Procuticle proteins and chitin-like material in the inner epicuticle of the Drosophila pupal cuticle

The inner (protein) epicuticle of the pupal cuticle of Drosophila is shown to contain at least two hydrophobic proteins (19 and 21 kD) that are also present in the outer procuticle lamellae. An N-acetylglucosamine-containing carbohydrate is also present in the inner epicuticle. This represents the first attempt to characterize the non-lipid components of an insect epicuticle.     

Local motion and conformational changes in the cuticle of Clivia miniata Regel

The temperature dependence of the local diffusion of fluorescent molecular probes of various polarities (alkane, long-chain fatty acid, short-chain alcohol and fatty acid), all labelled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl in the cuticle of Clivia miniata Regel was studied by the technique of fluorescence recovery after photobleaching. The technique yields the coefficient of diffusion, D, in the plane of the cuticle over distances of some 10 m and the fraction, R, of mobile reporter molecules. The inner (more hydrophilic) and the outer (more hydrophobic) faces of the cuticle were studied separately by appropriate incubation. The value of D was found to depend sensitively on the polarity of the probe, the temperature and the position within the cuticle (outer hydrophobic or inner hydrophilic side). Depending on the type of probe, D increased (in the case of the alkane) or decreased (in the case of the alcohol) after removal of the (monomeric) waxes. The electron-spin-resonance (ESR) spectra of incorporated spin-labelled fatty-acid probes measured in the intact cuticle contained a major component similar to the spectrum recorded from the polymerized matrix from which waxes had been extracted, and a second component similar to the spectrum from the monomeric waxes. At low temperatures, the ESR spectra from labels at two different chain positions corresponded to chain motion which was slow on the ESR timescale. At high temperatures, the spectral component from the monomeric waxes indicated chain motions in the motional narrowing regime which were of an essentially isotropic nature.No evidence was found for a liquid-crystalline lipid phase such as found for the polar lipids in cell membranes, nor was there evidence for a sharp, thermotropic, lipid-phase transition either in the cuticle or in the waxes. Experiments with oriented samples did not demonstrate the presence of large domains with a uniform orientation of the lipid chains relative to the cuticular layers. The diffusion measurements and spin-label studies provide evidence for conformational changes of the cuticle extending over the whole temperature range studied (10° C to 70° C). These conformational changes are attributed to phase-separation processes within the cuticle. The phase separation in extracted waxes extended over a similar broad temperature range. This indicates that the transitions in the cuticle are largely determined by these components. At higher temperatures, however, the chain mobility in the regions of monomeric wax was considerably greater than that in the polymerized matrix. The experimental results strongly indicate that all three layers of the Clivia cuticle exhibit a multilamellar structure of alternatingly stacked, highly hydrophobic layers of welldefined thickness (5±0.5 nm) and more disordered layers of variable (4 to 15 nm) thickness. The lamellae are wellordered and extend over the whole leaf in the cuticle proper but are split-up into small domains in the inner and the external cuticular layer. Furthermore, changes of the molecular transport properties caused by the influence of ozone exerted during the growth of the plant were studied. We found that the diffusion coefficient increased both in the outer and the inner layer of the cuticle. A particularly large increase, by about a factor of three, was found for alkane diffusion in the hydrophobic outer face, pointing to defects in the polymerized matrix.Abbreviations MX-membrane polymer matrix membrane (or monomeric wax-depleted cuticle) - ESR electron-spin resonance - n-SASL n-(4,4-dimethyl-N-oxy-2-oxazolidinyl)-stearic acid - NBD 7-nitrobenz-2-oxa-1,3-diazol-4-yl The present work was made possible by a grant from the Bayerische Umweltministerium. Additional support by the Fonds der Chemischen Industrie is gratefully acknowledged. We are most grateful for very helpful discussions with Professor H. Ziegler, Professor J. Schönherr and Dr. M. Riederer from the Institut für Botanik, Technische Universität München, FRG.     

A proposed solution to a fine-structural puzzle: The organization of gill cuticle in a crayfish (Panulirus)

Crayfish gill cuticle is approximately 2 μm thick and comprises an epicuticle and an endocuticle, which is subdivided into outer and inner layers. Sections demonstrate indistinct lamellae in the outer endocuticle and vertically striated lamellae in the inner endocuticle. Microfibrils cannot be seen in sections. Difficulties in interpretation of the fibrous architecture of the cuticle from thin sections have been overcome by examining tilted series of micrographs of sections and also by making freeze-fracture replicas of the cuticle, which reveal the microfibrils clearly. A model for the endocuticle based on a helicoidal configuration of microfibrillar laminae is proposed and the vertically striated structures seen in sections of the outer layer are accounted for by including regular rows of particles oriented perpendicular to microfibrils. The model is compared with cuticles and coverings reported from other invertebrates.     

The pupal cuticle of Drosophila: biphasic synthesis of pupal cuticle proteins in vivo and in vitro in response to 20-hydroxyecdysone

We investigated the synthesis and localization of Drosophila pupal cuticle proteins by immunochemical techniques using both a complex antiserum and monoclonal antibodies. A set of low molecular weight (15,000-25,000) pupal cuticle proteins are synthesized by the imaginal disk epithelium before pupation. After pupation, synthesis of the low molecular weight proteins ceases and a set of unrelated high molecular weight proteins (40,000-82,000) are synthesized and incorporated into the pupal cuticle. Ultrastructural changes in the cuticle deposited before and after pupation correlate with the switch in cuticle protein synthesis. A similar biphasic accumulation of low and high molecular weight pupal cuticle proteins is also seen in imaginal discs cultured in vitro. The low molecular weight pupal cuticle proteins accumulate in response to a pulse of the insect steroid hormone 20-hydroxyecdysone and begin to appear 6 h after the withdrawal of the hormone from the culture medium. The high molecular weight pupal cuticle proteins accumulate later in culture; a second pulse of hormone appears to be necessary for the accumulation of two of these proteins.     

Pupal and larval cuticle proteins of Drosophila melanogaster

Proteins, soluble in 7 M urea, were extracted from third-instar larval and pupal cuticles of Drosophila melanogaster. Both extracts contain a limited number of polypeptides resolved by one- or two-dimensional electrophoresis. The five major larval proteins have low molecular weights (less than 20000) and are not glycosylated. The major pupal cuticle proteins fall into two size classes: two with apparent molecular weights of 56K and 82K and four with molecular weights between 15K and 25K. The proteins with high apparent molecular weights are glycosylated. In nondenaturing gels, no components of the larval and pupal cuticle extracts comigrate. One-dimensional "fingerprints" indicate that cuticle proteins from these two stages have unique primary structures. Immunological results indicate that the major low molecular weight larval and pupal cuticle proteins are comprised of two families of proteins that share antigenic determinants. The high molecular weight pupal cuticle proteins are immunologically unrelated to the low molecular weight components. We conclude that the pupal and larval proteins are encoded in part by multigene families that have arisen by gene duplication and evolutionary divergence.     

An assembly zone antigen of the insect cuticle

The assembly zone is a morphologically distinct region in the insect integument that lies between the epidermis and its principal secretory product, the lamellate cuticle. Despite its central location in the process of cuticle formation, little is known about its structure or function. Using various antisera we have shown that in Drosophila melanogaster larvae and pupae the assembly zone is antigenically distinct from the overlying lamellate cuticle. This observation suggests that this region does not contain lamellae in the process of assembling but rather is a stable and permeable matrix through which lamellar components travel in the process of cuticle formation. Curiously an antigen present in the assembly zone was also contained in the moulting gel, indicating a heretofore unsuspected chemical relationship between these two materials.     

The cuticle of Caenorhabditis elegans. II. Stage-specific changes in ultrastructure and protein composition during postembryonic development

The cuticle of the free-living nematode Caenorhabditis elegans is a proteinaceous extracellular structure that is replaced at each of four postembryonic molts by the underlying hypodermis. The cuticles of the adult and three juvenile stages (L1, Dauer larva, L4) have been compared ultrastructurally and biochemically. Each cuticle has an annulated surface and comprises two main layers, an inner basal layer and an outer cortical layer. The adult cuticle has an additional clear layer which separates the basal and cortical layers and is traversed by regularly arranged columns of electron-dense material. The fine structure of the cortical layer is similar in cuticles from different stages while that of the basal layer is stage specific. Purified cuticles were obtained by sonication and treatment with sodium dodecyl sulfate (SDS) and their component proteins solubilized with a sulfhydryl reducing agent. The degree of cuticle solubility is stage specific and the insoluble structures for each cuticle were localized by electron microscopy. Analysis of ³⁵S-labeled soluble cuticle proteins by SDS-polyacrylamide gel electrophoresis yields unique banding patterns for each stage. Most proteins are of high molecular weight (100–200 K) and are restricted to particular stages. Sixteen of the nineteen major proteins characterized are specifically degraded by bacterial collagenase. The results indicate that the different molts are not reiterative, but require the integration of both unique and shared gene functions. The potential use of stage-specific cuticle differences to identify and characterize regulatory genes controlling cuticle-type switching during development is discussed.     

The structure of the cuticle and sheath of the infective juvenile of Trichostrongylus colubriformis

The infective third-stage juvenile of Trichostrongylus colubriformis is surrounded by its own cuticle as well as the incompletely moulted cuticle of the second-stage juvenile, which is referred to as the sheath. The sheath comprises an outer epicuticle, an amorphous cortical zone, a fibrous basal zone and an inner electron-dense layer. The basal zone of the sheath consists of three layers of fibres; the fibres are parallel within each layer, but the fibre direction of the middle layer is at an angle to that of the inner and outer layers. The cuticle comprises a complex outer epicuticle, an amorphous cortical zone and a striated basal zone. The lateral alae of the cuticle and the sheath are aligned and overlie the lateral hypodermal cords. The lateral alae of the sheath consist of two wing-like expansions of the cortical zone with associated specializations of the inner electron-dense layer which form a groove. The cuticular lateral alae consist of two tube-like expansions of the cortical zone. The lateral alar complex of the cuticle and the sheath may maximise locomotory efficiency and prevent rotation of the juvenile within the sheath.     

In vivo and in vitro effects of the nonsteroidal ecdysteroid agonist tebufenozide on cuticle formation in Spodoptera exigua: An ultrastructural approach

To evaluate the ecdysteroid-like mode of action of tebufenozide (RH-5992), the effects on the fine structure of the integument in last- and third-instar larvae of the beet armyworm, Spodoptera exigua, and on cuticle formation in cultured imaginal wing discs, were studied. After 3 h of treatment with tebufenozide, the first signs of a normal moult were observed in treated larvae. A few hours later, ecdysial space formation and secretion of a new epicuticle were started. Furthermore, the new cuticle was incomplete in treated larvae; the new procuticle was absent or contained only a very low number of lamellae. In addition, epidermal cells showed many vacuoles and symptoms of degeneration with increase in time. Only a few lamellae of the old procuticle were digested, and normal ecdysis was inhibited which led to the presence of a double cuticle within 24–48 h after treatment. Similarly, cultured discs were stimulated to deposit a new cuticle within 12 h after cultivation in a medium containing tebufenozide. Our observations in treated S. exigua larvae on the one hand and in imaginal discs cultured with tebufenozide on the other hand are indicative of a hyperecdysteroid action, and confirm that the moult accelerating mode of action of tebufenozide resulted in a forced, untimely synthesis of cuticle by activation of epidermal or epithelial cells, and that its ecdysis inhibitory activity is mediated by its effect on post-apolysis processes. © 1996 Wiley-Liss, Inc.     

Ultrastructure and cuticle formation of the carapace in the myodocopan ostracod exemplified by Euphilomedes japonica (Crustacea: Ostracoda)

The ultrastructure and formation of the cuticle of a myodocopan ostracod, Euphilomedes japonica, are investigated utilizing scanning and transmission electron microscopy. The outer lamella cuticle consists of four layers; epicuticle, exocuticle, endocuticle, and membranous layer like in the cuticle of other arthropods. The exocuticle and endocuticle are well-calcified and the organic matrix develops within the both cuticles. The outermost layer of new cuticle (epicuticle) is secreted first and the inner layers (exocuticle, endocuticle and membranous layer) are added proximally in the pre-, and postmoult stages. The calcification takes place in the whole area of carapace at the same time together with the synthesis of organic matrix within the endocuticle. This study demonstrates that the ultrastructure and formation of the cuticle in myodocopans are different from those in podocopans, and that the myodocopan carapaces have achieved a structural diversity for adaptation to different lifestyles.     

Structure of the cuticle of the alpine weta,Hemideina maori (Saussure) (Orthoptera: Stenopelmatidae)

Sclerotized cuticle segments from the thorax, dorsal abdomen, and ventral abdomen of the alpine, weta Hemideina maori (Saussure) (Orthoptera: Stenopelmatidae) were examined by light microscopy and by scanning and transmission electron microscopy. An epicuticle, exocuticle (outer and inner), mesocuticle, endocuticle, and deposition layer are present in transverse sections. The epicuticle is further composed of a cuticulin layer and inner epicuticle, the latter being finely laminated and containing narrow wax canals that terminate below the cuticle surface. Openings to dermal gland ducts are visible on the surface as are large setae and smaller sensory pegs. Frozen fractured cuticle reveals the presence of horizontal ducts or channels that run laterally within the cuticle. The structure of weta cuticle is compared with that of the common house cricket and arthropods in general.     

The integument in troglobitic and epigean woodlice (Isopoda: Oniscidea): a comparative ultrastructural study

We compared the ultrastructure and the relative thickness of the integumental cuticle in several species of troglobitic and non-troglobitic woodlice. Measurements of tergal cuticle thickness on histological sections demonstrated that the cuticles in non-troglobites are thicker than those in troglobites of similar body sizes. As revealed by scanning electron microscopy, the endocuticles in troglobites consist of more numerous and thinner lamellae compared to cuticles of similar thickness in non-troglobites. Similar differences in the number and thickness of cuticular lamellae were not found in the exocuticle. As demonstrated by transmission electron microscopy of the epicuticles in troglobitic and non-troglobitic woodlice, the simple inner epicuticle is thinner relative to the total epicuticle thickness in troglobites, but this is not the case for the outer epicuticle. Outer epicuticles consisting of different numbers of sublayers can be found in troglobites as well as in non-troglobites and more complex outer epicuticles are not characteristic of representatives of any of the two ecological groups. Our results indicate that the thickness and structure of the integumental cuticle are important for evolutionary success in the subterranean environment. Nevertheless, the cuticles of troglobites are diverse in their ultrastructural features, likely reflecting different lifestyles of various troglobites.     

Isolation and characterization of outer membranes of Bacteroides thetaiotaomicron grown on different carbohydrates.

To determine whether certain outer membrane proteins are associated with growth of Bacteroides thetaiotaomicron on polysaccharides, we developed a procedure for separating outer membranes from inner membranes by sucrose density centrifugation. Cell extracts in 10% (wt/vol) sucrose-10 mM HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.4) were separated into two fractions on a two-step (37 and 70% [wt/vol]) sucrose gradient. These fractions were further resolved into outer membranes (p = 1.21 g/cm3) and inner membranes (p = 1.14 g/cm3) on sucrose gradients. About 20 to 26% of the total 3-hydroxy fatty acids from lipopolysaccharide and 2 to 3% of the total cellular succinate dehydrogenase activity were recovered in the outer membrane preparation. The inner membrane preparation contained 22 to 49% of the total succinate dehydrogenase activity and 2 to 3% of the total 3-hydroxy fatty acids from lipopolysaccharide. Outer membranes contained a lower concentration of protein (0.34 mg/mg [dry weight]) than did the inner membranes (0.68 mg/mg [dry weight]). Molecular weights of inner membrane polypeptides ranged from 11,000 to 133,000. The most prominent polypeptides had molecular weights ranging from 11,000 to 26,000. In contrast, the molecular weights of outer membrane polypeptides ranged from 17,000 to 117,000. The most prominent polypeptides had molecular weights ranging from 42,000 to 117,000. There were several polypeptides in the outer membranes of bacteria grown on polysaccharides (chondroitin sulfate, arabinogalactan, or polygalacturonic acid) which were not detected or were not as prominent in outer membranes of bacteria grown on monosaccharide components of these polysaccharides.     

Fine structure of the cuticle of the desert scorpion, Hadrurus arizonensis.

The structure of the sclerite and intersegmental cuticle of the opithosoma of the desert scorpion, Hadrurus arizonensis, has been examined by transmission electron microscopy. The sclerite cuticle contains a four-layered epicuticle, a hyaline exocuticle, an inner exocuticle and an endocuticle. The outer part of the hyaline exocuticle and the whole of the inner exocuticle are constructed of helicoidally arranged planes of microfibrils. Within the endocuticle, the overall architecture is not helicoidal as previously assumed, but consists of bundles of microfibrils oriented horizontally and vertically. Microbibrils of the inner exocuticle and the endocutile are seen as simple unstained rods, but those of the hyaline exocuticle are electron dense rods with an unstained central core. The intersegmental cuticle contains a four-layered epicuticle and a procuticle. In detail, its fine structure differs in most respects from that of the sclerite cuticle. Electron microscopy reveals that hyaline exocuticle, previously assumed to be continuous from sclerite to intersegmental membrane, is absent in the latter.     

Structural Organization of Murine Intracisternal A Particles

Isolated murine intracisternal A particles have a distinctive set of structural properties as compared to the recognized oncogenic ribonucleic acid tumor viruses. A particles consist of two closely approximated concentric shells. The outer shell is largely, but not entirely, susceptible to treatment with detergents such as deoxycholate and Triton X-100. The inner shell is resistant to these agents and is stabilized against solubilization by sodium dodecyl sulfate as a result of disulfide bonding between its constituent proteins. The major A particle structural protein (molecular weight approximately 70,000) is contained in the inner shell. A protein component with a molecular weight of about 80,000 may be preferentially concentrated in sites of firm attachment between the inner and outer shells.     

The electron microscopy of the human hair follicle. II. The hair cuticle

1. During the early differentiation of the cuticle the cell membranes smooth out and the cells become closely attached over most of their surface. The change seems to be due to a layer of cement which forms between them. The plasma membranes also increase in density. 2. The decreased membrane activity of the cuticle cells may prevent a phagocytosis of the melanocyte processes and thus account for the non-pigmentation of the cuticle. 3. The flattening and imbrication of the cuticle may possibly be explained by a zipper-like spread of cell contacts. 4. Keratinisation of the cuticle occurs at a late stage in its development; the keratin formed is an amorphous type, similar to the gamma-fraction of the cortex which is produced at a similar level. 5. Keratinisation is accompanied by the formation of complex intercellular layers similar to structures observed in the inner root sheath (see Part 3). 6. In the final stage of keratinisation the remaining cytoplasm condenses with the result that the cell is divided into a laminated structure with an outer keratinised layer and an inner layer, which is insoluble in keratinolytic solvents.     

Modulation of spore adhesion of the hyperparasitic bacterium Pasteuria penetrans to nematode cuticle

S.B. Sharma and K.G. Davies. 1997. Monoclonal antibodies (mAb) raised to the cuticule surface of second-stage juveniles (J2) of the nematode Heterodern cajani were partially characterized by immunofluorescence and Western blot analysis. Five antigens with relative molecular weights (M_r) 55, 80, 110, 180 and 210 kDa were identified with six mAb. Pasteuria spores, originating from the same population of H. cajani to which the antibodies were raised, were tested for their ability to attach to J2, which had been pretreated with each of the mAb. Monoclonal antibody HC/129 was found to reduce spore attachment by 42%, whereas HC/145 increased spore attachment by 124%. This is the first record of an antibody binding to the cuticle and increasing spore attachment, and suggests that components of the cuticle involved in inhibiting spore attachment may be masking the Pasteuria receptor present on the cuticle.     

Studies on mitochondrial proteins I. Separation and characterization by polyacrylamide gel electrophoresis

Rat liver mitochondria were fractionated into inner and outer membranes and soluble intermembrane space and matrix. The protein components of these fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mitochondria contained at least 20 components ranging in molecular weights from 10 000 to 140 000. Inner membranes differed markedly from outer membranes both in number of components and size distribution. The intermembrane space contained a few polypeptide species. These were of low molecular weight. The matrix was characterized by a high molecular weight component (130 000) which comprised 30% of this fraction. A major carbohydrate-containing polypeptide with an approximate molecular weight of 93 000 was detected in outer membrane preparations.     

Synthesis of the major adult cuticle proteins of Drosophila melanogaster during hypoderm differentiation

Differentiating imaginal hypodermal cells of Drosophila melanogaster form adult cuticle during the second half of the pupal stage (about 40 to 93 hr postpupariation). A group of proteins with molecular weights of 23,000, 20,000, and 14,000 is identified as putative major wing cuticle proteins with the following biological properties: These proteins are abundant components of cuticle and are major synthetic products of cuticle-secreting hypodermal cells. They are leucine-rich and methionine-free and are the most prominent proteins of this type synthesized by wing hypoderm at 65 hr, during the period of procuticle formation. Electron microscopic autoradiography shows that leucine-rich, methionine-free proteins specifically localize to the apical cell surface and newly secreted cuticle of 65-hr wing cells. This strongly suggests the export of these proteins to the cuticle. Lastly, these proteins undergo a reduction in extractability just after eclosion, during the period of cuticle protein crosslinking (sclerotization). The synthesis of these major hypoderm proteins is temporally regulated in development. In wing cells, the 14-kDa proteins are synthesized first, from 53 to 78 hr, and the 20- and 23-kDa proteins are synthesized from 63 to 93 hr. The pattern of synthesis for these proteins is similar in abdominal cells but delayed by 6 to 10 hr. Two-dimensional gel electrophoresis shows that each of the 23-, 20-, and 14-kDa size classes contains at least two component polypeptides. Patterns of protein synthesis in cells of the imaginal hypodermis are regulated in a precise temporal sequence during the production of adult cuticle. Their study yields a useful system for the analysis of molecular events in gene control and cell differentiation.