Hexagonal columnar liquid crystal in the cells secreting spider silk

The liquid crystallinity of spider dragline silk dope is thought to be important for both the spinning process and the extreme mechanical properties of the final thread. Although the formation of the liquid crystalline units is poorly understood, it has been suggested that spider silk proteins are secreted in a random coil and then aggregate end-to-end into rod-shaped units to form supramolecular liquid crystals. However, evidence presented here from transmission electron microscopy indicates that coat protein of the dragline silk of a Nephila spider is stored as hexagonal columnar liquid crystals within the intracellular secretory vesicles. This implies that this component is already folded into short rods within the gland cells and forms molecular rather than supramolecular liquid crystals.     

Spinning an elastic ribbon of spider silk

The Sicarid spider Loxosceles laeta spins broad but very thin ribbons of elastic silk that it uses to form a retreat and to capture prey. A structural investigation into this spider's silk and spinning apparatus shows that these ribbons are spun from a gland homologous to the major ampullate gland of orb web spiders. The Loxosceles gland is constructed from the same basic parts (separate transverse zones in the gland, a duct and spigot) as other spider silk glands but construction details are highly specialized. These differences are thought to relate to different ways of spinning silk in the two groups of spiders. Loxosceles uses conventional die extrusion, feeding a liquid dope (spinning solution) to the slit-like die to form a flat ribbon, while orb web spiders use an extrusion process in which the silk dope is processed in an elongated duct to produce a cylindrical thread. This is achieved by the combination of an initial internal draw down, well inside the duct, and a final draw down, after the silk has left the spigot. The spinning mechanism in Loxosceles may be more ancestral.     

Spinneret Microstructure of the Silk Spinning Apparatus in the Crab Spider, Misumenops tricuspidatus (Araneae: Thomisidae)

The silk spinning apparatus in the crab spider, Misumenops tricuspidatus was studied with the field emission scanning electron microscope (FESEM) and the main microstructural characteristics of the silk glands are presented. In spite of the fact that the crab spiders do not spin webs to trap a prey, they also have silk apparatus even though the functions are not fully defined. The crab spider, Misumenops tricuspidatus possesses only three types of silk glands which connected through the typical spinning tubes on the spinnerets. The spinning apparatus of Misumenops closely corresponds to that of wandering spiders such as jumping spiders or wolf spiders except some local variations. Anterior spinnerets comprise 2 pairs of the ampullates and 48 (±5) pairs of pyriform glands. Another 2 pairs of ampullate glands and nearly 20 (±3) pairs of aciniform glands were connected on the middle spinnerets. Additional 50 (±5) pairs of the aciniform glands were connected on the posterior spinnerets. The aggregate glands and the flagelliform glands which have the function of sticky capture thread production in orb‐web spiders as well as the tubuliform glands for cocoon production in females were not developed at both sexes of this spider, characteristically.     

Liquid crystals and flow elongation in a spider's silk production line

Our observations on whole mounted major ampullate silk glands suggested that the thread is drawn from a hyperbolic die using a pre-orientated lyotropic liquid crystalline feedstock. Polarizing microscopy of the gland''s duct revealed two liquid crystalline optical textures: a curved pattern in the feedstock within the ampulla of the gland and, later in the secretory pathway, the cellular texture previously identified in synthetic nematic liquid crystals. The behaviour of droplet inclusions within the silk feedstock indicated that elongational flow at a low shear rate occurs in the gland''s duct and may be important in producing an axial molecular orientation before the final thread is drawn. Our observations suggested that the structure of the spider''s silk production pathway and the liquid crystalline feedstock are both involved in defining the exceptional mechanical properties of spider dragline silk.     

Review structure of silk by raman spectromicroscopy: from the spinning glands to the fibers

Raman spectroscopy has long been proved to be a useful tool to study the conformation of protein-based materials such as silk. Thanks to recent developments, linearly polarized Raman spectromicroscopy has appeared very efficient to characterize the molecular structure of native single silk fibers and spinning dopes because it can provide information relative to the protein secondary structure, molecular orientation, and amino acid composition. This review will describe recent advances in the study of the structure of silk by Raman spectromicroscopy. A particular emphasis is put on the spider dragline and silkworm cocoon threads, other fibers spun by orb-weaving spiders, the spinning dope contained in their silk glands and the effect of mechanical deformation. Taken together, the results of the literature show that Raman spectromicroscopy is particularly efficient to investigate all aspects of silk structure and production. The data provided can lead to a better understanding of the structure of the silk dope, transformations occurring during the spinning process, and structure and mechanical properties of native fibers.     

Microstructure of the silk apparatus in the coelotine spider Paracoelotes spinivulva (Araneae: Amaurobiidae)

The microstructural characteristics of the silk‐spinning apparatus and its ecological significance in the coelotine spider Paracoelotes spinivulva were examined by field emission scanning electron microscopy, with the goal of understanding the properties and the evolutionary origins of these silk constructs. The silk apparatuses of this spider were composed of four basic types of silk‐spinning spigot (ampullate, pyriform, aciniform and tubuliform), which connected with typical silk glands in the abdominal cavity. Of the three pairs of spinnerets, the posterior pairs were highly elongated along the body axis. Anterior spinnerets comprised two pairs of ampullate glands and approximately 70–80 pairs of pyriform glands in both sexes. Middle spinnerets had one to two pairs of ampullate spigots, three pairs of tubuliform spigots in females, and 50–60 (female) or 80–90 (male) pairs of aciniform spigots. An additional two pairs of tubuliform spigots in females and 70–80 (female) or 100–120 (male) pairs of aciniform spigots were counted on the spinning surfaces of the posterior spinnerets in both sexes. Although the coelotine spiders use their silk to catch prey, P. spinivulva characteristically do not have a typical “triad” spigot, including a flagelliform and two aggregate spigots, for capture thread production.     

Microstructure of the silk apparatus of the comb-footed spider, Achaearanea tepidariorum (Araneae: Theridiidae)

The microstructural organization of the silk‐spinning apparatus of the comb‐footed spider, Achaearanea tepidariorum, was observed by using a field emission scanning electron microscope. The silk glands of the spider were classified into six groups: ampullate, tubuliform, flagelliform, aggregate, aciniform and pyriform glands. Among these, three types of silk glands, the ampullate, pyriform and aciniform glands, occur only in female spiders. One (adult) or two (subadult) pairs of major ampullate glands send secretory ductules to the anterior spinnerets, and another pair of minor ampullate glands supply the median spinnerets. Three pairs of tubuliform glands in female spiders send secretory ductules to the median (one pair) and posterior (two pairs) spinnerets. Furthermore, one pair of flagelliform glands and two pairs of aggregate glands together supply the posterior spinnerets, and form a characteristic spinning structure known as a “triad” spigot. In male spiders, this combined apparatus of the flagelliform and the aggregate spigots for capture thread production is not apparent, instead only a non‐functional remnant of this triad spigot is present. In addition, the aciniform glands send ductules to the median (two pairs) and the posterior spinnerets (12–16 pairs), and the pyriform glands feed silk into the anterior spinnerets (90–100 pairs in females and 45–50 pairs in males).     

Silk production in a spider involves acid bath treatment

We studied physiological conditions during the spinning of dragline silk by the garden cross spider, Araneus diadematus. Silk is converted from the liquid feedstock in the gland into a solid thread via a tapering tubular duct and exits at a spigot. The distal part of the tubule appears specialized for ion transport and the management of the pH inside the lumen. Thus, it appears that spider silk in vivo, like some industrial polymers in vitro, is spun through an acid bath.     

Biology of spider silk.

Studies are beginning to show that spider silk can be highly variable in chemical composition and mechanical properties. Clearly, both external and internal conditions affect silk production and thus the mechanical properties of the finished thread. An argument can be made that silk is optimised for a wide range of conditions rather than maximised for strength or toughness. Moreover, it seems that the spider is able to induce rapid and temporary adaptations of silk properties.     

蜘蛛丝的组成结构与生物学功能

蜘蛛是纺丝种类最多的一种节肢动物,目前共发现有8种丝腺,各纺出具有不同生物学功能的丝纤维,可分别用于织网、捕食、逃避、扩散、织制卵袋等行为活动。蜘蛛丝是一种天然的动物蛋白纤维,是随蜘蛛4亿年进化的结果,也是为蜘蛛的生存与繁殖所设计的,蜘蛛丝的适应与进化使蜘蛛丝具有多样化的生物学功能。但蜘蛛不是唯一能纺丝的节肢动物,除蛛形纲以外,还有其它很多节肢动物,如昆虫纲和多足纲的动物都有具有丝腺,能纺出一种或多种丝蛋白纤维。本文将以昆虫作为比较来概述蜘蛛丝腺的起源与种类,蜘蛛丝的化学组成、结构、种类与其生物学功能。     

Plasticity in major ampullate silk production in relation to spider phylogeny and ecology

Spider major ampullate silk is a high-performance biomaterial that has received much attention. However, most studies ignore plasticity in silk properties. A better understanding of silk plasticity could clarify the relative importance of chemical composition versus processing of silk dope for silk properties. It could also provide insight into how control of silk properties relates to spider ecology and silk uses. We compared silk plasticity (defined as variation in the properties of silk spun by a spider under different conditions) between three spider clades in relation to their anatomy and silk biochemistry. We found that silk plasticity exists in RTA clade and orbicularian spiders, two clades that differ in their silk biochemistry. Orbiculariae seem less dependent on external spinning conditions. They probably use a valve in their spinning duct to control friction forces and speed during spinning. Our results suggest that plasticity results from different processing of the silk dope in the spinning duct. Orbicularian spiders seem to display better control of silk properties, perhaps in relation to their more complex spinning duct valve.     

Fine structural aspects of silk secretion in a spider. II. Conduction in the pyriform glands

The excretory duct of pyriform glands in Araneus diadematus is connected to the secretory sac through an intermediary cell ring. Apices of these cells bear thick, long microvilli and cytoplasmic extensions containing microtubules in bundles, some of which are derived from normal basal bodies. These finger-like extensions lie between the cuticular intima and the secretory product; they are thought to protect the intima and to initiate moulding of the silk thread. Structural features of the duct cells suggest that the latter play a role in the control of the water content of the silk glue which is restricted to the last portion of the duct where numerous nerve endings are inserted between cells. It is evident that duct structure and chemical and physical characteristics of silk are correlated in all spider silk glands.     

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring ^1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel ³. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences ⁴. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] ^5,6, tubuliform [synthesizes egg case silk] ^7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] ⁹ and pyriform [produces attachment disc silk] ¹⁰. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.     

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.     

Diversity of molecular transformations involved in the formation of spider silks

Spiders that spin orb webs secrete seven types of silk. Although the spinning process of the dragline thread is beginning to be understood, the molecular events that occur in spiders' opisthosomal glands, which produce the other fibers, are unknown due to a lack of data regarding their initial and final structures. Taking advantage of the efficiency of Raman spectromicroscopy in investigating micrometer-sized biological samples, we have determined the secondary structure of proteins in the complete set of glands of the orb-weaving spider Nephila clavipes. The major and minor ampullate silks in the sac of their glands have identical secondary structures typical of natively unfolded proteins. Spidroins are converted into fibers containing highly oriented β-sheets. The capture spiral represents a distinct structural singleton. The proteins are highly disordered prior to spinning and undergo no molecular change or alignment upon spinning. The cylindrical, aciniform, and piriform proteins are folded in their initial state with a predominance of α-helices, but whereas the cylindrical gland forms a fiber similar to the major ampullate thread, the aciniform and piriform glands produce fibers dominated by moderately oriented β-sheets and α-helices. The conformation of the proteins before spinning is related to intrinsic characteristics of their primary structure. Proteins that are unfolded in the gland have repeat sequences composed of submotifs and display no sequence regions with aggregation propensity. By contrast, the folded proteins have neither submotifs nor aggregation-prone sequence regions. Taken together, the Raman data show a remarkable diversity of molecular transformations occurring upon spinning.     

Biomechanical variation of silk links spinning plasticity to spider web function

Spider silk is renowned for its high tensile strength, extensibility and toughness. However, the variability of these material properties has largely been ignored, especially at the intra-specific level. Yet, this variation could help us understand the function of spider webs. It may also point to the mechanisms used by spiders to control their silk production, which could be exploited to expand the potential range of applications for silk. In this study, we focus on variation of silk properties within different regions of cobwebs spun by the common house spider, Achaearanea tepidariorum. The cobweb is composed of supporting threads that function to maintain the web shape and hold spiders and prey, and of sticky gumfooted threads that adhere to insects during prey capture. Overall, structural properties, especially thread diameter, are more variable than intrinsic material properties, which may reflect past directional selection on certain silk performance. Supporting threads are thicker and able to bear higher loads, both before deforming permanently and before breaking, compared with sticky gumfooted threads. This may facilitate the function of supporting threads through sustained periods of time. In contrast, sticky gumfooted threads are more elastic, which may reduce the forces that prey apply to webs and allow them to contact multiple sticky capture threads. Therefore, our study suggests that spiders actively modify silk material properties during spinning in ways that enhance web function.     

Primary structure elements of spider dragline silks and their contribution to protein solubility

Spider silk proteins have mainly been investigated with regard to their contribution to mechanical properties of the silk thread. However, little is known about the molecular mechanisms of silk assembly. As a first step toward characterizing this process, we aimed to identify primary structure elements of the garden spider's (Araneus diadematus) major dragline silk proteins ADF-3 and ADF-4 that determine protein solubility. In addition, we investigated the influence of conditions involved in mediating natural thread assembly on protein aggregation. Genes encoding spider silk-like proteins were generated using a cloning strategy, which is based on a combination of synthetic DNA modules and PCR-amplified authentic gene sequences. Comparing secondary structure, solubility, and aggregation properties of the synthesized proteins revealed that single primary structure elements have diverse influences on protein characteristics. Repetitive regions representing the largest part of dragline silk proteins determined the solubility of the synthetic proteins, which differed greatly between constructs derived from ADF-3 and ADF-4. Factors, such as acidification and increases in phosphate concentration, which promote silk assembly in vivo generally decreased silk protein solubility in vitro. Strikingly, this effect was pronounced in engineered proteins comprising the carboxyl-terminal nonrepetitive regions of ADF-3 or ADF-4, indicating that these regions might play an important role in initiating assembly of spider silk proteins.     

Structural conformation of spidroin in solution: a synchrotron radiation circular dichroism study

Spider silk is made and spun in a complex process that tightly controls the conversion from soluble protein to insoluble fiber. The mechanical properties of the silk fiber are modulated to suit the needs of the spider by various factors in the animal's spinning process. In the major ampullate (MA) gland, the silk proteins are secreted and stored in the lumen of the ampulla. A particular structural fold and functional activity is determined by the spidroins' amino acid sequences as well as the gland's environment. The transition from this liquid stage to the solid fiber is thought to involve the conversion of a predominantly unordered structure to a structure rich in beta-sheet as well as the extraction of water. Circular dichroism provides a quick and versatile method for examining the secondary structure of silk solutions and studying the effects of various conditions. Here we present the relatively novel technique of synchrotron radiation based circular dichroism as a tool for investigating biomolecular structures. Specifically we analyze, in a series of example studies on structural transitions induced in liquid silk, the type of information accessible from this technique and any artifacts that might arise in studying self-assembling systems.     

From EST sequence to spider silk spinning: identification and molecular characterisation of Nephila senegalensis major ampullate gland peroxidase NsPox

Spider dragline silk is renowned as one of the toughest materials of its kind. In nature, spider silks are spun out of aqueous solutions under environmental conditions. This is in contrast to production of most synthetic fibres, where hazardous solvents, high temperatures and pressure are used. In order to identify some of the chemical processes involved in spider silk spinning, we have produced a collection of cDNA sequences from specific regions of Nephila senegalensis major ampullate gland. We examined in detail the sequence and expression of a putative Nephila senegalensis peroxidase gene (NsPox) from our EST collection. NsPox encodes a protein with similarity to Drosophila melanogaster and Aedes aegypti peroxidases. Northern analysis and in situ localisation experiments revealed that NsPox is expressed in major and minor ampullate glands of the spider where the main components of the dragline silk are produced. We suggest that NsPox plays a role in dragline silk fibre formation and/or processing.