cuticleDB: a relational database of Arthropod cuticular proteins

Background  

The insect exoskeleton or cuticle is a bi-partite composite of proteins and chitin that provides protective, skeletal and structural functions. Little information is available about the molecular structure of this important complex that exhibits a helicoidal architecture. Scores of sequences of cuticular proteins have been obtained from direct protein sequencing, from cDNAs, and from genomic analyses.     

The cuticle of the nematode Caenorhabditis elegans: A complex collagen structure

The cuticle of the nematode Caenorhabditis elegans forms the barrier between the animal and its environment. In addition to being a protective layer, it is an exoskeleton which is important in maintaining and defining the normal shape of the nematode. The cuticle is an extracellular matrix consisting predominantly of small collagen-like proteins that are extensively crosslinked. Although it also contains other protein and non-protein compounds that undoubtedly play a significant part in its function, the specific role of collagen in cuticle structure and morphology is considered here. The C. elegans genome contains between 50 and 150 collagen genes, most of which are believed to encode cuticular collagens. Mutations that result in cuticular defects and grossly altered body form have been identified in more than 40 genes. Six of these genes are now known to encode cuticular collagens, a finding that confirms the importance of this group of structural proteins to the formation of the cuticle and the role of the cuticle as an exoskeleton in shaping the worm. It is likely that many more of the genes identified by mutations giving altered body form, will be collagen genes. Mutations in the cuticular collagen genes provide a powerful tool for investigating the mechanisms by which this group of proteins interact to form the nematode cuticle.     

Fine structure of the <Emphasis Type="Italic">Arabidopsis</Emphasis> stem cuticle: effects of fixation and changes over development

Main conclusion

The Arabidopsis cuticle, as observed by electron microscopy, consists primarily of the cutin/cutan matrix. The cuticle possesses a complex substructure, which is correlated with the presence of intracuticular waxes. The plant cuticle is composed of an insoluble polyester, cutin, and organic solvent soluble cuticular waxes, which are embedded within and coat the surface of the cutin matrix. How these components are arranged in the cuticle is not well understood. The Arabidopsis cuticle is commonly understood as ‘amorphous,’ lacking in ultrastructural features, and is often observed as a thin (~80–100 nm) electron-dense layer on the surface of the cell wall. To examine this cuticle in more detail, we examined cuticles from both rapidly elongating and mature sections of the stem and compared the preservation of the cuticles using conventional chemical fixation methods and high-pressure freezing/freeze-substitution (HPF/FS). We found that HPF/FS preparation revealed a complex cuticle substructure, which was more evident in older stems. We also found that the cuticle increases in thickness with development, indicating an accretion of polymeric material, likely in the form of the non-hydrolyzable polymer, cutan. When wax was extracted by chloroform immersion prior to sample preparation, the contribution of waxes to cuticle morphology was revealed. Overall, the electron-dense cuticle layer was still visible but there was loss of the cuticle substructure. Furthermore, the cuticle of cer6, a wax-deficient mutant, also lacked this substructure, suggesting that these fine striations were dependent on the presence of cuticular waxes. Our findings show that HPF/FS preparation can better preserve plant cuticles, but also provide new insights into the fine structure of the Arabidopsis cuticle.

     

Exoskeleton anchoring to tendon cells and muscles in molting isopod crustaceans

Specialized mechanical connection between exoskeleton and underlying muscles in arthropods is a complex network of interconnected matrix constituents, junctions and associated cytoskeletal elements, which provides prominent mechanical attachment of the epidermis to the cuticle and transmits muscle tensions to the exoskeleton. This linkage involves anchoring of the complex extracellular matrix composing the cuticle to the apical membrane of tendon cells and linking of tendon cells to muscles basally. The ultrastructural arhitecture of these attachment complexes during molting is an important issue in relation to integument integrity maintenance in the course of cuticle replacement and in relation to movement ability. The aim of this work was to determine the ultrastructural organization of exoskeleton - muscles attachment complexes in the molting terrestrial isopod crustaceans, in the stage when integumental epithelium is covered by both, the newly forming cuticle and the old detached cuticle. We show that the old exoskeleton is extensively mechanically connected to the underlying epithelium in the regions of muscle attachment sites by massive arrays of fibers in adult premolt Ligia italica and in prehatching embryos and premolt marsupial mancas of Porcellio scaber. Fibers expand from the tendon cells, traverse the new cuticle and ecdysal space and protrude into the distal layers of the detached cuticle. They likely serve as final anchoring sites before exuviation and may be involved in animal movements in this stage. Tendon cells in the prehatching embryo and in marsupial mancas display a substantial apicobasally oriented transcellular arrays of microtubules, evidently engaged in myotendinous junctions and in apical anchoring of the cuticular matrix. The structural framework of musculoskeletal linkage is basically established in described intramarsupial developmental stages, suggesting its involvement in animal motility within the marsupium.     

The essential role of bursicon during <Emphasis Type="Italic">Drosophila</Emphasis>development

Background  

The protective external cuticle of insects does not accommodate growth during development. To compensate for this, the insect life cycle is punctuated by a series of molts. During the molt, a new and larger cuticle is produced underneath the old cuticle. Replacement of the smaller, old cuticle culminates with ecdysis, a stereotyped sequence of shedding behaviors. Following each ecdysis, the new cuticle must expand and harden. Studies from a variety of insect species indicate that this cuticle hardening is regulated by the neuropeptide bursicon. However, genetic evidence from Drosophila melanogaster only supports such a role for bursicon after the final ecdysis, when the adult fly emerges. The research presented here investigates the role that bursicon has at stages of Drosophila development which precede adult ecdysis.     

Effects of exogenous N-acetylhexosaminidase on the structure and mineralization of the post-ecdysial exoskeleton of the blue crab, Callinectes sapidus

A cuticular glycosidase with characteristics of N-acetyl-β- -hexosaminidase (HexNAcase) was identified in post-ecdysial crab cuticle. Its appearance coincided with changes in cuticular glycoproteins and the onset of mineralization. To test if HexNAcase might be the causative agent in the alteration of the glycans and initiation of calcification, newly molted crab cuticle was treated with exogenous HexNAcase. Treating cuticular extracts from crabs at 0 h post-ecdysis with exogenous HexNAcase mimicked those changes observed in vivo. Specifically, the enzyme decreased the concanavalin A affinity of an 83-kDa glycoprotein that binds to calcite crystals in vitro. Treating pieces of 0 h post-ecdysial cuticle with HexNAcase rendered them capable of nucleating calcite in vitro (similar to 5 h post-ecdysial cuticle), while untreated, 0 h controls remained uncalcified. The data imply a role of the cuticular HexNAcase-like enzyme in the initiation of calcite nucleation in the newly formed exoskeleton.     

Purification and Structural Determination of a Phosphorylated Peptide with Anti-calcification and Chitin-binding Activities in the Exoskeleton of the Crayfish,Procambarus clarkii

Organic matrices in calcified hard tissues have been considered to control calcification. A matrix peptide, designated CAP-1, was extracted and purified by anionexchange and reverse-phase high performance liquid chromatographies from the exoskeleton of the crayfish, Procambarus clarkii. The amino acid sequence of CAP-1 was determined by mass spectral and sequence analyses of the intact peptide and its enzymatically digested peptides. CAP-1 consisted of 78 amino acid residues, including a phosphoserine residue, and was rich in acidic amino acid residues. CAP-1 had a RebersRiddiford consensus sequence, which is conserved in cuticle proteins from many arthropods. CAP-1 inhibited precipitation of calcium carbonate in an in vitro anticalcification assay dose-dependently, and completelyinhibited it at 3×10^-7 M. CAP-1 also showed chitinbinding ability, indicating that this molecule was bifunctional and played an important role in formation of the exoskeleton.     

The Caenorhabditis elegans Patched domain protein PTR-4 is required for proper organization of the precuticular apical extracellular matrix

The Patched-related superfamily of transmembrane proteins can transport lipids or other hydrophobic molecules across cell membranes. While the Hedgehog receptor Patched has been intensively studied, much less is known about the biological roles of other Patched-related family members. Caenorhabditis elegans has a large number of Patched-related proteins, despite lacking a canonical Hedgehog pathway. Here, we show that PTR-4 promotes the assembly of the precuticle apical extracellular matrix, a transient and molecularly distinct matrix that precedes and patterns the later collagenous cuticle or exoskeleton. ptr-4 mutants share many phenotypes with precuticle mutants, including defects in eggshell dissolution, tube shaping, alae (cuticle ridge) structure, molting, and cuticle barrier function. PTR-4 localizes to the apical side of a subset of outward-facing epithelia, in a cyclical manner that peaks when precuticle matrix is present. Finally, PTR-4 is required to limit the accumulation of the lipocalin LPR-3 and to properly localize the Zona Pellucida domain protein LET-653 within the precuticle. We propose that PTR-4 transports lipids or other hydrophobic components that help to organize the precuticle and that the cuticle and molting defects seen in ptr-4 mutants result at least in part from earlier disorganization of the precuticle.     

Mechanical properties of calcified exoskeleton from the neotropical millipede, Nyssodesmus python

The calcified exoskeleton of millipedes plays a crucial role in resisting large forces developed during burrowing locomotion. I measured morphological and mechanical properties of cuticle from the neotropical forest floor millipede, Nyssodesmus python (Diplopoda: Polydesmidae), which ranges in body mass from 2 to 7 g. Scaling of thickness of the cuticle with respect to body mass followed predictions of geometric similarity. Both fracture strength and Young's modulus increased with body mass in females but not in males. In spite of their smaller size, male millipedes were still stronger, on average, than female millipedes. Mean fracture strength of millipede cuticle was 124 MPa, and Young's modulus was 17 GPa. Both of these values exceed measurements from typical insect cuticle, suggesting that calcium salts may play a role in stiffening and strengthening the millipede exoskeleton. Because of the high density of calcified millipede cuticle (1660 kg/m³), stiffness and strength relative to body weight remain comparable to values for other insect cuticles. These results corroborate a previous hypothesis that absolute not specific strength and stiffness have been selective factors in the evolution of millipede cuticle, and that bulkiness of the exoskeleton has been minimized through the deposition of calcium salts.     

Formation of the hindgut cuticular lining during embryonic development of Porcellio scaber (Crustacea,Isopoda)

The hindgut and foregut in terrestrial isopod crustaceans are ectodermal parts of the digestive system and are lined by cuticle, an apical extracellular matrix secreted by epithelial cells. Morphogenesis of the digestive system was reported in previous studies, but differentiation of the gut cuticle was not followed in detail. This study is focused on ultrastructural analyses of hindgut apical matrices and cuticle in selected intramarsupial developmental stages of the terrestrial isopod Porcellio scaber in comparison to adult animals to obtain data on the hindgut cuticular lining differentiation. Our results show that in late embryos of stages 16 and 18 the apical matrix in the hindgut consists of loose material overlaid by a thin intensely ruffled electron dense lamina facing the lumen. The ultrastructural resemblance to the embryonic epidermal matrices described in several arthropods suggests a common principle in chitinous matrix differentiation. The hindgut matrix in the prehatching embryo of stage 19 shows characteristics of the hindgut cuticle, specifically alignment to the apical epithelial surface and a prominent electron dense layer of epicuticle. In the preceding embryonic stage – stage 18 – an electron dense lamina, closely apposed to the apical cell membrane, is evident and is considered as the first epicuticle formation. In marsupial mancae the advanced features of the hindgut cuticle and epithelium are evident: a more prominent epicuticular layer, formation of cuticular spines and an extensive apical labyrinth. In comparison to the hindgut cuticle of adults, the hindgut cuticle of marsupial manca and in particular the electron dense epicuticular layer are much thinner and the difference between cuticle architecture in the anterior chamber and in the papillate region is not yet distinguishable. Differences from the hindgut cuticle in adults imply not fully developed structure and function of the hindgut cuticle in marsupial manca, possibly related also to different environments, as mancae develop in marsupial fluid. Bacteria, evenly distributed within the homogenous electron dense material in the hindgut lumen, were observed only in one specimen of early marsupial manca. The morphological features of gut cuticle renewal are evident in the late marsupial mancae, and are similar to those observed in the exoskeleton.     

Damage,repair and regeneration in insect cuticle: The story so far,and possibilities for the future

The exoskeleton of an insect can contain countless specializations across an individual, across developmental stages, and across the class Insecta. Hence, the exoskeleton's building material cuticle must perform a vast variety of functions. Cuticle displays a wide range of material properties which are determined by several known factors: the amount and orientation of the chitin fibres, the constituents and degree of cross-linking and hydration of the protein matrix, the relative amounts of exo- and endocuticle, and the shape of the structures themselves. In comparison to other natural materials such as wood and mammal bone, relatively few investigations into the mechanical properties of insect cuticle have been carried out. Of these, very few have focussed on the need for repair and its effectiveness at restoring mechanical stability to the cuticle. Insect body parts are often subject to prolonged repeated cyclic loads when running and flying, as well as more extreme “emergency” behaviours necessary for survival such as jumping, wedging (squeezing through small holes) and righting (when overturned). What effects have these actions on the cuticle itself? How close to the limits of failure does an insect push its body parts? Can an insect recover from minor or major damage to its exoskeleton “bones”? No current research has answered these questions conclusively.     

Surface EMG pattern recognition for real-time control of a wrist exoskeleton

Background  

Surface electromyography (sEMG) signals have been used in numerous studies for the classification of hand gestures and movements and successfully implemented in the position control of different prosthetic hands for amputees. sEMG could also potentially be used for controlling wearable devices which could assist persons with reduced muscle mass, such as those suffering from sarcopenia. While using sEMG for position control, estimation of the intended torque of the user could also provide sufficient information for an effective force control of the hand prosthesis or assistive device. This paper presents the use of pattern recognition to estimate the torque applied by a human wrist and its real-time implementation to control a novel two degree of freedom wrist exoskeleton prototype (WEP), which was specifically developed for this work.     

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.     

A Simple Exoskeleton That Assists Plantarflexion Can Reduce the Metabolic Cost of Human Walking

Background

Even though walking can be sustained for great distances, considerable energy is required for plantarflexion around the instant of opposite leg heel contact. Different groups attempted to reduce metabolic cost with exoskeletons but none could achieve a reduction beyond the level of walking without exoskeleton, possibly because there is no consensus on the optimal actuation timing. The main research question of our study was whether it is possible to obtain a higher reduction in metabolic cost by tuning the actuation timing.

Methodology/Principal Findings

We measured metabolic cost by means of respiratory gas analysis. Test subjects walked with a simple pneumatic exoskeleton that assists plantarflexion with different actuation timings. We found that the exoskeleton can reduce metabolic cost by 0.18±0.06 W kg⁻¹ or 6±2% (standard error of the mean) (p = 0.019) below the cost of walking without exoskeleton if actuation starts just before opposite leg heel contact.

Conclusions/Significance

The optimum timing that we found concurs with the prediction from a mathematical model of walking. While the present exoskeleton was not ambulant, measurements of joint kinetics reveal that the required power could be recycled from knee extension deceleration work that occurs naturally during walking. This demonstrates that it is theoretically possible to build future ambulant exoskeletons that reduce metabolic cost, without power supply restrictions.     

Regulation of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> body size and male tail development by the novel gene <Emphasis Type="Italic">lon-8</Emphasis>

Background  

In C. elegans and other nematode species, body size is determined by the composition of the extracellular cuticle as well as by the nuclear DNA content of the underlying hypodermis. Mutants that are defective in these processes can exhibit either a short or a long body size phenotype. Several mutations that give a long body size (Lon) phenotype have been characterized and found to be regulated by the DBL-1/TGF-β pathway, that controls post-embryonic growth and male tail development.     

Surface structure,model and mechanism of an insect integument adapted to be damaged easily

Background  

Several sawfly larvae of the Tenthredinidae (Hymenoptera) are called easy bleeders because their whole body integument, except the head capsule, disrupts very easily at a given spot, under a slight mechanical stress at this spot. The exuding haemolymph droplet acts as a feeding deterrent towards invertebrate predators. The present study aimed to describe the cuticle surface, to consider it from a mechanistic point of view, and to discuss potential consequences of the integument surface in the predator-prey relationships.     

The organic preservation of fossil arthropods: an experimental study

Modern arthropod cuticles consist of chitin fibres in a protein matrix, but those of fossil arthropods with an organic exoskeleton, particularly older than Tertiary, contain a dominant aliphatic component. This apparent contradiction was examined by subjecting modern cockroach, scorpion and shrimp cuticle to artificial maturation (350 degrees C/700 bars/24 h) following various chemical treatments, and analysing the products with pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). Analysis of artificially matured untreated cuticle yielded moieties related to phenols and alkylated substituents, pyridines, pyrroles and possibly indenes (derived from chitin). n-Alkyl amides, C16 and C18 fatty acids and alkane/alk-1-ene homologues ranging from C9 to C19 were also generated, the last indicating the presence of an n-alkyl component, similar in composition to that encountered in fossil arthropods. Similar pyrolysates were obtained from matured pure C16 and C18 fatty acids. Py-GC/MS of cuticles matured after lipid extraction and hydrolysis did not yield any aliphatic polymer. This provides direct experimental evidence that lipids incorporated from the cuticle were the source of aliphatic polymer. This process of in situ polymerization appears to account for most of the fossil record of terrestrial arthropods as well as marine arthropods that lacked a biomineralized exoskeleton.     

Exoskeletal cuticle differentiation during intramarsupial development of Porcellio scaber (Crustacea: Isopoda)

Exoskeletal crustacean cuticle is a calcified apical extracellular matrix of epidermal cells, illustrating the chitin-based organic scaffold for biomineralization. Studies of cuticle formation during molting reveal significant dynamics and complexity of the assembly processes, while cuticle formation during embryogenesis is poorly investigated. This study reveals in the terrestrial isopod Porcellio scaber, the ultrastructural organization of the differentiating precuticular matrices and exoskeletal cuticles during embryonic and larval intramarsupial development. The composition of the epidermal matrices was obtained by WGA lectin labelling and EDXS analysis. At least two precuticular matrices, consisting of loosely arranged material with overlying electron dense lamina, are secreted by the epidermis in the mid-stage embryo. The prehatching embryo is the earliest developmental stage with a cuticular matrix consisting of an epicuticle and a procuticle, displaying WGA binding and forming cuticular scales. In newly hatched marsupial larva manca, a new cuticle is formed and calcium sequestration in the cuticle is evident. Progression of larval development leads to the cuticle thickening, structural differentiation of cuticular layers and prominent cuticle calcification. Morphological characteristics of exoskeleton renewal in marsupial manca are described. Elaborated cuticle in marsupial larvae indicates the importance of the exoskeleton in protection and support of the larval body in the marsupium and during the release of larvae in the external environment.     

The Distribution of GYR- and YLP-Like Motifs in Drosophila Suggests a General Role in Cuticle Assembly and Other Protein-Protein Interactions

Background

Arthropod cuticle is composed predominantly of a self-assembling matrix of chitin and protein. Genes encoding structural cuticular proteins are remarkably abundant in arthropod genomes, yet there has been no systematic survey of conserved motifs across cuticular protein families.

Methodology/Principal Findings

Two short sequence motifs with conserved tyrosines were identified in Drosophila cuticular proteins that were similar to the GYR and YLP Interpro domains. These motifs were found in members of the CPR, Tweedle, CPF/CPFL, and (in Anopheles gambiae) CPLCG cuticular protein families, and the Dusky/Miniature family of cuticle-associated proteins. Tweedle proteins have a characteristic motif architecture that is shared with the Drosophila protein GCR1 and its orthologs in other species, suggesting that GCR1 is also cuticular. A resilin repeat, which has been shown to confer elasticity, matched one of the motifs; a number of other Drosophila proteins of unknown function exhibit a motif architecture similar to that of resilin. The motifs were also present in some proteins of the peritrophic matrix and the eggshell, suggesting molecular convergence among distinct extracellular matrices. More surprisingly, gene regulation, development, and proteolysis were statistically over-represented ontology terms for all non-cuticular matches in Drosophila. Searches against other arthropod genomes indicate that the motifs are taxonomically widespread.

Conclusions

This survey suggests a more general definition for GYR and YLP motifs and reveals their contribution to several types of extracellular matrix. They may define sites of protein interaction with DNA or other proteins, based on ontology analysis. These results can help guide experimental studies on the biochemistry of cuticle assembly.     

Consistency of the Neighbor-Net Algorithm

Background  

Neighbor-Net is a novel method for phylogenetic analysis that is currently being widely used in areas such as virology, bacteriology, and plant evolution. Given an input distance matrix, Neighbor-Net produces a phylogenetic network, a generalization of an evolutionary or phylogenetic tree which allows the graphical representation of conflicting phylogenetic signals.