Morphology and Composition of the Spider Major Ampullate Gland

Jul 9, 2013 - Spider silk is made of unique proteins—spidroins—secreted and stored as a protein solution (dope) in specialized glands. The major a...
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Morphology and Composition of the Spider Major Ampullate Gland and Dragline Silk Marlene Andersson,† Lena Holm,† Yvonne Ridderstråle,† Jan Johansson,†,‡,§ and Anna Rising*,†,‡ †

Department of Anatomy, Physiology, and Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Box 575, 751 23 Uppsala, Sweden ‡ Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet, Novum, fifth floor, 141 86 Stockholm, Sweden § Institute of Mathematics and Natural Sciences, Tallinn University, Narva mnt 25, 101 20 Tallinn, Estonia S Supporting Information *

ABSTRACT: Spider silk is made of unique proteins spidroinssecreted and stored as a protein solution (dope) in specialized glands. The major ampullate gland, source of the dragline silk, is composed of a tail, a sac and an elongated duct. For this gland, several different types of epithelial cells and granules have been described, but it is largely unknown how they correlate with spidroin production. It is also not settled what parts of the large spidroins end up in the final silk, and it has been suggested that the N-terminal domain (NT) is lacking. Here we show that NT is present in the dope and throughout dragline silk fibers, including the skin layer, and that the major ampullate tail and sac consist of three different and sharply demarcated zones (A−C), each with a distinct epithelial cell type. Finally, we show that spidroins are produced in the A and B zone epithelia, while the C zone granules lack spidroins.

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INTRODUCTION Spider silk proteinsspidroinsare attractive for biomedical and technical applications because of the extraordinary properties of spider silk in terms of strength, flexibility, and biocompatibility.1,2 Spiders are, however, inherently difficult to house and breed, and the silk is tedious to harvest. Large-scale production of artificial spider silk is an alternative that requires heterologous production of spidroins, an endeavor that is ongoing in several laboratories.3−6 For example, our group has generated recombinant miniature spidroins that self-assemble spontaneously, but the goal to reproduce the mechanical properties of native spider silk is unmet.6 Surprisingly, quite little is known about the details of the spiders’ silk glands, the natural spidroin production and spinning processes, as well as the architecture and composition of silk fibers.7,8 To enable future generation of artificial silk fibers with properties that equal those of native spider silk, we need to gain a better understanding of the natural silk production and spinning processes. Spiders produce their silk in specialized glands located in the opisthosoma (abdomen). There are several different types of glands, each responsible for producing a specific type of silk.9 Up to seven different types of silk glands have been described; the number depends on the genus and gender studied (Table 1). In the Orbiculariae family (orb weaving spiders) there are seven different types of glands. These include major ampullate, minor ampullate, flagelliform, aciniform, pyriform, aggregate, and tubuliform glands (also referred to as cylindrical glands).10 The major ampullate gland is most extensively studied due to its relatively large size and the attractive properties of its © 2013 American Chemical Society

Table 1. Silk Glands and Their Silks in the Four Different Species Studied Herein type of gland, and silk produced

Ad

Nc

Ts

Ea

major ampullate, dragline silk minor ampullate, temporary scaffolding aciniform, pray wrapping and sperm webs (male) pyriform, attachment points tubuliform, egg cocoon silk (female) aggregate, aqueous coating/glue flagelliform, capture-spiral silk

+ + + + + + +

+ + + + + + +

+ + + + + − −

+ + + + + − −

Abbreviations: Araneus diadematus (Ad), Nephila clavipes (Nc), Tegenaria sp (Ts), Euprosthenops australis (Ea). Plus sign (+) indicates presence of gland, minus sign (−) indicates absence of gland.

product, the dragline silk. In line with this, most attempts to produce artificial spider silk has employed genes that code for parts of the major ampullate spidroins (MaSp’s). MaSp’s are large proteins (about 3500 amino acid residues long) and consist of an N-terminal domain (NT, ∼130 amino acid residues), an extensive repetitive part and a C-terminal domain (CT, ∼ amino acid 110 residues).11−13 The nature of the repetitive part determines the mechanical properties of the fiber, and the CT is thought to be important for spontaneous fiber formation.12 The NT has been shown to act as a pHdependent relay, promoting solubility of the spider silk proteins at pH 7 and rapid fiber formation at pH 6,14 and the molecular Received: June 19, 2013 Published: July 9, 2013 2945

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Figure 2A) and Western blot (S-Figure 2B) on dissolved silk from E. australis, and (iii) TEM immunohistochemistry on dragline silk from E. australis and fibers formed from recombinantly produced miniature spidroins composed of NT, repetitive regions and CT (NT4RepCT)14 as positive control (S-Figure 3) using B. mori silk and recombinant fibers lacking NT (4RepCT)14 as negative controls. In none of these experiments did the antibody bind to the negative controls, while it recognized all expected targets. For SDS-PAGE and Western blot, E. australis silk fibers were dissolved in hexafluoroispropanol (HFIP). HFIP was added to the silk, the solution was sonicated for 15 min, heated to 50 °C for 5 min, and incubated at room temperature overnight (ON). Next, SDS was added to a final concentration of 2%, and the samples were heated at 94 °C for 5 min, after which the HFIP was evaporated under airflow and put in a desiccator ON. The samples were dissolved in SDS-PAGE sample buffer without SDS and heated at 94 °C for 5 min before loading on an SDS-PAGE gel (12%). The two miniature spidroins NT4RepCT and 4RepCT fused with thioredoxin and hexa-His tags were expressed in Escherichia coli for 2 hours at 25 °C and subsequently purified on a metal affinity column, liberated from fusion tags, after which fibers were formed at room temperature according to Askarieh et al.14 Immuno-TEM of Fibers. For immuno-TEM, native E. australis silk fibers and recombinantly produced NT4RepCT and 4RepCT fibers were fixed for 24 h at room temperature in 2.5% paraformaldehyde and 0.2% (v/v) glutaraldehyde, dehydrated in graded series of ethanol and embedded in Lowicryl (K4M Kit, Polysciences Inc.) after which they were sectioned (20−60 nm) in an LKB ultrotome and mounted on nickel grids. Sections were subjected to immunohistochemical analysis using the NT antibodies as primary antibody and 10 nm goldlabeled goat antirabbit secondary antibodies (EM.GAR10, BBInternational). Grids were negatively stained with uranyl acetate. Tissue Processing, Microscopy and Immunohistochemistry. Spiders were anesthetized with CO2 gas and sacrificed by severing at the pedicle. Some spiders were first silked for 5 min and then sacrificed after 30 min. Dissection was carried out in 67 mM sodium phosphate buffer at pH 7.2, or in a modified (5% CO2, 26 mM HCO3−, 10 mM glucose) pH 7.4 spider Ringer solution.23 Some opisthosomas were fixed and embedded directly after removal of the exoskeleton, while others were dissected so that the major ampullate glands could be isolated before fixation. Tissues for light microscopy (LM) were immersion fixed in 2.5% (v/v) glutaraldehyde in 67 mM phosphate buffer, pH 7.2, for 24 h at 4 °C and subsequently rinsed with phosphate buffer, pH 7.2. After fixation, tissues were dehydrated using increasing concentrations of ethanol, infiltrated, and embedded in a water-soluble glycol methacrylate (Leica Historesin embedding kit). Glass knives (Leica) in a microtome (Leica RM 2165) were used to cut 2 μm sections. Sections floating on Millipore water were recovered on glass slides, dried at 37 °C and stained with hematoxylin-eosin (HE) or Azure blue. Since spider silk gland tissues have not been studied on Historesin sections previously, both HE and Azure blue were used to be able to distinguish between the different glands and cell types, and to ensure that differences could be observed with multiple stains. The glutaraldehyde fixation method and embedding in Historesin for LM, as opposed to the commonly used paraformaldehyde or formalin fixation and embedding in paraffin,24,25 allows thinner sectioning (2 μm compared to 4−6 μm) and better morphological resolution of the sections. Major ampullate glands for TEM were fixed in 2.5% (v/v) glutaraldehyde in phosphate buffer pH 7.2−7.4, for 24 h, at room temperature, postfixed in 1% osmium tetroxide, dehydrated in graded series of ethanol, embedded in EPON (Agar100 Resin Kit, Agar Scientific Ldt.), and sections of 20−60 nm were mounted on copper grids. Grids were stained with uranyl acetate and lead citrate. For TEM immunolabeling, major ampullate glands were fixed in 2.5% paraformaldehyde and 0.2% (v/v) glutaraldehyde for 24 h at room temperature, dehydrated in graded series of ethanol, and embedded in Lowicryl. Sections (20−60 nm) were mounted on gold or nickel grids. Sections were incubated with anti-NT antibodies and detected with goat antirabbit 10 nm gold secondary antibodies (EM.GAR10,

basis for this change involves complex transitions between monomeric and dimeric NT structures.14−17 CT has been shown to be present in the final dragline silk,18 but the final localization of NT is not unambiguously determined. Peptide fragments originating from TuSp1 NT were found by mass spectrometry analysis of a trypsinated egg case silk fiber.19 Sequence analysis by Edman degradation of dragline silk fibers, however, showed peptides originating only from the repetitive region.20 Herein we use methods of preparing spider tissues for light and confocal microscopy, which give high-quality sections for morphology studies and the possibility of whole gland scanning, respectively. The anatomy, morphology and ultrastructure of the major ampullate glands of four distantly related21,22 (Figure 1) species from the Araneidae, Agelenidae,

Figure 1. Schematic spider evolutionary tree showing the relationship of the four species studied, given in italics in the bottom row. E. australis and Tegenaria sp. both belong to the RTA clade, while A. diadematus and N. clavipes belong to the Orbiculariae. These two groups diverged approximately 230 million years ago (MYA).21,22 A. diadematus and N. clavipes are both orb weavers that diverged approximately 175 MYA,21,22 while Tegenaria sp. are funnel weavers and E. australis are nursery web spiders.

Nephilidae, and Pisauridae families as well as the overall morphology of the opisthosomas of two spider species from the Araneidae and Agelenidae families are studied. Furthermore, by using immuno transmission electron microscopy (TEM), the localization of the NT in major ampullate silk glands and dragline silk of Euprosthenops australis is determined.

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MATERIALS AND METHODS

Animals. Ten adult female and male Tegenaria sp. and 10 adult female Araneus diadematus were collected in Sweden during August and September. Eight adult female E. australis were collected in South Africa during December and January, and 10 adult female Nephila clavipes were collected in Florida, USA during September to November. Spiders were kept in individual containers and fed flies, crickets, and water. Generation and Analysis of NT Antibodies. Rabbits were immunized with recombinantly produced NT protein from E. australis15 by Capra Science AB, Sweden. IgG antibodies were purified from rabbit serum using a protein A sepharose column, after which NT antibodies were further captured on an NHS activated HiTrap Affinity column (Pharmacia) with bound recombinant NT. The specificity of the polyclonal NT antiserum was tested using several methods: (i) Dot blots on polyvinylidene difluoride (PVDF) membranes of major ampullate dope from E. australis as positive control and hemolymph as negative control (S-Figure 1), (ii) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (S2946

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Figure 2. Sagittal section of opisthosoma of female Tegenaria sp. stained with HE. Spinnerets (S) are visible to the lower left. Major ampullate (MA), aciniform (Ac), tubuliform (Tu), and pyriform glands (Py) are discerned. Egg cells are present close to the major ampullate gland. Hemolymph (H) and adipose tissue surrounds the silk glands and the hemolymph portion stains intensely eosinophilic. Arrows show examples of sharp borders between cell types in different glands. Scale bar = 100 μm.

Figure 3. Schematic image of major ampullate gland, showing, from left to right, the tail, sac, funnel, and s-shaped duct (having three limbs). The three different zones (A−C) discussed herein are indicated.

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BBInternational). Grids were stained with uranyl acetate and analyzed with a Hitachi H 7100 instrument. For confocal microscopy, major ampullate glands of female N. clavipes were immersion fixed in 4% paraformaldehyde for 4 h at room temperature, after which they were stained with 4′,6-diamidino-2phenylindole (DAPI, Invitrogen), 20 μg/mL in phosphate buffered saline (PBS) for 1 h. Subsequently, the samples were subjected to optical clearing.26 Optical clearing before scanning made it possible to scan the whole gland despite the significant depth of the sample (>1 mm). Briefly, the glands were incubated for 30 min intervals in increasing concentrations (10%, 25%, 50% an 97%) of thiodiethanol (TDE) in PBS, stored in 97% TDE overnight and then mounted in 97% TDE in PBS, in 35 mm glass bottom microwell dishes (MatTek part no:P35G-1.5-14-C) with a coverslip on top. Samples were scanned using a Leica TCS SP5 confocal and an inverted Leica DMI6000 microscope (Leica Microsystems GmbH, Wetzlar, Germany). A 10×/0.4 objective (dry) was used and the excitation wavelength was 405 nm with emission collection of 415−515 nm. Super-Z stage was used to enable scanning of the entire gland with 10 μm steps, the image stacks were obtained by collecting overlapping optical slices for the entire thickness of the preparation (approximately 1.2 mm) and Z-stack intensity compensation was used. Image format was 1024 × 1024 pixels and the frequency was 600 Hz. Data was analyzed by LAS AF software (Leica) and three-dimensional images created using the Imaris software (Bitplane).

RESULTS

Opisthosoma Histology. In sections of the spider opisthosoma, different silk gland types in different species were distinguished by their morphology, size, and staining affinities for HE (Figure 2) and Azure blue (S-Figure 4).9,10 Very sharp borders between epithelial cells with differently stained granules, described for the major ampullate gland in detail below, can also be seen in aciniform and pyriform glands (arrows, Figure 2). Hemolymph stains intensely pink with HE (Figure 2) and light blue with Azure blue (S-Figure 4), and egg cells are very large and stain light pink-purple with HE (Figure 2) and light blue with Azure blue (S-Figure 4). The Major Ampullate Tail and Sac Is Composed of Three Cell Types. The major ampullate gland consists of three distinct regions:27 a tail region, a sac region, and an s-shaped duct (Figure 3). We used optical clearing26 of whole glands, which allowed us, for the first time, to build a three-dimensional image of an entire major ampullate gland (S-Figure 5A and B). Our light and electron micrographs reveal that there are three different cell types localized to three separate zones in the major ampullate tail and sac in Tegenaria sp., A. diadematus, E. australis, and N. clavipes (Figures 4, 5, and 6). The tail and the beginning of the sac (denoted zone A, Figure 3) are composed 2947

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Figure 4. Azure blue stained sagittal section of A. diadematus opisthosoma, showing the major ampullate gland tail and sac and the three different cell types characteristic of zone A-C. Note the very sharp border between the zones (arrow). All cell types have basally positioned nuclei (arrow heads). The black box in the inset indicates the magnified area. Scale bar = 30 μm.

Figure 6. TEM cross-section of E. australis major ampullate tail and sac simple columnar epithelium. In all three parts of the figure, the lumen (Lu) is to the left and basal parts of the cell to the right. Three cell types are seen (zones A, B, and C) in tail and sac of an E. australis major ampullate gland. Cells of all zones have basal nuclei (arrow heads) and there are microvilli (MV) at the apical cell membrane. Zone A cells are around 45 μm tall. Granules of different sizes and electron densities can be found throughout these cells. Granules are bigger toward the basal part. There is an extensive rough endoplasmic reticulum (arrows). In zone B, cells are up to 60 μm tall and contain granules of different sizes and electron densities. In zone C, cells are up to 60 μm tall and granules are quite similar in size and very electron dense. Scale bars = 5 μm.

of one type of cells, while the middle and the distal parts of the sac are composed of a second and third type of cells (zones B and C, Figure 3), respectively. All three cell types form a single tall columnar epithelium. The borders between the different cell types are very sharp (Figure 4). The secretions of the three zones differ in staining intensity and form distinct layers in the lumen (Figure 5). In zone A, cellular granules are big, somewhat irregular in shape and are larger in the basal portion of the cell (around 5 μm) than in the apical portion of the cell (around 2 μm) and stain weakly with HE and Azure blue (Figure 5). Zone A nuclei are located in the basal part of the cell, and the content in the glandular lumen is weakly stained (Figure 5). In zone B, the cells are filled with smaller, more evenly sized granules (around 3 μm) that stain light pink with HE and light blue with Azure blue. Nuclei are located at the basal portion of the cell and the luminal content close to the apical cell membrane stains similarly to granular contents (labeled II, Figure 5). In zone C, granules are small (around 2− 3 μm), homogeneous, and intensely stained with HE and Azure blue. Nuclei are basally located, and a layer at the apical cell membrane stains as the intracellular granules (labeled III, Figure 5). The three cell types and their characteristic granules discerned by LM (Figure 5) can be distinguished also by TEM (Figure 6). In zone A, the cells are about 45 μm tall and contain granules of different electron densities and sizes. In line with the LM results, larger granules are present in the basal portion of the cell, and smaller granules closer to the apical cell membrane. In zone B, cells are taller than in zone A (around 60

Figure 5. Histology of major ampullate tail and sac epithelium. Three cell-types are discerned in sections stained with HE (top three rows) and Azure blue (lower row). In each column, epithelium from zones A−C in major ampullate glands from four different spider species, Tegenaria sp., N. clavipes, E. australis, and A. diadematus (from top to bottom), are shown. Note the three layers (I, II, and III) in the luminal content, added by zone A, B, and C, respectively. Nuclei are basally positioned in all three zones (arrow heads). Lumen (Lu). Scale bars = 20 μm.

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μm), and their nuclei are more basally positioned. Granules vary in size, shape, and electron density throughout the cell. Here there are clearly visible tight junctions at the apical end of the cells (S-Figure 6), and there is more electron dense content in the gland lumen compared to zone A. A thin electron dense layer (∼0.5 μm) is present close to the apical part of the epithelium (Figure 7). In zone C, the third cell type is about 60

appearance, apical of where the acellular cuticular intima starts (S-Figure 7). The cuticular intima surrounds the lumen all along the duct to the spinneret. It is composed of alternating electron dense and electron lucent areas (Figure 8), giving a

Figure 8. Ultrastructure of the major ampullate duct. Upper: TEM cross-section of the duct of an E. australis major ampullate gland. The cuticular intima (arrow) lines the lumen, and microvilli (arrow heads) are found at the cell surfaces. Boxes indicate the location of the magnifications shown in the lower row. Lower left: Small vesicles are seen in between the microvilli and the cuticular intima. Lower right: The cuticular intima displays a pore-like structure, with alternating electron dense (D) and electron lucent (L) areas. Scale bars: top = 5 μm, bottom = 200 nm.

pore like appearance previously described by Bell and Peakall.27 There are abundant microvilli lining the epithelial cells of the duct (Figure 8). The duct decreases in diameter from the funnel to the spinneret (S-Figure 8) and the cells lining the third limb of the duct differ from those lining limbs 1 and 2, forming a taller single layered epithelium (S-Figure 8). A similar electron dense layer as can be seen in the lumen of zones B and C is present also in the lumen of the duct (Figure 9).

Figure 7. NT is present in granules (G) in zones A and B, but not in zone C. TEM cross-section of the A zone (top), B zone (middle), and C zone (bottom) of an E. australis major ampullate gland immunolabeled for NT, using secondary antibodies with 10 nm gold particles. Granules in both the A and B zones contain NT, while granules in zone C do not. The lumen (Lu) of the gland in all zones contains NT. In the B and C zones, a thin electron dense layer (ED) is present close to the apical cell membrane (arrows point at the cell membrane). Also this layer contains NT. Microvilli (MV). Scale bars = 0.5 μm.

μm tall, and the nuclei are located basally. Granules are evenly sized and very electron dense. The bulk of the luminal contents is in general more electron dense than seen in zone B. The electron dense luminal layer identified in zone B is also present in zone C. In all three zones, the apical cell membrane forms microvilli. Zone A cells contain abundant rough endoplasmic reticulum (rER) and plenty of mitochondria associated with the rER. The rER is less abundant in zone B cells, and difficult to find in zone C cells (Figure 6). We did not observe any significant differences in the appearance of the epithelial cells of spiders that were silked prior to sacrifice and those that were not. Prolonged silking may result in larger morphological differences, as has been previously described by, e.g., Plazaola et al.28 The Major Ampullate Funnel and Duct. The major ampullate sac is connected to the duct via a funnel (Figure 3, SFigure 7). The appearance of the tissue in the funnel is different from that of the sac and tail, and also from that of the duct (see further below). In the funnel, there are basally located nuclei, and the structures closest to the lumen display a fibrillar

Figure 9. TEM cross-section of the duct of an E. australis major ampullate gland immuno-labeled for NT (detected with 10 nm gold particles). Cuticular intima to the left (striated), lumen (Lu) to the right. No gold particles are seen in the cuticular intima, while the content of the lumen including the electron dense layer (ED) contains gold particles and thereby NT. Scale bar is 0.5 μm.

Distribution of the NT in Major Ampullate Silk Glands and Silk Fibers. TEM on immuno-labeled silk glands and fibers show that the NT is present in zone A and B granules, in the lumen of the gland and the duct, as well as in the final fiber (Figures 7, 9, and 10). In zone A, all granules contain the NT and in zone B most granules contain the NT (Figure 7), while cells in zone C contain no granules that stain positively with NT antibodies (Figure 7). The dope (including the electron 2949

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electron dense and appears as a distinct continuous layer through the funnel and duct to the final fiber where it forms an outer layer (skin) that surrounds the A-zone derived core (Figures 7, 9, and 10). Faster polymerization of the skin layer than the core has been proposed to prevent mixing of the two layers in the duct.34 By contrast, we could not detect a specific layer added by the cells in zone C by TEM. The granules of these cells are more electron dense than any component of the dope and/or fiber. Furthermore, since the B-zone secretion contains NT, while the C-zone granules does not (cf. below), it is unlikely that the C-zone adds material to the luminal dope. Instead the cells in the C-zone may produce membrane bound components that are retained in the microvilli of the cells, which, like the granules, stain intensely with HE and Azure blue (Figure 5). By using recombinant NT from E. australis MaSp1 we generated an antiserum that specifically recognizes the NT (SFigures 1−3). The antibody enabled us to unequivocally show that the NT is present in granules of cells in zone A and B (Figure 7), in the dope (Figure 9), and in the dragline silk (Figure 10). The electron dense granules of zone C, by contrast, lack NT (Figure 7) and therefore likely lack spidroins altogether. The architecture of the dragline silk fiber has been debated, suggesting that it may vary between species but probably also reflecting difficulties in preserving native morphology after preparation for structural studies and/or that different experimental techniques were used. The presence of from two to five layers has been proposed, as well as the complete lack of a layered structure.34−42 The localization of MaSps in the dragline silk fiber has previously been determined by using either antibodies specific for the MaSp1 and MaSp2 CT,18 respectively, or antibodies that recognize the repetitive parts of MaSp1 and MaSp2, respectively.34,35 MaSp1 and MaSp2 were shown to be present in a core region, surrounded by a thin layer of unknown composition (skin)18,34,35 Our observations in the tail, sac, duct, and fiber (Figures 5−7, 9, and 10) are compatible with a two-layered model of the silk fiber in which the A-zone produces the core, and the B-zone the skin. The distribution of the spidroin NT in the fiber is homogeneous, which suggests a different distribution of spidroins than previously reported,35 and our data show that the skin layer contains spidroins. The reasons for the discrepancy between our results and those reported by Sponner et al.,18,34,35 where MaSp1 and MaSp2 could not be detected in the skin of the fiber, is not evident. The CT is less conserved between spidroins than the NT, and CT antibodies may thus not recognize CTs from tentative MaSp isoforms. The repetitive region is characteristic for different spidroins, but it is always composed of similar alanine- and glycine-rich repeats, which makes it difficult to predict the binding of repetitive antibodies to other spidroin types and isoforms. The NT, on the other hand, is the most conserved part of the spidroin,11,21,22 and our NT antibody may therefore recognize the NT from other spidroin types and from previously unknown MaSp isoforms.

Figure 10. TEM cross-section of an E. australis dragline silk fiber immuno-labeled for NT. The gold particles (10 nm) are evenly distributed. An electron dense thin outer layer (arrows) of the fiber is visible. Scale bar = 200 nm.

dense layer) in the lumen of the tail, sac and duct contains the NT. As expected, the cuticular intima in the duct shows no detectable NT (Figure 9). The electron dense layer forms the skin in the final fiber, while the material produced by zone A forms the core. In the dragline silk fiber from E. australis, the NT is evenly distributed in the core and is also present in the outer layer (skin) (Figure 10). There was no noticeable difference in the localization of the NT between individuals that had undergone silking before sacrifice, and those that had not. In Western blot of dissolved silk fibers from E. australis, the NT is detected in large molecular weight proteins (S-Figure 2B). Additionally, the NT antibody detects a band corresponding to a molecular weight around 45 kDa. This protein could potentially correspond to the recently discovered short dragline silk protein MaSp1s.29

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DISCUSSION A commonly held view of the major ampullate tail and sac morphology is that there are two zones (A and B) representing two different epithelial cell types. Numerous studies on spiders from different families support the presence of one cell type in the tail and another one in the sac.8,9,25,30−32 Notably, several orb weaving species, among them Nephila sp., and Tegenaria sp. (both studied herein), are reported to have two cell types that secrete two different substances into the lumen of the major ampullate gland.8,9,30,32 However, there are also a few reports on three separate cell type regions in the tail and sac of orb weaving spiders.32,33 We show that the tail and sac of the major ampullate glands of distantly related Araneidae, Nephilidae, Agelenidae and Pisauridae species (A. diadematus, N. clavipes, Tegenaria sp., and E. australis), harbor three types of epithelial cells appearing in distinct zones (A, B, and C). The respective cell types have different heights, specific positions of nuclei, and contain granules of different sizes, contents, and distribution (Figures 4−7). Thus, the presence of three different cell types in the tail and sac is likely a common feature of major ampullate glands of Entelegynae spiders (Figure 1). The bulk of the material found in the lumen of the tail, sac, and the duct originates from zone A, in line with earlier reports that show that the bulk of the silk is synthesized in the tail of the gland.27,28 The B-zone secretion is added to the A-zone material, thus forming two layers in the dope as determined by both LM and TEM (Figures 4−7). The B-zone secretion is

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CONCLUSIONS The major ampullate tail and sac of four distantly related spider species consist of three cell types confined to three separate zones, A−C. The A and B zones secrete spidroins, while the C zone does not. The N-terminal domain of major ampullate spider silk proteins is present and evenly distributed in both the skin and core of the dragline fiber, as shown by immuno-TEM. 2950

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In order to properly mimic the mechanical properties of dragline silk in artificial replicas, future studies should focus on getting a deeper understanding of the physiological and molecular mechanisms acting in the silk production system.

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ASSOCIATED CONTENT

S Supporting Information *

Analyses of NT antibodies, Azure blue-stained section of A. diadematus opisthosoma, 3D confocal microscopy images of an N. clavipes major ampullate gland, transmission electron micrograph of zone B epithelium with clearly visible tight junctions, DAPI-stained confocal image and HE-stained light microscopy image of the funnel region of an N. clavipes major ampullate gland, and cross-section of the three limbs of the sshaped duct from an A. diadematus major ampullate gland stained with Azure blue. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] or [email protected]. Tel: +46 709 744 888. Author Contributions

M.A. and A.R. performed experiments; M.A., A.R., and J.J. wrote the manuscript; all authors analyzed data and commented on the manuscript. Notes

The authors declare the following competing financial interest(s): A.R. and J.J. are shareholders in Spiber Technologies AB.

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ACKNOWLEDGMENTS The authors would like to thank Astri and John Leroy for collecting E. australis, Kerstin Nordling and Erik Hermansson at the Dept. of NVS at Karolinska Institutet, Dr. Gunilla T. Westermark at the Dept. of Medical Cell Biology at Uppsala University, Dr. Kay Thomas at the Rockefeller University’s Bioimaging Center, Dr. Thomas Huber at the Rockefeller University, and Gunilla Ericson-Forslund at the Dept. of Anatomy, Physiology, and Biochemistry, Swedish University of Agricultural Sciences for exellent technical support and constructive scientific discussions. This work was funded by the Swedish Research Council, Spiber Technologies AB and Karolinska Institutets Forskningsstiftelser. A.R. and M.A. were supported by Nicholson Exchange Program and a Spiber Award, respectively.

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ABBREVIATIONS MaSps, major ampullate spidroins; NT, N-terminal domain; CT, C-terminal domain; TEM, transmission electron microscopy; HFIP, hexafluoroisopropanol; MYA, million years ago; LM, light microscopy; TDE, thiodiethanol; HE, hematoxylineosin; DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate buffered saline; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF, polyvinylidene diflouride.

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REFERENCES

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