In the footsteps of sea stars: deciphering the catalogue of proteins involved in underwater temporary adhesion

Abstract

Sea stars adhere strongly but temporarily to underwater substrata via the secretion of a blend of proteins, forming an adhesive footprint that they leave on the surface after detachment. Their tube feet enclose a duo-gland adhesive system comprising two types of adhesive cells, contributing different layers of the footprint and de-adhesive cells. In this study, we characterized the catalogue of sea star footprint proteins (Sfps) in the species Asterias rubens to gain insights in their potential function. We identified 16 Sfps and mapped their expression to type 1 and/or type 2 adhesive cells or to de-adhesive cells by double fluorescent in situ hybridization. Based on their cellular expression pattern and their conserved functional domains, we propose that the identified Sfps serve different functions during attachment, with two Sfps coupling to the surface, six providing cohesive strength and the rest forming a binding matrix. Immunolabelling of footprints with antibodies directed against one protein of each category confirmed these roles. A de-adhesive gland cell-specific astacin-like proteinase presumably weakens the bond between the adhesive material and the tube foot surface during detachment. Overall, we provide a model for temporary adhesion in sea stars, including a comprehensive list of the proteins involved.

Keywords: Asteroidea; Echinodermata; duo-gland adhesive system; proteomics and transcriptomics; sea star footprint protein; tube feet.

Figure 1.

Sea stars use their multiple tube feet to temporarily but strongly attach to many substrata. Here, a sea star of the species Asterias rubens crawling over mussels.

Figure 2.

Stratified organization of the gland cells in the tube foot adhesive epidermis. (a) Schematic representation of a longitudinal section through a radial epidermal strip located between two adjacent connective tissue laminae (adapted from Hennebert et al. [3]; not to scale). The distal surface of the tube foot is at the bottom of the drawing. (b–d) TEM images taken at the same magnification and showing different areas of the adhesive epidermis corresponding to the areas illustrated in the drawing. Secretory cells are false coloured according to the schematic representation. (b) Proximal part showing cell bodies of AC1. (c) Middle part showing cell bodies of AC2 and DAC. (d) Distal part showing support cells and the apical processes of duo-gland cells (AC1, AC2 and DAC). (e–g) High magnification on AC1, AC2 and DAC at the distal part of the adhesive epidermis. Scale bars: (d) 5 µm, (e–g) 1 µm. AC1, type 1 adhesive gland cell; AC2, type 2 adhesive gland cell; CTL, connective tissue layer; DAC, de-adhesive gland cell; M, mucus gland; N, nerve plexus; SC, support cell.

Figure 3.

Localization of mRNAs coding for Sfps in the tube foot epidermis of A. rubens visualized by double ISH. (a) Schematic drawing and (b) bright field picture of a longitudinal tube foot section. Boxed area indicates the approximate area of double ISH images. (c–n) Localization of mRNAs coding for the AC1-specific protein Sfp1 (d,g,j,m; in green) and those coding for AC1-specific Sfp5 (c,e), AC2-specific Sfp8 (f,h), DAC-specific Astacin-like Sfp (i,k) and AC1/2 expressed Sfp10 (l,n) (in red). (o–q) Localization of the transcripts coding for the AC2-specific Sfp8 (in red) and AC1/2 expressed Sfp10 (in green). The nuclei were stained with DAPI (in blue). When different probes were available for a single Sfp, they were identified by a number between brackets (e.g. Sfp8(1); see electronic supplementary material, table S2). Schematic drawing in (a) modified after Santos et al. [12]. Scale bars: 50 µm.

Figure 4.

Conserved domain architecture of Sfps. The proteins were grouped according to their secreting cell: AC1 only, AC2 only, both adhesive gland cell types (AC1 + AC2), or DAC. AC1, type 1 adhesive gland cell; AC2, type 2 adhesive gland cell; DAC, de-adhesive gland cell; A2M, alpha-2-macroglobulin; A2M BRD, alpha-2-macroglobulin bait region domain; A2M rcpt-bd, alpha-2-macroglobulin receptor-binding domain; A2M TED, alpha-macroglobulin-like thioester domain; CUB, complement C1r/C1 s, uEGF, BMP1 domain; C8, domain of eight conserved cysteine residues; disorder pred., areas of predicted disorder with no stable secondary structure; D-Gal lectin, D-Galactose binding lectin domain; EGF, epidermal growth factor-like domain; FAMeT, Farnesoic acid O-methyltransferase domain; FA58C, coagulation factor 5/8 type C-terminal domain; incomplete seq, start and/or end of sequence is unknown; TIL/TILa, trypsin inhibitor-like cysteine-rich domain; vWF C, von Willebrand factor type C domain; vWF D, von Willebrand factor type D domain; WAP, whey acidic protein domain.

Figure 5.

Immunofluorescence localization of selected Sfps in tube foot sections of Asterias rubens. Tube foot longitudinal sections were labelled with antibodies directed against (a) Sfp7/8, (b) Sfp10 and (c) Astacin-like Sfp (labelling in green). Scale bars: 50 µm.

Figure 6.

Immunofluorescence localization of selected Sfps in the footprints of Asterias rubens. Footprints were labelled with antibodies directed against Sfp7/8 (a,b) and Astacin-like Sfp (c,d). Scale bars: 100 µm (a,c) and 30 µm (b,d).

Figure 7.

Double immuno- and lectin-labelling on footprints of Asterias rubens. Footprint labelled with antibodies directed against Sfp1β (a), with the lectin WGA (b) and overlay image (c). Footprint labelled with antibodies directed against Sfp7/8 (d), with WGA (e) and overlay image (f). Scale bars: 20 µm (a–c) and 100 µm (d–f).