Silk Formation Research Articles

1H, 15N and 13C resonance assignments of eggcase silk protein 3.

Spider silk is a high-performance biomaterial known for its outstanding combination of strength and flexibility. Among the six distinct types of spider silk, eggcase silk stands out as it is exclusively produced from the tubuliform gland, playing a specialized role in offspring protection. In the spider species Latrodectus hesperus, eggcase silk is spun from a large spidroin complex, including the major silk component tubuliform spidroin 1 (TuSp1) and at least six different minor silk components. One of these minor components is eggcase protein 3 (ECP3), a small silk protein of 11.8kDa that lacks the typical spidroin architecture. ECP3 shows very limited homology to all known spidroins. In this study, we report nearly complete backbone and side-chain resonance assignments of ECP3 as a basis for studying the structural mechanisms involved in eggcase silk formation.

Dimerization and liquid-liquid phase separation of the nonrepetitive domains of pyriform spidroin 1 controls the pyriform silk formation

Spiders spin high performance silks with diverse mechanical properties for specific biological functions. Of these spider silk types, pyriform silk stands out as a unique combination of wet glue and dry fibers. Investigation of self-assembly process of spider silk proteins is necessary for elucidating the silk formation mechanism. However, the functions of nonrepetitive domains in the silk formation of pyriform spidroins from liquid proteins to solid fibers are still unclear, making it difficult to achieve efficient biomimetic preparations of pyriform silk with good mechanical properties. In this study, we investigate the roles of the N-linker repeat (NLR) and both terminal domains of pyriform spidroin 1 (PySp1) in the silk formation. We demonstrate for the first time that the PySp1 NLR alone is sufficient to self-assemble into high strength fibers. Moreover, we showed that the ability to promote the pyriform silk formation by the addition of the NLR. We also found that the pH-sensitive dimerization property for N-terminal domain and the liquid-liquid phase separation (LLPS) coupled with acidification triggers the self-assembly mediated by the C-terminal domain. Overall, our results provide new insight into the role of nonrepetitive domains in the pyriform silk formation mechanism and the basis for producing new protein-based materials derived from spider pyriform silk.

Regionalization of cell types in silk glands of Larinioides sclopetarius suggest that spider silk fibers are complex layered structures

In order to produce artificial silk fibers with properties that match the native spider silk we likely need to closely mimic the spinning process as well as fiber architecture and composition. To increase our understanding of the structure and function of the different silk glands of the orb weaver Larinioides sclopetarius, we used resin sections for detailed morphology, paraffin embedded sections for a variety of different histological stainings, and a histochemical method for localization of carbonic anhydrase activity. Our results show that all silk glands, except the tubuliform glands, are composed of two or more columnar epithelial cell types, some of which have not been described previously. We observed distinct regionalization of the cell types indicating sequential addition of secretory products during silk formation. This means that the major ampullate, minor ampullate, aciniform type II, and piriform silk fibers most likely are layered and that each layer has a specific composition. Furthermore, a substance that stains positive for polysaccharides may be added to the silk in all glands except in the type I aciniform glands. Active carbonic anhydrase was found in all silk glands and/or ducts except in the type I aciniform and tubuliform glands, with the strongest staining in aggregate glands and their ductal nodules. Carbonic anhydrase plays an important role in the generation of a pH gradient in the major ampullate glands, and our results suggest that some other glands may also harbor pH gradients.

Domain swap facilitates structural transitions of spider silk protein C-terminal domains.

Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.

Larval and Pupal Silk Variation in the Indian Meal Moth (Plodia interpunctella): the Impact of Overcrowding and Temperature

The Indian meal moth (Plodia interpunctella) is becoming an important biological model to study silk. While there are many potential applications to materials science and medicine, many aspects of silk production in this species remain unknown. Here we characterize the silk of P. interpunctella by measuring the width of larval wandering and pupal silk strands and find that the latter is significantly thicker than the former. We also report intraspecific variation in pupal silk production in our lab-reared colony with a very small number of individuals forgoing pupal silk production entirely (< 4%). Overcrowding had no effect on silk formation, but exposure to elevated temperature reduced pupal silk production.

A molecular atlas reveals the tri-sectional spinning mechanism of spider dragline silk

The process of natural silk production in the spider major ampullate (Ma) gland endows dragline silk with extraordinary mechanical properties and the potential for biomimetic applications. However, the precise genetic roles of the Ma gland during this process remain unknown. Here, we performed a systematic molecular atlas of dragline silk production through a high-quality genome assembly for the golden orb-weaving spider Trichonephila clavata and a multiomics approach to defining the Ma gland tri-sectional architecture: Tail, Sac, and Duct. We uncovered a hierarchical biosynthesis of spidroins, organic acids, lipids, and chitin in the sectionalized Ma gland dedicated to fine silk constitution. The ordered secretion of spidroins was achieved by the synergetic regulation of epigenetic and ceRNA signatures for genomic group-distributed spidroin genes. Single-cellular and spatial RNA profiling identified ten cell types with partitioned functional division determining the tri-sectional organization of the Ma gland. Convergence analysis and genetic manipulation further validated that this tri-sectional architecture of the silk gland was analogous across Arthropoda and inextricably linked with silk formation. Collectively, our study provides multidimensional data that significantly expand the knowledge of spider dragline silk generation and ultimately benefit innovation in spider-inspired fibers.

Azadirachtin inhibits the development and metabolism of the silk glands of Spodoptera frugiperda and affects spinning behavior.

Spodoptera frugiperda is a major agricultural pest, and the dispersal of its larvae by spinning silk is one of the causes of crop damage. At present, there are relatively few reports of pest control that affect larvae spinning silk. In this study, the effect of spinning behavior of the S. frugiperda larvae was investigated through a series of experiments. The 3rd instar larvae of S. frugiperda were exposed to azadirachtin, and the pathological changes in the silk glands of S. frugiperda and the differences in their metabolites were analyzed by scanning electron microscopy, histological sectioning, transmission electron microscopy and metabolomics. The results showed that azadirachtin could affect the silk gland of S. frugiperda. After 48 h of treatment with azadirachtin, the silk gland lumen of S. frugiperda appeared vacuolated. KEGG showed that 31 different metabolites were identified, of which 12 were upregulated and 19 were downregulated. These metabolites were enriched in 15 different metabolic pathways, which indicated that the silk gland of S. frugiperda was closely related to the formation of fatty acids and energy metabolism for the silk formation process. This study provides a preliminary report of the effect of azadirachtin on the spinning behavior of the S. frugiperda larvae. Metabolomic results indicated that histidine, glycine and leucine, which are related to serine protein synthesis, were down-regulated. Azadirachtin can damage the silk glands of S. frugiperda and thus affect spinning behavior. This provides the basis for the control of S. frugiperda by spinning silk. © 2022 Society of Chemical Industry.

Structure of silk I (Bombyx mori silk fibroin before spinning) in the dry and hydrated states studied using 13C solid-state NMR spectroscopy

Nowadays, much attention has been paid to Bombyx mori silk fibroin (SF) by many researchers because of excellent physical properties and biocompatibility. These superior properties originate from the structure of SF and therefore, the structural analysis is a key to clarify the superiority. Here we concentrated on silk I structure (SF structure before spinning). We showed that silk I* (the structure of (GAGAGS)n which is a main part of SF) is a repeated type II β-turn, neither α-helix nor random coil, from the conformation-dependent 13C NMR chemical shift data. This conclusion is different from that obtained using IR by many researchers. Next, the formation of silk I* structure was investigated at molecular level using 13C solid-state NMR spectroscopy. Three kinds of 13C INEPT, CP/MAS and DD/MAS NMR spectra were observed for SF, [3-13C]Ser- and [3-13C]Tyr-SF, the crystalline fraction obtained by chymotrypsin treatment of SF and their model peptide with silk I structures in the dry and hydrated states. Especially, the presence of the sequences containing Tyr, (((GX)m1GY)m2 where X = A or V) with random coil conformations adjacent to (GAGAGS)n is an essence to get water-soluble SF and the formation of silk I* structure of (GAGAGS)n.

Characterization of the genome and silk-gland transcriptomes of Darwin's bark spider (Caerostris darwini).

Natural silks crafted by spiders comprise some of the most versatile materials known. Artificial silks-based on the sequences of their natural brethren-replicate some desirable biophysical properties and are increasingly utilized in commercial and medical applications today. To characterize the repertoire of protein sequences giving silks their biophysical properties and to determine the set of expressed genes across each unique silk gland contributing to the formation of natural silks, we report here draft genomic and transcriptomic assemblies of Darwin's bark spider, Caerostris darwini, an orb-weaving spider whose dragline is one of the toughest known biomaterials on Earth. We identify at least 31 putative spidroin genes, with expansion of multiple spidroin gene classes relative to the golden orb-weaver, Trichonephila clavipes. We observed substantial sharing of spidroin repetitive sequence motifs between species as well as new motifs unique to C. darwini. Comparative gene expression analyses across six silk gland isolates in females plus a composite isolate of all silk glands in males demonstrated gland and sex-specific expression of spidroins, facilitating putative assignment of novel spidroin genes to classes. Broad expression of spidroins across silk gland types suggests that silks emanating from a given gland represent composite materials to a greater extent than previously appreciated. We hypothesize that the extraordinary toughness of C. darwini major ampullate dragline silk may relate to the unique protein composition of major ampullate spidroins, combined with the relatively high expression of stretchy flagelliform spidroins whose union into a single fiber may be aided by novel motifs and cassettes that act as molecule-binding helices. Our assemblies extend the catalog of sequences and sets of expressed genes that confer the unique biophysical properties observed in natural silks.

C-Terminal Domains of Spider Silk Proteins Having Divergent Structures but Conserved Functional Roles.

Spider silk is self-assembled from silk proteins or spidroins. C-terminal domains (CTDs) of various types of spidroins are relatively conserved in amino acid sequences and are suggested to adopt similar structures and perform similar functional roles in spidroin storage and silk formation. Here, we solved the structure of the CTD from a capture-spiral silk protein (CTDFl) and characterized its stability and fibril formation in the presence and absence of a reducing agent at different pH values. CTDFl adopts a dimeric structure with 8 helices, but the CTDs of other types of spidroins exist in a domain-swapped dimeric structure with 10 helices. Despite the structural differences, CTDFl is pH-responsive in stability and fibril formation, similar to the CTDs from minor and major ampullate spidroins. Thus, the functional role of CTDs in silk fiber formation seems conserved. Comparing wild-type CTDFl and its mutants, we found that the pH-responsive behavior results from the protonation of H76, which is conserved from different spider species. In addition, the fibril formation rate of CTDFl correlates with its instability, suggesting that structural changes are involved in fibril formation.

Enhanced formation of bioactive and strong silk-bioglass hybrid materials through organic-inorganic mutual molecular nucleation induction and templating.

Materials based on silk fibroin (SF) are important for many biomedical applications due to their excellent biocompatibility and tunable biodegradability. However, the insufficient mechanical strength and low bioactivity of these materials have limited their applications. For silk hydrogels, slow gelation is also a crucial problem. In this work, a simple approach is developed to address these challenging problems all at once. By mixing SF solution with bioglass (BG) sol, instant gelation of silk is induced, the storage modulus of the hydrogel and the compressive modulus of the aerogel are significantly enhanced. The formation of a complex of SF and tetraethyl orthosilicate (TEOS), either through hydrogen bonding or TEOS condensation on SF, facilitated the aggregation of SF and, on the other hand, created active sites for the condensation of TEOS and BG formation on the surface of silk nanofibrils. The resultant hybrid gels have much higher capacity for biomineralization, indicating their higher bioactivity, compared with the pristine silk gels. This organic (SF)-inorganic (BG) mutual nucleation induction and templating can be used for a general approach to produce bioactive silk materials of various formats not limited to gels and may also inspire the formation of other functional protein-BG hybrid materials.

Evaluation of Blended NPS Fertilizer Rates and Inter Row Spacing on Yield Components and Yield of Maize (Zea Mays L.)

Corn is an important cereal crop in Ethiopia due to its use as a source of food security. However, its productivity is limited by insufficient application of the NPS fertilizer and different row spacing. A field trial was carried out to assess the effects of the application of different NPS fertilizer quantities and inter row spacing on the growth, yield components and yield of maize and the cost-benefit analysis of the NPS compound fertilizer application on the yield of maize in the main growing season 2019/2020.The study was arranged in a factorial combination of five levels of NPS fertilizers (0, 50, 100, 150 and 200 kg NPS ha-1) and four inter row spacing (55 cm, 65 cm, 75 cm and 85 cm). in a randomized complete block design with three replications. The consequence showed the main result of the NPS fertilizer had a highly significant (p &lt;0.01) effect on days up to 50% anthesis, days up to 50% silk formation, 90% physiological maturity, leaf area, leaf area index, number of plants at harvest, the number of grains per ear was determined from the main effects of NPS fertilizer of 200 kg NPS ha-1. The interaction effects of NPS and row spacing have highly significant (p &lt;0.01) effects on the number of ears per plant, number of ears per hectare, ear length, agronomic effectiveness and grain yield were obtained when using 150 kg. measured NPS ha-1 at 75 cm row spacing. The highest economic (91,608 Birr ha-1) and a higher MRR (1745%) resulted from the 150 kg NPS ha-1 and 75 cm row spacing. Thus, it should be noted that the application of 150 kg NPS ha-1 with a row spacing of 75 cm was both agronomic and cost-effective for the grain yield of the Melkassa-II in the study area.

Preparation of Natural Silk Nanofiber/Graphene Conductive Film

The use of natural silk nanofibers (SNFs) in flexible materials has been widely studied in recent years. However, the reported preparation methods are not suitable for commercial consideration. We report a method for rapid preparation of silk nanofibers in water. Silk nanofibers were mixed with graphene to prepare composite conductive silk films (CSF) with good flexibility and conductivity. Micro-morphology shows that graphene is embedded and modified between silk nanofibers to form a stable structure. Infrared analysis showed that graphene compounds do not alter silk formation, especially in the stable silk structure. Resistance tests show that the process is most effective when the ratio of SNFs to graphene is 1:3. This study offers a new approach to fabricating bioelectric devices.

Fiber Formation and Mechanical Properties of Bombyx mori Silk Are Regulated by Vacuolar-Type ATPase.

The mechanism of silk fiber formation in silkworms, Bombyx mori, is of particular scientific interest because it is closely related to the mechanical properties of silk fibers. However, there are still substantial knowledge gaps in understanding the details of this mechanism. Studies have found a pH gradient in the silk gland of silkworms. A vacuolar-type ATPase (V-ATPase) is thought to be involved in establishing this pH gradient. Although it is reported that the pH gradient plays a role in silk fibrillogenesis, the direct relationship between V-ATPase and silk mechanical properties is unclear. Thus, this study aims to clarify this relationship. We found that V-ATPase is highly and stably expressed in the anterior silk gland (ASG) and maintains the pH gradient and the fine structure of ASG. Inhibition of V-ATPase activity increased the β-sheet content and crystallinity of silk fibers. Tensile testing showed that the mechanical properties of silk fibers improved after inhibiting V-ATPase activity. All the data suggest that V-ATPase is a key factor in regulating silk fibrillogenesis and is related to the final mechanical properties of the silk fibers. V-ATPase is a potential target for silk mechanical property improvement.

Critical role of minor eggcase silk component in promoting spidroin chain alignment and strong fiber formation

Significance Our manuscript is focused on the structural and functional role of minor silk component TuSp2 in spider eggcase silk formation. We present the structure of TuSp2 core domain and reveal how this domain renders main component TuSp1 more favorable for molecular chain alignment, which is critical for strong fiber formation. We further designed a series of biomimetic minispidroin complexes and showed that the artificial silk fiber can surpass its native counterpart in strength significantly. This study represents a distinct advance in understanding the critical role of the minor component in silk formation. The strategy developed in this study also throws light on spinning other types of artificial silk fibers with predictable physical properties.

1H, 15N and 13C resonance assignments of a repetitive domain of tubuliform spidroin 2.

Spider silk is renowned for its excellent mechanical properties. Among six types of silk and one silk glue produced by different abdominal glands for various purposes, tubuliform (eggcase) silk is unique due to its high serine and low glycine content. Eggcase silk is spun from at least two spidroins, tubuliform spidroin 1 (TuSp1) and TuSp2. TuSp1 and TuSp2 were identified as the major and the minor components in tubuliform glands, respectively. TuSp2 consists of multiple repetitive (RP) domains with short terminal tails and shares very limited homology to all known spidroins.Here we report backbone and side chain resonance assignments of TuSp2-RP as a basis for structural and functional studies on eggcase silk formation.

Mutation in Bombyx mori fibrohexamerin (P25) gene causes reorganization of rough endoplasmic reticulum in posterior silk gland cells and alters morphology of fibroin secretory globules in the silk gland lumen

Larvae of many lepidopteran species produce a mixture of secretory proteins, known as silk, for building protective shelters and cocoons. Silk consists of a water-insoluble silk filament core produced in the posterior silk gland (PSG) and a sticky hydrophilic coating produced by the middle silk gland (MSG). In Bombyx mori, the fiber core comprises three proteins: heavy chain fibroin (Fib-H), light chain fibroin (Fib-L) and fibrohexamerin (Fhx, previously referred to as P25). To learn more about the role of Fhx, we used transcription activator-like effector nuclease (TALEN) mutagenesis and prepared a homozygous line with a null mutation in the Fhx gene. Our characterization of cocoon morphology and silk quality showed that the mutation had very little effect. However, a detailed inspection of the secretory cells in the posterior silk gland (PSG) of mid-last-instar mutant larvae revealed temporary changes in the morphology of the endoplasmic reticulum. We also observed a morphological difference in fibroin secretory globules stored in the PSG lumen of Fhx mutants, which suggests that their fibroin complexes have a slightly lower solubility. Finally, we performed an LC-MS-based quantitative proteomic analysis comparing mutant and wild-type (wt) cocoon proteins and found a high abundance of a 16 kDa secretory protein likely involved in fibroin solubility. Overall, our study shows that whilst Fhx is dispensable for silk formation, it contributes to the stability of fibroin complexes during intracellular transport and affects the morphology of fibroin secretory globules in the PSG lumen.

Nano-fishnet formation of silk controlled by Arginine density

Silk fiber is renowned for its superb mechanical properties, such as over 7 times the toughness of Kevlar 49 Fibre. As the spider silk is tougher than any man-made fiber, there is a lot to be learned from spider silk. Recently, it has been reported that a large portion of the properties of silk is from naturally formed nano-fishnet structures of silk, but neither its formation mechanism nor its formation condition has been explained. Here, we show how the formation and disappearance of nano-fishnet of silk is determined by humidity, and how the humidity-dependency of nano-fishnet formation can be overcome by changing density of Arginine through sequence mutation. We demonstrate that the nano-fishnet-structured silk exhibits higher strength and toughness than its counterparts. This information on controllable nano-fishnet formation of silk is expected to pave the way for development of protein and synthetic fiber design. Statement of significanceSilk fibers are a very interesting material in that it exhibits superb mechanical properties such as 7 times the toughness of Kevlar 49 Fibre, despite being only composed of proteins. Therefore, it is important that we understand the principle of its high mechanical properties so that it may be applied in designing synthetic fibers. Recently, it has been reported that a large portion of its mechanical property comes from its nano-fishnet structures, but no detailed explanation on the condition or mechanism of formation. Through molecular dynamic simulations, we simulated the nano-fishnet formation of silk and analyzed the condition and mechanism behind it, and showed how the formation of nano-fishnet structures could be controlled by changing the density of Arginine residues. Our study provides information on fiber enhancement mechanism that could be applied to synthetic and protein fiber design.

Morphological description and statistical characteristic simulation of cocoon filament size curve

Computer simulation of silk reeling is an important means to understand structure and formation of raw silk, and simulation to generate cocoon filament size curve is the premise of computer simulation of reeling silk. In this paper, according to unique pattern of cocoon filament size curve that is “thin-thick-thin, the last part is the thinnest”, a simulation and generation method of cocoon filament size curve with adjustable morphological is obtained by analyzing the characteristic point values and statistical data of cocoon filament size curve, such as head end size, maximum size and its position on cocoon filament, length of cocoon filament. Through a lot of simulation practice and analysis on statistical characteristics of cocoon filament size curve, it is considered that the proposed generation method has good applicability.

Self-assembly of tubuliform spidroins driven by hydrophobic interactions among terminal domains

Spider silk has remarkable physical and biocompatible properties. Investigation of structure-function relationship and self-assembly process of spidroins is necessary for uncovering the mechanism of silk fiber formation. Nevertheless, how the terminal domains initiate self-assembly of soluble tubuliform spidroins to form solid eggcase silk is still not fully understood. Here we investigate the roles of both terminal domains of tubuliform spidroin 1 (TuSp1) in the silk fiber formation. We found that interactions among the terminal domains drive rapid TuSp1 self-assembly and fiber formation, which is insensitive to pH changes from 6.0 to 7.0. These interactions also contribute to the spidroin chain alignment in fiber formation upon shear-force exposure. Structural analysis and site-directed mutagenesis identified eight critical surface-exposed residues involved in hydrophobic interactions among terminal domains. Spidroins with single-point mutations of these residues fail to form intermediate micelle-like structures. The structural docking model indicates that multiple terminal domains of TuSp1 may interact with each other based on hydrophobic interactions and surface complementarity, which may lead to forming the surface of the micelle-like structure. Our results provide new insights into the structural mechanism of eggcase silk formation and the basis for designing and producing novel biomaterials derived from spider eggcase silk.