent to which they move is negatively correlated with the amount of cell-ECM adhesion. However, the motion that was observe is distinct from crawling cell behaviors that are used to generate directed cell migration in cell culture. This analysis of cell-substrate mechanical coupling shows that substrate movement is positively correlated with cell movement, suggesting that the substrate is deformed in the same direction as cell movement. This is the opposite of what is observed in crawling cells, where cells pull themselves over the substrate, deforming the substrate in the opposite direction to cell movement (Goodwin, 2016).

The data support the hypothesis that cell-ECM adhesion plays a key role in transmitting tension between AS cells, since tissue response to laser-induced release of tension was perturbed when cell-ECM adhesion was modulated. This finding is in agreement with recent work which showed that clusters of cells in culture exhibit greater correlation between cell-cell forces in opposing cell junctions and, therefore, more force transmission across cells when integrin-mediated adhesion is downregulated (Ng, 2014). Cell culture models have also been used to examine the relationship between traction exerted at cell-ECM adhesions and tension experienced at cell-cell adhesions (Maruthamuthu, 2011). In cell pairs the ratio between traction and tension has been shown to be constant, and thus changes to traction result in changes to tension. However, as the number of cell contacts increases, this ratio changes: traction exerted by cells adhering to multiple other cells decreases, while tension at cell-cell interfaces is enhanced (Maruthamuthu, 2011). How the relationship between forces exerted by cells on the ECM and tension experienced across cell membranes changes in the context of a tissue remains unclear. This study examined the effects of changing cell-ECM adhesion on stresses experienced at cell-cell contacts within a simple epithelium. While it was not possible to measure the magnitude of tension across cells or of traction exerted by cells within an intact, living embryo, it was found that changes to cell-ECM adhesion (and thus potentially to traction forces) result in differential responses to laser ablation at the level of cell-cell contacts. These findings suggest that in the context of a living tissue, there may be a strong relationship between the traction exerted by cells and the tension experienced across their junctions, as observed in cell culture models (Goodwin, 2016).

By tracking the movement of FALS during cell oscillations and laser ablation experiments, this study provides evidence that cell-ECM adhesion can resist apically generated forces. Specifically, it was found that wild-type variations in FALS morphology can predict differences in apical behaviors: in the presence of larger FALS, cell membranes exhibit less displacement during oscillations and less recoil during laser ablation experiments. These data provide strong evidence for two main conclusions: firstly, that the apical and basal parts of the cell are mechanically coupled, and secondly, that FALS act as tethers to the ECM to resist apically generated forces. It is proposed that this mechanical coupling is achieved through the actin cytoskeleton, given that both cell-cell and cell-ECM adhesion associated with F-actin, and that F-actin is present along the entire cortex of AS cells. However, it is also possible that other cytoskeletal components play a role. For example, microtubules mediate flattening and elongation of AS cells in early embryogenesis, and in Caenorhabditis elegans, morphogenesis of adjacent tissues is mechanically coupled via hemidesmosomes and intermediate filaments. Apical-basal mechanical coupling in AS cells could be achieved through the combined efforts of different cytoskeletal networks; future studies will be needed to characterize the entity that mediates this mechanical coupling (Goodwin, 2016).

If cell-ECM adhesions provide tethering to the ECM, then AS cells would exert forces on their substrate, leading to deformation. This was visualized by developing a tool inspired by TFM to measure deformations of the underlying yolk cell membrane. The movements of AS cells and the yolk membrane were found to be correlated, suggesting that cells are mechanically coupled to the underlying substrate. Furthermore, this correlation is lost in the absence of integrins, as predicted by downregulating cell-ECM adhesion in multicellular clusters. These findings confirm that cell-generated forces pull and deform the underlying substrate in vivo, and that tethers connected to the ECM through FALS are required for mechanical coupling between cells and the substrate. Overall, these findings suggest that a balance of cell-cell and cell-ECM force transmission previously described in cell culture may play a role in the generation of tension across AS cells (Goodwin, 2016).

Based on these results, a model is proposed whereby integrins act as tethers to the ECM and resist apically generated forces, thereby mediating force transmission between neighboring cells and to the substrate. As a result, cell-ECM adhesion controls displacement and mechanics of AS cells, which may have consequences for tissue contraction and closure. DC requires precise timing and coordination of many different cell behaviors; disrupting any one of these can lead to closure defects. By examining large-scale cell movements in the AS, it was found that cell displacement toward the dorsal midline during the slow phase of contraction is increased in mys / mutants. Previous studies of mys / mutants revealed that in the slow phase of closure, inner cells of the AS contract more rapidly than in the wild-type. It is speculated that this increased rate of contraction could be due to a loss of basal tethering; this would also lead to increased cell displacement toward the midline. Evidently this increased rate of contraction is not maintained, as mys / embryos experience delayed or failed closure; defects later in closure likely arise from loss of adhesion between tissue layers, leading to tears between the AS and the epidermis. When cell-ECM adhesion is increased, more tethering results in a downregulation of cell-cell force transmission, which could hinder contraction of the AS and lead to delayed or failed DC. In line with this, it was found that cell displacement toward the dorsal midline is reduced in Talin(E1777A) and Rap1-CA mutant embryos (Goodwin, 2016).

Animal morphogenesis and particularly DC are dependent upon the cellular mechanisms that generate biomechanical forces and, subsequently, on the proper translation of these forces into changes in tissue architecture. This study provides insight into how forces are transmitted within and between cells. It is proposed that cell-ECM adhesion must be fine-tuned to allow for proper force transmission, which could play a role in establishing optimal levels of tension across the AS during tissue contraction. Given the ubiquitous presence of cell-ECM adhesions in epithelia, the ability of integrins to modulate force transmission may represent a fundamental, conserved feature of animal development (Goodwin, 2016).


PS1 integrin is expressed primarily on the presumptive dorsal wing epithelium, and the PS2 integrin (alpha PS2 beta PS) is found almost exclusively on the ventral epithelium. Immediately after pupariation, the central wing pouch evaginates, folding along its center to appose the epithelia that will secrete the dorsal and ventral surfaces of the adult wing blade. Both of the PS integrins are required to maintain the close apposition of the dorsal and ventral wing epithelia during morphogenesis (Brower, 1989).

Wing disc ultrastructure is correlated with the distribution of the beta chain of integrin, laminin A, and filamentous actin for each key stage of pupal development. Integrin is present on the basal surface of intervein cells but not on vein cells whereas laminin A is absent from the basal surfaces of intervein cells but is present on vein cells. Laminin is not a ligand for integrin in this context. During apposition and adhesion stages integrin is broadly distributed over the basal and lateral surfaces of intervein cells but subsequently becomes localized to small basal foci. Basal adherens-type junctions are first evident during the adhesion stage and become closely associated with the cytoskeleton during the separation stage. Basal junction formation occurs in two discrete steps; intercellular connections are established first and junction/cytoskeletal connections are formed about 20 hours later (Fristrom, 1993). Drosophila serum response factor, otherwise known as Blistered is confined to intervein cells (Montagne, 1996).

A network of cell-cell contacts mediated by adherens junctions and cell-extracellular matrix contacts defines the architecture of the Drosophila ommatidium. This network is built incrementally; contacts established early in eye development typically persist into adulthood. Photoreceptor apical surfaces are involuted into the retinal epithelium and are subsequently elaborated to form the photosensitive rhabdomeres (see The Drosophila Adult Ommatidium: Illustration and explanation with Quicktime animation). Rhabdomeres become aligned to the ommatidial optical axis via their anchorage to the retinal floor at the cone cell plate, a specialized nexus of cell-cell and cell-extracellular matrix contacts. Several eye phenotypes trace their origin to the structural failure of the cone cell plate (Longley, 1995).

Thin, a Trim32 ortholog, is essential for myofibril stability and is required for the integrity of the costamere in Drosophila

Myofibril stability is required for normal muscle function and maintenance. Mutations that disrupt myofibril stability result in individuals who develop progressive muscle wasting, or muscular dystrophy, and premature mortality. This study presents investigations of the Drosophila l(2)thin [l(2)tn] mutant. The "thin" phenotype exhibits features of the human muscular disease phenotype in that tn mutant larvae show progressive muscular degeneration. Loss-of-function and rescue experiments determined that l(2)tn is allelic to the tn locus [previously annotated as both CG15105 and another b-box affiliate (abba)]. tn encodes a TRIM (tripartite motif) containing protein highly expressed in skeletal muscle and is orthologous to the human limb-girdle muscular dystrophy type 2H disease gene Trim32. Thin protein is localized at the Z-disk in muscle, but l(2)tn mutants showed no genetic interaction with mutants affecting the Z-line-associated protein muscle LIM protein 84B. l(2)tn, along with loss-of-function mutants generated for tn, showed no relative mislocalization of the Z-disk proteins alpha-Actinin and muscle LIM protein 84B. In contrast, tn mutants had significant disorganization of the costameric orthologs beta-integrin, Spectrin, Talin, and Vinculin, and the initial description for the costamere, a key muscle stability complex, in Drosophila is presented. These studies demonstrate that myofibrils progressively unbundle in flies that lack Thin function through progressive costamere breakdown. Due to the high conservation of these structures in animals, this study demonstrates a previously unknown role for TRIM32 proteins in myofibril stability (LaBeau-DiMenna, 2012).


Drosophila alpha-actinin in ovarian follicle cells is regulated by EGFR and Dpp signalling and required for cytoskeletal remodelling; alpha-actinin is involved in the assembly of a β-integrin-dependent adhesion site and Enabled localization

α-Actinin is an evolutionarily conserved actin filament crosslinking protein with functions in both muscle and non-muscle cells. In non-muscle cells, interactions between α-actinin and its many binding partners regulate cell adhesion and motility. In Drosophila, one non-muscle and two muscle-specific α-actinin isoforms are produced by alternative splicing of a single gene. In wild-type ovaries, α-actinin is ubiquitously expressed. The non-muscle α-actinin mutant ActnΔ233, which is viable and fertile, lacks α-actinin expression in ovarian germline cells, while somatic follicle cells express α-actinin at late oogenesis. This latter population of α-actinin, termed FC-α-actinin, is shown to be absent from the dorsoanterior follicle cells, and evidence is presented that this is the result of a negative regulation by combined Epidermal growth factor receptor (EGFR) and Decapentaplegic signalling. Furthermore, EGFR signalling increases the F-actin bundling activity of ectopically expressed muscle-specific α-actinin. A novel morphogenetic event in the follicle cells is described that occurs during egg elongation. This event involves a transient repolarisation of the basal actin fibres and the assembly of a posterior β-integrin-dependent adhesion site accumulating α-actinin and Enabled. Clonal analysis using Actn null alleles demonstrated that although α-actinin is not necessary for actin fibre formation or maintenance, the cytoskeletal remodelling is perturbed, and Enabled does not localise in the posterior adhesion site. Nevertheless, epithelial morphogenesis proceeded normally. This work provides the first evidence that α-actinin is involved in the organisation of the cytoskeleton in a non-muscle tissue in Drosophila (Wahlström, 2006).

To understand how α-actinin is involved in the function of the follicle cells at late oogenesis, α-actinin localisation was studied in wild-type follicle cells at stages 10-14. For detection, used the monoclonal antibody MAC276, which recognises all three α-actinin isoforms was used, along with a staining protocol that does not allow simultaneous labelling of F-actin with phalloidin. The follicle cells are polarised with the apical side facing the germline and the basal side facing the epithelial sheath surrounding each string of developing egg chambers. At the time of egg chamber assembly, the basal surface of the follicle cells acquires a layer of stress fibre-like actin bundles, which is maintained throughout oogenesis. At stage 10A, α-actinin was localised at the cell cortex (not shown) and was especially abundant in the basal actin fibres. At stage 10B/11, the evenly stained actin fibres began to reorganise, and by stage 11, a distinct patch of α-actinin accumulation was detected in the posterior part of the cell. This pattern was seen in all main body follicle cells, i.e. ventral follicle cells and dorsal cells posterior to the dorsal appendages. In the dorsoanterior follicle cells, α-actinin was expressed at lower levels and showed less distinct localisation patterns. α-Actinin was also detected at the roof cell apices of the elongating dorsal appendages. At the end of stage 12, the basal α-actinin pattern in the main body follicle cells was reorganised again. The accumulation at the posterior end of the cell gradually dispersed, and at stage 13, α-actinin was concentrated at the lateral cell margins. The central actin fibres were less strongly labelled. By stage 14, when the basal actin fibres have disappeared, α-actinin displayed a cortical localisation (Wahlström, 2006).

The lateral stripes of α-actinin in the follicle cells at stage 13 correspond to the previously described adhesion sites shown to contain β-integrin and Ena. Integrins are transmembrane receptors for ligands in the extracellular matrix (ECM), and they mediate adhesion between the cell and the ECM. Ena is the sole Drosophila member of the conserved family of Ena/VASP proteins, which act as positive regulators of actin filament assembly. Co-localisation studies of α-actinin and Ena revealed a complete overlap in the basal cytoskeleton, including the posterior patch, during stages 11 and 12. At stage 13, there was also extensive co-localisation, although Ena appeared to be located closer to the cell margin than α-actinin. Thus, both α-actinin and Ena accumulate in a transient adhesion site-like structure that forms at the onset of egg elongation (Wahlström, 2006).

The basal stress fibres are aligned perpendicular to the A/P axis of the oocyte between stages 7 and 10, but then a phase of slight disorganisation occurs before the perpendicular alignment is reassumed by stage 13. The disorganised phase correlates with the relocalisation of α-actinin and Ena observed in the basal cytoskeleton. The remodelling could also be recognised by phalloidin-staining of the actin fibres, although they indeed appeared quite irregular in most cells. In several cells, they are polarised in the A/P direction, and they often also converge in a denser patch of F-actin, which overlaps with the posterior patch containing Ena and α-actinin. Thus, egg elongation involves an organised repolarisation of the basal actin fibres (Wahlström, 2006).

Analysis of the α-actinin expression pattern in the non-muscle mutant ActnΔ233 revealed that at least two separate populations of α-actinin are present in the follicle cells. α-Actinin produced from an mRNA that is transcribed from the upstream promoter (NC-α-actinin) is ubiquitously expressed in the egg chamber. The second α-actinin population, FC-α-actinin, corresponds to α-actinin that is present in certain non-muscle cells of all examined non-muscle-specific α-actinin mutants. FC-α-actinin is most likely produced from an mRNA transcribed from the downstream promoter and may include both non-muscle α-actinin and adult muscle-specific α-actinin. However, an analysis using isoform-specific antibodies or a complete sequencing of the mRNAs expressed in the egg chamber will be required in order to clarify this issue. FC-α-actinin protein was expressed in the main body follicle cells starting from stage 10, but excluded from the dorsoanterior cells. The dorsoanterior cells are patterned by the EGFR and Dpp signalling pathways, and the results showed that these two pathways together downregulate FC-α-actinin expression, but not the expression of NC-α-actinin (Wahlström, 2006).

The dorsoanterior and main body follicle cells undergo very different morphogenetic changes. The dorsoanterior cells elongate in the apicobasal direction and migrate (Dorman, 2004), an event that did not seem to require α-actinin. In contrast, the main body follicle cells flatten and expand their surfaces. These events are expected to involve different sets of cytoskeletal regulators, of which very little is yet known. The formation of a dense layer of basal actin fibres in the main body follicle cells may include upregulation of proteins known to be involved in stress fibre formation, such as α-actinin. It has been shown that the dorsal midline cells upregulate basal E-cadherin and FasIII, indicating increased cell-cell adhesion. These cells also lose their basal actin fibres, which may explain why less α-actinin, i.e., only NC-α-actinin, is expressed in these cells (Wahlström, 2006).

Throughout oogenesis, the basal cytoskeleton is organised into actin fibres aligned in parallel. Variation has been noted in the actin fibre polarity at the late stages of oogenesis. These observations are extended by showing that the basal cytoskeleton undergoes an organised remodelling during the final stages of oogenesis. The rapid increase in oocyte volume during nurse cell dumping at stage 11 requires that the follicle cells expand their surfaces in order to maintain a coherent epithelium. This process involves a transient change in the polarity of the basal actin fibres, from a perpendicular to a parallel orientation relative to the A/P axis of the egg chamber, and the assembly of a transient structure that accumulates α-actinin and Ena. The association of this structure with an accumulation of β-integrin and its dependence on integrin adhesion demonstrate that the cytoskeletal reorganisation is linked to a remodelling of integrin-based adhesion sites. The fact that α-actinin and β-integrin did not show a strict co-localisation is in good agreement with studies on mammalian cells showing that integrin, but not α-actinin, is present in nascent adhesion sites termed focal complexes. α-Actinin accumulation in the adhesion site occurs later, as the focal complexes mature into focal adhesions (Zaidel-Bar, 2003). The signal that induces the remodelling of the basal cytoskeleton remains to be identified. An intriguing possibility is that the mechanical stress applied to the epithelium during nurse cell dumping is transduced into biochemical signals that result in the observed reorganisation. Two different mechanisms are known to mediate mechanotransduction: stretch-activated ion channels or conformational changes within cell-matrix adhesion sites (Wahlström, 2006).

The current view is that the parallel basal actin fibres shape the oocyte during egg elongation by preventing axial expansion. However, since the basal actin fibres repolarise during egg elongation, the current model does not adequately explain how the oocyte acquires its final shape. The fact that Ena accumulates in the posterior of the cell during egg elongation suggests that a mechanism involving localised actin polymerisation and directed cell growth may also contribute to shaping the oocyte. It has been reported that egg elongation is blocked by mutations in the genes encoding α-integrin, β-integrin, the adhesion site components talin or tensin, the receptor tyrosine phosphatase Dlar or the ECM component Laminin A. In the case of β-integrin and Dlar, it has been shown that the actin fibre polarity is disturbed (Bateman, 2001; Frydman, 2001), and this has been suggested to be the cause of the short egg phenotype. However, the data presented in this work give reason to speculate that defective adhesion between the follicle cells and the ECM might play a role as well (Wahlström, 2006).

To explore the function of α-actinin in the main body follicle cells, clones of cells lacking α-actinin were generated. This experiment unexpectedly revealed that α-actinin is not required for the formation or maintenance of the basal actin fibres. Previous studies, relying on the introduction of truncated α-actinin molecules into cultured mammalian cells, have suggested that α-actinin is important for stress fibre maintenance. Furthermore, examination of transformed cells expressing different levels of α-actinin showed that cells with low α-actinin levels had poorly developed stress fibres, an effect was not observe in this study. It is possible that the follicle cell basal actin fibres are not true contractile stress fibres and therefore do not depend on α-actinin. Alternatively, an alternative pathway for stress fibre assembly that is independent of α-actinin might be activated in the follicle cells following removal of α-actinin (Wahlström, 2006 and references therein).

The clonal analysis revealed that while α-actinin was not necessary for the lateral accumulation of Ena at stage 13, it was cell-autonomously required for the posterior localisation of Ena at stages 11 and 12. The reason for this could be that α-actinin is specifically required for recruiting Ena to the posterior adhesion site, perhaps by recruiting their common binding partner zyxin. Alternatively, the posterior adhesion site may not form at all. The latter possibility is supported by observations on mosaic stage 10B/11 egg chambers that are in the process of assembling the posterior adhesion site. While wild-type cells are in the process of translocating Ena towards the posterior, neighbouring cells lacking α-actinin still showed a lateral Ena pattern. At stage 12/13, the lateral adhesion sites are assembled earlier in the mutant cells than in the wild-type cells, perhaps because the mutant cells had not reorganised their cytoskeleton to the same extent as the wild-type cells had. Thus, these results clearly demonstrate that adhesion site remodelling is altered in the absence of α-actinin. However, in contrast to the cells lacking β-integrin, the Actn mutant cells appear to maintain their adhesion to the ECM, since they appear equally well spread as the wild-type cells at stage 13 (Wahlström, 2006).

The results are in agreement with the current view that vertebrate α-actinin is involved in adhesion site disassembly. This is a strictly regulated process that involves signalling by phosphoinositides, tyrosine phosphorylation and proteolytic cleavage of individual components. α-Actinin is one of the targets for these activities. Phosphorylation of α-actinin by focal adhesion kinase (FAK) reduces α-actinin’s affinity for F-actin and regulates the activity of FAK itself, PtdIns(3,4,5)-P3 binding to α-actinin disrupts α-actinin binding to β-integrin and F-actin, and cleavage of α-actinin by calpain has been associated with cell shape changes in certain cell types. It has also been shown that α-actinin is essential for maintaining the link between the adhesion site and the stress fibre. This conclusion was reached based on an experiment showing that laser-mediated inactivation of α-actinin located in an adhesion site resulted in stress fibre detachment from the adhesion site. In the Drosophila follicle cells, α-actinin is clearly not required for actin fibre attachment. However, by the laser-mediated inactivation, α-actinin was removed from an adhesion site, whereas in the Actn null mutant follicle cells, the adhesion sites never contained α-actinin. Considering the large number of proteins that interact with α-actinin, it is expected that a signal targeted at α-actinin indirectly affects many other proteins and processes as well. An adhesion site lacking α-actinin may well be functional, but it may respond differently to various signals that induce adhesion site remodelling (Wahlström, 2006).

Interestingly, even very large clones of Actn mutant cells had no negative effects on egg morphology. This indicates that proper cytoskeletal remodelling and posterior localisation of Ena is not necessary for egg elongation. Apparently, the expansion of the main body follicle cells in the Actn mutant cells occurs by an alternative mechanism that is not dependent on α-actinin. This raises the question of whether the wild-type remodelling mechanism would become important under some specific conditions not prevailing in the laboratory. The impact of the environment on the development of mutant phenotypes has been well documented in the slime mould Dictyostelium discoideum. Lack of α-actinin results in only minor alterations in cellular functions and did not reduce viability. However, when the cells were grown under conditions resembling their natural habitat, specific developmental defects appeared (Wahlström, 2006).

This study contributes new data to the field of cytoskeletal dynamics in Drosophila follicle cells. A surprisingly complex regulation was undercovered underlaying α-actinin expression in the follicle cells. The basal cytoskeleton of the main body follicle cells undergoes an organised remodelling during egg elongation, and α-actinin is required in this process. This observation provides the first identified phenotype in a Drosophila non-muscle tissue lacking α-actinin. The fact that both loss of α-actinin and overexpression of α-actinin results in very distinct cellular phenotypes suggests that the follicular epithelium could serve as a very useful in vivo system for further studies on mechanisms that regulate α-actinin function and activity. Furthermore, the cytoskeletal remodelling may provide an easily accessible and genetically tractable model for studies on adhesion dynamics in vivo (Wahlström, 2006).

JNK signaling controls border cell cluster integrity and collective cell migration: Involvement of β-Integrin

Collective cell movement is a mechanism for invasion identified in many developmental events. Examples include the movement of lateral-line neurons in Zebrafish, cells in the inner blastocyst, and metastasis of epithelial tumors. One key model to study collective migration is the movement of border cell clusters in Drosophila. Drosophila egg chambers contain 15 nurse cells and a single oocyte surrounded by somatic follicle cells. At their anterior end, polar cells recruit several neighboring follicle cells to form the border cell cluster. By stage 9, and over 6 hr, border cells migrate as a cohort between nurse cells toward the oocyte. The specification and directionality of border cell movement are regulated by hormonal and signaling mechanisms. However, how border cells are held together while they migrate is not known. This study shows that negative-feedback loop controlling JNK activity regulates border cell cluster integrity. JNK signaling modulates contacts between border cells and between border cells and substratum to sustain collective migration by regulating several effectors including the polarity factor Bazooka and the cytoskeletal adaptor D-Paxillin. A role for the JNK pathway is anticipated in controlling collective cell movements in other morphogenetic and clinical models (Llense, 2008).

In an analysis of the mechanisms regulating the expression of puckered (puc), the gene encoding the Drosophila Jun N-terminal kinase (JNK) dual-specificity phosphatase (DSP) (Martin-Blanco, 1998) regulatory sequences (PG2) were uncovered directing its expression to border cells. PG2 expands across the first and second introns of puc, in which the pucB48 insertion is located. This expression is also observed in puc enhancer (pucB48) and protein trap lines (Llense, 2008).

JNKs represent a signaling hub with pivotal functions in cell proliferation, differentiation, and death. JNKs are inactivated by DSPs, and transcriptional induction of DSP expression is well documented as a negative-feedback mechanism. In Drosophila, this loop modulates JNK activity in processes such as epithelial expansion and overexpression of dominant-negative constructs relies on JNK signaling. Further, Puc overexpression leads to inhibition of JNK activity. Thus, Puc implements a negative-feedback loop in border cells (Llense, 2008).

Defects caused by the loss of JNK function in border cells included cluster dissociation and impaired motility. Instead of collectively following a leader cell, JNK-minus border cells autonomously disperse at the late step of migration, with most exhibiting long cellular extensions (LCEs) and actin-rich protrusions. JNK signaling does not affect polar cell specification or border cell recruitment (Llense, 2008).

Dissociation phenotypes are also observed in JNK-specific but not ERK-specific loss-of-function conditions for D-Fos, a major MAPK target, thereby ruling out potential interference via ERK. Indeed, reduced D-Fos suppresses border cell migration defects induced by elevated JNK activity (Llense, 2008).

Does JNK act in a linear pathway or does it target multiple independent effectors simultaneously to produce a multifaceted phenotype? Cells that migrate as part of a group cling firmly to each other while adhering transiently to the substrate. So, during migration, border cells show apicobasal polarity and remain attached to one another and to polar cells. Cell contacts are enriched in the adherens junctions (AJs) components, DE-Cadherin and Armadillo (β-Catenin). In electron microscopy (EM) preparations, border cells are tightly bound, whereas interfaces between border and nurse cells exhibit multiple interdigitations (Llense, 2008).

In JNK-minus conditions, namely after Puc overexpression or in bsk (JNK) clones, cell polarity is disrupted and only remnants of apical markers, such as Bazooka (Baz), are present. Adhesion is impaired, and DE-Cadherin and Armadillo are downregulated. Reduction of JNK activity also resulted in β-Integrin accumulation at ectopic actin-rich protrusions. These also accumulate MyoVI, consistent with its role in force generation. In summary, upon depletion of JNK activity, border cells lose apicobasal polarity and progress into a mesenchymal phenotype. Indeed, EM preparations show that border-border cell contacts are less tight than wild-type cell contacts and cell membranes detach from each other at multiple sites. The end result is a cluster with multiple leading edges and residual cell-cell contacts (Llense, 2008).

How does the JNK pathway become activated in border cells? Rho, Rac, and Cdc42 GTPases are potential candidates. Loss of Rac completely abolishes border cell migration. However, phenotypes for RhoA and Cdc42 expression of dominant-negative forms -- RhoADN and Cdc42DN) closely resemble JNK-minus induced dissociation. Furthermore, in Cdc42DN, polarity, cell contacts, and redistribution of substrate adhesion and motor markers are similarly affected. Most importantly, reporters of JNK activity such as Jun phosphorylation and the expression of the pucB48 transgene are also downregulated. Null cdc42 MARCM clones display the same phenotype, although frequency and penetrancy were very low. Therefore, a role for other GTPases, such as RhoA, in JNK activation cannot be ruled out (Llense, 2008).

Border cell clusters deficient for Baz (BazRNAi) resemble JNK loss of function (which leads to Baz downregulation) and exhibit dissociation and downregulation of DE-Cadherin. Thus, Baz, a critical landmark of epithelial polarity, could serve as an effector for the control of border-border cell contacts. To test this, Baz was overexpressed in cells lacking JNK activity (or expressing Cdc42DN); Baz was strongly rescued cluster integrity and DE-Cadherin expression (Llense, 2008).

Epithelial cells use a specialized repertoire of integrin receptors to mediate contacts and migration. However, border cells lacking β-Integrin were still able to adopt a leading migratory position, although the effect of complete removal of integrins from the cluster has not been reported (Llense, 2008).

Interestingly, β-Integrin antibodies reveal a rosette staining in border cell clusters that colocalize with AJ markers. Thus, β-Integrin could participate in the stabilization or strengthening of cell contacts, as shown for amnioserosa and larval epithelial cells in Drosophila, mammalian keratinocytes, and carcinoma cell clusters. Furthermore, β-Integrin, after JNK inactivation, strikingly accumulates at the front of LCEs suggesting a second function in cell invasiveness, as observed in leukocytes (Llense, 2008).

Direct evidence for β-Integrin involvement in border cell migration was obtained by RNAi in a sensitized JNK-minus condition. The expression of β-Integrin dsRNAs in border cells reduced β-Integrin levels but did not cause migration or integrity defects. However, in the presence of Puc, β-Integrin RNAi led to a strong enhancement of cluster dissociation and prevented the full extension of LCEs, which become mostly blunted. Moreover, an adhesion dominant negative (diβ) integrin chimera showed weak, but reproducible, dissociation phenotypes. Thus, β-Integrin turns out to participate in, first, the stabilization of border-border cell contacts and, second, the promotion of LCEs extension. The integrin countereceptors that facilitate border cell attachment and invasiveness are not yet known (Llense, 2008).

D-Paxillin was present in border cell contacts but was downregulated in JNK-minus conditions. Genomic-profiling analyses of JNK mutants suggests a transcriptional control of D-Paxillin expression. However, other options, such as subcellular relocation after phosphorylation, could also explain why D-Paxillin may be absent from JNK-minus border cells. Expression in border cells of two different D-Paxillin dsRNA lines was found to result in JNK loss-of-function-like dissociation, DE-Cadherin downregulation and β-Integrin accumulation at LCEs. Expression of a Talin RNAi line does not produce any migration phenotype, although it impairs follicle epithelia integrity (Llense, 2008).

In migratory leukocytes, PKA-mediated integrin phosphorylation prevents Paxillin accumulation at the leading front. Paxillin-integrin interactions in lateral positions lead to the inhibition of Rac, whose activation is thus spatially limited to the leading edge where it induces lamellipodia. Consequently, D-Paxillin might stabilize β-Integrin in border-border cell contacts. Its absence, in JNK-minus conditions, would lead in lateral and trailing cells to Rac activation, dissociation of border-border cell contacts, and extension of β-Integrin-rich ectopic lamellipodia. Indeed, the PKA-RII subunit is expressed in border cells, and border cells mutant for PKA show migration defects (Llense, 2008).

Interestingly, D-Paxillin overexpression rescued the border cell defect resulting from loss of JNK activity (or expression of Cdc42DN). DE-Cadherin relocated to border-border cell contacts, and β-Integrin expression was partially eliminated from residual LCEs. D-Paxillin overexpression alone had no effects (Llense, 2008).

It was further asked whether the control of cell polarity and cytoskeletal adaptor proteins by JNK were related. Paxillin expression was strongly reduced in baz mutant conditions, whereas Baz expression was only slightly affected by interference in Paxillin expression (Llense, 2008).

JNK signaling regulates border cells clustering by controlling at least two key elements, cell polarity (Baz) and cytoskeletal adaptor proteins (D-Paxillin), and as a consequence cell-cell contacts and cell-substrate attachments. Interestingly, the overexpression of Hindsight (Hnt), a target and negative regulator of JNK, results in similar defects to those caused by inhibition of JNK. Because re-expression of a variety of proteins (Baz, D-Paxillin, DE-Cadherin, and Armadillo) can rescue the dissociation phenotype and given that each time rescue is achieved, DE-Cadherin and Armadillo expression are restored, a plausible explanation for the effects observed with JNK-minus and Hnt overexpression is that there is an overall loss of multiple cell-cell adhesion complexes. The restoration of any of them would provide sufficient cell-cell adhesion to enable the cluster to move as a collective (Llense, 2008).

The individual migratory abilities of JNK-minus border cells could be partially explained by the observed β-Integrin relocalization to LCEs (border-nurse cell contacts). Alternatively, border cells could have lost their capacity to respond to positional gradients leading to random outward movements. Border cells use PVF and EGF to guide their migration. Blocking PVR and EGFR does not reduce the ability of border cells to extend protrusions but abolishes their directionality, with protrusions now extending in all directions. However, in these conditions, border cell clusters do not dissociate, thereby ruling out the possibility that dissociation in JNK mutants is due only to loss of directional guidance. A directionality index (DI) can be calculated. A DI of 0 indicates equal numbers of protrusions extending forward and backward. A DI of 1 indicates that cells only extend protrusions in the direction of migration. This study found a DI of 0.59 for wild-type clusters. In the absence of JNK, however, clusters show a DI ranging from -0.2 to 0, suggesting that JNK-minus border cells are blind to positional cues. This fact accounts for recently described synergistic effects of JNK and PVR signaling on border cells (Llense, 2008).

This model makes a significant prediction: JNK hyperactivation should increase adhesiveness and eventually block migration. Accordingly, ut was observed that the overexpresssion of a constitutively active form of Hep, the overexpresssion of a constitutively active form of Misshapen, or loss of function clones of puc resulted in nonmigratory and strongly compacted clusters. Occasionally, the death of a number of border cells was observed (Llense, 2008).

So far, the molecular and cellular study of collective versus individual migration both in developmental and cancer models has mainly focused on the analysis of structural elements. The identification of the JNK cascade as a key determinant of migratory responses in border cells could have an important impact in the understanding of collective movements. Border cell migration could serve as a good model for studying migratory transitions, thus impacting on the understanding of cancer metastasis and invasiveness, during which so little is known about the signaling mechanisms controlling migratory behavior (Llense, 2008).

Integrins regulate epithelial cell differentiation by modulating Notch activity

Coordinating exit from the cell cycle with differentiation is critical for proper development and tissue homeostasis. Failure to do so can lead to aberrant organogenesis and tumorigenesis. However, little is known about the developmental signals that regulate the cell cycle exit-to-differentiation switch. Signals downstream of two key developmental pathways, Notch and Salvador-Warts-Hippo (SWH), and of myosin activity regulate this switch during the development of the follicle cell epithelium of the Drosophila ovary. This study identified a fourth player, the integrin signaling pathway. Elimination of integrin function blocks mitosis-to-endocycle switch and differentiation in posterior follicle cells (PFCs), via regulation of the CDK inhibitor Dacapo. In addition, integrin mutant PFCs show defective Notch signalling and endocytosis. Furthermore, integrins act in PFCs by modulating the activity of the Notch pathway, as reducing the amount of Hairless, the major antagonist of Notch, or misexpressing Notch intracellular domain rescues the cell cycle and differentiation defects. Altogether, these findings reveal a direct involvement of integrin signalling on the spatial and temporal regulation of epithelial cell differentiation during development (Gomez-Lamarca, 2014).

ECM stiffness regulates glial migration in Drosophila and mammalian glioma models

Cell migration is an important feature of glial cells. This study used the Drosophila eye disc to decipher the molecular network controlling glial migration. Glial motility was stimulated by pan-glial PDGF receptor (PVR) activation, and several genes acting downstream of PVR were identified. Drosophila lox is a non-essential gene encoding a secreted protein that stiffens the extracellular matrix (ECM). Glial-specific knockdown of integrin results in ECM softening. Moreover, it was shown that lox expression is regulated by integrin signaling and vice versa, suggesting that a positive-feedback loop ensures a rigid ECM in the vicinity of migrating cells. The general implication of this model was tested in a mammalian glioma model, where a Lox-specific inhibitor unraveled a clear impact of ECM rigidity in glioma cell migration (Kim, 2014).

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

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