Localization of the cellular origin of sea star adhesive proteins
using in situ hybridization
Elise
1
Hennebert ,
1Laboratory
Birgit
2
Lengerer ,
Mélanie
1
Demeuldre ,
Peter
2
Ladurner ,
Patrick
1
Flammang
of Biology of Marine Organisms and Biomimetics, Research Institute for Biosciences, University of Mons, 7000 Mons, Belgium
2Institute of Zoology, University of Innsbruck, 6020 Innsbruck, Austria
Background
A
Tube feet
B
C
Sea stars are able to adhere to various surfaces using an adhesive material secreted by their
tube feet. This material is produced by two types of adhesive cells localized in the tube foot
distal part, the disc (Figs 1,2; Hennebert et al. 2008). Recently, proteins extracted from the
adhesive material were analyzed using tandem mass spectrometry and the resulting data were
compared to the translated tube foot transcriptome. For each of the 296 proteins identified in
the transcriptome, the exponentially modified Protein Abundance Index (emPAI; Ishihama et al.
2005), which estimates the relative abundance of proteins in a mixture, was calculated. Among
the 20 major proteins on the basis of emPAI values, only 2 (cofilin-like and muscle-specific
protein 20-like), were identified in the NCBInr database. No homology was found for the 18
other proteins, which therefore correspond to potential major novel adhesive proteins. The aim
of my short-term scientific mission at the Institute of Zoology (University of Innsbruck, Austria)
was to use the technique of in situ hybridization to confirm the role of some of these proteins in
sea star adhesion through their localization in the adhesive cells.
Figure 1. Photographs showing a sea star of the species Asterias rubens (A) and its tube feet (B). (C) Longitudinal section through a tube foot stained with
Heidenhain’s azan trichrome showing the stem (S) covered by a non-adhesive epidermis (NAE) and the disc (D) presenting a thick adhesive epidermis (AE).
Methods
The complementary DNA sequences coding for these
proteins picked up in the tube foot transcriptome were
used to design specific oligonucleotide probes. These
probes were used in whole tube feet in situ hybridization
to label the corresponding mRNA in the cells (Lengerer et
al. 2014).
Results
For the 4 proteins investigated, a specific labelling at the
base of the tube foot disc, at the level of the nucleuscontaining cell bodies of adhesive cells (Fig. 2B-F).
However, the labelling appears to be restricted to the
outer layer of the disc, probably resulting from the
difficulty encountered by the probes to penetrate
through all the thickness of the disc (Fig. 2C). For
controls, in situ hybridizations were also performed on
tube feet using specific probes for actin, a protein
abundantly expressed in all the cells, and for a major
protein of the mucus secreted by the sea stars. For actin,
a labelling was observed in the entirety of the stem
epidermis as well as in the base of the disc, confirming
the expression of this protein in all the tube foot cells
(Fig. 2G). For the mucus protein, the labelling is
restricted to some scattered spots in the stem epidermis
(Fig. 2H, I).
Figure 2. In situ hybridization on Asterias rubens tube feet using probes designed on the basis of cDNA sequences available in the tube foot
transcriptome (A,B,F,G,H: general views of the tube foot showing the disc (D) made up of an adhesive epidermis and the stem (S) covered by
a non-adhesive epidermis; D,E,I: detailed views of the base of the disc epidermis (D,E) and of the stem epidermis (I); C: upper view of a tube
foot). (A) negative control for the cDNA comp199 coding for the first major protein in adhesive footprints, (B) specific probe for the cDNA
comp199 coding for the first major protein in adhesive footprints, (C,D) specific probe for the cDNA comp43 coding for the second major
protein in adhesive footprints, (E) specific probe for the cDNA comp1698 coding for the third major protein in adhesive footprints, (F) specific
probe for the cDNA comp6449 coding for the fourth major protein in adhesive footprints, (G) specific probe for the cDNA comp5 coding for
actin, (H,I) specific probe for the cDNA comp24506 coding for one major protein in mucus.
Discussion and perspectives
These results clearly demonstrate that the proteins identified as major components in adhesive footprints using mass spectrometry are adhesive
proteins. In a near future, the technique of in situ hybridization and will be applied to the other major Sfps. These proteins, once their
involvement in adhesion confirmed, will be analyzed by RT-PCR in order to obtain their complete sequence and the combined results will be the
subject of a publication.
References:
Hennebert E, Viville P, Lazzaroni R, Flammang P (2008) Micro- and nanostructure of the adhesive material secreted by the tube feet of the sea star Asterias rubens. J Struct Biol 164(1): 108-118; Ishihama Y et al. (2005) Exponentially modified protein abundance index (emPAI) for estimation of absolute
protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 4(9): 1265–1272; Lengerer B et al. (2014) Biological adhesion of the flatworm Macrostomum lignano relies on a duo-gland system and is mediated by a cell type-specific intermediate filament protein
Front Zool 11:12.