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Immunolocalization of specific keratin associated beta-proteins (beta-keratins) in the adhesive setae of Gekko gecko
Tissue and Cell 45 (2013) 231–240
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Immunolocalization of specific keratin associated beta-proteins (beta-keratins) in the adhesive setae of Gekko gecko Lorenzo Alibardi ∗ Comparative Histolab and Dipartimento di Biologia, University of Bologna, Bologna, Italy
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Article history: Received 1 October 2012 Received in revised form 25 January 2013 Accepted 28 January 2013 Available online 29 April 2013 Keywords: Geckos Adhesive pads Beta-proteins Immunoblotting Immunocytochemistry
a b s t r a c t The previous identification of 21 proteins in the digital setae transcriptome of Gekko gecko, 2 alphakeratins of 52–53 kDa and 19 beta-proteins (beta-keratins) of 10–21 kDa, has indicated that most of setal corneous proteins are cysteine-rich. The production of specific antibodies for two of the main beta-protein subfamilies expressed in gecko setae has allowed the ultrastructural localization of two beta-proteins indicated as Ge-cprp-9 (cysteine-rich) and Ge-gprp-6 (glycine-rich). Only Ge-cprp-9, representing most of the 16 cysteine-rich beta-proteins, is present in the oberhautchen, setae and in the terminal spatula where adhesion takes place, supporting the previous expression study. Instead, the glycine-rich betaproteins (Ge-gprp-6), representing the 3 glycine-rich beta-proteins of digital epidermis is only present in the stiff beta-layer of the digital scales and in the thin beta layer of the pad lamella sustaining the setae. Ge-cprp-9 is representative for most of the remaining 15 cys-rich proteins (Ge-cprp 1–16) and may have a structural and functional role in the process of adhesion. Most of the cysteine-rich setal proteins have a net positive charge and it is here hypothesized that these proteins may induce the formation of dipoles at the surface interface between the spatula and the substrate, enhancing the van der Waals forces and therefore adhesion to the substrate. The selection and improvement of these proteins during the evolution of geckos may have represented a successful factor for the survival and ecological adaptations of these climbing lizards. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The climbing ability of many geckos and few other lizard species allow these reptiles the exploitation of an arboreal lifestyle otherwise denied to other lizards (Maderson, 1970; Bauer, 1998; Russel, 1986, 2002; Russel and Johnson, 2007). The ability to climb derives from some anatomical and functional adaptations but it is mainly centered on the evolution of microscopic devices located on modified digital scales, the pad lamellae that form on their surface the adhesive setae (Fig. 1A and B). The latter are bristles of 0.5–3 m wide and 8–120 m long in different species, which endings (spatulae) are able to instantaneously stick on a substrate through mainly the action of van der Waals interactions and be released from the substrate by variation of the attachment angle of the spatula (Autumn et al., 2000, 2002; Autumn and Peattie, 2002; Autumn and Gavish, 2012). Detailed morphological studies have shown that the setae of the adhesive pads are an outstanding modification of the oberhautchen layer, the more external layer of the epidermis present in lizards and snakes (Maderson, 1964, 1970; Ernst and
∗ Corresponding author. Fax: +39 0512094286. E-mail address: [email protected] 0040-8166/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tice.2013.01.002
Ruibal, 1966; Hiller, 1972; Alibardi, 1999, 2009). These studies have shown that the small spinulae of initial cells of the oberhautchen layer in both developing or regenerating pad lamellae growth and enlarge within the cytoplasm the cells of the clear (or granulated) layer. The identification of the proteins making the setae has required numerous biochemical and immunological analysis (Alibardi and Toni, 2005; Rizzo et al., 2006; Toni et al., 2007) but has been in part solved by using molecular biology techniques (Dalla Valle et al., 2007; Hallahan et al., 2009). The latter studies have indicated that only 3 glycine-rich beta-proteins (Ge-gprp-6, 7, 8) of 17–21 kDa are present in the setal transcriptome of G. gekko while at least 16 cysteine-rich beta-proteins of 10-kDa (Ge-cp-1–16) form most of the expressed corneous proteins of the setae (Fig. 1, Table 1). The latter studies have indicated that in three species of geckos (Tarentola mauritanica, Hemidactylus turcicus and Gecko gekko) the proteins isolated from their digits belong to two main subfamilies: glycine-rich or High Glycine proteins (HgG, cysteine < 4%, glycine > 20%) and cysteine-rich or High Glycine–Cysteine Proteins (HgGC, cysteine > 10%, glycine < 18%) (Dalla Valle et al., 2007; Toni et al., 2007; Hallahan et al., 2009; Alibardi, 2009, Table 1). Another short beta-protein (87 amino acids), indicates as serine–treoninerich (Ge-strp-1) and little expressed was also found in the pad
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Fig. 1. Schematic drawings representing a digit (A) with the apical claw and the pad (green) made of lamellae which section is shown in (B). The central image shows a manual alignment of the sequences of all the beta-proteins detected from the transcriptome of G. gekko (Hallahan et al., 2009) distinguished in three glycine-rich beta-proteins and 16 cysteine-rich beta-proteins (Glycines are colored in red, Cysteines in green, Proline in blue and Serines in yellow). The high homologous central region, the core-box, is boxed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
lamellae (Hallahan et al., 2009). In the latter study, although the presence of the two alpha-keratins of 52–53 kDa has been detected in the epidermis of pad lamellae, their specific localization in setae is unlikely as indicated from the absence of any immunolabeling for alpha-keratins in setae using immunocytochemistry. Despite the above information it remains unknown which of the 16 cysteine-rich beta-proteins is truly localized in G. gekko setae and in particular in the terminal spatula. Therefore in the present study we have done the ultrastructural immunolocalization for some of these proteins like it has been successfully done for the setae of the climbing iguanid lizard A. carolinensis (Alibardi et al., 2012; Alibardi, 2012a) and other geckos (Alibardi, 2012b). Previous in situ hybridization analysis indicated that glycinerich beta-proteins were also expressed in the beta-layer of gecko pad lamellae although also cys-rich beta-proteins might also be revealed by the probes utilized, directed to the conserved coding region of the gene (Dalla Valle et al., 2007; Alibardi et al., 2007). The knowledge of the specific proteins forming the setae and the spatulae in geckos is important for further analysis of the chemical physical characteristics of these proteins that give rise to setal flexibility and spatular adhesiveness. Recent studies on the lizard Anolis carolinensis and three species of geckos have identified
two main cysteine-rich beta-proteins forming the setae and spatulae, out of the 17 total cysteine-rich beta proteins present in the genome of this species (Dalla Valle et al., 2010; Alibardi et al., 2012; Alibardi, 2012a,b). Using two epitope-specific generated antibodies the present study shows the immunolocalization of gecko-specific beta-proteins at the ultrastructural level, and allows the hypothesis that the mechanism of adhesion depends not only from the setal nano-dimensional and physical properties of the setae but also from their electrical charge. 2. Materials and methods 2.1. Antibody selection The epitopes utilized in the present study to produce specific antibodies were selected on the different core-box regions of two representative beta-proteins out of the remaining sequences (Fig. 2) that we previously isolated (see Fig. 5 in Hallahan et al., 2009, and Fig. 13 in Alibardi, 2009). As it is seen in Fig. 2, the amino acid similarity among all the core boxes in the 19 betaproteins isolated from the setae varies from 50 to 95%. The epitopes were selected from two beta-proteins highly expressed in the setae
Table 1 Amino acid composition of the 19 beta-proteins and 2 ␣-keratins characterized in G. gekko transcriptome (Hallahan et al., 2009). HgG (High Glycine beta proteins, red color); HgGC (High Glycine Cysteine beta proteins, green color; ␣k, alpha-keratins, orange color).
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Fig. 2. Amino acid sequence similarity (%ages) among the core-boxes of the 16 cysteine-rich (green) versus the three glycine-rich (pink) beta-proteins of the gecko transcriptome. The changes in amino acids in comparison to the selected epitopes, Ge-cprp-9 in the left colum and Ge-gprp-6 in the right column, are colored in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
that were considered representative for most of the other proteins (see Fig. 5 in Hallahan et al., 2009). One epitope is present in the small cysteine-rich beta-protein Ge-cprp-9 (SEVTIQPPPCTVVVPGPVLA; AN, ABU98602.1) containing 117 amino acids for a deduced MW of 13.1 kDa, and the specific antibody directed to it was produced in rabbit. The other epitope (AEVLIQPPPSVVTLPGPILS; AN ABU98593.1, 60% epitope identity with Ge-cprp-9 epitope) is present in the larger glycine-rich protein Ge-gprp-6, a protein of 176 amino acids with a deduced MW of 16.9 kDa, and the antibody was made in guinea pig. The peptides and the antibodies production, including their affinity purification, were obtained through a Biotechnology Company (Thermofisher Openbiosystems, USA), and small aliquotes were supplied from Dr. Peter Newiarowsky, University of Akron, OH, USA.
for alpha- and beta-keratins (GE, Healthcare, Milan, Italy). Briefly, for western blotting, the proteins separated in SDS-PAGE were transferred to a polyvinylidene difluoride membrane using a blotting apparatus (Biorad). After Western blot, membranes were stained with Ponceau red to verify the protein transfer, the membranes were destained in buffer, and then incubated with primary antibodies directed against the epitopes of Ge-cprp-9 and Gegprp-6 (diluted 1:2000–3000). In controls the primary antibodies were omitted. After rinsing, the membranes were incubated with HRP-conjugated anti-Rabbit (for Ge-cprp-9) or anti-guinea pig (for Ge-cprp-6) secondary antibodies (1:20,000), and the detection was performed using the enhanced chemioluminescence procedure developed by the manufacturer (ECS, EuroClone).
2.3. Immunocytochemistry 2.2. Western blotting The antibodies were tested on proteins extracted from molts of the digits of G. gekko using the method by Sybert et al. (1985). Briefly, the molts were homogenized in 8 M urea/50 mM Tris–HCl (pH 7.6)/0.1 M 2-mercaptoethanol/1 mM dithiothreithol/1 mM phenylmethylsulphonyl fluoride. The particulate matter obtained after homogenization was removed by centrifugation at 10,000 × g for 10 min, and protein concentration of the surnatant was assayed by the Lowry or Bradford methods. The electrophoresis analysis of the extracted proteins was carried out after 20 g of proteins were loaded in each electrophoretic lane, and the proteins were separated in 15% of SDS-polyacrylamide gels (SDS-PAGE) as previously indicated (Alibardi et al., 2012). Marker proteins were in the 7–100 kDa range of molecular weight
The experiments were conducted on three adults of Tokay gecko (Gekko gecko), purchased in authorized pet shops, as reported in previous studies (Alibardi, 2009, 2012a,b). Tips of the digits of sacrificed animals, containing the claw and the adhesive pads were immediately fixed for 5–8 h in cold (0–4 ◦ C) 4% paraformaldehyde in 0.1 M Phosphate buffer at pH 7.4, dehydrated in ethanol, and embedded in Bioacryl Resin at 0–4 ◦ C under UV light for 2 days. Tissues were sectioned with an ultramicrotome to obtain 2–4 m thick sections that were stained with 0.5% Toluidine blue for light microscopy analysis. For light microscopy immunocytochemistry the sections were incubated overnight at 0–4 ◦ C or for 4 h at room temperature with the antibodies diluted 1: 200 in Buffer (Tris 0.05 M at pH 7.6 containing 1% BSA). The sections were rinsed in buffer and incubated for 60 min at room temperature with a fluorescein-conjugated anti-rabbit or anti-guinea pig
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antibodies (FITC, Sigma, diluted 1:100), rinsed in the buffer, mounted in Fluoroshield anti-fading medium (Sigma), and observed under a fluorescence microscope equipped with a fluorescein filter and a digital camera. Selected sections of 60–90 nm thickness collected on Nickel grids, were used for the immunogold electron microscopic analysis. The sections were incubated overnight at 0–4 ◦ C in the primary antibodies diluted in 0.05 M Tris–HCl buffer at pH 7.6, containing 1% Cold Water Fish Gelatin. In controls, the primary antibody was omitted in the overnight incubation. The sections were rinsed in buffer and incubated for 1 hour at room temperature with anti-rabbit or anti-guinea pig Gold-conjugated secondary antibody for beta-proteins detection (Sigma, 10 nm gold particles). In some immunoreactions both the antibodies were used on the same section in double-labeling immunodetection. The primary rabbit antibody was detected using gold particles of 5 nm, while the guinea pig primary antibody was detected using gold particles of 15 nm. Grids were rinsed in buffer, dried and stained for 5 minutes with 2% uranyl acetate, and then observed under the electron microscope Philips CM-100 or Zeiss C10. 3. Results 3.1. Western blots Two main immunoreactive bands for the Ge-gprp-6 epitope were found at 18 and 25 kDa and a minor band appeared at 30 kDa, and much weaker bands around 45 kDa (Fig. 3, first lane, G6). A single large band at 14–16 kDa was instead seen for the detection of the Ge-cprp-9 epitope (Fig. 3, second lane, G9). No labeled bands were seen in the controls for CG6 and CG9 (last two lanes in Fig. 3). 3.2. Immunocytochemistry The epidermis of normal scales in geckos consists in a dead corneous layer composed by a pale beta-layer and a denser alpha-layer, followed by a viable epidermis made of one–two irregular layers of keratinocytes in resting phase or more stratified keratinocytes layers during epidermal renewal (data not shown, but see details in Maderson, 1964, 1966). Immunocytochemical analysis of setae showed that mainly setae were immunofluorescent with the Ge-cprp-9 antibody (Fig. 4A) while only the corneous beta-layer but not the setae appeared immunofluorescent for the Ge-gprp-6 antibody (Fig. 4B). Controls were completely unlabeled (Fig. 4C). Another antibody directed to the unique beta-protein Ge-sprp-1 protein did not produce any labeling in both light and ultrastructural immunocytochemistry, and this beta-protein is not further considered in the present study.
Fig. 3. Western blot results of protein extract from the molts of digital pads. Lane 1 for Ge-gprp-6 (G6), lane 2 for Ge-cprp-9 (G9), lane 3 for the control of G6 (CG6), and lane 4 for the control of G9 (CG9). The numbers on the right indicate the indicative molecular weight in kilodaltons (kDa) (see text for descriptions).
While in other body scales in G. gecko a random sampling of the skin generally yielded the epidermis in resting or early renewal stage, the epidermis of pad lamellae was often in renewal stage histology. In the latter condition, the outer setal generation was localized at the apical tip of the lamella and the inner setae generation was forming underneath the outer corneous layer (Fig. 5A). This common feature indicated a frequent cycling of the epidermis in pad lamellae. In fact, in case the epidermis was in resting stage, no inner setae formation was seen underneath the corneous layer of the pad lamella (arrowhead in Fig. 5A). The inner setae appear as bristles extending from the basal oberhautchen layer toward the outer corneous layer. The details of the inner setae of G. gecko showed that they were partially pigmented (forming dark bands) and that they were branched toward their apex, in a region contacting the layer of clear cells. The cytoplasm of clear cells was interspersed among the elongating setae (Fig. 5B). The ultrastructural immunogold labeling for the Ge-cprp-9 protein showed a labeled oberhautchen layer forming the base of setae, followed by a cytoplasm region devoid of corneous material but with a loose keratin meshwork (asterisk in Fig. 5C). This non-cornified region was followed underneath by a deep dense layer with the typical, mottled beta-keratin pattern of electronpale mixed to electron-denser material, and was diffusely labeled or unlabeled with this antibody. The beta-layer was followed underneath by 2–4 narrow and more electrondense corneocytes forming the mesos-region (Fig. 5C), and then by thicker corneocytes of the alpha-layer (data not shown).
Fig. 4. Immunofluorescence of pad lamella for Ge-cprp-9 (Gc9 in A), for Ge-gprp-6 (Gg6 in B), and of a control section (CO). Bar in all pictures is 10 m. (A) Shows immunoreactive setae (se) and immunonegative beta-layer and epidermis (e). (B) Shows that only the corneous beta-layer (c) at the base of setae (se) is labeled while the setae are immunonegative. (C) Shows no labeling (se, setae position; c, corneous layer position) in a control section.
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Fig. 5. Light microscopy view of setal morphology (A and B) and ultrastructural immunocytochemistry for Ge-csrp-9 antibody (Gc9 in C–F). (A) Section of a pad lamella showing the inner setae (is) and the apical free margin. Bar, 15 m. (B) details of some setae which base is in the narrow oberhautchen and tips form branches (arrow), Bar, 5 m. (C) Sectioned corneous layer sustaining the setae (the arrow indicates a cross-sectioned setae) that comprises a labeled oberhautchen, an intermediate pale region (asterisks), a lower beta-layer (b), and the mesos-layer (m). Bar, 150 nm. (D) Four cross-sectioned setae (se) are intensely labeled while the surrounding spaces that were occupied from the clear layer cytoplasm are immunonegative. Bar, 100 nm. (E) Detail on setae showing gold particles mostly present over darker bundles or material present inside the setae (arrow). Bar, 100 nm. (F) control section (CO) on bundles (arrow) within a seta that are immunonegative. Bar, 100 nm.
Both the base of setae attached to the oberhautchen cells as well as the stalk or intermediate region of the setae and the branching region, were intensely immunolabeled with the Ge-cprp-9 antibody (Fig. 5D). The gold particles were especially or exclusively concentrated over the denser areas of the setae and less frequently over electron-paler areas, an indication that other components are present in these regions (Fig. 5E). No labeling was detected in the cytoplasm of cells forming the clear layer, in the inter-setae spaces or in other epidermal and dermal tissues using the employed antibody. Also the
controls were immuno-negative (Fig. 5F). Although with variable intensity, also the branching region of the setae up to the terminal spatula showed immunolabeling for Ge-cprp-9 (Fig. 6A). In particular, gold particles were seen both inside the setae and on their surface. At the proximal base of pad lamellae where setae became smaller, or in the oberhautchen layer of the other non specialized, digital scales, the setae shortened and transformed in spinulated pointing structures. Immunolabeling for Ge-cprp-9 was present in spinulae, and in the electron-dense and paler areas of the beta-layer (Fig. 6B).
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Fig. 6. Immunogold labeling for Ge-cprp-9 (Gc9, (A) and (B)), for Ge-gprp-6 (Gg6, (C)), and double labeling (Gecprp-9 + Gedprp-6, (D)). (A) terminal part of branched setae (se) with immunolabeled spatulae (arrows). Bar, 100 nm. (B) Immunolabeled mature beta (b) and superficial oberhautchen spinulae (sp) of a digital scale. Bar, 150 nm. (C) Immunolabeling only present over the beta-layer (b) of digit scale while the merged superficial oberhautchen and spinulae (sp) and the mesos layer (m) are not labeled. Bar, 150 nm. (D) The double labeling shows the smaller particles (5 nm detecting Gc9) over the oberhautchen and spinulae (sp) while the larger gold particles (15 nm detecting Gc6) labele only the underlying beta-layer (b). Bar, 100 nm.
A sharp difference was observed using the Ge-gprp-6 antibody on both pad lamellae and normal digital scales. In fact, this antibody specifically labeled only the beta-layer but not the oberhautchen layer merged to the beta-layer and its spinulae in the digital scales or in the setae of the pad lamellae (Fig. 6C). The double-labeling immunolocalization (5 nm gold particles for Ge-cprp-9 and 15 nm gold particles for Ge-gprp-6) confirmed that the oberhautchen only immunolocalized Ge-cprp-9 (Fig. 6D). In pad lamellae immunostained with the Ge-gprp-6 antibody the only labeled substructure was the thin beta-layer present below the pale region (asterisks in Fig. 7A–C), exactly the reverse localization seen using the Ge-cprp-9 antibody (compare Fig. 5C and Fig. 7A and B). The labeling in the beta-layer was mainly seen over the paler
areas among the smaller electron-dense areas were filaments of 7–8 nm thickness were often seen (Fig. 7C). The oberhautchen, pale layer, mesos and alpha-layers were immunonegative for the Gegprp-6 antibody. In particular, no labeling for Ge-gprp-6 was seen at the base, intermediate and apical regions of setae, suggesting absence of this epitope. 4. Discussion 4.1. Beta-protein distribution in setae The present study has shown the specific skin localization of representative highly expressed cysteine-rich and glycine-rich
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Fig. 7. Immunogold labeling for Ge-gprp-6 (Gg6) in pad lamellae. (A) Immunogold particles are only seen over the beta-layer (b) but are absent in the pale layer (asterisks) and its keratin bundles (k) or in the oberautchen (o) and setae (se). Also the underlying mesos-layer is negative (m). Bar, 150 nm. (B) higher magnification image of mature epidermal layers showing immunonegative setal base (se) merged with the oberhautchen (o), the intermediate (asterisk) layer containing a keratin bundles (k), and the labeled beta-layer (b) confining with an unlabeled mesos cells (m). Bar, 100 nm. (C) Higher magnification of the beta-layer (b) located underneath the intermediate layer (asterisk) showing that most gold particles decorate the paler areas. Bar, 100 nm.
beta-proteins that were characterized in previous studies in scales of the digits and of the specialized adhesive pad lamellae in G. gecko (Fig. 1A, Table 1, see Refs. Hallahan et al., 2009; Alibardi, 2009). The western blot analysis has indicated that the employed antibodies indeed recognize proteins in the expected range, although with a MW difference of 1–3 kDa from the deduced proteins (14–15 kDa vs deduced 11.3 kDa for Ge-cprp-9, and the 18 kDa band vs 17.6 kDa of the deduced Ge-gprp-6). The presence of other bands at 25 and 30 kDa may indicate form of aggregation or fragmentation of these proteins, as it frequently occurs using the employed extractive methods (Alibardi and Toni, 2005; Rizzo et al., 2006; Toni et al., 2007). Another possibility is that the antibody recognizes the corebox of the other two glycine-rich beta-proteins (Ge-gprp-7 and Ge-gprp-8) that possess a deduced MW respectively of 19 and 20.7 kDa (Alibardi, 2009). The ultrastructural study has indicated that while in mature normal scales in all body areas the oberhautchen layer is merged with the beta-layer (Fig. 8A), in most of the outer surface of pad lamellae, except toward the basal (hinge) and apical (tip)
regions, the oberhautchen layer is separated by a non-corneous layer from the beta-layer (asterisks in Fig. 8B). Although the antibodies were directed against the core-box regions of the two proteins, which showed a certain sequence similarity (50–95%, see Fig. 2), the immunological results clearly indicates that Gecprp-9 and Ge-gprp-6 proteins are localized in two different layers of the digital scales and adhesive pads of G. gecko (Fig. 9). In particular Ge-cprp-9, representing the cysteine-rich beta-proteins (Fig. 1), appears mainly localized in the oberhautchen and setae, including their surface, while Ge-gprp-6, a glycine-rich betaprotein, appears exclusive of the beta-layer of digital scales. Since the setae and the oberhautchen layer are much more developed then the beta-layer in pad lamellae of geckos, this explains why cysteine-rich beta-proteins are more abundant then glycinerich beta-proteins in the setal transcriptome (Hallahan et al., 2009). The present study has found that Ge-cprp-9 (19.7% of cysteine, 17.9% proline, and 12.8% glycine, see Table 1) is a major protein of spinulae, setae and of their spatula ending. The comparison of
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Fig. 8. Drawing illustrating the structure of the spinulated oberhautchen-beta-layer present in normal scales of digits or other body regions (A) in comparison to the structure of the oberhautchen-beta-layer found in the modified scales of pad lamellae (B). While in normal scales the oberautchen (ob) is merged with a relatively thick beta-layer (; arrow in (A)) in pad lamellae most of oberhautchen form long setae (arrow in (B)) while a non-cornified layer (asterisks) divides the oberhautchen from a thinner beta-layer along most of the outer scale surface (except by the tip and at the base where the oberhautchen and beta-layer are merged like in normal scales).
Ge-cprp-9 core-box with that of the remaining 15 sequences of cys-rich beta-proteins and of the three sequences of glycine-rich beta-proteins (Fig. 2, left column) however suggests that numerous of these proteins should also be recognized by this antibody. In fact, the amino acid identity of core-boxes varies from 70 to 90% for the cysteine-rich types so that also other of these beta-proteins are likely present in the setae. Differently, the lower identity of the core-box in the glycine-rich types (50–60%, see Fig. 2, left column at the bottom) suggests these proteins are less recognized from the Ge-cprp-9 antibody. The specific role for each one of these different beta-proteins in setae formation, structural and material properties for adhesion remains to be evaluated. The localization of cysteine-rich beta-proteins in the oberhautchen, spinulae and setae of G. gecko confirms similar findings using other antibodies against cysteine-rich beta-proteins in
geckos (G. gekko, T. mauritanica and H. turcicus, Alibardi, 2012b) as well as in the lizards A. carolinensis and A. lineatopus (Alibardi et al., 2012; Alibardi, 2012a). Conversely, the Ge-gprp-6 antibody very likely recognizes all the three glycine-rich proteins since the identity is 85–95% (Fig. 2, right column) while this antibody should recognize with lower specificity the cysteine-rich proteins since the identity falls to 55–70% in the cysteine-rich beta-proteins. Ge-gprp-6 contains 20.5% of glycine, 10.2% proline, but only 3.4% in cysteine (Table 1). Therefore all the three glycine-rich proteins are likely localized in the thin beta-layer present in the setae but mainly in the thicker betalayer present in normal digital scales (Fig. 8 A), or even more in the thick beta-layer of scales in other body areas such as in the head and tail. In conclusion, while glycine-rich beta-proteins appear localized in hard epidermal layers (beta-layer) those rich in cysteine appear more typical of epidermal layers where flexibility (oberhautchen and spjnulae or setae) is required. The disulphide bonds are therefore most likely utilized for building a deformable meshwork in the oberhautchen cells (recalling the elastin molecular structure) while the glycine-rich proteins form a resistant, hydrophobic and chromophobic texture in the beta-layer. Future chemical physical studies on the specific properties of these proteins will clarify this issue. 4.2. Hypothesis on the influence of Ge-cprp-9-like beta-proteins on setal adhesion The fine immunolocalization of specific beta-proteins in gecko setae and their surface allows for a (qualitative) hypothesis on the possible role of these proteins on the mechanism of adhesion. Since gecko adhesion is mainly due to van der Waals forces (Autumn et al., 2000, 2002; Autumn and Peattie, 2002; Autumn and Gavish, 2012) it is believed that the production of bio-inspired artificial devices with similar shape, dimension, organization and spacing of natural gecko setae and spatulae can replaced the biological material represented by gecko proteins (Berengueres et al., 2007; Ge et al., 2007; Seth et al., 2007). Like in previous studies on the
Fig. 9. Drawing illustrating the hypothetical influence of charged proteins on the setae surface. (A) Shows the net positive charges on the different beta-proteins of gecko setae (HgGCs and HgGs). In (B) the progressive details of the setae are illustrated starting from the hand or foot with the digital pads (1), sectioned and enlarged in 2 to show the pad lamella, which setae are enlarged in 3 to show the various layers of the epidermis and the terminal branching ramification of setae contacting the substratum (␣, alpha-layer; , beta-layer; ba, basal layer, , mesos-layer; obj, oberhautchen; sb, supra-basal layer). In 4 details of the localization of Ge-cprp-9 in setae/spatulae and oberhautchen, and of Ge-gprp-6 in the underlying beta-layer (separated by the intermediate layer, asterisks) are shown. The positively charged surfaces attract water while the charged spatula can induce temporary dipoles on its surface that influence van der Waals forces (see text).
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immunolocalization of setal proteins in A. carolinensis, T. mauritanica, and H. turcicus (Alibardi, 2012a,b), it appears that the deduced proteins in the setae and spatular ends of G. gekko may be electrically charged. In fact, assuming that these cysteine-rich proteins are major proteins of the setae, the Protparam Program at http://www.web. expasy.org/protparam/ for Ge-cprp-9 and for most of the other High Glycine Cysteine beta-proteins shows that they are positively charged (Fig. 9A). We also assume that most of these charges, 2–6 positive charges in each protein multiply for billions of these proteins present on the surface of setae, can produce a charged surface of contact with the substrate. The presence along the setae surface of proteins with the same charge (positive as indicated in Fig. 9B4) impedes that the setae aggregate during motion since they are repelled one to another by electrostatic charges of the same sign. In particular, the surface of the spatula with its charged molecules can interact with the substrate in 2 different ways: electrostatically for long-range distances (micrometers) and through van der Waals forces for short-range distances such as nanometers or Angstroms (Autumn et al., 2000, 2002; Autumn and Peattie, 2002; Autumn and Gavish, 2012). Initially the electrostatic forces (attractive) may prevail but as the spatula and the substrate get closer, below 10 Angstroms, van der Waals forces may become prevalent, especially if the charged proteins can induce dipoles on the surface of contact. The presence of interposed water molecules between the betaproteins of the spatula and the substrate that may be polarized increases the number of induced dipoles and therefore further enhances van der Waal forces and adhesion. It has been shown that the increase of relative humidity enhances gecko adhesion (Sun et al., 2005; Huber et al., 2005; Niewiarowski et al., 2008; Puthoff et al., 2010). Charged beta-proteins on the surface of both oberhautchen and the beta-layer can also trap water dipolar molecules producing capillary forces (Fig. 9B4). Capillary forces may aid adhesion in ways that are still not clear (Pesika et al., 2009). Furthermore, spatular material made of cysteine–glycine-rich beta-proteins may partially adsorb water to become softer when the humidity increases, and the spatula becomes more pliable to increases the surface of contact and therefore enhances adhesion (Huber et al., 2005; Niewiarowski et al., 2008; Pesika et al., 2009; Prowse et al., 2011; Puthoff et al., 2010). The hypothetical influence of electrically charged proteins on van der Waals interactions and material hydration affecting adhesion remains however to be experimentally demonstrated. In conclusion, the present study suggests that the largely unexplored chemical physical properties of the setal proteins of geckos are important for understanding the mechanism of adhesion. Future artificial adhesive materials should possess not only the microscopical features but also similar chemical physical properties present in the natural gecko cysteine-rich beta-proteins. Aside other anatomical–physiological features during gecko evolution (Russel, 1986, 2002; Bauer, 1998; Russel and Johnson, 2007), the selection of genes coding for the beta-proteins produced in the oberhautchen layer, the more external corneous layer of the epidermis, has represented a successful factor for the survival and radiation of these climbing lizards.
Acknowledgments The study was in part self supported (Comparative Histolab) and with a contribution of the RFO 2010 from the University of Bologna. I thank Dr. Peter Niewiarowsky, University of Akron, USA, for sending me some small amount of the antibodies against the epitopes selected against the core-box region of the Tokay gecko. Dr. Anna
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Segalla, University of Padova, helped with the electrophoretic and western blot analysis.
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Expression of beta-keratin mRNAs and proline-uptake in epidermal cells of growing scales and pad lamellae of gecko lizards. J. Anat. 211, 104–116. Alibardi, L., Segalla, A., Dalla Valle, L., 2012. Distribution of specific keratin associated beta-proteins (beta-keratins) in the epidermis of the lizard Anolis carolinensis helps to clarify the process of cornification in lepidosaurians. J. Exp. Zool. 318B, 388–403. Autumn, K., Liang, Y.A., Hsieh, S.T., Zesch, W., Chan, W.P., Kenny, T.W., Fearing, R., Full, R.J., 2000. Adhesive force of a single gecko foot-hair. Nature 405, 681–685. Autumn, K., Sitti, M., Liang, Y.A., Peattie, A.M., Hansen, W.R., Sponberg, S., Kenny, T.W., Fearing, R., Israelachvili, J.N., Full, R.J., 2002. Evidence for van der Waals adhesion in gecko setae. Proc. Natl. Acad. Sci. U.S.A. 99, 12252–12256. Autumn, K., Peattie, A.M., 2002. Mechanisms of adhesion in geckos. Integr. Comp. Biol. 42, 1081–1090. Autumn, K., Gavish, N., 2012. Gecko adhesion: evolutionary nanotechnology. Phil. Trans. R. Soc. Lond. A 366, 1575–1590. Bauer, A.M., 1998. Morphology of the adhesive tail tips of carphodactyline geckos (Reptilia: Diplodactylidae). J. Morphol. 35, 41–58. Berengueres, J., Saito, S., Tadakuma, K., 2007. Structural properties of a scaled gecko foot-hair. Bioinspiration Biomimetics 2, 1–8. Dalla Valle, L., Nardi, A., Toffolo, V., Niero, C., Toni, M., Alibardi, L., 2007. Cloning and characterization of scale beta-keratins in the differentiating epidermis of geckos show they are glycine–proline–serine-rich proteins with a central motif homologous to avian beta-keratins. Dev. Dyn. 236, 374–388. Dalla Valle, L., Nardi, A., Bonazza, G., Zuccal, C., Emeera, D., Alibardi, L., 2010. Forty keratin-associated -proteins (-keratins) form the hard layers of scales, claws and adhesive pads in the green anole lizard, Anolis carolinensis. J. Exp. Zool. 314B, 11–32. Ernst, V., Ruibal, R., 1966. The structure and development of the digital lamellae of lizards. J. Morphol. 120, 233–266. Ge, L., Sethi, S., Ajayan, P.M., Dhinojawala, A., 2007. Carbon nanotube-based synthetic gecko tapes. PNAS 104, 10792–10795. Hallahan, D.L., Keiper-Hrynko, N.M., Shang, T.Q., Ganzke, T.S., Toni, M., Dalla Valle, L., Alibardi, L., 2009. Analysis of gene expression in gecko digital adhesive pads indicates significant production of cysteine- and glycine-rich beta-keratins. J. Exp. Zool. 312B, 58–73. Hiller, U., 1972. Licht- und elektronenmikroskopische untersuchungen zur haftborstenentwicklung bei Tarentola mauritanica L. (Reptilia, Gekkonidae). Zoomorphology 73, 263–278. Huber, G., Mantz, H., Spolenak, R., Mecke, K., Jacobs, K., Gorb, S.N., Arzt, E., 2005. Evidence for capillarity contributions to gecko adhesion from single spatula nanomechanical measurerements. PNAS 102, 16293–16296. Maderson, P.F.A., 1964. Keratinized epidermal derivatives as an aid to climbing in gekkonid lizards. Nature 203, 780–781. Maderson, P.F.A., 1966. Histological changes in the epidermis of the Tokay (Gekko gecko) during the sloughing cycle. J. Morphol. 119, 39–50. Maderson, P.F.A., 1970. Lizard glands and lizard hands: models for evolutionary study. Forma Funct. 3, 179–204. Niewiarowski, P.H., Lopez, S., Ge, L., Hagan, E., Dhinojawala, A., 2008. Sticky gecko feet: the role of temperature and humidity. Plos ONE 3, e2192. Pesika, N.S., Zeng, H., Kristiansen, K., Zhao, B., Tian, Y., Autumn, K., Israelachvili, J., 2009. Geko adhesion pad: a smart surface? J. Phys. Condens. Matter. 21, 464132. Prowse, M., Wilkinson, M., Puthhoff, J.B., Mayer, G., Autumn, K., 2011. Effects of humidity on the mechanical properties of gecko setae. Acta Biomater. 2, 733–738. Puthoff, J.B., Prowse, M.S., Wilkinson, M., Autumn, K., 2010. Changes in material properties explain the effects of humidity on gecko adhesion. J. Exp. Biol. 213, 3699–3704. Rizzo, N.W., Gardner, K.H., Walls, D.J., Keiper-Hrynko, N.M., Ganzke, T.S., Hallahan, D.L., 2006. Characterization of the structure and composition of gecko adhesive setae. J. R. Soc. Interface 3, 441–451. Russel, A., 1986. The morphological basis of weight-bearing in the scansors of the tokay gecko (Reptilia: sauria). Can. J. Zool. 64, 948–955. Russel, A.P., 2002. Integrative functional morphology of the gekkotan adhesive system (Reptilia, Gekkota). Int. Comp. Biol. 42, 1154–1163.
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Immunolocalization of specific keratin associated beta-proteins (beta-keratins) in the adhesive setae of Gekko gecko Lorenzo Alibardi ∗ Comparative Histolab and Dipartimento di Biologia, University of Bologna, Bologna, Italy
a r t i c l e
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Article history: Received 1 October 2012 Received in revised form 25 January 2013 Accepted 28 January 2013 Available online 29 April 2013 Keywords: Geckos Adhesive pads Beta-proteins Immunoblotting Immunocytochemistry
a b s t r a c t The previous identification of 21 proteins in the digital setae transcriptome of Gekko gecko, 2 alphakeratins of 52–53 kDa and 19 beta-proteins (beta-keratins) of 10–21 kDa, has indicated that most of setal corneous proteins are cysteine-rich. The production of specific antibodies for two of the main beta-protein subfamilies expressed in gecko setae has allowed the ultrastructural localization of two beta-proteins indicated as Ge-cprp-9 (cysteine-rich) and Ge-gprp-6 (glycine-rich). Only Ge-cprp-9, representing most of the 16 cysteine-rich beta-proteins, is present in the oberhautchen, setae and in the terminal spatula where adhesion takes place, supporting the previous expression study. Instead, the glycine-rich betaproteins (Ge-gprp-6), representing the 3 glycine-rich beta-proteins of digital epidermis is only present in the stiff beta-layer of the digital scales and in the thin beta layer of the pad lamella sustaining the setae. Ge-cprp-9 is representative for most of the remaining 15 cys-rich proteins (Ge-cprp 1–16) and may have a structural and functional role in the process of adhesion. Most of the cysteine-rich setal proteins have a net positive charge and it is here hypothesized that these proteins may induce the formation of dipoles at the surface interface between the spatula and the substrate, enhancing the van der Waals forces and therefore adhesion to the substrate. The selection and improvement of these proteins during the evolution of geckos may have represented a successful factor for the survival and ecological adaptations of these climbing lizards. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The climbing ability of many geckos and few other lizard species allow these reptiles the exploitation of an arboreal lifestyle otherwise denied to other lizards (Maderson, 1970; Bauer, 1998; Russel, 1986, 2002; Russel and Johnson, 2007). The ability to climb derives from some anatomical and functional adaptations but it is mainly centered on the evolution of microscopic devices located on modified digital scales, the pad lamellae that form on their surface the adhesive setae (Fig. 1A and B). The latter are bristles of 0.5–3 m wide and 8–120 m long in different species, which endings (spatulae) are able to instantaneously stick on a substrate through mainly the action of van der Waals interactions and be released from the substrate by variation of the attachment angle of the spatula (Autumn et al., 2000, 2002; Autumn and Peattie, 2002; Autumn and Gavish, 2012). Detailed morphological studies have shown that the setae of the adhesive pads are an outstanding modification of the oberhautchen layer, the more external layer of the epidermis present in lizards and snakes (Maderson, 1964, 1970; Ernst and
∗ Corresponding author. Fax: +39 0512094286. E-mail address: [email protected] 0040-8166/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tice.2013.01.002
Ruibal, 1966; Hiller, 1972; Alibardi, 1999, 2009). These studies have shown that the small spinulae of initial cells of the oberhautchen layer in both developing or regenerating pad lamellae growth and enlarge within the cytoplasm the cells of the clear (or granulated) layer. The identification of the proteins making the setae has required numerous biochemical and immunological analysis (Alibardi and Toni, 2005; Rizzo et al., 2006; Toni et al., 2007) but has been in part solved by using molecular biology techniques (Dalla Valle et al., 2007; Hallahan et al., 2009). The latter studies have indicated that only 3 glycine-rich beta-proteins (Ge-gprp-6, 7, 8) of 17–21 kDa are present in the setal transcriptome of G. gekko while at least 16 cysteine-rich beta-proteins of 10-kDa (Ge-cp-1–16) form most of the expressed corneous proteins of the setae (Fig. 1, Table 1). The latter studies have indicated that in three species of geckos (Tarentola mauritanica, Hemidactylus turcicus and Gecko gekko) the proteins isolated from their digits belong to two main subfamilies: glycine-rich or High Glycine proteins (HgG, cysteine < 4%, glycine > 20%) and cysteine-rich or High Glycine–Cysteine Proteins (HgGC, cysteine > 10%, glycine < 18%) (Dalla Valle et al., 2007; Toni et al., 2007; Hallahan et al., 2009; Alibardi, 2009, Table 1). Another short beta-protein (87 amino acids), indicates as serine–treoninerich (Ge-strp-1) and little expressed was also found in the pad
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Fig. 1. Schematic drawings representing a digit (A) with the apical claw and the pad (green) made of lamellae which section is shown in (B). The central image shows a manual alignment of the sequences of all the beta-proteins detected from the transcriptome of G. gekko (Hallahan et al., 2009) distinguished in three glycine-rich beta-proteins and 16 cysteine-rich beta-proteins (Glycines are colored in red, Cysteines in green, Proline in blue and Serines in yellow). The high homologous central region, the core-box, is boxed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
lamellae (Hallahan et al., 2009). In the latter study, although the presence of the two alpha-keratins of 52–53 kDa has been detected in the epidermis of pad lamellae, their specific localization in setae is unlikely as indicated from the absence of any immunolabeling for alpha-keratins in setae using immunocytochemistry. Despite the above information it remains unknown which of the 16 cysteine-rich beta-proteins is truly localized in G. gekko setae and in particular in the terminal spatula. Therefore in the present study we have done the ultrastructural immunolocalization for some of these proteins like it has been successfully done for the setae of the climbing iguanid lizard A. carolinensis (Alibardi et al., 2012; Alibardi, 2012a) and other geckos (Alibardi, 2012b). Previous in situ hybridization analysis indicated that glycinerich beta-proteins were also expressed in the beta-layer of gecko pad lamellae although also cys-rich beta-proteins might also be revealed by the probes utilized, directed to the conserved coding region of the gene (Dalla Valle et al., 2007; Alibardi et al., 2007). The knowledge of the specific proteins forming the setae and the spatulae in geckos is important for further analysis of the chemical physical characteristics of these proteins that give rise to setal flexibility and spatular adhesiveness. Recent studies on the lizard Anolis carolinensis and three species of geckos have identified
two main cysteine-rich beta-proteins forming the setae and spatulae, out of the 17 total cysteine-rich beta proteins present in the genome of this species (Dalla Valle et al., 2010; Alibardi et al., 2012; Alibardi, 2012a,b). Using two epitope-specific generated antibodies the present study shows the immunolocalization of gecko-specific beta-proteins at the ultrastructural level, and allows the hypothesis that the mechanism of adhesion depends not only from the setal nano-dimensional and physical properties of the setae but also from their electrical charge. 2. Materials and methods 2.1. Antibody selection The epitopes utilized in the present study to produce specific antibodies were selected on the different core-box regions of two representative beta-proteins out of the remaining sequences (Fig. 2) that we previously isolated (see Fig. 5 in Hallahan et al., 2009, and Fig. 13 in Alibardi, 2009). As it is seen in Fig. 2, the amino acid similarity among all the core boxes in the 19 betaproteins isolated from the setae varies from 50 to 95%. The epitopes were selected from two beta-proteins highly expressed in the setae
Table 1 Amino acid composition of the 19 beta-proteins and 2 ␣-keratins characterized in G. gekko transcriptome (Hallahan et al., 2009). HgG (High Glycine beta proteins, red color); HgGC (High Glycine Cysteine beta proteins, green color; ␣k, alpha-keratins, orange color).
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Fig. 2. Amino acid sequence similarity (%ages) among the core-boxes of the 16 cysteine-rich (green) versus the three glycine-rich (pink) beta-proteins of the gecko transcriptome. The changes in amino acids in comparison to the selected epitopes, Ge-cprp-9 in the left colum and Ge-gprp-6 in the right column, are colored in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
that were considered representative for most of the other proteins (see Fig. 5 in Hallahan et al., 2009). One epitope is present in the small cysteine-rich beta-protein Ge-cprp-9 (SEVTIQPPPCTVVVPGPVLA; AN, ABU98602.1) containing 117 amino acids for a deduced MW of 13.1 kDa, and the specific antibody directed to it was produced in rabbit. The other epitope (AEVLIQPPPSVVTLPGPILS; AN ABU98593.1, 60% epitope identity with Ge-cprp-9 epitope) is present in the larger glycine-rich protein Ge-gprp-6, a protein of 176 amino acids with a deduced MW of 16.9 kDa, and the antibody was made in guinea pig. The peptides and the antibodies production, including their affinity purification, were obtained through a Biotechnology Company (Thermofisher Openbiosystems, USA), and small aliquotes were supplied from Dr. Peter Newiarowsky, University of Akron, OH, USA.
for alpha- and beta-keratins (GE, Healthcare, Milan, Italy). Briefly, for western blotting, the proteins separated in SDS-PAGE were transferred to a polyvinylidene difluoride membrane using a blotting apparatus (Biorad). After Western blot, membranes were stained with Ponceau red to verify the protein transfer, the membranes were destained in buffer, and then incubated with primary antibodies directed against the epitopes of Ge-cprp-9 and Gegprp-6 (diluted 1:2000–3000). In controls the primary antibodies were omitted. After rinsing, the membranes were incubated with HRP-conjugated anti-Rabbit (for Ge-cprp-9) or anti-guinea pig (for Ge-cprp-6) secondary antibodies (1:20,000), and the detection was performed using the enhanced chemioluminescence procedure developed by the manufacturer (ECS, EuroClone).
2.3. Immunocytochemistry 2.2. Western blotting The antibodies were tested on proteins extracted from molts of the digits of G. gekko using the method by Sybert et al. (1985). Briefly, the molts were homogenized in 8 M urea/50 mM Tris–HCl (pH 7.6)/0.1 M 2-mercaptoethanol/1 mM dithiothreithol/1 mM phenylmethylsulphonyl fluoride. The particulate matter obtained after homogenization was removed by centrifugation at 10,000 × g for 10 min, and protein concentration of the surnatant was assayed by the Lowry or Bradford methods. The electrophoresis analysis of the extracted proteins was carried out after 20 g of proteins were loaded in each electrophoretic lane, and the proteins were separated in 15% of SDS-polyacrylamide gels (SDS-PAGE) as previously indicated (Alibardi et al., 2012). Marker proteins were in the 7–100 kDa range of molecular weight
The experiments were conducted on three adults of Tokay gecko (Gekko gecko), purchased in authorized pet shops, as reported in previous studies (Alibardi, 2009, 2012a,b). Tips of the digits of sacrificed animals, containing the claw and the adhesive pads were immediately fixed for 5–8 h in cold (0–4 ◦ C) 4% paraformaldehyde in 0.1 M Phosphate buffer at pH 7.4, dehydrated in ethanol, and embedded in Bioacryl Resin at 0–4 ◦ C under UV light for 2 days. Tissues were sectioned with an ultramicrotome to obtain 2–4 m thick sections that were stained with 0.5% Toluidine blue for light microscopy analysis. For light microscopy immunocytochemistry the sections were incubated overnight at 0–4 ◦ C or for 4 h at room temperature with the antibodies diluted 1: 200 in Buffer (Tris 0.05 M at pH 7.6 containing 1% BSA). The sections were rinsed in buffer and incubated for 60 min at room temperature with a fluorescein-conjugated anti-rabbit or anti-guinea pig
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antibodies (FITC, Sigma, diluted 1:100), rinsed in the buffer, mounted in Fluoroshield anti-fading medium (Sigma), and observed under a fluorescence microscope equipped with a fluorescein filter and a digital camera. Selected sections of 60–90 nm thickness collected on Nickel grids, were used for the immunogold electron microscopic analysis. The sections were incubated overnight at 0–4 ◦ C in the primary antibodies diluted in 0.05 M Tris–HCl buffer at pH 7.6, containing 1% Cold Water Fish Gelatin. In controls, the primary antibody was omitted in the overnight incubation. The sections were rinsed in buffer and incubated for 1 hour at room temperature with anti-rabbit or anti-guinea pig Gold-conjugated secondary antibody for beta-proteins detection (Sigma, 10 nm gold particles). In some immunoreactions both the antibodies were used on the same section in double-labeling immunodetection. The primary rabbit antibody was detected using gold particles of 5 nm, while the guinea pig primary antibody was detected using gold particles of 15 nm. Grids were rinsed in buffer, dried and stained for 5 minutes with 2% uranyl acetate, and then observed under the electron microscope Philips CM-100 or Zeiss C10. 3. Results 3.1. Western blots Two main immunoreactive bands for the Ge-gprp-6 epitope were found at 18 and 25 kDa and a minor band appeared at 30 kDa, and much weaker bands around 45 kDa (Fig. 3, first lane, G6). A single large band at 14–16 kDa was instead seen for the detection of the Ge-cprp-9 epitope (Fig. 3, second lane, G9). No labeled bands were seen in the controls for CG6 and CG9 (last two lanes in Fig. 3). 3.2. Immunocytochemistry The epidermis of normal scales in geckos consists in a dead corneous layer composed by a pale beta-layer and a denser alpha-layer, followed by a viable epidermis made of one–two irregular layers of keratinocytes in resting phase or more stratified keratinocytes layers during epidermal renewal (data not shown, but see details in Maderson, 1964, 1966). Immunocytochemical analysis of setae showed that mainly setae were immunofluorescent with the Ge-cprp-9 antibody (Fig. 4A) while only the corneous beta-layer but not the setae appeared immunofluorescent for the Ge-gprp-6 antibody (Fig. 4B). Controls were completely unlabeled (Fig. 4C). Another antibody directed to the unique beta-protein Ge-sprp-1 protein did not produce any labeling in both light and ultrastructural immunocytochemistry, and this beta-protein is not further considered in the present study.
Fig. 3. Western blot results of protein extract from the molts of digital pads. Lane 1 for Ge-gprp-6 (G6), lane 2 for Ge-cprp-9 (G9), lane 3 for the control of G6 (CG6), and lane 4 for the control of G9 (CG9). The numbers on the right indicate the indicative molecular weight in kilodaltons (kDa) (see text for descriptions).
While in other body scales in G. gecko a random sampling of the skin generally yielded the epidermis in resting or early renewal stage, the epidermis of pad lamellae was often in renewal stage histology. In the latter condition, the outer setal generation was localized at the apical tip of the lamella and the inner setae generation was forming underneath the outer corneous layer (Fig. 5A). This common feature indicated a frequent cycling of the epidermis in pad lamellae. In fact, in case the epidermis was in resting stage, no inner setae formation was seen underneath the corneous layer of the pad lamella (arrowhead in Fig. 5A). The inner setae appear as bristles extending from the basal oberhautchen layer toward the outer corneous layer. The details of the inner setae of G. gecko showed that they were partially pigmented (forming dark bands) and that they were branched toward their apex, in a region contacting the layer of clear cells. The cytoplasm of clear cells was interspersed among the elongating setae (Fig. 5B). The ultrastructural immunogold labeling for the Ge-cprp-9 protein showed a labeled oberhautchen layer forming the base of setae, followed by a cytoplasm region devoid of corneous material but with a loose keratin meshwork (asterisk in Fig. 5C). This non-cornified region was followed underneath by a deep dense layer with the typical, mottled beta-keratin pattern of electronpale mixed to electron-denser material, and was diffusely labeled or unlabeled with this antibody. The beta-layer was followed underneath by 2–4 narrow and more electrondense corneocytes forming the mesos-region (Fig. 5C), and then by thicker corneocytes of the alpha-layer (data not shown).
Fig. 4. Immunofluorescence of pad lamella for Ge-cprp-9 (Gc9 in A), for Ge-gprp-6 (Gg6 in B), and of a control section (CO). Bar in all pictures is 10 m. (A) Shows immunoreactive setae (se) and immunonegative beta-layer and epidermis (e). (B) Shows that only the corneous beta-layer (c) at the base of setae (se) is labeled while the setae are immunonegative. (C) Shows no labeling (se, setae position; c, corneous layer position) in a control section.
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Fig. 5. Light microscopy view of setal morphology (A and B) and ultrastructural immunocytochemistry for Ge-csrp-9 antibody (Gc9 in C–F). (A) Section of a pad lamella showing the inner setae (is) and the apical free margin. Bar, 15 m. (B) details of some setae which base is in the narrow oberhautchen and tips form branches (arrow), Bar, 5 m. (C) Sectioned corneous layer sustaining the setae (the arrow indicates a cross-sectioned setae) that comprises a labeled oberhautchen, an intermediate pale region (asterisks), a lower beta-layer (b), and the mesos-layer (m). Bar, 150 nm. (D) Four cross-sectioned setae (se) are intensely labeled while the surrounding spaces that were occupied from the clear layer cytoplasm are immunonegative. Bar, 100 nm. (E) Detail on setae showing gold particles mostly present over darker bundles or material present inside the setae (arrow). Bar, 100 nm. (F) control section (CO) on bundles (arrow) within a seta that are immunonegative. Bar, 100 nm.
Both the base of setae attached to the oberhautchen cells as well as the stalk or intermediate region of the setae and the branching region, were intensely immunolabeled with the Ge-cprp-9 antibody (Fig. 5D). The gold particles were especially or exclusively concentrated over the denser areas of the setae and less frequently over electron-paler areas, an indication that other components are present in these regions (Fig. 5E). No labeling was detected in the cytoplasm of cells forming the clear layer, in the inter-setae spaces or in other epidermal and dermal tissues using the employed antibody. Also the
controls were immuno-negative (Fig. 5F). Although with variable intensity, also the branching region of the setae up to the terminal spatula showed immunolabeling for Ge-cprp-9 (Fig. 6A). In particular, gold particles were seen both inside the setae and on their surface. At the proximal base of pad lamellae where setae became smaller, or in the oberhautchen layer of the other non specialized, digital scales, the setae shortened and transformed in spinulated pointing structures. Immunolabeling for Ge-cprp-9 was present in spinulae, and in the electron-dense and paler areas of the beta-layer (Fig. 6B).
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Fig. 6. Immunogold labeling for Ge-cprp-9 (Gc9, (A) and (B)), for Ge-gprp-6 (Gg6, (C)), and double labeling (Gecprp-9 + Gedprp-6, (D)). (A) terminal part of branched setae (se) with immunolabeled spatulae (arrows). Bar, 100 nm. (B) Immunolabeled mature beta (b) and superficial oberhautchen spinulae (sp) of a digital scale. Bar, 150 nm. (C) Immunolabeling only present over the beta-layer (b) of digit scale while the merged superficial oberhautchen and spinulae (sp) and the mesos layer (m) are not labeled. Bar, 150 nm. (D) The double labeling shows the smaller particles (5 nm detecting Gc9) over the oberhautchen and spinulae (sp) while the larger gold particles (15 nm detecting Gc6) labele only the underlying beta-layer (b). Bar, 100 nm.
A sharp difference was observed using the Ge-gprp-6 antibody on both pad lamellae and normal digital scales. In fact, this antibody specifically labeled only the beta-layer but not the oberhautchen layer merged to the beta-layer and its spinulae in the digital scales or in the setae of the pad lamellae (Fig. 6C). The double-labeling immunolocalization (5 nm gold particles for Ge-cprp-9 and 15 nm gold particles for Ge-gprp-6) confirmed that the oberhautchen only immunolocalized Ge-cprp-9 (Fig. 6D). In pad lamellae immunostained with the Ge-gprp-6 antibody the only labeled substructure was the thin beta-layer present below the pale region (asterisks in Fig. 7A–C), exactly the reverse localization seen using the Ge-cprp-9 antibody (compare Fig. 5C and Fig. 7A and B). The labeling in the beta-layer was mainly seen over the paler
areas among the smaller electron-dense areas were filaments of 7–8 nm thickness were often seen (Fig. 7C). The oberhautchen, pale layer, mesos and alpha-layers were immunonegative for the Gegprp-6 antibody. In particular, no labeling for Ge-gprp-6 was seen at the base, intermediate and apical regions of setae, suggesting absence of this epitope. 4. Discussion 4.1. Beta-protein distribution in setae The present study has shown the specific skin localization of representative highly expressed cysteine-rich and glycine-rich
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Fig. 7. Immunogold labeling for Ge-gprp-6 (Gg6) in pad lamellae. (A) Immunogold particles are only seen over the beta-layer (b) but are absent in the pale layer (asterisks) and its keratin bundles (k) or in the oberautchen (o) and setae (se). Also the underlying mesos-layer is negative (m). Bar, 150 nm. (B) higher magnification image of mature epidermal layers showing immunonegative setal base (se) merged with the oberhautchen (o), the intermediate (asterisk) layer containing a keratin bundles (k), and the labeled beta-layer (b) confining with an unlabeled mesos cells (m). Bar, 100 nm. (C) Higher magnification of the beta-layer (b) located underneath the intermediate layer (asterisk) showing that most gold particles decorate the paler areas. Bar, 100 nm.
beta-proteins that were characterized in previous studies in scales of the digits and of the specialized adhesive pad lamellae in G. gecko (Fig. 1A, Table 1, see Refs. Hallahan et al., 2009; Alibardi, 2009). The western blot analysis has indicated that the employed antibodies indeed recognize proteins in the expected range, although with a MW difference of 1–3 kDa from the deduced proteins (14–15 kDa vs deduced 11.3 kDa for Ge-cprp-9, and the 18 kDa band vs 17.6 kDa of the deduced Ge-gprp-6). The presence of other bands at 25 and 30 kDa may indicate form of aggregation or fragmentation of these proteins, as it frequently occurs using the employed extractive methods (Alibardi and Toni, 2005; Rizzo et al., 2006; Toni et al., 2007). Another possibility is that the antibody recognizes the corebox of the other two glycine-rich beta-proteins (Ge-gprp-7 and Ge-gprp-8) that possess a deduced MW respectively of 19 and 20.7 kDa (Alibardi, 2009). The ultrastructural study has indicated that while in mature normal scales in all body areas the oberhautchen layer is merged with the beta-layer (Fig. 8A), in most of the outer surface of pad lamellae, except toward the basal (hinge) and apical (tip)
regions, the oberhautchen layer is separated by a non-corneous layer from the beta-layer (asterisks in Fig. 8B). Although the antibodies were directed against the core-box regions of the two proteins, which showed a certain sequence similarity (50–95%, see Fig. 2), the immunological results clearly indicates that Gecprp-9 and Ge-gprp-6 proteins are localized in two different layers of the digital scales and adhesive pads of G. gecko (Fig. 9). In particular Ge-cprp-9, representing the cysteine-rich beta-proteins (Fig. 1), appears mainly localized in the oberhautchen and setae, including their surface, while Ge-gprp-6, a glycine-rich betaprotein, appears exclusive of the beta-layer of digital scales. Since the setae and the oberhautchen layer are much more developed then the beta-layer in pad lamellae of geckos, this explains why cysteine-rich beta-proteins are more abundant then glycinerich beta-proteins in the setal transcriptome (Hallahan et al., 2009). The present study has found that Ge-cprp-9 (19.7% of cysteine, 17.9% proline, and 12.8% glycine, see Table 1) is a major protein of spinulae, setae and of their spatula ending. The comparison of
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Fig. 8. Drawing illustrating the structure of the spinulated oberhautchen-beta-layer present in normal scales of digits or other body regions (A) in comparison to the structure of the oberhautchen-beta-layer found in the modified scales of pad lamellae (B). While in normal scales the oberautchen (ob) is merged with a relatively thick beta-layer (; arrow in (A)) in pad lamellae most of oberhautchen form long setae (arrow in (B)) while a non-cornified layer (asterisks) divides the oberhautchen from a thinner beta-layer along most of the outer scale surface (except by the tip and at the base where the oberhautchen and beta-layer are merged like in normal scales).
Ge-cprp-9 core-box with that of the remaining 15 sequences of cys-rich beta-proteins and of the three sequences of glycine-rich beta-proteins (Fig. 2, left column) however suggests that numerous of these proteins should also be recognized by this antibody. In fact, the amino acid identity of core-boxes varies from 70 to 90% for the cysteine-rich types so that also other of these beta-proteins are likely present in the setae. Differently, the lower identity of the core-box in the glycine-rich types (50–60%, see Fig. 2, left column at the bottom) suggests these proteins are less recognized from the Ge-cprp-9 antibody. The specific role for each one of these different beta-proteins in setae formation, structural and material properties for adhesion remains to be evaluated. The localization of cysteine-rich beta-proteins in the oberhautchen, spinulae and setae of G. gecko confirms similar findings using other antibodies against cysteine-rich beta-proteins in
geckos (G. gekko, T. mauritanica and H. turcicus, Alibardi, 2012b) as well as in the lizards A. carolinensis and A. lineatopus (Alibardi et al., 2012; Alibardi, 2012a). Conversely, the Ge-gprp-6 antibody very likely recognizes all the three glycine-rich proteins since the identity is 85–95% (Fig. 2, right column) while this antibody should recognize with lower specificity the cysteine-rich proteins since the identity falls to 55–70% in the cysteine-rich beta-proteins. Ge-gprp-6 contains 20.5% of glycine, 10.2% proline, but only 3.4% in cysteine (Table 1). Therefore all the three glycine-rich proteins are likely localized in the thin beta-layer present in the setae but mainly in the thicker betalayer present in normal digital scales (Fig. 8 A), or even more in the thick beta-layer of scales in other body areas such as in the head and tail. In conclusion, while glycine-rich beta-proteins appear localized in hard epidermal layers (beta-layer) those rich in cysteine appear more typical of epidermal layers where flexibility (oberhautchen and spjnulae or setae) is required. The disulphide bonds are therefore most likely utilized for building a deformable meshwork in the oberhautchen cells (recalling the elastin molecular structure) while the glycine-rich proteins form a resistant, hydrophobic and chromophobic texture in the beta-layer. Future chemical physical studies on the specific properties of these proteins will clarify this issue. 4.2. Hypothesis on the influence of Ge-cprp-9-like beta-proteins on setal adhesion The fine immunolocalization of specific beta-proteins in gecko setae and their surface allows for a (qualitative) hypothesis on the possible role of these proteins on the mechanism of adhesion. Since gecko adhesion is mainly due to van der Waals forces (Autumn et al., 2000, 2002; Autumn and Peattie, 2002; Autumn and Gavish, 2012) it is believed that the production of bio-inspired artificial devices with similar shape, dimension, organization and spacing of natural gecko setae and spatulae can replaced the biological material represented by gecko proteins (Berengueres et al., 2007; Ge et al., 2007; Seth et al., 2007). Like in previous studies on the
Fig. 9. Drawing illustrating the hypothetical influence of charged proteins on the setae surface. (A) Shows the net positive charges on the different beta-proteins of gecko setae (HgGCs and HgGs). In (B) the progressive details of the setae are illustrated starting from the hand or foot with the digital pads (1), sectioned and enlarged in 2 to show the pad lamella, which setae are enlarged in 3 to show the various layers of the epidermis and the terminal branching ramification of setae contacting the substratum (␣, alpha-layer; , beta-layer; ba, basal layer, , mesos-layer; obj, oberhautchen; sb, supra-basal layer). In 4 details of the localization of Ge-cprp-9 in setae/spatulae and oberhautchen, and of Ge-gprp-6 in the underlying beta-layer (separated by the intermediate layer, asterisks) are shown. The positively charged surfaces attract water while the charged spatula can induce temporary dipoles on its surface that influence van der Waals forces (see text).
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immunolocalization of setal proteins in A. carolinensis, T. mauritanica, and H. turcicus (Alibardi, 2012a,b), it appears that the deduced proteins in the setae and spatular ends of G. gekko may be electrically charged. In fact, assuming that these cysteine-rich proteins are major proteins of the setae, the Protparam Program at http://www.web. expasy.org/protparam/ for Ge-cprp-9 and for most of the other High Glycine Cysteine beta-proteins shows that they are positively charged (Fig. 9A). We also assume that most of these charges, 2–6 positive charges in each protein multiply for billions of these proteins present on the surface of setae, can produce a charged surface of contact with the substrate. The presence along the setae surface of proteins with the same charge (positive as indicated in Fig. 9B4) impedes that the setae aggregate during motion since they are repelled one to another by electrostatic charges of the same sign. In particular, the surface of the spatula with its charged molecules can interact with the substrate in 2 different ways: electrostatically for long-range distances (micrometers) and through van der Waals forces for short-range distances such as nanometers or Angstroms (Autumn et al., 2000, 2002; Autumn and Peattie, 2002; Autumn and Gavish, 2012). Initially the electrostatic forces (attractive) may prevail but as the spatula and the substrate get closer, below 10 Angstroms, van der Waals forces may become prevalent, especially if the charged proteins can induce dipoles on the surface of contact. The presence of interposed water molecules between the betaproteins of the spatula and the substrate that may be polarized increases the number of induced dipoles and therefore further enhances van der Waal forces and adhesion. It has been shown that the increase of relative humidity enhances gecko adhesion (Sun et al., 2005; Huber et al., 2005; Niewiarowski et al., 2008; Puthoff et al., 2010). Charged beta-proteins on the surface of both oberhautchen and the beta-layer can also trap water dipolar molecules producing capillary forces (Fig. 9B4). Capillary forces may aid adhesion in ways that are still not clear (Pesika et al., 2009). Furthermore, spatular material made of cysteine–glycine-rich beta-proteins may partially adsorb water to become softer when the humidity increases, and the spatula becomes more pliable to increases the surface of contact and therefore enhances adhesion (Huber et al., 2005; Niewiarowski et al., 2008; Pesika et al., 2009; Prowse et al., 2011; Puthoff et al., 2010). The hypothetical influence of electrically charged proteins on van der Waals interactions and material hydration affecting adhesion remains however to be experimentally demonstrated. In conclusion, the present study suggests that the largely unexplored chemical physical properties of the setal proteins of geckos are important for understanding the mechanism of adhesion. Future artificial adhesive materials should possess not only the microscopical features but also similar chemical physical properties present in the natural gecko cysteine-rich beta-proteins. Aside other anatomical–physiological features during gecko evolution (Russel, 1986, 2002; Bauer, 1998; Russel and Johnson, 2007), the selection of genes coding for the beta-proteins produced in the oberhautchen layer, the more external corneous layer of the epidermis, has represented a successful factor for the survival and radiation of these climbing lizards.
Acknowledgments The study was in part self supported (Comparative Histolab) and with a contribution of the RFO 2010 from the University of Bologna. I thank Dr. Peter Niewiarowsky, University of Akron, USA, for sending me some small amount of the antibodies against the epitopes selected against the core-box region of the Tokay gecko. Dr. Anna
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Segalla, University of Padova, helped with the electrophoretic and western blot analysis.
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