Background: The shells of various Haliotis species have served as models of invertebrate biomineralization and physical shell properties for more than 20 years. A focus of this research has been the nacreous inner layer of the shell with its conspicuous arrangement of aragonite platelets, resembling in cross-section a brick-and-mortar wall. In comparison, the outer, less stable, calcitic prismatic layer has received much less attention. One of the first molluscan shell proteins to be characterized at the molecular level was Lustrin A, a component of the nacreous organic matrix of Haliotis rufescens. This was soon followed by the C-type lectin perlucin and the growth factor-binding perlustrin, both isolated from H. laevigata nacre, and the crystal growth-modulating AP7 and AP24, isolated from H. rufescens nacre. Mass spectrometry-based proteomics was subsequently applied to to Haliotis biomineralization research with the analysis of the H. asinina shell matrix and yielded 14 different shell-associated proteins. That study was the most comprehensive for a Haliotis species to date. Methods: The shell proteomes of nacre and prismatic layer of the marine gastropod Haliotis laevigata were analyzed combining mass spectrometry-based proteomics and next generation sequencing. Results: We identified 297 proteins from the nacreous shell layer and 350 proteins from the prismatic shell layer from the green lip abalone H. laevigata. Considering the overlap between the two sets we identified a total of 448 proteins. Fifty-one nacre proteins and 43 prismatic layer proteins were defined as major proteins based on their abundance at more than 0.2% of the total. The remaining proteins occurred at low abundance and may not play any significant role in shell fabrication. The overlap of major proteins between the two shell layers was 17, amounting to a total of 77 major proteins. Conclusions: The H. laevigata shell proteome shares moderate sequence similarity at the protein level with other gastropod, bivalve and more distantly related invertebrate biomineralising proteomes. Features conserved in H. laevigata and other molluscan shell proteomes include short repetitive sequences of low complexity predicted to lack intrinsic three-dimensional structure, and domains such as tyrosinase, chitin-binding, and carbonic anhydrase. This catalogue of H. laevigata shell proteins represents the most comprehensive for a haliotid and should support future efforts to elucidate the molecular mechanisms of shell assembly.

Figure S1. SDS-PAGE of shell organic matrix. This figure in jpg format shows a SDS-PAGE comparison between the nacre acid-soluble fraction obtained with different protocols A, B and C, and comparison of prismatic layer acid-insoluble fractions A and B. Similar amounts of matrix (ca. 200Â Iźg) were applied to each lane. (JPG 1284Â kb)

Peptides, nacre acid-insoluble, protocol B. See legend to Additional file 17. (XLSX 547Â kb)

Peptides, nacre acid-soluble, protocol C. See legend to Additional file 17. (XLSX 339Â kb)

ProteinGroups, prismatic layer acid-soluble, protocol B. See legend to Additional file 6. (XLSX 53Â kb)

ProteinGroups, nacre acid-soluble, protocol A. See legend to Additional file 6. (XLSX 167Â kb)

Mimicry of the tough natural composite nacre in future bioengineering requires knowledge of the biomineralisation process. The insoluble organic matrix isolated from the shell of the gastropod Haliotis laevigata was characterised by protein chemistry, topographical and mechanical measurements. Demineralisation of nacre in dilute acetic acid or ethylenediaminetetraacetic acid revealed a set of soluble proteins and the insoluble matrix. The insoluble matrix contains a chitin core and firmly attached proteins, which could be removed by sodium dodecyl sulfate and glycerol indicating a hydrophobic interaction. Atomic force microscopy images of the native insoluble matrix showed a filamentous network with pores or holes, where the filaments showed globular attachments of different sizes, possibly the attached protein molecules. During direct observation of protein degradation imaged by atomic force microscopy the insoluble matrix gets smooth and flat indicating the removal of the attached proteins by proteases. We propose a model of protein coated chitin filaments for the insoluble matrix of nacre. Mechanical measurements by force mapping revealed a Young's modulus depending on the hydration state of the organic layers. The fully hydrated organic matrix has an elastic modulus below 1 MPa comparable to some hydrogels.

Mineral growth in nacre and other CaCO3-containing biominerals is controlled by biopolymers. Water-soluble proteins were extracted from nacre of the sea snail Haliotis laevigata by dissolving the mineral phase with 6% acetic acid. The influence of this protein mixture on CaCO3 precipitation rates was investigated at different concentrations. A well-established assay for measuring the pH-value during CaCO3 precipitation with and without protein additives was extended by calculating maximum precipitation rates from the pH-values. It could be shown that precipitation rates are greatly influenced by the mixtures of water-soluble nacre proteins. At very low protein concentrations (0.02μg/ml) a rate enhancement in comparison to the pure supersaturated calcium carbonate solution by a factor of 1.4 was observed. At higher protein concentrations, a strong inhibitory effect occurred, with total inhibition at concentrations of 1.0μg/ml and higher. Two unspecific proteins (bovine serum albumin and lysozyme) showed little or no modification of precipitation rates. In vivo, the function of the strong inhibition of CaCO3 precipitation by nacre proteins at higher concentrations is presumably to prevent uncontrolled crystallization in the extrapallial fluid. The rate-enhancing capability of proteins at low concentrations may be explained by the presence of acidic and/or hydrophilic moieties.

During the growth process of the nacre layer in abalone shells, aragonite platelets are built into a preformed organic matrix. This matrix consists of chitin and different proteins that may affect the morphology and the crystal structure of the developed platelets. The organic matrix can be divided into two fractions: the soluble and the insoluble matrix. The soluble matrix has influence on the precipitation rates of calcium carbonate crystals and also the insoluble matrix affects crystal growth. In this work we investigated the collective influence of soluble and insoluble matrix in a crystallization device which contained the insoluble matrix and was flowed through by CaCl2 and NaHCO3 solutions. The presence of 0.02 mu g/mL soluble matrix promoted the growth of flat CaCO3 crystals on the surface layers of the insoluble matrix of Haliotis laevigata. The crystals could be identified as aragonite using electron diffraction in the transmission electron microscope (TEM). Furthermore the addition of 1 mu g/mL soluble matrix inhibited largely crystal growth on the surface of the insoluble matrix. Our findings indicate that components of the soluble matrix are required for a controlled nucleation and growth of flat aragonite crystals on the insoluble matrix.

Mouse embryonic fibroblasts explore the chemical suitability before spreading on a given substrate. We find this early phase of cell spreading to be characterized by transient adhesion patches with a typical mean size of (1.0 ± 0.4) µm and a lifetime of (33 ± 12) s. Eventually, these patches fuse to initiate extensive spreading of the cell. We monitor cell adhesion using reflection interference contrast and total internal reflection fluorescence microscopy. Digital time lapse movies are analysed employing spatio-temporal correlation functions of adhesion patterns. Correlation length and time can be scaled to obtain a master curve at the fusion point.