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Integrins are the major family of transmembrane proteins that mediate cell-matrix adhesion and have a critical role in epithelial morphogenesis. Integrin function largely depends on the indirect connection of the integrin cytoplasmic tail to the actin cytoskeleton through an intracellular protein network, the integrin adhesome. What is currently unknown is the role of individual integrin adhesome components in epithelia dynamic reorganization. Drosophila egg chamber consists of the oocyte encircled by a monolayer of somatic follicle epithelial cells that undergo specific cell shape changes. Egg chamber morphogenesis depends on a developmental array of cell-cell and cell-matrix signalling events. Recent elegant work on the role of integrins in the Drosophila egg chamber has indicated their essential role in the early stages of oogenesis when the pre-follicle cells assemble into the follicle epithelium. Here, we have focused on the functional requirement of two key integrin adhesome components, Parvin and Integrin-Linked Kinase (ILK). Both proteins are expressed in the developing ovary from pupae to the adult stage and display enriched expression in terminal filament and stalk cells, while their genetic removal from early germaria results in severe disruption of the subsequent oogenesis, leading to female sterility. Combining genetic mosaic analysis of available null alleles for both Parvin and Ilk with conditional rescue utilizing the UAS/Gal4 system, we found that Parvin and ILK are required in pre-follicle cells for germline cyst encapsulation and stalk cell morphogenesis. Collectively, we have uncovered novel developmental functions for both Parvin and ILK, which closely synergize with integrins in epithelia.

Transgelins are a conserved family of actin-binding proteins involved in cytoskeletal remodeling, cell contractility, and cell shape. In both mammals and Drosophila, three genes encode transgelin proteins. Transgelins exhibit a broad and overlapping expression pattern, which has obscured the precise identification of their role in development. Here, we report the first systematic developmental analysis of all Drosophila transgelin proteins, namely, Mp20, CG5023, and Chd64 in the living organism. Drosophila transgelins display overall higher sequence identity with mammalian TAGLN-3 and TAGLN-2 than with TAGLN. Detailed examination in different developmental stages revealed that Mp20 and CG5023 are predominantly expressed in mesodermal tissues with the onset of myogenesis and accumulate in the cytoplasm of all somatic muscles and heart in the late embryo. Notably, at postembryonic developmental stages, Mp20 and CG5023 are detected in the gut’s circumferential muscles with distinct subcellular localization: Z-lines for Mp20 and sarcomere and nucleus for CG5023. Only CG5023 is strongly detected in the adult fly in the abdominal, leg, and synchronous thoracic muscles. Chd64 protein is primarily expressed in endodermal and ectodermal tissues and has a dual subcellular localization in the cytoplasm and the nucleus. During the larval–pupae transition, Chd64 is expressed in the brain, eye, legs, halteres, and wings. In contrast, in the adult fly, Chd64 is expressed in epithelia, including the alimentary tract and genitalia. Based on the non-overlapping tissue expression, we predict that Mp20 and CG5023 mostly cooperate to modulate muscle function, whereas Chd64 has distinct roles in epithelial, neuronal, and endodermal tissues.

Eukaryotic 5’−3’ mRNA decay plays important roles during development and in response to stress, regulating gene expression post-transcriptionally. In Caenorhabditis elegans, deficiency of DCAP-1/DCP1, the essential co-factor of the major cytoplasmic mRNA decapping enzyme, impacts normal development, stress survival and ageing. Here, we show that overexpression of dcap-1 in neurons of worms is sufficient to increase lifespan through the function of the insulin/IGF-like signaling and its effector DAF-16/FOXO transcription factor. Neuronal DCAP-1 affects basal levels of INS-7, an ageing-related insulin-like peptide, which acts in the intestine to determine lifespan. Short-lived dcap-1 mutants exhibit a neurosecretion-dependent upregulation of intestinal ins-7 transcription, and diminished nuclear localization of DAF-16/FOXO. Moreover, neuronal overexpression of DCP1 in Drosophila melanogaster confers longevity in adults, while neuronal DCP1 deficiency shortens lifespan and affects wing morphogenesis, cell non-autonomously. Our genetic analysis in two model-organisms suggests a critical and conserved function of DCAP-1/DCP1 in developmental events and lifespan modulation.

Integrin-linked kinase (ILK), PINCH and Parvin proteins form the IPP-complex that has been established as a core component of the integrin-actin link. Our recent genetic studies on Drosophila parvin, reveal that loss of function mutant defects phenocopy those observed upon loss of ILK or PINCH in the muscle and the wing, strengthening the notion that these proteins function together in the organism. Our work identified that ILK is necessary and sufficient for parvin subcellular localization, corroborating previous data indicating a direct association between these two proteins. Further genetic epistasis analysis of the IPP-complex assembly at integrin adhesion sites reveals that depending on the cell context each component is required differently. At the muscle attachment sites of the embryo, ILK is placed upstream in the hierarchy of genetic interactions required for the IPP-complex assembly. By contrast, in the wing epithelium the three proteins are mutually interdependent. Finally, we uncovered a novel property for the CH1-domain of parvin: its recruitment at the integrin-containing junctions in an ILK-dependent manner. Apparently, this ability of the CH1-domain is controlled by the inter-CH linker region. Thus, an intramolecular interaction within parvin could serve as a putative regulatory mechanism controlling the ILK-Parvin interaction.

The mammalian focal adhesion kinase (FAK) family of non-receptor protein-tyrosine kinases has been implicated in controlling a multitude of cellular responses to the engagement of cell-surface integrins and G-protein-coupled receptors. The high level of sequence conservation between the mammalian proteins and the Drosophila homologue of FAK, Fak56,suggested that it would have similar functions. However, we show here that Drosophila Fak56 is not essential for integrin functions in adhesion,migration or signaling in vivo. Furthermore, animals lacking Fak56 are viable and fertile, demonstrating that Fak56 is not essential for other developmental or physiological functions. Despite this, overexpressed Fak56 is a potent inhibitor of integrins binding to the extracellular matrix, suggesting that Fak56 may play a subtle role in the negative regulation of integrin adhesion.

Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human β1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK–green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.

It is well known that activated prophenoloxidase (proPO) plays an important role in cuticular melanization and sclerotization. In addition, studies dealing with immune response of insects suggest that phenoloxidase (PO) is also critical in the defense reactions of insects against invaders. proPO is activated by elicitors derived from microbial cell wall components such as peptidoglycan, beta-1,3-glucan, and lipopolysaccharide (LPS). According to our recent studies we proposed a model clarifying the role of PO in both cellular and humoral immune responses. LPS triggers Ceratitis capitata hemocytes via induced protein tyrosine phosphorylation to release biologically active molecules, including p47 and proPO-activators. Furthermore, hemocytes in response to LPS facilitate clearance of LPS from the hemocoel of medfly. The effector molecules involved in the LPS clearance are hemocyte surface-associated p47 (mp47), soluble p47 (sp47), activated proPO, and tyrosine. A similar LPS clearance system in the integument of medfly in vitro was also demonstrated. According to our data, the proposed mechanism for LPS clearance from hemocoel and from integument is the crosslinking of LPS to p47 or certain integumental proteins via the intermediacy of reactive tyrosine derivatives generated by PO activity, as is the case for cuticular protein-chitin crosslinks during sclerotization. We also demonstrated that metabolites of the eumelanin biosynthesis and not melanin itself or N-acetyldopamine (NADA), the key precursor of sclerotizing agent, were necessary for the immune responses by hemocytes and integument.

Integrin-linked kinase (ILK), PINCH and Parvin constitute the tripartite IPP-complex that maintains the integrin-actin link at embryonic muscle attachment sites (MASs) in Drosophila. Here we showed that parvin null mutations in Drosophila exhibit defects in muscle adhesion, similar to ILK and PINCH mutants. Furthermore, the identical muscle phenotype of the triple mutant, which for the first time in any organism removed the entire IPP-complex function, genetically demonstrated that parvin, ILK and PINCH function synergistically. This is consistent with the tight localization of the tripartite complex at sites of integrin adhesion, namely MASs in the developing embryo and focal contact-like structures in the wing epithelium. Parvin contains tandem unconventional Calponin-Homology (CH) domains separated by a linker sequence, and a less well conserved N-terminal region. In vivo structure-function analysis revealed that all the domains are essential for parvin function, whereas recruitment at integrin adhesion sites is mediated by two localization signals: one located within the CH2-domain as previously reported, and a second novel signal within the CH1 domain. Interestingly, this site is masked by the linker region between the two CH-domains, suggesting a regulatory mechanism to control parvin localization. Finally, whereas in muscles only ILK controls the stability and localization of both PINCH and parvin, in the wing epithelium the three proteins mutually depend on each other. Thus molecular differences exist in the assembly properties of IPP-complex in specific tissues during development, where differential modulation of the integrin connection to cytoskeleton is required.

Integrin-linked kinase (ILK) is an essential component of a multiprotein complex that links actin to the plasma membrane. Here, we have used a genetic approach to examine the molecular interactions that are essential for the assembly of this ILK-containing complex at Drosophila muscle attachment sites (MASs). We show that, downstream of integrins, talin plays a decisive role in the recruitment of three proteins: ILK, PINCH and paxillin. The accumulation of ILK at MASs appears to follow an amplification mechanism, suggesting that numerous binding sites are generated by minimal levels of the upstream integrin and talin effectors. This property suggests that ILK functions as an essential hub in the assembly of its partner proteins at sites of integrin adhesion. We found that PINCH stability, and its subcellular localization at MASs, depends upon ILK function, but that ILK stability and localization is not dependent upon PINCH. An in vivo structure–function analysis of ILK demonstrated that each ILK domain has sufficient information for its independent recruitment at embryonic MASs, whereas at later developmental stages only the kinase domain was effectively recruited. Our data strengthen the view that the ILK complex is assembled sequentially at sites of integrin adhesion by employing multiple molecular interactions, which collectively stabilize the integrin–actin link.

Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, β5 and β6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In toto, our findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development.

Parvin is a putative F-actin binding protein important for integrin-mediated cell adhesion. Here we used overexpression of Drosophila Parvin to uncover its functions in different tissues in vivo. Parvin overexpression caused major defects reminiscent of metastatic cancer cells in developing epithelia, including apoptosis, alterations in cell shape, basal extrusion and invasion. These defects were closely correlated with abnormalities in the organization of F-actin at the basal epithelial surface and of integrin-matrix adhesion sites. In wing epithelium, overexpressed Parvin triggered increased Rho1 protein levels, predominantly at the basal side, whereas in the developing eye it caused a rough eye phenotype and severely disrupted F-actin filaments at the retina floor of pigment cells. We identified genes that suppressed these Parvin-induced dominant effects, depending on the cell type. Co-expression of both ILK and the apoptosis inhibitor DIAP1 blocked Parvin-induced lethality and apoptosis and partially ameliorated cell delamination in epithelia, but did not rescue the elevated Rho1 levels, the abnormal organization of F-actin in the wing and the assembly of integrin-matrix adhesion sites. The rough eye phenotype was suppressed by coexpression of either PTEN or Wech, or by knock-down of Xrp1. Two main conclusions can be drawn from our studies: (1), high levels of cytoplasmic Parvin are toxic in epithelial cells; (2) Parvin in a dose dependent manner affects the organization of actin cytoskeleton in both wing and eye epithelia, independently of its role as a structural component of the ILK-PINCH-Parvin complex that mediates the integrin-actin link. Thus, distinct genetic interactions of Parvin occur in different cell types and second site modifier screens are required to uncover such genetic circuits.

Recent genetic studies in the worm Caenorhabditis elegans and fruitfly Drosophila have revealed the essential role integrin-linked kinase plays in integrin adhesion — but it apparently acts in this role as an adaptor rather than a kinase.

We report the functional characterization of the Drosophila ortholog of tensin, a protein implicated in linking integrins to the cytoskeleton and signaling pathways. A tensin null was generated and is viable with wing blisters, a phenotype characteristic of loss of integrin adhesion. In tensin mutants, mechanical abrasion is required during wing expansion to cause wing blisters, suggesting that tensin strengthens integrin adhesion. The localization of tensin requires integrins, talin, and integrin-linked kinase. The N-terminal domain and C-terminal PTB domain of tensin provide essential recruitment signals. The intervening SH2 domain is not localized on its own. We suggest a model where tensin is recruited to sites of integrin adhesion via its PTB and N-terminal domains, localizing the SH2 domain so that it can interact with phosphotyrosine-containing proteins, which stabilize the integrin link to the cytoskeleton.

Summary Cytoskeleton-mediated forces regulate the assembly and function of integrin adhesions; however, the underlying mechanisms remain unclear. The tripartite IPP complex, comprising ILK, Parvin, and PINCH, mediates the integrin-actin link at Drosophila embryo muscle attachment sites (MASs). Here, we demonstrate a developmentally earlier function for the IPP complex: to reinforce integrin-extracellular matrix (ECM) adhesion in response to tension. In IPP-complex mutants, the integrin-ECM linkage at MASs breaks in response to intense muscle contractility. Mechanistically, the IPP complex is required to relay force-elicited signals that decelerate integrin turnover at the plasma membrane so that the integrin immobile fraction is adequate to withstand tension. Epistasis analysis shows that alleviation of muscle contractility, downregulation of endocytosis, and enhanced integrin binding to the ECM are sufficient to restore integrin-ECM adhesion and maintain integrin-adhesome organization in IPP-complex mutants. Our findings reveal a role for the IPP complex as an essential mechanosensitive regulatory switch of integrin turnover in vivo.