Multiscale X-ray study of Bacillus subtilis biofilms reveals interlinked structural hierarchy and elemental heterogeneity.

Biofilms are multicellular microbial communities that encase themselves in an extracellular matrix (ECM) of secreted biopolymers and attach to surfaces and interfaces. Bacterial biofilms are detrimental in hospital and industrial settings, but they can be beneficial, for example, in agricultural as well as in food technology contexts. An essential property of biofilms that grants them with increased survival relative to planktonic cells is phenotypic heterogeneity, the division of the biofilm population into functionally distinct subgroups of cells. Phenotypic heterogeneity in biofilms can be traced to the cellular level; however, the molecular structures and elemental distribution across whole biofilms, as well as possible linkages between them, remain unexplored. Mapping X-ray diffraction across intact biofilms in time and space, we revealed the dominant structural features in Bacillus subtilis biofilms, stemming from matrix components, spores, and water. By simultaneously following the X-ray fluorescence signal of biofilms and isolated matrix components, we discovered that the ECM preferentially binds calcium ions over other metal ions, specifically, zinc, manganese, and iron. These ions, remaining free to flow below macroscopic wrinkles that act as water channels, eventually accumulate and may possibly lead to sporulation. The possible link between ECM properties, regulation of metal ion distribution, and sporulation across whole, intact biofilms unravels the importance of molecular-level heterogeneity in shaping biofilm physiology and development.

Three-dimensional structural interrelations between cells, extracellular matrix, and mineral in normally mineralizing avian leg tendon.

The spatial-temporal relationship between cells, extracellular matrices, and mineral deposits is fundamental for an improved understanding of mineralization mechanisms in vertebrate tissues. By utilizing focused ion beam-scanning electron microscopy with serial surface imaging, normally mineralizing avian tendons have been studied with nanometer resolution in three dimensions with volumes exceeding tens of micrometers in range. These parameters are necessary to yield sufficiently fine ultrastructural details while providing a comprehensive overview of the interrelationships between the tissue structural constituents. Investigation reveals a complex lacuno-canalicular network in highly mineralized tendon regions, where ∼100 nm diameter canaliculi emanating from cell (tenocyte) lacunae surround extracellular collagen fibril bundles. Canaliculi are linked to smaller channels of ∼40 nm diameter, occupying spaces between fibrils. Close to the tendon mineralization front, calcium-rich deposits appear between the fibrils and, with time, mineral propagates along and within them. These close associations between tenocytes, tenocyte lacunae, canaliculi, small channels, collagen, and mineral suggest a concept for the mineralization process, where ions and/or mineral precursors may be transported through spaces between fibrils before they crystallize along the surface of and within the fibrils.

The spider cuticle: a remarkable material toolbox for functional diversity.

Engineered systems are typically based on a large variety of materials differing in composition and processing to provide the desired functionality. Nature, however, has evolved materials that are used for a wide range of functional challenges with minimal compositional changes. The exoskeletal cuticle of spiders, as well as of other arthropods such as insects and crustaceans, is based on a combination of chitin, protein, water and small amounts of organic cross-linkers or minerals. Spiders use it to obtain mechanical support structures and lever systems for locomotion, protection from adverse environmental influences, tools for piercing, cutting and interlocking, auxiliary structures for the transmission and filtering of sensory information, structural colours, transparent lenses for light manipulation and more. This paper illustrates the 'design space' of a single type of composite with varying internal architecture and its remarkable capability to serve a diversity of functions. This article is part of the theme issue 'Bio-derived and bioinspired sustainable advanced materials for emerging technologies (part 1)'.

Spatiotemporal Measurement of Osmotic Pressures by FRET Imaging.

Osmotic pressures (OPs) play essential roles in biological processes and numerous technological applications. However, the measurement of OP in situ with spatiotemporal resolution has not been achieved so far. Herein, we introduce a novel kind of OP sensor based on liposomes loaded with water-soluble fluorescent dyes exhibiting resonance energy transfer (FRET). The liposomes experience volume changes in response to OP due to water outflux. The FRET efficiency depends on the average distance between the entrapped dyes and thus provides a direct measure of the OP surrounding each liposome. The sensors exhibit high sensitivity to OP in the biologically relevant range of 0-0.3 MPa in aqueous solutions of salt, small organic molecules, and macromolecules. With the help of FRET microscopy, we demonstrate the feasibility of spatiotemporal OP imaging, which can be a promising new tool to investigate phenomena involving OPs and their dynamics in biology and technology.

Quantification of sheet nacre morphogenesis using X-ray nanotomography and deep learning.

High-resolution three-dimensional imaging is key to our understanding of biological tissue formation and function. Recent developments in synchrotron-based X-Ray tomography techniques provide unprecedented morphological information on relatively large sample volumes with a spatial resolution better than 50 nm. However, the analysis of the generated data, in particular image segmentation - separation into structure and background - still presents a significant challenge, especially when considering complex biomineralized structures that exhibit hierarchical arrangement of their constituents across many length scales - from millimeters down to nanometers. In the present work, synchrotron-based holographic nano-tomography data are combined with state-of-the-art machine learning methods to image and analyze the nacreous architecture in the bivalve Unio pictorum in 3D. Using kinetic and thermodynamic considerations known from physics of materials, the obtained spatial information is then used to provide a quantitative description of the structural and topological evolution of nacre during shell formation. Ultimately, this study establishes a workflow for high-resolution three-dimensional analysis of fine highly-mineralized biological tissues while providing a detailed analytical view on nacre morphogenesis.

Cryo-FIB-SEM as a promising tool for localizing proteins in 3D.

Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) is an invaluable tool to visualize the 3D architecture of cell constituents and map cell networks. Recently, amorphous ice embedding techniques have been associated with FIB-SEM to ensure that the biological material remains as close as possible to its native state. Here we have vitrified human HeLa cells and directly imaged them by cryo-FIB-SEM with the secondary electron InLens detector at cryogenic temperature and without any staining. Image stacks were aligned and processed by denoising, removal of ion beam milling artefacts and local charge imbalance. Images were assembled into a 3D volume and the major cell constituents were modelled. The data illustrate the power of the workflow to provide a detailed view of the internal architecture of the fully hydrated, close-to-native, entire HeLa cell. In addition, we have studied the feasibility of combining cryo-FIB-SEM imaging with live-cell protein detection. We demonstrate that internalized gold particles can be visualized by detecting back scattered primary electrons at low kV while simultaneously acquiring signals from the secondary electron detector to image major cell features. Furthermore, gold-conjugated antibodies directed against RNA polymerase II could be observed in the endo-lysosomal pathway while labelling of the enzyme in the nucleus was not detected, a shortcoming likely due to the inadequacy between the size of the gold particles and the voxel size. With further refinements, this method promises to have a variety of applications where the goal is to localize cellular antigens while visualizing the entire native cell in three dimensions.

Unraveling the Rapid Assembly Process of Stiff Cellulosic Fibers from Mistletoe Berries.

The mucilaginous viscin tissue within mistletoe berries possesses an extraordinary ability to be rapidly processed under ambient conditions into stiff cellulosic fibers (>14 GPa) through simple mechanical drawing. This rapid and extreme transformation process is hydration-dependent and involves an astonishing >200-fold increase in length, providing a relevant role model for efforts to produce advanced composites from cellulose-based structures such as cellulose nanocrystals or cellulose nanofibrils. Using a combination of in situ polarized light microscopy, synchrotron X-ray scattering, and humidity-controlled mechanical analysis, we examine here the dynamic transition of a viscin cell bundle from hydrogel-like tissues to high-performance fibers. Our findings indicate a massive phase transition in which cellulose microfibrils containing high-aspect-ratio crystalline domains undergo dramatic reorganization, facilitated by a water-responsive noncellulosic matrix. Transition from an aligned, yet flowing state to a stiff fiber is likely triggered by rapid water loss below 45% relative humidity. These findings not only help understanding the adaptive success of mistletoe but may also be relevant for the development of new facile processing methods for next-generation cellulosic composites.

Co-incorporation of alkali metal ions during amorphous calcium carbonate precipitation and their stabilizing effect.

Calcium carbonate formation has been studied extensively due to its central role in biomineralization and geochemistry. Specifically, the effect of additives incorporated during the formation process has been described in several works related to inorganic, small organic, molecular or macromolecular additives. However, in these previous experiments the presence of counter ions and their possible role has been mostly disregarded. Co-incorporation of counter ions into calcite at low supersaturations has been studied in detail but their incorporation in and effect on the formation and stability of the amorphous phase, which precedes the formation of the crystalline phase at high supersaturations, has not been studied. To address this, we have investigated the incorporation of alkali metal ions into the amorphous phase using various carbonate salts as a carbonate source. We show that the incorporation is the highest for Rb+ with the highest measured value being 5.8 at% Rb+/(Rb+ + Ca2+). The extent of ion incorporation follows the ion size of Rb+ > K+ > Na+ > Li+ which is opposite to that observed in calcite formed at low supersaturation. The presence of these ions in the amorphous phase increases the crystallization temperature, which can be shifted by as much as 200 °C depending on the concentration of alkali metal ions incorporated. However, the lifetime of ACC in solution was similar for all the different carbonate sources.

Mobility of hydrous species in amorphous calcium/magnesium carbonates.

Amorphous calcium carbonate (ACC) is commonly found in many biological materials. As ACC readily crystallizes into calcite, stabilizers, such as anions, cations or macromolecules, often occur to avoid or delay unwanted crystallization. In biogenic ACC, magnesium is commonly present as one of the stabilizing agents. It is generally thought that the presence of mobile water in ACC is responsible for its limited stability and that the strong interaction of Mg2+ with water stabilizes the amorphous structure by retarding dehydration of ACC. To test this hypothesis, we studied the mobility of hydrous species in the model materials ACC, amorphous magnesium carbonate (AMC) and amorphous calcium/magnesium carbonate (ACMC), using quasi elastic neutron scattering (QENS) which is highly sensitive to the dynamics of H atoms. We discovered that hydrous species in the considered amorphous materials consist of water and hydroxide ions, as magnesium ions are incorporated in a ratio of 1 to about 0.6 with OH-. Surprisingly, we found that there is no evidence of translational diffusion of water and hydroxides when calcium is present in the samples, showing that hydrous species are highly static. However, we did observe diffusion of water in AMC with similar dynamics to that found for water in clays. Our results suggest that Mg2+-water interactions alone are not the only reason for the high stability of AMC and ACMC. The stabilizing effect of Mg ions, in addition to Mg-water binding, is likely to be caused by binding to hydroxide in amorphous calcium carbonates. In fact, the incorporation of hydroxides into the amorphous phase results in a mineral composition that is incompatible with any of the known Ca/Mg-carbonate crystal phases, requiring large scale phase separation to reach the composition of even the basic magnesium carbonate minerals artinite and hydromagnesite.

Ultrastructural, material and crystallographic description of endophytic masses - A possible damage response in shark and ray tessellated calcified cartilage.

The cartilaginous endoskeletons of elasmobranchs (sharks and rays) are reinforced superficially by minute, mineralized tiles, called tesserae. Unlike the bony skeletons of other vertebrates, elasmobranch skeletons have limited healing capability and their tissues' mechanisms for avoiding damage or managing it when it does occur are largely unknown. Here we describe an aberrant type of mineralized elasmobranch skeletal tissue called endophytic masses (EPMs), which grow into the uncalcified cartilage of the skeleton, but exhibit a strikingly different morphology compared to tesserae and other elasmobranch calcified tissues. We use materials and biological tissue characterization techniques, including computed tomography, electron and light microscopy, X-ray and Raman spectroscopy and histology to characterize the morphology, ultrastructure and chemical composition of tesserae-associated EPMs in different elasmobranch species. EPMs appear to develop between and in intimate association with tesserae, but lack the lines of periodic growth and varying mineral density characteristic of tesserae. EPMs are mineral-dominated (high mineral and low organic content), comprised of birefringent bundles of large calcium phosphate crystals (likely brushite) aligned end to end in long strings. Both tesserae and EPMs appear to develop in a type-2 collagen-based matrix, but in contrast to tesserae, all chondrocytes embedded or in contact with EPMs are dead and mineralized. The differences outlined between EPMs and tesserae demonstrate them to be distinct tissues. We discuss several possible reasons for EPM development, including tissue reinforcement, repair, and disruptions of mineralization processes, within the context of elasmobranch skeletal biology as well as damage responses of other vertebrate mineralized tissues.

Control of Polymorph Selection in Amorphous Calcium Carbonate Crystallization by Poly(Aspartic Acid): Two Different Mechanisms.

Poly(aspartic acid) (pAsp) is known to stabilize amorphous calcium carbonate (ACC) and affect its crystallization pathways. However, little is known about the mechanisms behind these phenomena. Here it is shown that ACC is stabilized by pAsp molecules in the solution rather than by the amount of pAsp incorporated into the ACC bulk, and that the effect of pAsp on the polymorph selection is entirely different at low and high concentration of pAsp. At low concentrations, pAsp is more effective in inhibiting the nucleation and growth of vaterite than calcite. At high concentrations, when calcite formation is prevented, the crystallization of vaterite proceeds via a pseudomorphic transformation of ACC nanospheres, where vaterite nucleates on the surface of ACC nanospheres and grows by a local transformation of the bulk ACC phase. These results shed some light on the function of pAsp during an ACC-mediated biomineralization process and provide an explanation for the presence of metastable vaterite at conditions where calcite is thermodynamically favored.

Neutron reflectometry yields distance-dependent structures of nanometric polymer brushes interacting across water.

The interaction between surfaces displaying end-grafted hydrophilic polymer brushes plays important roles in biology and in many wet-technological applications. In this context, the conformation of the brushes upon their mutual approach is crucial, because it affects interaction forces and the brushes' shear-tribological properties. While this aspect has been addressed by theory, experimental data on polymer conformations under confinement are difficult to obtain. Here, we study interacting planar brushes of hydrophilic polymers with defined length and grafting density. Via ellipsometry and neutron reflectometry we obtain pressure-distance curves and determine distance-dependent polymer conformations in terms of brush compression and reciprocative interpenetration. While the pressure-distance curves are satisfactorily described by the Alexander-de-Gennes model, the pronounced brush interpenetration as seen by neutron reflectometry motivates detailed simulation-based studies capable of treating brush interpenetration on a quantitative level.

Cooperative behavior of a sacrificial bond network and elastic framework in providing self-healing capacity in mussel byssal threads.

The dissipative and self-healing properties of mussel byssal threads are critical for their function as anchoring fibers in wave-battered habitats and central to their emergence as an exciting model system for bio-inspired polymers. Much is now understood about the structure-function relationships defining this remarkable proteinaceous bio-fiber; however, the molecular mechanisms underlying the distinctive tough, viscoelastic and self-healing behavior are still unclear. Here, we investigate elastic and dissipative contributions from the primary load-bearing proteins in the distal region of byssal threads (the preCols) using X-ray diffraction (XRD) combined with in situ tensile testing. Specifically, we identified cross ß-sheet structure in the preCol flanking domains that functions as an elastic framework, providing hidden length. Dissipative behavior was associated with a strain-rate dependent phase transition of a sacrificial network stabilized by strong, reversible cross-links. Based on these findings, we posit a new model for byssal thread deformation and self-healing.

Layered growth of crayfish gastrolith: about the stability of amorphous calcium carbonate and role of additives.

Previous studies on pre-molt gastroliths have shown a typical onion-like morphology of layers of amorphous mineral (mostly calcium carbonate) and chitin, resulting from the continuous deposition and densification of amorphous mineral spheres on a chitin-matrix during time. To investigate the consequences of this layered growth on the local structure and composition of the gastrolith, we performed spatially-resolved Raman, X-ray and SEM-EDS analysis on complete pre-molt gastrolith cross-sections. Results show that especially the abundance of inorganic phosphate, phosphoenolpyruvate (PEP)/citrate and proteins is not uniform throughout the organ but changes from layer to layer. Based on these results we can conclude that ACC stabilization in the gastrolith takes place by more than one compound and not by only one of these additives.

Selecting for function: solution synthesis of magnetic nanopropellers.

We show that we can select magnetically steerable nanopropellers from a set of carbon coated aggregates of magnetic nanoparticles using weak homogeneous rotating magnetic fields. The carbon coating can be functionalized, enabling a wide range of applications. Despite their arbitrary shape, all nanostructures propel parallel to the vector of rotation of the magnetic field. We use a simple theoretical model to find experimental conditions to select nanopropellers which are predominantly smaller than previously published ones.

ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis.

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates' biomineralizing cells, yet, little is known on ROCK's role in invertebrates' biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

The magnetosome membrane protein, MmsF, is a major regulator of magnetite biomineralization in Magnetospirillum magneticum AMB-1.

Magnetotactic bacteria (MTB) use magnetosomes, membrane-bound crystals of magnetite or greigite, for navigation along geomagnetic fields. In Magnetospirillum magneticum sp. AMB-1, and other MTB, a magnetosome gene island (MAI) is essential for every step of magnetosome formation. An 8-gene region of the MAI encodes several factors implicated in control of crystal size and morphology in previous genetic and proteomic studies. We show that these factors play a minor role in magnetite biomineralization in vivo. In contrast, MmsF, a previously uncharacterized magnetosome membrane protein encoded within the same region plays a dominant role in defining crystal size and morphology and is sufficient for restoring magnetite synthesis in the absence of the other major biomineralization candidates. In addition, we show that the 18 genes of the mamAB gene cluster of the MAI are sufficient for the formation of an immature magnetosome organelle. Addition of MmsF to these 18 genes leads to a significant enhancement of magnetite biomineralization and an increase in the cellular magnetic response. These results define a new biomineralization protein and lay down the foundation for the design of autonomous gene cassettes for the transfer of the magnetic phenotype in other bacteria.

Submicron-Sized In Situ Osmotic Pressure Sensors for In Vitro Applications in Biology.

Physical forces are important cues in determining the development and the normal function of biological tissues. While forces generated by molecular motors have been widely studied, forces resulting from osmotic gradients have been less considered in this context. A possible reason is the lack of direct in situ measurement methods that can be applied to cell and organ culture systems. Herein, novel kinds of resonance energy transfer (FRET)-based liposomal sensors are developed, so that their sensing range and sensitivity can be adjusted to satisfy physiological osmotic conditions. Several types of sensors are prepared, either based on polyethylene glycol- (PEG)ylated liposomes with steric stabilization and stealth property or on crosslinked liposomes capable of enduring relatively harsh environments for liposomes (e.g., in the presence of biosurfactants). The sensors are demonstrated to be effective in the measurement of osmotic pressures in pre-osteoblastic in vitro cell culture systems by means of FRET microscopy. This development paves the way toward the in situ sensing of osmotic pressures in biological culture systems.

Subcanalicular Nanochannel Volume Is Inversely Correlated With Calcium Content in Human Cortical Bone.

The spatial distribution of mineralization density is an important signature of bone growth and remodeling processes, and its alterations are often related to disease. The extracellular matrix of some vertebrate mineralized tissues is known to be perfused by a lacunocanalicular network (LCN), a fluid-filled unmineralized structure that harbors osteocytes and their fine processes and transports extracellular fluid and its constituents. The current report provides evidence for structural and compositional heterogeneity at an even smaller, subcanalicular scale. The work reveals an extensive unmineralized three-dimensional (3D) network of nanochannels (~30 nm in diameter) penetrating the mineralized extracellular matrix of human femoral cortical bone and encompassing a greater volume fraction and surface area than these same parameters of the canaliculi comprising the LCN. The present study combines high-resolution focused ion beam-scanning electron microscopy (FIB-SEM) to investigate bone ultrastructure in 3D with quantitative backscattered electron imaging (qBEI) to estimate local bone mineral content. The presence of nanochannels has been found to impact qBEI measurements fundamentally, such that volume percentage (vol%) of nanochannels correlates inversely with weight percentage (wt%) of calcium. This mathematical relationship between nanochannel vol% and calcium wt% suggests that the nanochannels could potentially provide space for ion and small molecule transport throughout the bone matrix. Collectively, these data propose a reinterpretation of qBEI measurements, accounting for nanochannel presence in human bone tissue in addition to collagen and mineral. Further, the results yield insight into bone mineralization processes at the nanometer scale and present the possibility for a potential role of the nanochannel system in permitting ion and small molecule diffusion throughout the extracellular matrix. Such a possible function could thereby lead to the sequestration or occlusion of the ions and small molecules within the extracellular matrix. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

A joint proteomic and genomic investigation provides insights into the mechanism of calcification in coccolithophores.

Coccolithophores are globally abundant, calcifying microalgae that have profound effects on marine biogeochemical cycles, the climate, and life in the oceans. They are characterized by a cell wall of CaCO3 scales called coccoliths, which may contribute to their ecological success. The intricate morphologies of coccoliths are of interest for biomimetic materials synthesis. Despite the global impact of coccolithophore calcification, we know little about the molecular machinery underpinning coccolithophore biology. Working on the model Emiliania huxleyi, a globally distributed bloom-former, we deploy a range of proteomic strategies to identify coccolithogenesis-related proteins. These analyses are supported by a new genome, with gene models derived from long-read transcriptome sequencing, which revealed many novel proteins specific to the calcifying haptophytes. Our experiments provide insights into proteins involved in various aspects of coccolithogenesis. Our improved genome, complemented with transcriptomic and proteomic data, constitutes a new resource for investigating fundamental aspects of coccolithophore biology.