Refine
Document Type
- Article (9)
Language
- English (9)
Is part of the Bibliography
- yes (9)
Keywords
- biomineralization (2)
- mollusc shell (2)
- Chitin (1)
- Chitin Synthesis (1)
- Larval Shell (1)
- Mollusc Shell (1)
- Shell Formation (1)
- amino acids (1)
- bending strength (1)
- biomechanics (1)
Scientific Unit
Mechanical properties of plants and underlying structure-property relationships are important for agricultural purposes as well as for biomimetic concepts. In this study, the effect of mechanical stimulation on morphology and bending properties of the stalk was investigated for Sorghum bicolor (Poaceae), a widely used drought-tolerant biomass grass. An experimental set-up allowing for defined growth and mechanical perturbation (flexing) during a defined growth period was designed. Mechanical properties of individual internodes of the stalk were determined by three-point bending tests. We found that the three investigated lines showed differences in their general bending strength in the non-stimulated condition. However, similar high range of bending strength values was measured for all plant lines after they underwent the mechanical stimulation procedure. The anatomy of internode cross-sections was examined to evaluate structure-property relationships. An increased thickness of the outer sclerenchymatous tissue was observed for internodes with higher bending strength values. Dried internodes fail under lower strains but showed higher bending strength. These findings show that mechanosensitivity in sorghum is dependent on genetic as well as environmental factors. The experimental system presented here offers new straight-forward possibilities for S. bicolor line selection for applications requiring mechanical strength of the stalk.
A microfluidic biosensor with surface acoustic wave technology was used in this study to monitor the interaction of calcium carbonate with standard carboxylate self-assembled monolayer sensor chips. Different fluids, with and without biomolecular components, were investigated. The pH-dependent surface interactions of two bio-inspired cationic peptides, AS8 and ES9, which are similar to an extracellular domain of the chitin synthase involved in mollusc shell formation, were also investigated in a biological buffer system. A range of experimental conditions are described that are suitable to study non-covalent molecular interactions in the presence of ionic substances, such as, mineral precursors below the solubility equilibrium. The peptide ES9, equal to the mollusc chitin synthase epitope, is less sensitive to changes in pH than its counterpart AS8 with a penta-lysine core, which lacks the flanking acidic residues. This study demonstrates the extraordinary potential of microfluidic surface acoustic wave biosensors to significantly expand our experimental capabilities for studying the principles underlying biomineralization in vitro.
Datasets from a slow carbonate vapor diffusion and mineral precipitation protocol for Dictyostelium ECM and cellulose stalks show examples for composite materials obtained by an in vitro approach, which differs substantially from the in vivo approach reported in The Journal of Structural Biology, doi: 10.1016/j.jsb.2016.03.015 [1]. Methods for obtaining the datasets include bright field transmitted light microscopy, fluorescence microscopy, LC-PolScope birefringence microscopy, variable pressure scanning electron microscopy (VP-SEM/ESEM), and Raman imaging spectroscopy.
Background: Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain. Results: Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-Dglucosamine (UDP-GlcNAc). The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 μM -10 μM) dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges. Conclusion: Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types.
Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro . First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 mg/ml and higher, the inhibitory effect was observed predominantly for HCO 3 − as the first ionic interaction partner, but not necessarily for Ca 2+ . The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.
BACKGROUND: Mollusc shells are commonly investigated using high-resolution imaging techniques based on cryo-fixation. Less detailed information is available regarding the light-optical properties. Sea shells of Haliotis pulcherina were embedded for polishing in defined orientations in order to investigate the interface between prismatic calcite and nacreous aragonite by standard materialographic methods. A polished thin section of the interface was prepared with a defined thickness of 60 µm for quantitative birefringence analysis using polarized light and LC-PolScope microscopy. Scanning electron microscopy images were obtained for comparison. In order to study structural-mechanical relationships, nanoindentation experiments were performed.RESULTS:Incident light microscopy revealed a super-structure in semi-transparent regions of the polished cross-section under a defined angle. This super-structure is not visible in transmitted birefringence analysis due to the blurred polarization of small nacre platelets and numerous organic interfaces. The relative orientation and homogeneity of calcite prisms was directly identified, some of them with their optical axes exactly normal to the imaging plane. Co-oriented "prism colonies" were identified by polarized light analyses. The nacreous super-structure was also visualized by secondary electron imaging under defined angles. The domains of the super-structure were interpreted to consist of crystallographically aligned platelet stacks. Nanoindentation experiments showed that mechanical properties changed with the same periodicity as the domain size.CONCLUSIONS:In this study, we have demonstrated that insights into the growth mechanisms of nacre can be obtained by conventional light-optical methods. For example, we observed super-structures formed by co-oriented nacre platelets as previously identified using X-ray Photo-electron Emission Microscopy (X-PEEM) [Gilbert et al., Journal of the American Chemical Society 2008, 130:17519--17527]. Polarized optical microscopy revealed unprecedented super-structures in the calcitic shell part. This bears, in principle, the potential for in vivo studies, which might be useful for investigating the growth modes of nacre and other shell types.
The size, morphology and species-specific texture of mollusc shell biominerals is one of the unresolved questions in nature. In search of molecular control principles, chitin has been identified by Weiner and Traub (FEBS Lett. 1980, 111 : 311-316) as one of the organic compounds with a defined co-organization with mineral phases. Chitin fibers can be aligned with certain mineralogical axes of crystalline calcium carbonate in a speciesspecific manner. These original observations motivated the functional characterization of chitin forming enzymes in molluscs. The full-length cDNA cloning of mollusc chitin synthases identified unique myosin domains as part of the biological control system. The potential impact of molecular motors and other conserved domains of these complex transmembrane enzymes on the evolution of shell biomineralization is investigated and discussed in this article.
We here present the nucleation and growth of calcium carbonate under the influence of synthetic peptides on topographically patterned poly(dimethylsiloxane) (PDMS) substrates, which have a controlled density of defects between the wrinkles. Experiments with two lysine-rich peptides derived from the extracellular conserved domain E22 of the mollusc chitin synthase Ar-CS1, AKKKKKAS (AS8) and EEKKKKKES (ES9) on these substrates showed their influence on the calcium carbonate morphology. A transition from polycrystalline composites to single crystalline phases was achieved with the peptide AS8 by changing the pH of the buffer solution. We analyzed three different pH values as previous experiments showed that E22 interacts with aragonite biominerals more strongly at pH 7.75 than at pH 9.0. At any given pH, crystals appeared in characteristic morphologies only on wrinkled substrates, and did not occur on the flat, wrinkle-free PDMS substrate. These results suggest that these wrinkled substrates could be useful for controlling the morphologies of other mineral/peptide and mineral/protein composites. In nature, these templates are formed enzymatically by glycosyltransferases containing pH-sensitive epitopes, similar to the peptides investigated here. Our in vitro test systems may be useful to gain understanding of the formation of distinct 3D morphologies in mollusc shells in response to local pH shifts during the mineralization of organic templates.