Biology subjects

Bertinetti, L.

Publications and source records attributed to Bertinetti, L..

13 recordsLinked to original sources

bioRxiv2026

A fatty acid-binding protein links lipid handling to chitin synthase-dependent cuticle formation and nanoscale organization

Background: The insect cuticle is a chitin-containing extracellular matrix whose formation requires precise coordination of chitin biosynthesis, fibril organization, and post-synthetic maturation. Although lipids are essential components of the cuticle, the molecular mechanisms linking lipid transport to chitin deposition remain poorly understood. Here we identify TcFabp8, a fatty acid-binding protein in Tribolium castaneum, as a functionally important regulator of chitin synthase 1 (Chs1)-dependent cuticle formation. Results: RNAi-mediated depletion of TcFABP8 caused molting defects, reduced cuticle thickness, loss of apical TcChs1 immunoreactivity, and reduced chitin deposition and organization. The finding that TcCHS1 transcript levels were unaffected by RNAi targeting TcFABP8 suggests that a post-transcriptional step, potentially involving vesicular TcChs1 transport, is affected, thereby impairing chitin synthesis and deposition. Synchrotron X-ray diffraction analysis of adult elytra revealed that TcFabp8 depletion preserved characteristic -chitin-associated scattering features, but altered chitin fibril accumulation, crystalline coherence, and the spatially ordered alignment of fibrils within the cuticular matrix. The TcFabp8 gene locus encodes splice variants yielding different N-terminal architectures: TcFabp8X1 carries a Sec/SPI signal peptide which is absent in TcFabp8X2 and TcFabp8X3. Immunocytochemistry using TcFabp8-specific antibodies detected intracellular signals in subapical cytoplasmic puncta and extracellular signals within the cuticle layers, which partially colocalized with Nile red fluorescence indicating lipid-rich environments. Conclusions: Together, our data demonstrate that TcFabp8 is a critical facilitator of TcChs1-dependent chitin deposition and extends functional connection between lipid transport and chitin biosynthesis in insects.

physiology↗

bioRxiv2026

Structural and chemical properties of insects' chitin-containing extracellular matrices

Insects body barriers rely on specialized extracellular matrices that protect against harmful environmental influences. The outer barrier is the cuticle, which is composed of chitin, cuticle proteins and lipids. The peritrophic matrix (PM) serves as an inner barrier lining the midgut epithelium. It is composed of chitin fibers that are organized by PM proteins. While cuticle and PM proteins have received considerable attention in the past, supramolecular organization and physicochemical properties of the chitin component - particularly of the PM - remain poorly understood. Here, we combine synchrotron-based X-ray diffraction data from the PMs of lepidopteran and coleopteran insects with RNA interference (RNAi), mass spectrometric and histochemical analyses of the PM from Tribolium castaneum to determine chitins allomorphic state and degree of acetylation. The chitin of the PM exhibits signatures characteristic of dihydrate {beta}-chitin along the entire midgut. In contrast, the cuticle is made of tightly packed -chitin nanofibrils. Mass spectrometry revealed that the PMs chitin is highly acetylated (>95%). RNAi silencing of gut-specific genes encoding chitin deacetylasesTcCDA6-9 further increases the degree of acetylation. Histochemical analyses staining chitin with different degrees of acetylation confirm the predominance of highly acetylated chitin in the PM. Notably, the larval cuticle has a layered organization with deacetylated chitin present in exo- and highly acetylated chitin in endocuticles. Depletion of both TcCDA1 or TcCDA2 impairs chitin deacetylation, which indicates that both proteins cooperate in their activity in the integument. These results establish fundamental principles of polysaccharide-based extracellular matrices, with broad implications for insect biology.

molecular biology↗

bioRxiv2026

Osmotic pressure gradients in E. coli biofilms revealed by in-situ sensors

Osmotic pressure has been known to play essential roles in living systems from single cells to complex tissues. However, direct in-situ measurements of osmotic pressures in biosystems have remained challenging, especially in complicated heterogeneous systems in which osmotic pressure gradients could exist and induce directed forces. Bacterial biofilms -- organized communities of bacteria encased in a self-produced extracellular matrix -- are a major mode of bacterial life. It has, however, remained unexplored how the osmotic pressure is distributed in the biofilm and how this distribution contributes to biofilm growth and activity. Here, liposomal nano-sensors are developed for the in-situ mapping of osmotic pressures at an unprecedented microscale resolution in real time using Escherichia coli. biofilm as a model system that develops at the surface of a hydrogel containing the nutrients. The measurements reveal osmotic pressure gradients with a radially increasing trend from the inner regions to the outer regions of the biofilm, which is associated with biofilm formation, morphology, and metabolism. The gradients likely contribute to mechanical properties, internal stresses, and nutrient transport. The sensor readouts also show that there is an osmotic pressure difference between the biofilm and the adjacent medium, which may promote biofilm expansion through matrix swelling and bacteria growth via water and nutrient uptake from the surroundings. Our novel approach based on in-situ osmotic pressure mapping in a growing biofilm reveals a sophisticated spatial regulation of physical forces, which may inspire new models and approaches in the field of mechanobiology.

biophysics↗

bioRxiv2026

A bacterial extracellular matrix protein forms a supramolecular metallogel

The microbial extracellular matrix (ECM) is a complex network of self-secreted biopolymers uniting the cells in biofilms, providing them with structural integrity, and contributing to their elevated resistance to antibiotic treatments. Recently, there is a growing realization that a regulated, bidirectional cross-talk of bacteria and ECM confers biofilms with tissue-like traits, however, the mechanisms of spatio-temporal self-organisation of ECM and its regulation are still poorly understood. In the model organism for biofilm formation Bacillus subtilis, TasA is the major protein component of the extracellular matrix. We recently showed that TasA, isolated in the form of stable and structured globules, assembles into elongated and ordered fibers via a donor-strand complementation mechanism. In this study, we discovered that in the presence of zinc metal ions, TasA is able to form hydrogels with > 97% water content. Electron- and atomic force-microscopies as well as small angle X-ray scattering measurements show that cross-linking with zinc ions induces a transition in TasA morphology from one-dimensional fibers to two-dimensional sheets. Electron paramagnetic resonance measurements then show that such a significant morphological shift is associated with molecular changes in the coordination environment of zinc ions, which lead to structural changes at the protein level. When assembling into macroscopic networks, TasA-Zn metallogels exhibit viscoelastic properties and a fast recovery following an excessive strain. These metallogels represent a novel class of bacterially-derived ECMs that form easily at room temperature without covalent crosslinking, and may be used as a natural matrix-mimics in biofilm models for infection studies.

biochemistry↗

bioRxiv2024

Longhorn Beetles form Structural Colour Using Calcium Phosphate Biominerals

Brilliant structural colors originating from diverse photonic crystals are found across many phyla, including the striking iridescent colors of beetles and butterflies, produced by three-dimensional photonic crystal structures in the specialized cuticular scales. However, the precise composition of these structures remains largely unknown, although it is key to unravelling colour production mechanisms and morphogenesis. The longhorn beetle Doliops similis displays vibrant green patterns on its otherwise dark elytra. These patterns are formed by arrays of minute scales that encompass a three-dimensional photonic crystal made of orderly packed nanospheres. We found that these nanospheres are composed of carbonated amorphous calcium phosphate biomineral. By accurately parameterizing the structure and calculating the refractive index deduced from the relative fractions of the organic and inorganic phases, we derived reflection wavelengths that match the observed green hue, demonstrating the biominerals role in colour production. Our study further reveals that biomineralization is widespread in the Lamiinae subfamily, with colour diversity achieved through variations in nanosphere size, packing, and composition. This study opens new avenues for developing bioinspired mineral-based optical devices with high refractive indices and defect-resistance, overcoming the shortcomings of current polymer-based designs.

molecular biology↗

bioRxiv2022

Actomyosin remodeling regulates biomineral formation, growth and morphology during eukaryote 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 ROCKs 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 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.

developmental biology↗

bioRxiv2022

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 FRET (resonance energy transfer)-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 PEGylated 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 towards the in-situ sensing of osmotic pressures in biological culture systems.

biophysics↗

bioRxiv2022

Gradients of orientation, composition and hydration of proteins for efficient light collection by the cornea of the horseshoe crab

The lateral eyes of the horseshoe crab, Limulus polyphemus, are the largest compound eyes within recent Arthropoda. While this visual system has been extensively described before, the precise mechanism allowing vision has remained controversial. Correlating quantitative refractive index (RI) mapping and detailed structural analysis, we demonstrate how gradients of RI in the cornea result from the hierarchical organization of chitin-protein fibers, heterogeneity in protein composition and bromine doping, as well as spatial variation in water content. Combining the realistic cornea structure and measured RI gradients with full-wave optical modelling and ray-tracing approaches, we show that the light collection mechanism depends on both refraction-based graded index (GRIN) optics and total internal reflection. The optical properties of the cornea are governed by different mechanisms at different hierarchical levels, demonstrating the remarkable versatility of arthropod cuticle. One-sentence summaryStructural hierarchy and protein hydration determine the optical performance of the cornea of L. polyphemus.

biophysics↗

bioRxiv2021

Spores and extracellular matrix components impart molecular order in Bacillus subtilis biofilms.

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 in agricultural 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 (XRD) 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 (XRF) 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 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. Significance StatementBiofilms are multicellular soft microbial communities that are able to colonize synthetic surfaces as well as living organisms. To survive sudden environmental changes and efficiently share their common resources, cells in a biofilm divide into subgroups with distinct functions, leading to phenotypic heterogeneity. Here, by studying intact biofilms by synchrotron X-ray diffraction and fluorescence, we revealed correlations between biofilm macroscopic architectural heterogeneity and the spatio-temporal distribution of extracellular matrix, spores, water and metal ions. Our findings demonstrate that biofilm heterogeneity is not only affected by local genetic expression and cellular differentiation, but also by passive effects resulting from the physicochemical properties of the molecules secreted by the cells, leading to differential distribution of nutrients that propagates through macroscopic length scales.

microbiology↗

bioRxiv2020

Three-dimensional interrelationship between osteocyte network and forming mineral during human bone remodeling

During bone remodeling, osteoblasts are known to deposit unmineralized collagenous tissue (osteoid), which mineralizes after some time lag. Some of the osteoblasts differentiate into osteocytes, forming a cell network within the lacunocanalicular network (LCN) of bone. To get more insight into the potential role of osteocytes in the mineralization process of osteoid, sites of bone formation were three-dimensionally imaged in nine forming human osteons using focused ion beam-scanning electron microscopy (FIB-SEM). In agreement with previous observations, the mineral concentration was found to gradually increase from the central Haversian canal towards preexisting mineralized bone. Most interestingly, a similar feature was discovered on a length scale more than 100-times smaller, whereby mineral concentration increased from the LCN, leaving around the canaliculi a zone virtually free of mineral, the size of which decreases with progressing mineralization. This suggests that the LCN controls mineral formation but not just by diffusion of mineralization precursors, which would lead to a continuous decrease of mineral concentration from the LCN. Our observation is, however, compatible with the codiffusion and reaction of precursors and inhibitors from the LCN into the bone matrix.

biophysics↗

bioRxiv2019

Hierarchically-structured metalloprotein composite coatings biofabricated from co-existing condensed liquid phases

Complex hierarchical structure governs emergent properties in soft biopolymeric materials; yet, the material processing involved remains poorly understood. Here, we investigated the multiscale structure and composition of the mussel byssus cuticle before, during and after formation to gain further insight into the processing of this hard, yet extensible metal cross-linked protein composite. Our findings reveal that the granular substructure crucial to the cuticles function as a wear-resistant coating of an extensible polymer fiber is pre-organized in condensed liquid phase secretory vesicles. These are separated into catechol-rich proto-granules enveloped in a sulfur-rich proto-matrix which fuses during secretion, forming the sub-structure of the cuticle. Metal ions are added subsequently in a site-specific way, with Fe contained in the sulfur-rich matrix and V being relegated to the granules, coordinated by catechol. We posit that this hierarchical structure self-organizes via phase separation of specific amphiphilic protein components within secretory vesicles, resulting in a meso-scale structuring, critical to the cuticles advanced function.

biophysics↗

bioRxiv2019

Three-dimensional Structural Interrelations between Cells, Extracellular Matrix and Mineral in Vertebrate Mineralization

The spatial-temporal relationship between cells, extracellular matrices and mineral deposits is fundamental for an improved understanding 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 microns in range. These parameters are necessary to yield fine ultrastructural details while encompassing tissue domains sufficient to provide a comprehensive overview of the interrelationships between the tissue structural constituents. Investigation reveals a novel complex cellular network in highly mineralized tendon aspects, 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 globules appear between the fibrils and, with time, mineral propagates along and within collagen. These close associations between tenocytes, canaliculi, small channels, collagen and mineral suggest a new 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.\n\nSignificance StatementThe basic mechanism by which vertebrate collagenous tissues are mineralized is still not fully elucidated, despite the importance of this process for skeletal formation and regeneration. Through three-dimensional imaging of the cellular network together with the extracellular matrix and mineral deposits, the present work investigates normally mineralizing avian leg tendon as a model system for vertebrates in general. The data support a mechanism where mineral ions and possible mineral precursors are initially present in interfibrillar collagen spaces and are subsequently translocated to neighboring collagen fibrils. Mineral particles then nucleate in association with collagen to form the well known collagen-mineral composite material of the skeleton.

bioengineering↗

bioRxiv2019

Epidermal cell surface structure and chitin-protein co-assembly determine fiber architecture in the Locust cuticle

The geometrical similarity of helicoidal fiber arrangement in many biological fibrous extracellular matrices, such as bone, plant cell wall or arthropod cuticle, to that of cholesteric liquid mesophases has led to the hypothesis that they may form passively through a mesophase precursor rather than by direct cellular control. In search of direct evidence to support or refute this hypothesis, here, we studied the process of cuticle formation in the tibia of the migratory locust, Locusta migratoria, where daily growth layers arise by the deposition of fiber arrangements alternating between unidirectional and helicoidal structures. Using FIB/SEM volume imaging and scanning X-ray scattering, we show that the epidermal cells determine an initial fiber orientation from which the final architecture emerges by the self-organized co-assembly of chitin and proteins. Fiber orientation in the locust cuticle is therefore determined by both active and passive processes.

biophysics↗