Luca Bertinetti

The microbial extracellular matrix (ECM) is a complex network of self-secreted biopolymers that unite cells in biofilms, providing them with structural integrity and protection from antimicrobials. Made of fibrillar proteins interspersed with polysaccharides, ECM material is often implicitly held responsible for biofilms’ hydrogel-like mechanical signature; the exact molecular mechanisms of the ECM assembly and their link to hydrogel formation, however, remain enigmatic. Here, we discover that in the presence of zinc ions, TasA, the major ECM protein of Bacillus subtilis, forms hydrogels that recapitulate the mechanical properties of whole biofilms. Electron and atomic force microscopies as well as small-angle X-ray scattering showed that cross-linking with zinc ions induced a transition in the TasA architecture from one-dimensional fibers to two-dimensional nanosheets. Electron paramagnetic resonance and X-ray diffraction data link this dramatic morphological shift to molecular changes in the coordination environment of zinc ions and protein structure. This study provides the first example and a novel molecular mechanism of supramolecular metallogelation by a native bacterial protein. Bridging between molecular processes, mesoscopic structure, and macroscopic material properties, it not only advances the fundamental understanding of ECM assembly in situ but also establishes artificial ECM-based (metallo-)hydrogels as matrix mimics for bacterial infection studies.

Insect’s 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 chitin’s allomorphic state and degree of acetylation. The chitin of the PM exhibits signatures characteristic of dihydrate β-chitin along the entire midgut. In contrast, the cuticle is made of tightly packed α-chitin nanofibrils. Mass spectrometry revealed that the PM’s 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.

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.

Herein, we use synchrotron micro-computed tomography to capture the three-dimensional architecture of the skeletal stereom in sea urchins for morphological quantification.

Adult female desert locusts ( Schistocerca gregaria ) dig underground to lay their eggs, ensuring optimal conditions for successful hatching. Digging is performed using the two pairs of oviposition valves at the tip of the female's abdomen. These valves are subjected to considerable shear forces during the repeated digging cycles, potentially leading to wear over time. The resilience of the valves is investigated by analyzing the relationship between digging experience and valve damage and wear throughout the female locust's life. The findings reveal the ability of the valves to withstand the significant shear forces encountered during digging. Despite this resilience, however, perceptible limitations in the valves’ mechanical durability against wear are observed. Toward the end of the female locust's life, the valves show substantial signs of wear, indicating effective performance but with limited longevity, i.e., a designated life span that enables successful oviposition for ca . four oviposition cycles. A comparison of the valve material with that of the animals’ mandibles, which are used continuously throughout their life and show remarkable wear‐resistance, further highlights the evolutionary adaptation of the valve materials to their specific function, suggesting a trade‐off between energetic investment and the sufficient, or “good‐enough”, performance that is required for survival.

For a better understanding of living tissues and materials, it is essential to study the intricate spatial relationship between cells and their surrounding tissue on the nanoscale, with a need for 3D, high‐resolution imaging techniques. In the case of bone, focused ion beam‐scanning electron microscopy (FIB‐SEM) operated in the backscattered electron (BSE) mode proves to be a suitable method to image mineralized areas with a nominal resolution of 5 nm. However, as clinically relevant samples are often resin‐embedded, the lack of atomic number (Z) contrast makes it difficult to distinguish the embedding material from unmineralized parts of the tissue, such as osteoid, in BSE images. Staining embedded samples with iodine vapor has been shown to be effective in revealing osteoid microstructure by 2D BSE imaging. Based on this idea, an iodine (Z = 53) staining protocol is developed for 3D imaging with FIB‐SEM, investigating how the amount of iodine and exposure time influences the imaging outcome. Bone samples stained with this protocol also remain compatible with confocal laser scanning microscopy to visualize the lacunocanalicular network. The proposed protocol can be applied for 3D imaging of tissues exhibiting mineralized and nonmineralized regions to study physiological and pathological biomineralization.

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.

Mussels (Mytilus edulis) adhere to hard surfaces in intertidal marine habitats with a porous underwater glue called the byssus plaque. The plaque is an established role model for bioinspired underwater glues and comprises at least six proteins, most of which are highly cationic and enriched in the post-translationally modified amino acid 3,4-dihydroxyphenylalanine (DOPA). While much is known about the chemistry of plaque adhesion, less is understood about the natural plaque formation process. Here, we investigated plaque structure and formation using 3D electron microscopic imaging, revealing that micro- and nanopores form spontaneously during secretion of protein-filled secretory vesicles. To better understand this process, we developed a method to purify intact secretory vesicles for in vitro assembly studies. We discovered that each vesicle contains a sulfate-associated fluid condensate consisting of ∼9 histidine- and/or DOPA-rich proteins, which are presumably the required ingredients for building a plaque. Rupturing vesicles under specific buffering conditions relevant for natural assembly led to controlled multiphase liquid-liquid phase separation (LLPS) of different proteins, resulting in formation of a continuous phase with coexisting droplets. Rapid coarsening of the droplet phase was arrested through pH-dependent cross-linking of the continuous phase, producing native-like solid porous "microplaques" with droplet proteins remaining as fluid condensates within the pores. Results indicate that histidine deprotonation and sulfates figure prominently in condensate cross-linking. Distilled concepts suggest that combining phase separation with tunable cross-linking kinetics could be effective for microfabricating hierarchically porous materials via self-assembly.

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.