Ingrid M. Weiss

Calcium and silicon mineralization in land plants: Transport, structure and function

Plant biomineralization involves calcium and silicon transport and mineralization. Respective analytical methods and case studies are listed. Calcium carbonate is deposited in cystoliths, calcium oxalate in idioblasts. Silicon is deposited in phytoliths. Biomineralization is a coordinated process.

The chitin synthase involved in marine bivalve mollusk shell formation contains a myosin domain

Chitin is a key component in mollusk nacre formation. However, the enzyme complex responsible for chitin deposition in the mollusk shell remained unknown. We cloned and characterized the chitin synthase of the marine bivalve mollusk Atrina rigida. We present here the first chitin synthase sequence from invertebrates containing an unconventional myosin motor head domain. We further show that a homologous gene for chitin synthase is expressed in the shell forming tissue of larval Mytilus galloprovincialis even in early embryonic stages. The new data presented here are the first clear‐cut indication for a functional role of cytoskeletal forces in the precisely controlled mineral deposition process of mollusk shell biogenesis.

The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z

BackgroundChitin 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.ResultsEnzymatic 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-D-glucosamine (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 shells edges.ConclusionProvided 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.

Imaging microtubules and kinesin decorated microtubules using tapping mode atomic force microscopy in fluids

Abstract The atomic force microscope has been used to investigate microtubules and kinesin decorated microtubules in aqueous solution adsorbed onto a solid substrate. The netto negatively charged microtubules did not adsorb to negatively charged solid surfaces but to glass covalently coated with the highly positively charged silane trimethoxysilylpropyldiethylenetriamine (DETA) or a lipid bilayer of 1,2-dipalmitoyl-3-dimethylammoniumpropane. Using electron beam deposited tips for microtubules adsorbed on DETA, single protofilaments could be observed showing that the resolution is up to 5 nm. Under conditions where the silane coated surfaces are hydrophobic, microtubules opened, presumably at the seam, whose stability is lower than that of the bonds between the other protofilaments. This led to a “sheet” with a width of about 100 nm firmly attached to the surface. Microtubules decorated with a stoichiometric low amount of kinesin molecules in the presence of the non-hydrolyzable ATP-analog 5′-adenylylimidodiphosphate could also be adsorbed onto silane-coated glass. Imaging was very stable and the molecules did not show any scan-induced deformation even after hundreds of scans with a scan frequency of 100 Hz.

The peacock's train (Pavo cristatus and Pavo cristatus mut. alba) I. structure, mechanics, and chemistry of the tail feather coverts

The feathers in the train of the peacock serve not for flying but for sexual display. They are long, slender beams loaded in bending by their own weight. An outer circular conical shell, the cortex, is filled by a closed foam of 7.6% relative density, the medulla, both of feather keratin. Outer diameter and thickness of the cortex decrease linearly from the body toward the tip. This self-similar geometry leads to a division of labor. The cortex (longitudinal Youngs modulus 3.3 GPa, transverse modulus 1 GPa) provides 96% of the longitudinal strength and bending rigidity of the feather. The medulla (Youngs modulus 10 MPa) provides 96% of the transverse compressive rigidity. Fracture stress of the cortex, both longitudinal and transverse, is 120 MPa.

Jewels in the Pearl

The recent discovery of the aragonite-specific protein complex of Pif97 and Pif80, has inspired a simple concept based on extracellular membranes that would explain the remote control of nacre formation and reunify the epitaxy, colloidal, and cellular concepts for nacre platelet morphology. In this model, cells contribute by providing an oriented and asymmetric chitinous membrane. This membrane is pinned to the more matured nacre in certain spots until the onset of nucleation. By diffusing in both directions through the chitinous membrane, low-molecular-weight acidic proteins are likely to fulfill an important role as Ca/H shuttles to avoid local acidification and balance mineralization gradients over the chitinous membrane on the nanoscale. Then, aragonite nucleation by Pif97/Pif80 complexes selectively on one side of this membrane could be a function of CO2 diffusion. In vivo, an intermittent contact of chitinous membranes with the previously formed nacre during nucleation, and the subsequent separation of this layer including chitin-silk phase separations while the crystals proceed towards the pre-existing layer, should be taken into account. Molluscs such as the abalone and the pearl oysters (Figure 1) probably earned the most ancient degrees in materials science—back in neoproterozoic times, once they had learned their lessons in the biochemistry of how to build a shell. A precisely controlled hierarchical architecture of organic (mainly chitin) and inorganic (mainly aragonite) layers is the secret behind the excellent mechanical performance of nacre. The genetic encoding of mollusc-shell architectures is one of the currently most challenging research areas. Molecular biochemical approaches complemented with RNAi (a tool to knock out expression of specific genes) based in vivo studies in the pearl oyster Pinctada fucata have now substantially increased our knowledge. Suzuki and colleagues have discovered two acidic nacre matrix proteins, Pif80 and Pif97, derived from a single polypeptide by post-translational modifications. Pif97 contains putative regulatory VWA (von Willebrand factor type A domain, a multifunctional regulatory protein) and chitin binding domains. Pif80 induces the formation of aragonite (Figure 2). Here I explain why Pif complexes are likely to challenge the existing concepts of nacre formation that have been developed in several laboratories worldwide over many years. Figure 1. Nacre from various species. Upper left, Abalone (Haliotis spec.), 12 cm in length, view towards the mantle epithelium–shell interface. Lower left, cross-section of Unio at 1000:1. Taken from ref. [3] , copyright : Schmidt, 1924. Upper right, carbon replica of fractured pearl oyster Pinctada. Taken from ref. [9] , copyright: Gr goire, 1957. Lower right, confocal fluorescence image of a chitin-binding protein in a cross-section of Mytilus (as previously described).

Native supported membranes on planar polymer supports and micro-particle supports

To bridge soft biological materials and hard inorganic materials is an interdisciplinary scientific challenge. Despite of experimental difficulties, the deposition of native biological membranes on supports is a straightforward strategy. This review provides an overview of advances in the fabrication and characterization of native biological membranes on planar polymer supports and micro-particles.

Covalent modification of chitin with silk-derivatives acts as an amphiphilic self-organizing template in nacre biomineralisation

Molluscs have a well-deserved reputation for being expert mineralizers of various shell types such as nacre. Nacre is defined as regularly arranged layers and stacks of approximately 0.5 microm thick aragonite platelets that are extracellularly formed within a complex mixture of organic matrix. The control of species-specific layer thickness by the animal is still enigmatic. Despite the recent findings on the periodic layer-by-layer structures of chitin layers and silk-like protein layers in nacre-type biominerals, little is known about how the interface is defined between two different layers. In this paper, we demonstrate the presence of covalently attached, hydrophobic amino acid side chains in the chitin matrix in the bivalve mollusc Mytilus galloprovincialis by the combination of infrared spectroscopy and mass spectroscopy. The accumulation of the modified chitin matrix at the interface is quantified by the critical aggregate concentration of the purified chitin matrix, which is approximately an order of magnitude smaller than that of pure chitin. Our finding suggests an active role of such chemically modified chito-oligosaccharides in the creation of a defined interface and guidance of the periodic matrix textures, which would result in unique material properties of natural mollusc shells.

Keratin homogeneity in the tail feathers of Pavo cristatus and Pavo cristatus mut. alba

The keratin structure in the cortex of peacocks’ feathers is studied by X-ray diffraction along the feather, from the calamus to the tip. It changes considerably over the first 5 cm close to the calamus and remains constant for about 1 m along the length of the feather. Close to the tip, the structure loses its high degree of order. We attribute the X-ray patterns to a shrinkage of a cylindrical arrangement of β-sheets, which is not fully formed initially. In the final structure, the crystalline beta-cores are fixed by the rest of the keratin molecule. The hydrophobic residues of the beta-core are locked into a zip-like arrangement. Structurally there is no difference between the blue and the white bird.

On the function of chitin synthase extracellular domains in biomineralization

Molluscs with various shell architectures evolved around 542-525 million years ago, as part of a larger phenomenon related to the diversification of metazoan phyla. Molluscs deposit minerals in a chitin matrix. The mollusc chitin is synthesized by transmembrane enzymes that contain several unique extracellular domains. Here we investigate the assembly mechanism of the chitin synthase Ar-CS1 via its extracellular domain ArCS1_E22. The corresponding transmembrane protein ArCS1_E22TM accumulates in membrane fractions of the expression host Dictyostelium discoideum. Soluble recombinant ArCS1_E22 proteins can be purified as monomers only at basic pH. According to confocal fluorescence microscopy experiments, immunolabeled ArCS1_E22 proteins adsorb preferably to aragonitic nacre platelets at pH 7.75. At pH 8.2 or pH 9.0 the fluorescence signal is less intense, indicating that protein-mineral interaction is reduced with increasing pH. Furthermore, ArCS1_E22 forms regular nanostructures on cationic substrates as revealed by atomic force microscopy (AFM) experiments on modified mica cleavage planes. These experiments suggest that the extracellular domain ArCS1_E22 is involved in regulating the multiple enzyme activities of Ar-CS1 such as chitin synthesis and myosin movements by interaction with mineral surfaces and eventually by protein assembly. The protein complexes could locally probe the status of mineralization according to pH unless ions and pCO2 are balanced with suitable buffer substances. Taking into account that the intact enzyme could act as a force sensor, the results presented here provide further evidence that shell formation is coordinated physiologically with precise adjustment of cellular activities to the structure, topography and stiffness at the mineralizing interface.