Protoplasma https://doi.org/10.1007/s00709-018-1270-9

ORIGINAL ARTICLE

Review: mapping proteins localized in adhesive setae of the tokay gecko and their possible influence on the mechanism of adhesion Lorenzo Alibardi 1,2 Received: 13 January 2018 / Accepted: 25 May 2018 # Springer-Verlag GmbH Austria, part of Springer Nature 2018

Abstract The digital adhesive pads that allow gecko lizards to climb vertical surfaces result from the modification of the oberhautchen layer of the epidermis in normal scales. This produces sticky filaments of 10–100 μm in length, called setae that are composed of various proteins. The prevalent types, termed corneous beta proteins (CBPs), have a low molecular weight (12–20 kDa) and contain a conserved central region of 34 amino acids with a beta-conformation. This determines their polymerization into long beta-filaments that aggregate into corneous beta-bundles that form the framework of setae. Previous studies showed that the prevalent CBPs in the setae of Gekko gecko are cysteine-rich and are distributed from the base to the tip of adhesive setae, called spatulae. The molecular analysis of these proteins, although the three-dimensional structure remains undetermined, indicates that most of them are charged positively and some contain aromatic amino acids. These characteristics may impede adhesion by causing the setae to stick together but may also potentiate the van der Waals interactions responsible for most of the adhesion process on hydrophobic or hydrophilic substrates. The review stresses that not only the nanostructural shape and the high number of setae present in adhesive pads but also the protein composition of setae influence the strength of adhesion to almost any type of substrate. Therefore, formulation of dry materials mimicking gecko adhesiveness should also consider the chemical nature of the polymers utilized to fabricate the future dry adhesives in order to obtain the highest performance. Keywords Gecko lizard . Adhesive pads . Setae . Protein composition . Immunolocalization

Gecko adhesion is based on epidermal specializations The gecko’s ability to stick on vertically oriented substrates, defeating gravitational forces and allowing movement in arboreal habitats, mainly derives from the modification of an epidermal layer called oberhautchen in some digital and tail scales, forming adhesive pads (scansors) of variable extension (Maderson 1970, 1971; Hiller 1972; Russel 1985, 2002; Bauer 1998; Alibardi 2009; Alibardi and Meyer-Rochow 2017). The epidermis of geckos is formed by alternating epidermal generations, and each generation comprises an Handling Editor: Douglas Chandler * Lorenzo Alibardi [email protected] 1

Comparative Histolab Padua, Bologna, Italy

2

Dipartimento di Biologia, Universita’ di Bologna, via Selmi 3, 40126, Universita’ di Bologna, Bologna, Italy

oberhautchen-beta-layers followed by alpha-layers. The tiny spinulae of the oberhautchen layer of gecko scales, 1–2 μm by 0.2–0.4 μm, forming the spinulated pattern of geckos, in the developing adhesive pads undergo a drastic growth in diameter and length (10–100 μm by 0.5–3 μm) to form long bristles termed setae (se, Fig. 1a–c). The growth of spinulae into setae is accomplished by the cooperation of the cytoskeleton of another epidermal layer, termed clear layer (cl), that is interfaced with the oberhautchen (ob) spinulae or setae, and belongs to the previous epidermal generation (Fig. 1b, d, e). Although the details of the cytological process are not completely known, in particular the specific cytoskeletal proteins involved, it is believed that the cytoplasm of clear cells molds the soft cytoplasm at the beginning of setae formation, forming cytoplasmic channels around the elongating setae, and their final branching into nanoscale endings termed spatulae (Figs. 1f, g and 2a–c; Hiller 1972; Alibardi 1997). This is also indicated from the different organization of the cytoskeleton and shape of keratohyalin-like granules present in clear cells of various species of lepidosaurian reptiles that form species-specific micro-

L. Alibardi Fig. 1 Light microscopic (a) and ultrastructural (b–f) detail of pad lamella in G. gecko. a Numerous pale setae rest upon a darker corneous layer (oberhautchen and beta-layer). The double arrow indicates that the corneous layer has detached from the scale (pad lamella) during sectioning. The arrowhead indicates the free margin. Bar 20 μm. b Detail at the base of setae featuring the oberhautchen, the electron-pale sub-layer, and the thin beta-layer (arrow). Bar 0.5 μm. c Free margin (arrows) at the very tip of the pad lamellae sustaining some setae. Bar 1 μm. d Detail of growing setae within the surrounding cytoplasm of clear cells (which plasma membrane is indicated by arrowheads). Bar 0.5 μm. e Forming branches and spatula endings (arrows) in the external cytoplasm of a clear cell containing sparse fibrous material. Bar 0.5 μm. f Crosssectioned seta at mid-level showing the dense corneous bundles (arrows) separated by electron-clear (matrix) material. The pale cytoplasm of a clear cell surrounds the setae. Bar 0.25 μm. g detail of mature spatulae (arrowhead). Bar 150 nm. a alpha-layer, cl clear cell cytoplasm, fi cytoskeletal fibrous material in clear cell, ob oberhautchen, pal pale sublayer, sc scale (pad lamella), se setae

ornamentation (Alibardi 1999). The fibrous material forming the cytoskeleton of clear cells surrounds the growing setae, likely providing mechanical support that shapes the setae as they grow (Fig. 2a–c). While the base and large portions of the setae along their length are made of compact bundles of corneous material alternated with paler areas of unknown composition (Figs. 1d, f and 2a–c), the spatula end is made of a compact mass of dense corneous material (Figs. 1f, g and 2c). Although the gene structure, mRNA expression, and composition of the main proteins forming the bulk of setae are known (Alibardi and Toni 2005; Dalla Valle et al. 2007; Toni et al. 2007; Alibardi et al. 2007; Hallahan et al. 2008; Alibardi 2009), the precise composition and organization of the proteins

composing the fibrous cytoskeleton of clear cells and of the spatula ends remains the main unsolved problem in setal cell biology. In setae, the oberhautchen layer is merged with a thinner beta-layer in comparison to the thicker beta-layer present in scales of other body regions. In particular, in G. gecko, a pale, intermediate sub-layer is present, likely containing lipids since it is electron-pale and unlabeled using antibodies against glycine-rich or cystine-rich corneous beta proteins (CBPs, Alibardi 2013a, b, c; Figs. 1b and 2b), the main components of setae (see later). This sub-layer, detected only in geckos so far (not in Anolis), may be functioning as a cushioning layer for improving the flexibility of the setae in the contact with the substrate. This sub-layer seems not to be continuous with the

Review: mapping proteins localized in adhesive setae of the tokay gecko and their possible influence on the...

Fig. 2 Schematic drawing of seta formation (a–c) and electrophoretic gel separation of setal proteins (d–g, G. gecko). a Initial formation of a spinula into the cytoplasm of clear cell with its cytoskeletal fibrous material located around the spinula (possibly shaping it). b Elongating seta with beginning of apical branching to form thinner setal endings. c Maturing setae with the formation of apical spatula. d Mono-dimensional electrophoretic separation with Ponceau staining of total setal proteins. e Two-dimensional

electrophoresis separation and Coomassie blue staining. This shows protein spots in the alpha region at 40–66 kDa (α, acidic) and beta region at 10–22 kDa (β, neutral-basic). f Western blotting after mono-dimensional separation using the beta-1 antibody that identifies a large band at 12–16 kDa (beta-region). g Two-dimensional electrophoretic gel, immune-stained with the beta-universal antibody (a CBP antibody) that reveals some protein spots in the beta-region (β)

pale areas present in the setae (Fig. 1f), but this specific point has not been fully resolved. It appears evident that oberhautchen cells in geckos, as representing the transition between the alpha-layer of the outer epidermal generation into the beta-layer of the inner epidermal generation (Maderson 1971; Alibardi 2009), can still produce lipids in addition to CBPs. The transformation of clear and also oberhautchen cells into a glandular layer of variable thickness has been described for other scales in geckos (Maderson 1970). In order to respond to some of the above questions, the present review represents the collection of the most recent data on the protein localization in setae of the tokay gecko (Gekko gecko) and also provides some (qualitative) hypotheses on the

possible influence of these proteins in the mechanism of adhesion, stressing the importance of the setae chemistry in addition to the physical shape of setae and their spatula ends.

Protein composition of setae Mono- and two-dimensional fractionation of protein extracts from the setae of the tokay gecko showed that the prevalent type comprises proteins formerly indicated as beta-keratins (Thorpe and Giddings 1981; Alibardi and Toni 2005; Rizzo et al. 2006; Toni et al. 2007). The small proteins are now indicated as corneous beta proteins (CBPs), and this change

L. Alibardi

of terminology and concept has been amply explained in other extensive publications (Calvaresi et al. 2016; Alibardi 2016a, b; Fig. 2c–f). Briefly, proteins formerly called beta-keratins are completely different from true keratins (intermediate filament keratins) and instead belong to the family of corneous proteins of the epidermal differentiation complex (EDC) of reptiles and birds (Strasser et al. 2014; Holthaus et al. 2015, 2017). Therefore, the term CBPs, instead of beta-keratins, will be used to refer to these proteins throughout the present review. Immunolocalization studies further support previous observations indicating that these proteins are produced and mature in the setae (see later). The other types of setal proteins, the (alpha)keratins or Intermediate Filament Keratins (IF-Keratins), are also detected in the epidermis isolated from digital scales or from molts (Rizzo et al. 2006; Toni et al. 2007). However, the immunolocalization studies have shown that while IF-keratins are present in the living and corneous beta-layer of body scales, including digit scales, they are little represented in the setae themselves (see later immunolocalization). Setal proteins are known in few gecko species, and the best known IF-keratins and CBPs derive from the detailed twodimensional gel electrophoretic separation (Toni et al. 2007), especially by the first transcriptome study (EST library) of digital setal proteins analyzed in the tokay gecko (Gekko gecko; Hallahan et al. 2008; Alibardi 2009, 2013b, c). The recent analysis of the genome in another gecko, Gekko japonicus, revealed an expansion of the number of CBPs (beta-keratins, 71 genes versus 23 genes in G. gecko) that appears most correlated to the formation of setae in the Japanese gecko (Liu et al. 2015). In particular, the smaller CBPs of 10 kDa and cysteine-rich variety (35 mainly clustered in a single locus out of 71 total CBP genes in G. japonicus) have expanded in number and appear to be present in setae (Liu et al. 2015). The studies on G. gecko have detected alpha-keratin of type I (acidic) of 52.7 kDa and one alpha-keratin type II (basic) of 64.1 kDa, and 19 CBPs of lower MW whose amino acid sequences are known (Hallahan et al. 2008). The latter are subdivided into cysteine-rich (Ge-cprp, 16 members) and glycine-rich (Ge-gprp, 3 members). One member (Ge-cprp9) represents the cysteine-rich subfamily, and another member (Ge-cprp-6) represents the glycine-rich subfamily, while only one member of serine-proline-rich CBPs has been found (Gesprp-1). Among the prevalent amino acids present in the smaller Ge-cprp9 protein are cysteines, prolines, serines, and glycines (Fig. 3), while glycine is prevalent over serine, proline, and cysteine in the larger Ge-gprp6 (Fig. 3). The latter are similar to those previously found in gecko scales (Dalla Valle et al. 2007), suggesting that they are localized in the beta-layer of the setae but are not setae specific. The analysis of the amino acid composition of CBPs of setae indicates that only one glycine-cysteine-rich protein (Ge-cprp16) has a 4% in

tyrosine content, 10.7% cysteine, and 18.0% of glycine, while the other 18 cysteine-rich beta proteins are poor in aromatic amino-acids, in particular phenylalanine and tyrosine (generally lower than 2%, see Alibardi 2013b, c). A higher but limited amount of phenylalanine (4.1–4.5%) and tyrosine (2.5–3.7%) is present in the three Ge-gprp CBPs detected in G. gecko, but these are not very abundant in setae. Finally, phenylalanine is present (3.4%), but tyrosine is quite high (10.3%) in the unique type of Ge-sprp-1 found in the setae, whose localization remains unknown (Fig. 3). Other studies using Raman spectroscopy indicated that cysteinerich proteins with aromatic groups of phenylalanine and tyrosine are present in the setae of G. gekko (Rizzo et al. 2006). This observation may indicate that other types of proteins aside from cysteine-rich CBPs are present in the setae or that immunogold studies did not detect these proteins yet (particularly Ge-sprp1 and Ge-cprp16, the two proteins containing aromatic amino acids). Other proteins, detected after electrophoretic separation (one- and two-dimensional), with intermediate MW between IF-keratins and CBPs, have not been characterized yet, but they represent a minor fraction in comparison to the above cited proteins. The importance of knowing the molecular composition and three-dimensional form of these proteins, not only as structural elements but also for the mechanism of adhesion, will become clearer after describing the interplay between electrical charges and van der Waals forces when spatulae are faced with adhesion to a substrate. The localization of the most representative proteins has been mainly obtained using TEMimmunogold labeling (Alibardi 2013a, b, c).

Setal lipids contribute to structural and functional properties of setae In addition to proteins, lipids are also present in setae, probably associated in the paler regions of the setae, among the darker corneous bundles and in the oberhautchen (Alibardi et al. 2011; Hsu et al. 2011; Jain et al. 2015; Fig. 1f). Total setae lipids in G. gecko constitute about 11% of setae fresh weight (Jain et al. 2015), and most of them are probably contained in the pale areas, as unbound lipids or lipoproteins (Fig. 1d, f; see later). These lipids seem to be released on the substrate as monolayer or bilayered lipid footprints (1– 2 nm thick), containing polar phospholipids with choline oriented toward the spatular surface and the apolar part of phospholipids oriented toward the surface of an apolar, water-free, and hydrophobic or hydrophilic substrate (Hsu et al. 2011; Niewiarowski et al. 2016; see later discussion). Whether the presence of lipids may create a hydrophobic spot for adhesion is a good possibility (sacrificial lipid layer, see Hsu et al., 2011), as it is discussed later in the mechanism of adhesion section.

Review: mapping proteins localized in adhesive setae of the tokay gecko and their possible influence on the...

Fig. 3 Figure showing the percentage of amino acids present in three representative CBPs of setae and one IF-keratin sequences (right). Arrows indicate the more abundant amino acids. Arrowheads indicate

some percentages of aromatic amino acids. At the bottom, the epitopes selected for the IF-keratins immune-localization are shown

These studies have also suggested that lipids of the footprints may derive from those released by clear cells over the setae before and during the shedding process, when clear cells degenerate (Fig. 2c). A contribution of these lipids from the electron-clear sub-layer between the oberhautchen and betalayers present at the base of the setae (Figs. 1b and 2b, c) has not been demonstrated. This sub-layer within the oberhautchen can be visualized at the light microscope using oil red O in the geckos Phelsuma dubia and G. gecko (Alibardi et al. 2011). The continuous release of lipids during the activity and movements of geckos would require the production of large amount of lipids, a process that can hardly be sustained only from clear-oberhautchen cells. Although some lipid reserves have been described in the connective tissues located underneath the pad lamellae of G. gecko (Alibardi et al. 2011), no secretory duct or mechanism of secretion has been demonstrated so far. Further studies have shown that a more complex mix of lipids is present in the setae themselves and includes phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, glycerides, cholesterol, and fatty acids (Jain et al. 2015). It remains unknown the specific molecular association and organization of the lipids in the matrix or pale regions of the setae, and their contribution to the flexibility of setae as the gecko moves the scansors over different surfaces (Russel 1985, 2002).

Immunolocalization indicates that setae mainly contain cysteine-rich proteins After the identification and characterization of the amino acid composition in setal proteins in G. gecko (Hallahan et al. 2008), specific antibodies against different epitopes of major CBPs of setae were produced and utilized for mapping these proteins on the setae at the optical and ultrastructural level using immunogold (Alibardi 2013b, c). Three antibodies against IF-keratins (AK2, type I and II keratins), which tag the epitopes reported in Fig. 3, have shown a low to almost absent labeling in setae from their base to the spatula tips, while they show some labeling in the alphalayer located underneath the setae (Figs. 4 and 5). This occurs especially using the antibody against basic IK-filaments (αII, Fig. 5b). From these observations, it is confirmed that IF(alpha)keratins are little represented in setae and that they are likely masked by the accumulation of CBPs that may reduce the availability of epitopes for immunolabeling. Therefore, most of the IF-keratins detected by electrophoresis likely derive from the alpha-layers included in the molts or in the entire epidermis of setae (Toni et al. 2007). Immunolocalization at the electron microscopic level of CBPs has instead revealed that they are truly localized in the beta-layer, and in setae in particular. While the distribution of Ge-cprp9 and Ge-gprp6 is known (Alibardi 2013b, c), no

L. Alibardi Fig. 4 Ultrastructural immunolabeling for IF-keratins of setae (a, b, d–g) and spatulae (c) in G. gecko. Bars represent 100 nm in all figures. a Detail on a AK2 low labeled corneous bundles of the periphery of a seta. b Diffuse AK2 labeling in the denser corneous bundles at the center of a seta. c Unlabeled setae and their spatula endings (arrowheads). d Very low labeling in a seta using the antibody against αI IF-keratin. e Poor labeling using an antibody against αII IF-keratin in a seta. f Detail of diffuse labeling for αII IF-keratin over dense bundles in a seta. g Unlabeled control section. cb corneous bundles, se setae

immunolabeling has been obtained for Ge-sprp1, for which localization in the setae, if any, remains unknown. This result may stem from the masking of the epitope or from the very low amount or even a true absence of the latter protein in setae, and represent an un-resolved point of research. According to a phylogenetic analysis, Ge-sprp-1 is the closest CBP to feather CBPs (Hallahan et al. 2008), small proteins that accumulate in elongating barbule cells (Sawyer et al. 2000). The antibody utilized against cysteine-rich proteins indicates that this type of CBP is present in the setae, from their base to the spatula tip, while the glycine-rich proteins are present in the beta-layer and little, if at all, in the setae (Alibardi 2013b,c). More recently developed antibodies, directed toward general or specific epitopes present in the three classes of G. gecko CBPs, have further shown the detailed localization of setal

CBPs (Figs. 6 and 7). Some of these antibodies tag the more conserved amino acid region present in these proteins, the central beta-region (underlined and modeled in threedimensions in Fig. 6, see Calvaresi et al. 2016). Other antibodies, developed against different epitopes and types of CBPs of the lizard Anolis carolinensis (cysteine-rich or glycine-rich CBPs, see Alibardi 2015, 2016c; Alibardi et al. 2012), or against snake epitopes for CBPs (Pantherophis guttatus, Sn-grp1, Alibardi 2014), have also been tested for cross-reactivity with G. gecko setae. Despite the presence of linear epitopes potentially cross-reactive with G. gecko proteins (Fig. 7), the immunolocalization has shown a poor or absent cross-reactivity with the latter antibodies (HgGC11, HgG5, HgGC3, Ac37, Ac39, Sn-grp1, Fig. 8). Conversely, an intense labeling of the dense corneous bundles present in

Review: mapping proteins localized in adhesive setae of the tokay gecko and their possible influence on the...

indicated in Fig. 9. While the main proteins and lipids forming the setae in G. gecko are relatively known, it remains to be clarified the specific CBPs present at the spatula tip, possibly associated with lipids in the electron-clear areas. In addition, it remains to be determined the nature of the cytoskeletal proteins forming the fibrous cytoskeleton present in the clear cells and likely involved in the growth of setae and molding the spatula.

Composition in addition to physical shape of setae affects adhesion

Fig. 5 Ultrastructural labeling of setae (a) and the underlying alpha-layer (b) in pads of G. gecko using the αII antibody against IF-keratin. Bars indicate 100 nm in the two figures. a The gold particles are present at the base of setae (se) and in the oberhautchen, but are unevenly present in the thin beta-layer (b). b Diffuse labeling over cells of the alpha layer (a) beneath the setae (location similar to Fig. 1a)

setae has been obtained using the core-box, beta-universal (also tagging the core box-region; Sawyer et al. 2000), and the pre-core-box antibodies (Figs. 7 and 8). However, since the latter antibodies recognize numerous protein species among the cysteine-rich CBPs of setae, this intense labeling provides only a general indication on the presence in setae of these proteins, including the Ge-srp-1, but does not indicate the specific localization of each one in the seta, and in the spatula ending in particular. The low to absent labeling by these antibodies, observed in the electron-pale areas located among the denser corneous bundles of setae, confirms that these regions contain other proteins and likely lipids, as also indicated in a recent chemical study on the lipids present in setae (Jain et al. 2015). Based on this immunological information, we can summarize the present knowledge on setal composition and mapping as

It is believed that polymers casted to replicate the geometric structure of gecko setae are capable of reproducing most of the amazing adhesive properties of geckos (Autumn and Peattie 2002; Huber et al. 2005; Berengueres et al. 2007; Autumn and Gravish 2008). These micro-structures work very well but lack versatility (see review by Niewiarowski et al. 2016). However, the chemistry of these polymers may also be important for adhesion (Alibardi 2013a,b; Badge et al. 2014). Data from Figs. 3 and 9, derived from computational predictions (using Protparam at http:// ca.expasy.org/tools/protparam. html.), indicate that while the detected IF-keratins are charged negatively, CBPs of setae are instead charged positively. This opposite charge likely determines the association between IFkeratins and CBPs, but the prevalent amount of CBPs with net positive charges (Fig. 9) over the scanty IF-keratins make the surface of setae and spatulae likely positively charged (Fig. 10a, a1). Another possibility is that the spatular ending prevalently contains CBPs with no net charge (O, uncharged) such as Ge-sprp-1 and Ge-cprp-16 (Figs. 9 and 10a, a2), but without the ultrastructural immunolocalization for these proteins, this remains a preliminary hypothesis. Other studies have also indicated that setae are neither hydrophobic nor hydrophilic (Badge et al. 2014). Using the Protparam program previously utilized for other proteins detected in A. carolinensis (immunopositive for HgGC10 and HgGC3 antibodies), we obtained a negative or neutral charge for gecko setae (Tarentola mauritanica and Hemydactylus turcicus), while they were indeed positive for A. carolinensis (Alibardi 2013a). Regardless, our more recent immunolocalization data using G. gecko specific antibodies indicate that setae are more likely positively charged. The presence of a charge of the same sign over setae surface prevents the setae to sticking together (Fig. 10a). In case of charged setae, the interaction between adhesive pads and substrate surfaces may be initially based on static charges as the pads get to within 1 to 1000 μm of the substrate surface (Fig. 10b, c). The induction of electrical charge on the substrate by charged setae can support the recent hypothesis on “contact electrification” as the main attractive force operating in setae adhesion, at least on dry substrates (Izadi et al.

L. Alibardi

Fig. 6 Modeling figures of one of the main setal CBPs (Ge-cprp-9). a Prediction (Pred, PSIPRED server) of the secondary structure for Gecprp-9, a representative member of the cysteine-rich beta proteins. The red line underlines the central stretch of amino acids forming the betasheet region (four yellow arrows, strands). b Three-dimensional model of

the central beta-sheet region of the Ge-cprp-9 protein with the four yellow arrows (details in Calvaresi et al. 2016) and the included random coil regions (light blue strings). The location of the N- and C- terminals are indicated

Fig. 7 Graphic representation of different linear epitopes, indicated with the one-letter code for amino acids, tagged by the antibodies on different regions (arrows) of three cysteine-rich CBPs (green), glycine-rich CBPs (red), and serine-cysteine-rich CBP (pale yellow), representative for most

of CBPs of setae. - means negative labeling; +/- poor labeling; ++ high to intense labeling. The different colors match the epitopes and the relative position within the three proteins

Review: mapping proteins localized in adhesive setae of the tokay gecko and their possible influence on the...

Fig. 8 Immunogold labeling (e, f, h, 10 nm gold particles) and immunogold-silver intensified labeling (a–d, g, i) for different CBP epitopes in setae of G. gecko. a Mid-level cross-sectioned seta, low labeled for HgG5 protein. Bar 0.5 μm. b Mid-level cross-sectioned seta, poorly labeled for HgGC3 beta-protein. Bar 0.5 μm. c Intensely labeled seta, mainly over the denser corneous bundles among the paler spaces, using the beta-universal antibody. Bar 0.5 μm. d Very high labeling of a seta using the pre-core box antibody. Bar 0.5 μm. e Labeled seta using the pre-core box antibody and the smaller gold

particles (10 nm) to demonstrate that the labeling is mostly present over the denser corneous bundles (cb) among the pale (matrix) areas. Bar 100 nm. f Terminal spatulae (arrows) with some labeling using the precore box antibody and 10 nm gold particles. Bar 50 nm. g Detail on two terminal setae (arrows) with spatular endings intensely labeled after silver intensification (pre-core box antibody). Bar 100 nm. h Poorly labeled setae branching using the HgGC3 antibody. Bar 100 nm. i Almost unlabeled terminal endings with spatula (arrows) using the HgG5 antibody. Bar 200 nm

2014). As the two surfaces, substrate and spatula endings come closer and enter in the range of action of van der Waals forces (below 10 Angstrom), the latter force may become more effective than electrical forces in the process of adhesion (Fig. 10b, c; Autumn and Peattie 2002; Autumn and Gravish 2008). The binding strength of van der Waals interactions decreases with the cube of the distance between atoms, while van der Waals forces increase when larger organic molecules are involved (Wagner et al. 2014). This is particularly important as Waals forces depend on the molecular size and type of polymers, including proteins. Their aromatic rings (present in tyrosine and phenylalanine) form a larger surface where electrons can move and produce dipoles in comparison to molecules formed by isolated or centered atoms as in inorganic compounds or in crystals (Wagner et al. 2014). The

larger the molecule, especially with aromatic rings where electrons are largely displaced by resonance, the stronger is the attraction to the surface (Wagner et al. 2014). The presence of proteins rich in tyrosine and phenylalanine in setae (Rizzo et al. 2006; Hallahan et al. 2008; Alibardi 2009) indicates that the regions where these aromatic amino acids are more concentrated may enhance van der Waals forces as suggested by experimental measurements for other molecules (Wagner et al. 2014). The presence of smaller molecule of lipids released on the setal surface (Hsu et al. 2011; Jain et al. 2015), their possible action to displace water molecules in the contact surface (Fig. 10c1), and their influence on the van der Waals attractions contributing to enhancing the adhesion remain to be specifically analyzed. It is also possible that lipids associated with

L. Alibardi Fig. 9 Schematic drawing listing the set of proteins (CBP and IFkeratins) of G. gecko (a, left column), and the structure with composition of a seta (b). a All CBPs are charged positively and IF-keratins negatively (but they are much less abundant in setae than CBPs). b The latest view of the microanatomy of a seta with indications on the general protein and lipid composition and localization. The matrix material includes proteins/lipoproteins (green) mixed to lipids (white spaces). Question marks indicate the main unresolved aspects on the biochemical composition of setae (see text)

proteins in the spatula determine the formation of a pliable corneous material that can be deformed, increasing the contact surface, during the interaction of the spatula with the substrate (see Fig. 4 modeling in Hsu et al. 2011). Among CBPs, Ge-gprp-6 and the other glycine-rich betaproteins of this subtype have the higher percentage of aromatic amino acids (phe and tyr, see Fig. 3) but are not present in the spatula. Cysteine-rich CBPs instead lack aromatic amino acids and appear most related to the building up of the seta corneous bundles structurally more than in the spatula (Fig. 9). According to the above hypothesis, only two CBPs richer in tyrosine and phenylalanine, Ge-sprp1 and Ge-cprp16, could contribute a stronger van der Waals attraction, but the lack of information on the fine localization of them does not allow determining their contribution to the mechanism of adhesion which remains speculative. Nevertheless, if phenylalanine and tyrosine were localized on the surface of these proteins on the spatula, they could exert a strong attractive van der Waals force. Therefore, the nature of the proteins selected in the setae during gecko evolution might have been influenced by the efficiency of adhesion. The mechanism of adhesion may involve multiple sources, including van der Waals forces on a neutral or charged substrate (Fig. 10a) for distances of less than 10 Å, or on coupling

electrical and van der Waals forces (Fig. 10b) in the presence of water or polar hydrated substrates (Fig. 10c), or of capillary forces. The independent or interactive contributions of these mechanisms need to be further analyzed. It is known that increase of humidity enhances adhesion (Huber et al. 2005; Sun et al. 2005; Niewiarowski et al. 2008, 2016; Puthoff et al. 2010), possibly by the induction of numerous water dipoles that enhance the formation of localized van der Waals contacts between setae and substrate (Fig. 10c, c1, d, d1).

Concluding remarks Adhesion to chemically different types of substrates demonstrates the versatility of gecko setae in obtaining adhesive forces capable to sustaining climbing in dry or wet conditions and over almost any substrate, neutral or polar, smooth or variably rough using single or combined effects generated from electrical, van der Waals, or capillary forces (Russel 1985, 2002; Huber et al. 2005; Autumn and Gravish 2008; Puthoff et al. 2010; Izadi et al. 2014; Niewiarowski et al. 2016). Some of the various processes that may influence adhesion, presented in Fig. 10, explain the ability of gecko setae to adhere to almost any substrate,

Review: mapping proteins localized in adhesive setae of the tokay gecko and their possible influence on the...

Fig. 10 Drawing summarizing the hypothetical contribution of charged or uncharged setae to the mechanism of adhesion. a Setae are covered with positive charges and surrounded by water (dipoles). The spatula can be charged (a1) or not (a2, uncharged, 0), and can interact with substrates of different nature, neutral, charged negatively or positively, coated with water or other polar molecules or lipids, and hydrophobic. Charged surfaces avoid collapsing/stickiness among setae. b, b1 Examples showing a charged spatula during approaching the dry substrate, initially prevail long range (e.g., 1 to 100 nm) static electrical forces in charged condition (b), or simply the mechanical movement of

progressive approximation in uncharged (b1) conditions. c, c1 Examples in case the surface of the substrate is coated by one layer of water dipoles or more layers of water molecules (higher relative humidity). In this case, water or other hydrophilic molecules form a further interactive effect in the charged case (c), or the hydrophobic spatula might even displace by repulsion the water molecules creating a hydrophobic surface for attachment (c1). d, d1 Possible interactive effects of charged (d) or uncharged (d1, 0) spatulae on different substrates where the spatula might enhance di-polarization of molecules

natural or artificial (Hubert et al. 2005; Niewiarowski et al. 2016). In fact, the interplay between electrical charges and van der Waals forces when spatula are faced with different substrates at different distances ensures adhesion on almost every substrate. The combination of weak forces (van der Waals) and stronger electrical forces (charges, Izadi et al. 2014) ensures the climbing activity of geckos over all substrates that they encounter in nature, rocks and trees, and to many man-made substrates (glass, concrete, plastic etc., see Autumn and Peattie 2002; Russel 2002; Huber et al. 2005; Berengueres et al. 2007; Niewiarowski et al. 2016). In conclusion, future cell and molecular biology studies on gecko adhesion will have to identify the specific proteins present in the cytoskeleton of clear cells and in the spatula (Fig. 9). In addition, molecular modeling is necessary to determine the presence and the localization of aromatic or charged amino acids on the molecules of different CBPs, and if they are exposed on the spatula surface to generate stronger van der Waals or electrical forces (Fig. 10). This information will allow a better understanding of the mechanisms of adhesion,

leading to the design of the best polymers adapted to fabricate the next generation of dry adhesives. Acknowledgments Study self-supported, in particular for the ultrastructural analysis (Comparative Histolab). A preliminary communication on the protein localization in gecko setae was presented during the Belnstein Symposium on September 2016, held in Postdam, Berlin, Germany. The comments of referees helped in improving the MS.

Compliance with ethical standards Conflict of interest interest.

The author declares that there is no conflict of

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