Using the atomic force microscope, we have investigated the nanoscale mechanical response of the attachment adhesive of the terrestrial alga Prasiola linearis (Prasiolales, Chlorophyta). We were able to locate and extend highly ordered mechanical structures directly from the natural adhesive matrix of the living plant. The in vivo mechanical response of the structured biopolymer often displayed the repetitive sawtooth force-extension characteristics of a material exhibiting high mechanical strength at the molecular level. Mechanical and histological evidence leads us to propose a mechanism for mechanical strength in our sample based on amyloid fibrils. These proteinaceous, pleated β-sheet complexes are usually associated with neurodegenerative diseases. However, we now conclude that the amyloid protein quaternary structures detected in our material should be considered as a possible generic mechanism for mechanical strength in natural adhesives.

No abstract

While biological systems are notorious for their complexity, nature sometimes displays mechanisms that are elegant in their simplicity. We have recently identified such a mechanism at work to enhance the mechanical properties of certain natural adhesives. The mechanism is simple because it utilizes a non-specific protein folding and subsequent aggregation process, now thought to be generic for any polypeptide under appropriate conditions. This non-specific folding forms proteinaceous crossed β-sheet amyloid fibrils, which are usually associated with neurodegenerative diseases. Here we show evidence for the beneficial mechanical characteristics of these fibrils discovered in natural adhesives. We suggest that amyloid protein quaternary structures should be considered as a possible generic mechanism for mechanical strength in a range of natural adhesives and other natural materials due to their many beneficial mechanical features and apparent ease of self-assembly.

SUMMARY The patterns of occurrence of photosynthetic pigments and fatty acids among seven available species (11 strains) of marine raphidophytes were determined and used as chemotaxonomic markers. All currently recognized genera of marine raphidophytes were included for analysis: that is, Chattonella, Fibrocapsa, Heterosigma, Olisthodiscus and Haramonas. The characteristic pigment composition was shown to be chlorophyll a, chlorophylls c1 and/or c2, fucoxanthin as the major carot‐enoid, β,β‐carotene and any or all of zeaxanthin, violaxanthin and an auroxanthin‐like pigment as the minor carotenoids. The carotenoid composition of all marine raphidophyte genera investigated was virtually the same, except in Fibrocapsa and Haramonas, which differed due to the occurrence of fucoxanthinol and 19′‐butanoyloxyfucoxanthin, respectively. These fucoxanthin derivatives, in addition to fucoxanthin, have potential chemotaxonomic use for differentiating the two species. In all 11 strains, 15 fatty acids (saturated, mono‐unsaturated and polyunsaturated) were determined. Significant taxonomic distinctions between genera were reflected by their fatty acid profiles. A rapid key for the differentiation of genera, in addition to morphological features, may be the absence of the 18:4 fatty acid in Olisthodiscus; presence of 18:5 in Heterosigma; the presence of fucoxanthinol in Fibrocapsa and presence of 19′‐butanoyloxyfucoxanthin in Haramonas.

The presence of ''proteinaceous b-sheet rich fibrillar structures'' and amyloidogenic material, has been alluded to extensively in the literature, in association with natural materials exhibiting superior mechanical strength per unit volume. Here we provide a clear experimental demonstration and explanation for why individual amyloid quaternary structures themselves have beneficial mechanical characteristics.

The composition and nanoscale mechanical characteristics of the adhesive from two species of subaerial green unicellular microalgae (Chlorophyta), Coccomyxa sp. and Glaphyrella trebouxiodes, have been studied using Raman spectroscopy, chemical staining, and atomic force microscopy (AFM). Raman spectroscopy confirmed the adhesive proteins of both species to be predominantly in ß-sheet conformations and composed of a number of hydrophobic amino acid residues. Chemical staining with Congo red and thioflavin-T dyes further confirmed the presence of amyloid-like structures. Probing the adhesives with AFM revealed highly ordered and repetitive mechanical responses indicative of highly ordered structures within the adhesive. The repetitive nature of the sawtooth response is typical of a ''sacrificial bond'' and ''hidden length'' mechanism, and what wepropose is the result of mechanical manipulation of individual molecules within an intermolecular ß-sheet that makes up the generic amyloid structure. The mechanical data show how amyloid provides cohesive strength to the adhesives, and this intrinsic mechanical property of an amyloid-based adhesive explains the ecological success of attachment of these subaerial microalgae on various surfaces in urban environments. It is unknown to what extent amyloid fibrils occur in algal adhesives, but we postulate that the amyloid structure could provide a widespread mechanism for mechanical strength.

We have investigated the surface structure of islet amyloid polypeptide (IAPP) fibrils and α-synuclein protofibrils in liquid by frequency modulation atomic force microscopy (FM-AFM).Angstrom-resolution FM-AFM imaging of isolated macromolecules in liquid is demonstrated for the first time. Individual β-strands aligned perpendicular to the fibril axis with a spacing of 0.5 nm are resolved in FM-AFM images, which confirms cross-β structure of IAPP fibrils in real-space.FM-AFM images also reveal the existence of 4 nm periodic domains along the axis of IAPP fibrils.Stripe features with 0.5 nm spacing are also found in images of α-synuclein protofibrils. However, in contrast to IAPP fibrils, the stripes are oriented 30º from the axis, suggesting the possibility of different β-strand alignment in protofibrils from that in mature fibrils or the regular arrangement of Thioflavin T molecules present during the fibril preparation aligned at the surface of the protofibrils.

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.