Effects of benzoylphenylurea on chitin synthesis and orientation in the cuticle of the Drosophila larva

Chitin is an essential constituent of the insect exoskeleton, the cuticle, which is an extracellular matrix (ECM) covering the animal. It is produced by the glycosyltransferase chitin synthase at the apical plasma membrane of epidermal and tracheal cells. To fulfil its role in cuticle elasticity and stiffness it associates with proteins, thereby adopting a stereotypic arrangement of helicoidally stacked sheets, which run parallel to the surface of the animal. One approach to understand the mechanisms of chitin synthesis and organisation is to dissect these processes genetically. However, since only a few genes coding for factors involved in chitin synthesis and organisation have been identified to date using the model arthropod Drosophila melanogaster insight arising from mutant analysis is rather limited. To collect new data on the role of chitin during insect cuticle differentiation, we have analysed the effects of chitin synthesis inhibitors on Drosophila embryogenesis. For this purpose, we have chosen the benzoylphenylurea diflubenzuron and lufenuron that are widely used as insect growth regulators. Our data allow mainly two important conclusions. First, correct organisation of chitin seems to directly depend on the amount of chitin synthesised. Second, chitin synthesis and organisation are cell-autonomous processes as insecticide-treated larvae display a mosaic of cuticle defects. As benzoylphenylurea are used not only as insecticides but also as anti-diabetic drugs, the study of their impact on Drosophila cuticle differentiation may be fruitful for understanding their mode of action on a cellular pathway that is seemingly conserved between vertebrates and invertebrates.     

Hormonal regulation of mummy is needed for apical extracellular matrix formation and epithelial morphogenesis in Drosophila

Many epithelia produce apical extracellular matrices (aECM) that are crucial for organ morphogenesis or physiology. Apical ECM formation relies on coordinated synthesis and modification of constituting components, to enable their subcellular targeting and extracellular assembly into functional matrices. The exoskeleton of Drosophila, the cuticle, is a stratified aECM containing ordered chitin polysaccharide lamellae and proteinaceous layers, and is suited for studies of molecular functions needed for aECM assembly. Here, we show that Drosophila mummy (mmy) mutants display defects in epithelial organisation in conjunction with aberrant deposition of the cuticle and an apical matrix needed for tracheal tubulogenesis. We find that mmy encodes the UDP-N-acetylglucosamine pyrophosphorylase, which catalyses the production of UDP-N-acetylglucosamine, an obligate substrate for chitin synthases as well as for protein glycosylation and GPI-anchor formation. Consequently, in mmy mutants GlcNAc-groups including chitin are severely reduced and modification and subcellular localisation of proteins designated for extracellular space is defective. Moreover, mmy expression is selectively upregulated in epithelia at the time they actively deposit aECM, and is altered by the moulting hormone 20-Hydroxyecdysone, suggesting that mmy is part of a developmental genetic programme to promote aECM formation.     

Obstructor A Organizes Matrix Assembly at the Apical Cell Surface to Promote Enzymatic Cuticle Maturation in Drosophila

Assembly and maturation of the apical extracellular matrix (aECM) is crucial for protecting organisms, but underlying molecular mechanisms remain poorly understood. Epidermal cells secrete proteins and enzymes that assemble at the apical cell surface to provide epithelial integrity and stability during developmental growth and upon tissue damage. We analyzed molecular mechanisms of aECM assembly and identified the conserved chitin-binding protein Obst-A (Obstructor A) as an essential regulator. We show in Drosophila that Obst-A is required to coordinate protein and chitin matrix packaging at the apical cell surface during development. Secreted by epidermal cells, the Obst-A protein is specifically enriched in the apical assembly zone where matrix components are packaged into their highly ordered architecture. In obst-A null mutant larvae, the assembly zone is strongly diminished, resulting in severe disturbance of matrix scaffold organization and impaired aECM integrity. Furthermore, enzymes that support aECM stability are mislocalized. As a biological consequence, cuticle architecture, integrity, and function are disturbed in obst-A mutants, finally resulting in immediate lethality upon wounding. Our studies identify a new core organizing center, the assembly zone that controls aECM assembly at the apical cell surface. We propose a genetically conserved molecular mechanism by which Obst-A forms a matrix scaffold to coordinate trafficking and localization of proteins and enzymes in the newly deposited aECM. This mechanism is essential for maturation and stabilization of the aECM in a growing and remodeling epithelial tissue as an outermost barrier.     

The apical plasma membrane of chitin‐synthesizing epithelia

Abstract Chitin is the second most abundant polysaccharide on earth. It is produced at the apical side of epidermal, tracheal, fore‐, and hindgut epithelial cells in insects as a central component of the protective and supporting extracellular cuticle. Chitin is also an important constituent of the midgut peritrophic matrix that encases the food supporting its digestion and protects the epithelium against invasion by possibly ingested pathogens. The enzyme producing chitin is a glycosyltransferase that resides in the apical plasma membrane forming a pore to extrude the chains of chitin into the extracellular space. The apical plasma membrane is not only a platform for chitin synthases but, probably through its shape and equipment with distinct factors, also plays an important role in orienting and organizing chitin fibers. Here, I review findings on the cellular and molecular constitution of the apical plasma membrane of chitin‐producing epithelia mainly focusing on work done in the fruit fly Drosophila melanogaster.     

Involvement of mind the gap in the organization of the tracheal apical extracellular matrix in Drosophila and Nilaparvata lugens

The tracheal apical extracellular matrix (aECM) is vital for expansion of the tracheal lumen and supports the normal structure of the lumen to guarantee air entry and circulation in insects. Although it has been found that some cuticular proteins are involved in the organization of the aECM, unidentified factors still exist. Here, we found that mind the gap (Mtg), a predicted chitin‐binding protein, is required for the normal formation of the apical chitin matrix of airway tubes in the model holometabolous insect Drosophila melanogaster. Similar to chitin, the Mtg protein was linearly arranged in the tracheal dorsal trunk of the tracheae in Drosophila. Decreased mtg expression in the tracheae seriously affected the viability of larvae and caused tracheal chitin spiral defects in some larvae. Analysis of mtg mutant showed that mtg was required for normal development of tracheae in embryos. Irregular taenidial folds of some mtg mutant embryos were found on either lateral view of tracheal dorsal trunk or internal view of transmission electron microscopy analysis. These abnormal tracheae were not fully filled with gas and accompanied by a reduction in tracheal width, which are characteristic phenotypes of tracheal aECM defects. Furthermore, in the hemimetabolous brown planthopper (BPH) Nilaparvata lugens, downregulation of NlCPAP1‐N (a homolog of mtg) also led to the formation of abnormal tracheal chitin spirals and death. These results suggest that mtg and its homolog are involved in the proper organization of the tracheal aECMs in flies and BPH, and that this function may be conserved in insects.     

A direct interaction between Cdc42 and vesicle-associated membrane protein 2 regulates SNARE-dependent insulin exocytosis

In pancreatic beta cells, insulin granule exocytosis is regulated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein (SNAP) receptor) proteins, and this is coupled to cortical F-actin reorganization via the Rho family GTPase Cdc42 by an unknown mechanism. We investigated interactions among the target SNARE protein Syntaxin 1A and the vesicle-associated membrane SNARE protein (VAMP2) with Cdc42 and compared these structural interactions with their functional importance to glucose-stimulated insulin secretion in MIN6 beta cells. Subcellular fractionation analyses revealed a parallel redistribution of Cdc42 and VAMP2 from the granule fraction to the plasma membrane in response to glucose that temporally corresponded with the glucose-induced activation of Cdc42. Moreover, within these fractions Cdc42 and VAMP2 were found to co-immunoprecipitate under basal and glucose-stimulated conditions, suggesting that they moved as a complex. Furthermore, VAMP2 bound both GST-Cdc42-GTPgammaS and GST-Cdc42-GDP, indicating that the Cdc42-VAMP2 complex could form under both cytosolic GDP-bound Cdc42 and plasma membrane GTP-bound Cdc42 conformational conditions. In vitro binding analyses showed that VAMP2 bound directly to Cdc42 and that a heterotrimeric complex with Syntaxin 1A could also be formed. Deletion analyses of VAMP2 revealed that only the N-terminal 28 residues were required for Cdc42 binding. Expression of this 28-residue VAMP2 peptide in MIN6 beta cells resulted in the specific impairment of glucose-stimulated insulin secretion, indicating a functional importance for the Cdc42-VAMP2 interaction. Taken together, these data suggest a mechanism whereby glucose activates Cdc42 to induce the targeting of intracellular Cdc42-VAMP2-insulin granule complexes to Syntaxin 1A at the plasma membrane.     

Involvement of chitin in exoskeleton morphogenesis in Drosophila melanogaster

Exoskeletons stabilize cell, tissue, and body morphology in many living organisms including fungi, plants, and arthropods. In insects, the exoskeleton, the cuticle, is produced by epidermal cells as a protein extracellular matrix containing lipids and the polysaccharide chitin, and its formation requires coordinated synthesis, distribution, and modification of these components. Eventually, the stepwise secretion and sorting of the cuticle material results in a layered structure comprising the envelope, the proteinaceous epicuticle, and the chitinous procuticle. To study the role of chitin during cuticle development, we analyzed the consequences of chitin absence in the embryo of Drosophila melanogaster caused by mutations in the Chitin Synthase-1 (CS-1) gene, called krotzkopf verkehrt (kkv). Our histological data confirm that chitin is essential for procuticle integrity and further demonstrate that an intact procuticle is important to assemble and to stabilize the chitin-less epicuticle. Moreover, the phenotype of CS-1/kkv mutant embryos indicates that chitin is required to attach the cuticle to the epidermal cells, thereby maintaining epidermal morphology. Finally, sclerotization and pigmentation, which are the last steps in cuticle differentiation, are impaired in tissues lacking CS-1/kkv function, suggesting that proper cuticle structure is crucial for the activity of the underlying enzymes.     

serpentine and vermiform encode matrix proteins with chitin binding and deacetylation domains that limit tracheal tube length in Drosophila

Many organs contain epithelial tubes that transport gases or liquids . Proper tube size and shape is crucial for organ function, but the mechanisms controlling tube diameter and length are poorly understood. Recent studies of tracheal (respiratory) tube morphogenesis in Drosophila show that chitin synthesis genes produce an expanding chitin cylinder in the apical (luminal) extracellular matrix (ECM) that coordinates the dilation of the surrounding epithelium . Here, we describe two genes involved in chitin modification, serpentine (serp) and vermiform (verm), mutations in which cause excessively long and tortuous tracheal tubes. The genes encode similar proteins with an LDL-receptor ligand binding motif and chitin binding and deacetylation domains. Both proteins are expressed and secreted during tube expansion and localize throughout the lumen in a chitin-dependent manner. Unlike previously characterized chitin pathway genes, serp and verm are not required for chitin synthesis or secretion but rather for its normal fibrillar structure. The mutations also affect structural properties of another chitinous matrix, epidermal cuticle. Our work demonstrates that chitin and the matrix proteins Serp and Verm limit tube elongation, and it suggests that tube length is controlled independently of diameter by modulating physical properties of the chitin ECM, presumably by N-deacetylation of chitin and conversion to chitosan.     

Drosophila Knickkopf and Retroactive are needed for epithelial tube growth and cuticle differentiation through their specific requirement for chitin filament organization

Precise epithelial tube diameters rely on coordinated cell shape changes and apical membrane enlargement during tube growth. Uniform tube expansion in the developing Drosophila trachea requires the assembly of a transient intraluminal chitin matrix, where chitin forms a broad cable that expands in accordance with lumen diameter growth. Like the chitinous procuticle, the tracheal luminal chitin cable displays a filamentous structure that presumably is important for matrix function. Here, we show that knickkopf (knk) and retroactive (rtv) are two new tube expansion mutants that fail to form filamentous chitin structures, both in the tracheal and cuticular chitin matrices. Mutations in knk and rtv are known to disrupt the embryonic cuticle, and our combined genetic analysis and chemical chitin inhibition experiments support the argument that Knk and Rtv specifically assist in chitin function. We show that Knk is an apical GPI-linked protein that acts at the plasma membrane. Subcellular mislocalization of Knk in previously identified tube expansion mutants that disrupt septate junction (SJ) proteins, further suggest that SJs promote chitinous matrix organization and uniform tube expansion by supporting polarized epithelial protein localization. We propose a model in which Knk and the predicted chitin-binding protein Rtv form membrane complexes essential for epithelial tubulogenesis and cuticle formation through their specific role in directing chitin filament assembly.     

Cuticle differentiation in the embryo of the amphipod crustacean Parhyale hawaiensis

The arthropod cuticle is a multilayered extracellular matrix produced by the epidermis during embryogenesis and moulting. Molecularly and histologically, cuticle differentiation has been extensively investigated in the embryo of the insect Drosophila melanogaster. To learn about the evolution of cuticle differentiation, we have studied the histology of cuticle differentiation during embryogenesis of the amphipod crustacean Parhyale hawaiensis, which had a common ancestor with Drosophila about 510 million years ago. The establishment of the layers of the Parhyale juvenile cuticle is largely governed by mechanisms observed in Drosophila, e.g. as in Drosophila, the synthesis and arrangement of chitin in the inner procuticle are separate processes. A major difference between the cuticle of Parhyale and Drosophila concerns the restructuring of the Parhyale dorsal epicuticle after deposition. In contrast to the uniform cuticle of the Drosophila larva, the Parhyale cuticle is subdivided into two regions, the ventral and the dorsal cuticles. Remarkably, the boundary between the ventral and dorsal cuticles is sharp suggesting active extracellular regionalisation. The present analysis of Parhyale cuticle differentiation should allow the characterisation of the cuticle-producing and -organising factors of Parhyale (by comparison with the branchiopod crustacean Daphnia pulex) in order to contribute to the elucidation of fundamental questions relevant to extracellular matrix organisation and differentiation. This work was supported by the German Research Foundation (DFG, grant number MO 1714/1-1).     

δ-Aminolevulinate synthase is required for apical transcellular barrier formation in the skin of the Drosophila larva

Animals construct a layered skin to prevent dehydration and pathogen entrance. The barrier function of the skin relies on the extensive cross-linking of specialised components. In insects, for instance, epidermal cells produce an apical extracellular cuticle that consists of a network of proteins, chitin and lipids. We have identified mutations in the Drosophila gene coding for the δ-aminolevulinate synthase (Alas) that cause massive water loss. The cuticle of alas mutant larvae detaches from the epidermis and its basal region is frayed suggesting that an Alas dependent pathway is needed to organise the contact between the cuticle and the epidermis and anchor the cuticle to the apical surface of epidermal cells. Concomitantly, reduction of Alas function results in weakening of the extracellular dityrosines network in the cuticle, whereas glutamyl-lysine isopeptide bonds are not affected. The lateral septate junctions of epidermal cells that serve as a paracellular plug are intact, as well. Taken together, we hypothesise that Alas activity, which initiates heme biosynthesis in the mitochondrion, is needed for the formation of a dityrosine-based barrier that confers resistance to the internal hydrostatic pressure protecting both the cuticle from transcellular infiltration of body fluid and the animal from dehydration. We conclude that at least two modules--an apical protein-chitin lattice and the lateral septate junctions, act in parallel to ensure Drosophila skin impermeability.     

The apical plasma membrane of Drosophila embryonic epithelia

The apical plasma membrane of epithelia presents the interface between organs and the external environment. It has biochemical activities distinct from those of the basal and lateral plasma membranes, as it accommodates the production and assembly of ordered apical matrices involved in organ protection and physiology and determines the microenvironment in the apical extracellular milieu. Here, we emphasise the importance of the apical plasma membrane in tissue differentiation, by mainly focussing on the embryo of the fruit fly Drosophila melanogaster, and discuss the principal organisation of the apical plasma membrane into repetitive subdomains of specific topologies and activities essential for epithelial function.     

Cuticle differentiation during Drosophila embryogenesis

The constitutive criterion for the evolutionary successful clade of ecdysozoans is a protective exoskeleton. In insects the exoskeleton, the so-called cuticle consists of three functional layers, the waterproof envelope, the proteinaceous epicuticle and the chitinous procuticle that are produced as an extracellular matrix by the underlying epidermal cells. Here, we present our electron-microscopic study of cuticle differentiation during embryogenesis in the fruit fly Drosophila melanogaster. We conclude that cuticle differentiation in the Drosophila embryo occurs in three phases. In the first phase, the layers are established. Interestingly, we find that establishment of the layers occurs partially simultaneously rather than in a strict sequential manner as previously proposed. In the second phase the cuticle thickens. Finally, in the third phase, when secretion of cuticle material has ceased, the chitin laminae acquire their typical orientation, and the epicuticle of the denticles and the head skeleton darken. Our work will help to understand the phenotypes of embryos mutant for genes encoding essential cuticle factors, in turn revealing mechanisms of cuticle differentiation.     

Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells

Targeted delivery of proteins to distinct plasma membrane domains is critical to the development and maintenance of polarity in epithelial cells. We used confocal and time-lapse total internal reflection fluorescence microscopy (TIR-FM) to study changes in localization and exocytic sites of post-Golgi transport intermediates (PGTIs) carrying GFP-tagged apical or basolateral membrane proteins during epithelial polarization. In non-polarized Madin Darby Canine Kidney (MDCK) cells, apical and basolateral PGTIs were present throughout the cytoplasm and were observed to fuse with the basal domain of the plasma membrane. During polarization, apical and basolateral PGTIs were restricted to different regions of the cytoplasm and their fusion with the basal membrane was completely abrogated. Quantitative analysis suggested that basolateral, but not apical, PGTIs fused with the lateral membrane in polarized cells, correlating with the restricted localization of Syntaxins 4 and 3 to lateral and apical membrane domains, respectively. Microtubule disruption induced Syntaxin 3 depolarization and fusion of apical PGTIs with the basal membrane, but affected neither the lateral localization of Syntaxin 4 or Sec6, nor promoted fusion of basolateral PGTIs with the basal membrane.     

Doc2beta is a novel Munc18c-interacting partner and positive effector of syntaxin 4-mediated exocytosis

The widely expressed Sec/Munc18 (SM) protein Munc18c is required for SNARE-mediated insulin granule exocytosis from islet beta cells and GLUT4 vesicle exocytosis in skeletal muscle and adipocytes. Although Munc18c function is known to involve binding to the t-SNARE Syntaxin 4, a paucity of Munc18c-binding proteins has restricted elucidation of the mechanism by which it facilitates these exocytosis events. Toward this end, we have identified the double C2 domain protein Doc2beta as a new binding partner for Munc18c. Unlike its granule/vesicle localization in neuronal cells, Doc2beta was found principally in the plasma membrane compartment in islet beta cells and adipocytes. Moreover, co-immunoprecipitation and GST interaction assays showed Doc2beta-Munc18c binding to be direct and complexes to be devoid of Syntaxin 4. Supporting the notion of Munc18c binding with Syntaxin 4 and Doc2beta in mutually exclusive complexes, in vitro competition with Syntaxin 4 effectively displaced Munc18c from binding to Doc2beta. The second C2 domain (C2B) of Doc2beta and an N-terminal region of Munc18c were sufficient to confer complex formation. Disruption of endogenous Munc18c-Doc2beta complexes by addition of the Doc2beta binding domain of Munc18c (residues 173-255) was found to selectively inhibit glucose-stimulated insulin release. Moreover, increased expression of Doc2beta enhanced glucose-stimulated insulin secretion by approximately 40%, whereas siRNA-mediated depletion of Doc2beta attenuated insulin release. All changes in secretion correlated with parallel alterations in VAMP2 granule docking with Syntaxin 4. Taken together, these data support a model wherein Munc18c transiently switches from association with Syntaxin 4 to association with Doc2beta at the plasma membrane to facilitate exocytosis.     

Drosophila Chitinase 2 is expressed in chitin producing organs for cuticle formation

The architecture of the outer body wall cuticle is fundamental to protect arthropods against invading pathogens and numerous other harmful stresses. Such robust cuticles are formed by parallel running chitin microfibrils. Molting and also local wounding leads to dynamic assembly and disassembly of the chitin-matrix throughout development. However, the underlying molecular mechanisms that organize proper chitin-matrix formation are poorly known. Recently we identified a key region for cuticle thickening at the apical cell surface, the cuticle assembly zone, where Obstructor-A (Obst-A) coordinates the formation of the chitin-matrix. Obst-A binds chitin and the deacetylase Serpentine (Serp) in a core complex, which is required for chitin-matrix maturation and preservation. Here we present evidence that Chitinase 2 (Cht2) could be essential for this molecular machinery. We show that Cht2 is expressed in the chitin-matrix of epidermis, trachea, and the digestive system. There, Cht2 is enriched at the apical cell surface and the dense chitin-matrix. We further show that in Cht2 knockdown larvae the assembly zone is rudimentary, preventing normal cuticle formation and pore canal organization. As sequence similarities of Cht2 and the core complex proteins indicate evolutionarily conserved molecular mechanisms, our findings suggest that Cht2 is involved in chitin formation also in other insects.     

Comparative cell response to artificial extracellular matrix proteins containing the RGD and CS5 cell-binding domains

This study addresses endothelial cell adhesion and spreading on a family of artificial extracellular matrix (aECM) proteins designed for application in small-diameter vascular grafts. The aECM proteins contain domains derived from elastin and from fibronectin. aECM 1 contains the RGD sequence from the tenth type III domain of fibronectin; aECM 3 contains the fibronectin CS5 cell-binding domain. Negative control proteins aECM 2 and 4 are scrambled versions of aECM 1 and 3, respectively. Competitive peptide inhibition studies and comparisons of positive and negative control proteins confirm that adhesion of HUVECs to aECM proteins 1 and 3 is sequence specific. When subjected to a normal detachment force of 780 pN, 3-fold more HUVECs remained adherent to aECM 1 than to aECM 3. HUVECs also spread more rapidly on aECM 1 than on aECM 3. These results (i) indicate that cellular responses to aECM proteins can be modulated through choice of cell-binding domain and (ii) recommend the RGD sequence for applications that require rapid endothelial cell spreading and matrix adhesion.     

Zona Pellucida Domain Proteins Remodel the Apical Compartment for Localized Cell Shape Changes

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Highlights? In Drosophila epidermis, shavenbaby controls ZPD gene expression ? Apical cell shape remodeling relies on the coordinated action of ZPD genes ? ZPD proteins define sub-regions in the apical extracellular matrix (aECM) ? ZPD products maintain localized apical membrane/aECM interactions.     

Obstructor-A is required for epithelial extracellular matrix dynamics, exoskeleton function, and tubulogenesis

The epidermis and internal tubular organs, such as gut and lungs, are exposed to a hostile environment. They form an extracellular matrix to provide epithelial integrity and to prevent contact with pathogens and toxins. In arthropods, the cuticle protects, shapes, and enables the functioning of organs. During development, cuticle matrix is shielded from premature degradation; however, underlying molecular mechanisms are poorly understood. Previously, we identified the conserved obstructor multigene-family, which encodes chitin-binding proteins. Here we show that Obstructor-A is required for extracellular matrix dynamics in cuticle forming organs. Loss of obstructor-A causes severe defects during cuticle molting, wound protection, tube expansion and larval growth control. We found that Obstructor-A interacts and forms a core complex with the polysaccharide chitin, the cuticle modifier Knickkopf and the chitin deacetylase Serpentine. Knickkopf protects chitin from chitinase-dependent degradation and deacetylase enzymes ensure extracellular matrix maturation. We provide evidence that Obstructor-A is required to control the presence of Knickkopf and Serpentine in the extracellular matrix. We propose a model suggesting that Obstructor-A coordinates the core complex for extracellular matrix protection from premature degradation. This mechanism enables exoskeletal molting, tube expansion, and epithelial integrity. The evolutionary conservation suggests a common role of Obstructor-A and homologs in coordinating extracellular matrix protection in epithelial tissues of chitinous invertebrates.