Mechanically functional amyloid fibrils in the adhesive of a marine invertebrate as revealed by Raman spectroscopy and atomic force microscopy

Abstract

Amyloid fibrils are primarily known in a pathogenic context for their association with a wide range of debilitating human diseases. Here we show a marine invertebrate (Entobdella soleae) utilizes functional amyloid fibrils comparable to those of a unicellular prokaryote (Escherichia coli). Thioflavin-T binding and Raman spectroscopy provided evidence for the presence of amyloid in the adhesive of Entobdella soleae. We elucidated that for these two very different organisms, amyloid fibrils provide adhesive and cohesive strength to their natural adhesives. Comparing the nanoscale mechanical responses of these fibrils with those of pathogenic amyloid by atomic force microscopy revealed that the molecular level origin of the cohesive strength was associated with the generic intermolecular β-sheet structure of amyloid fibrils. Functional adhesive residues were found only in the case of the functional amyloid. Atomic force microscopy provided a useful means to characterize the internal structural forces within individual amyloid fibrils and how these relate to the mechanical performance of both functional and pathogenic amyloid. The mechanistic link of amyloid-based cohesive and adhesive strength could be widespread amongst natural adhesives, irrespective of environment, providing a new strategy for biomimicry and a new source of materials for understanding the formation and stability of amyloid fibrils more generally.

Characterisation of Amyloid Nanostructures in the Natural Adhesive of Unicellular Subaerial Algae

“…In conclusion, we have established that the natural adhesives of two representative species of subaerial, unicellular microalgae exhibit the same underlying nanoscale repetitive sawtooth response to a tensile force as those measured for adhesives from multicellular subaerial algae [15], diatoms [12,13], a parasitic flatworm [29], and the bacterial amyloid-based biofilm, curli [29]. A large body of literature exists describing the structure and performance of natural adhesives, but often with an emphasis on the diversity of biological adhesives [22,63], rather than highlighting any shared underlying mechanisms.…”

“…To date there has been no identification of any protein from the family of structurally homologous proteins that would need to exist in order to explain the observed sawtooth responses of different dimensions found from an increasing number of different natural adhesives. Our preferred explanation is that the response is due to pulling on a functional form of amyloid in the adhesive [15,28,29], whereby sawtooths indicate the successive unravelling of linked small proteins from the intermolecular b-sheets making up the amyloid fibril structure. This is based on mechanical arguments linking a number of unusual measured mechanical responses to the generic amyloid core structure that cannot be explained by a large single molecule [28].…”

“…The algal autofluorescence due to the presence of chlorophyll was evident at 680 nm, as determined by exciting an unstained control sample at 488 nm and collecting the emission between 505 and 700 nm. Emission of thiofavin-T binding to a control sample of amyloid fibrils grown from a synthesized amyloid b-protein [25][26][27][28][29][30][31][32][33][34][35] (H-1192, Bachem, Merseyside, UK) was determined using the same procedure as described previously [15]. Sample binding by thioflavin-T was detected by directly staining algae attached to coverslips with 10 mM aqueous thioflavin-T for 5 mins before being washed in pure water.…”

Nanoscale characterization of the temporary adhesive of the sea urchin Paracentrotus lividus

“…Among marine adhesives, most studies have focused on permanent adhesives like those of mussels, barnacles and tubeworms, whereas the so-called nonpermanent, temporary or reversible adhesives have received much less attention. To our knowledge, the nanostructure of temporary adhesives has only been examined in a few organisms such as marine flatworms [1], barnacle cyprids [2–4], freshwater cnidaria [5] and echinoderms such as sea cucumbers [6] and sea stars [7–8]. This characterization was performed using atomic force microscopy (AFM), a technique that allows high-resolution images of soft biological materials to be obtained [9–10] as well as the nanomechanical properties.…”

“…Parasitic marine flatworms such as Entobdella solae temporarily attach to fish skin using anterior pads located in the head [1]. Adhesion is brought about by interaction between two kinds of glandular secretions which are extruded together to form the adhesive [11].…”