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Disulfide bond formation in secretory proteins occurs primarily in the endoplasmic reticulum (ER), where multiple enzyme families catalyze cysteine cross-linking. Quiescin sulfhydryl oxidase 1 (QSOX1) is an atypical disulfide catalyst, localized to the Golgi apparatus or secreted from cells. We examined the physiological function for extracellular catalysis of de novo disulfide bond formation by QSOX1. QSOX1 activity was required for incorporation of laminin into the extracellular matrix (ECM) synthesized by fibroblasts, and ECM produced without QSOX1 was defective in supporting cell-matrix adhesion. We developed an inhibitory monoclonal antibody against QSOX1 that could modulate ECM properties and undermine cell migration.

It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissue-remodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM–cell crosstalk. Because ECM proteolysis is an abundant biochemical process that is critical for tissue homoeostasis, these results improve our fundamental understanding its complexity and its impact on cell behavior.

Historically, multicellular bacterial communities, known as biofilms, have been thought to be held together solely by a self-produced extracellular matrix. Our study identified a novel mechanism maintaining Bacillus subtilis and Mycobacterium smegmatis biofilms—active production of calcite minerals. We studied, for the first time, the effects of mutants defective in biomineralization and calcite formation on biofilm development, resilience and morphology. We demonstrated that an intrinsic rise in carbon dioxide levels within the biofilm is a strong trigger for the initiation of calcite-dependent patterning. The calcite-dependent patterns provide resistance to environmental insults and increase the overall fitness of the microbial community. Our results suggest that it is highly feasible that the formation of mineral scaffolds plays a cardinal and conserved role in bacterial multicellularity. Biofilm development: Bolstering bacterial infrastructure Bacteria reinforce the three-dimensional structure of their biofilms with a mineralized mixture of amorphous and crystalline calcium. The soil bacterium Bacillus subtilis forms biofilms with numerous wrinkles, which scientists have generally attributed to the organization of the proteins and polysaccharides forming the underlying extracellular matrix. Ilana Kolodkin-Gal and colleagues at the Weizmann Institute of Science now reveal that these microbes also construct communities atop a robust interior layer of calcium carbonate. The researchers identified bacterial genes and environmental triggers that contribute to this mineralization process, and observed identical behavior in another, distantly related bacterial species. Their results suggest that this mineral matrix strengthens the integrity of the biofilm and allows it to support larger bacterial populations. This process may also reduce bacterial vulnerability to antibiotics, making it a potentially interesting target for fighting biofilm-associated infections.

The complex structures and morphologies of coral skeletons make it difficult to study the construction of individual skeleton components. This is especially true regarding micro-structures deposited during different stages of skeletogenesis. As such structures serve as the basis for subsequent skeleton development, they are often obscured by additional skeletal deposition and growth. Recently we described a new model system, coral-on-a-chip, for studying coral biology and skeletogenesis. This system utilizes micropropagates of the Indo-Pacific coral Pocillopora damicornis maintained within a microfluidic environment, allowing us to follow early stages of skeletogenesis over time and under controlled environmental conditions. Our findings reveal that, following settlement onto glass slides, the micropropagates initially form a thin, almost two dimensional skeleton, which subsequently develops into a robust three-dimensional form with features resembling those of the mother colony. Studying the early stages of skeleton accretion in our micropropagates using high-resolution scanning electron microscopy and Energy Dispersive X-ray Spectroscopy (EDS) revealed a magnesium-rich layer deposited directly on the glass surface. This layer, which has a typical Mg/Ca ratio of 1.43±0.78, forms a dense lattice with a typical pore size of 100 nm. Microscopic observations indicate that this lattice serves as the basis for subsequent growth of fibrous aragonite. Examination of the underside of a skeleton from a small P. damicornis colony growing on a glass surface revealed a similar high-magnesium lattice at the interface between the glass and aragonitic skeleton in association with fibrous aragonite deposition. These observations suggest a role for this magnesium-rich lattice in the deposition of the fibrous aragonite forming the bulk of the coral skeleton.

Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study, we probe the spatial relation of microvilli and T-cell receptors (TCRs), the major molecules responsible for antigen recognition on the T-cell membrane. To this end, an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced, based on two complementary superresolution microscopies. Strikingly, TCRs are found to be highly localized on microvilli, in both peripheral blood human T cells and differentiated effector T cells, and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli, immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form, with microvilli serving as anchors for specific T-cell surface molecules.

The glaucous appearance of wheat (Triticum aestivum) and barley (Hordeum vulgare) plants, that is the light bluish-gray look of flag leaf, stem, and spike surfaces, results from deposition of cuticular beta-diketone wax on their surfaces; this phenotype is associated with high yield, especially under drought conditions. Despite extensive genetic and biochemical characterization, the molecular genetic basis underlying the biosynthesis of beta-diketones remains unclear. Here, we discovered that the wheat W1 locus contains a metabolic gene cluster mediating beta-diketone biosynthesis. The cluster comprises genes encoding proteins of several families including type-III polyketide synthases, hydrolases, and cytochrome P450s related to known fatty acid hydroxylases. The cluster region was identified in both genetic and physical maps of glaucous and glossy tetraploid wheat, demonstrating entirely different haplotypes in these accessions. Complementary evidence obtained through gene silencing in planta and heterologous expression in bacteria supports a model for a b-diketone biosynthesis pathway involving members of these three protein families. Mutations in homologous genes were identified in the barley eceriferum mutants defective in b-diketone biosynthesis, demonstrating a gene cluster also in the b-diketone biosynthesis Cer-cqu locus in barley. Hence, our findings open new opportunities to breed major cereal crops for surface features that impact yield and stress response.

Impaired salivary gland (SG) function leading to oral diseases is relatively common with no adequate solution. Previously, tissue engineering of SG had been proposed to overcome this morbidity, however, not yet clinically available. Multiwall inorganic (tungsten disulfide [WS2]) nanotubes (INT-WS2) and fullerene-like nanoparticles (IF-WS2) have many potential medical applications. A yet unexplored venue application is their interaction with SG, and therefore, our aim was to test the biocompatibility of INT/IF-WS2 with the A5 and rat submandibular cells (RSC) SG cells. The cells were cultured and subjected after 1 day to different concentrations of INT-WS2 and were compared to control groups. Growth curves, trypan blue viability test, and carboxyfluorescein succinimidyl ester (CFSE) proliferation assay were obtained. Furthermore, cells morphology and interaction with the nanoparticles were observed by light microscopy, scanning electron microscopy and transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy. The results showed no significant differences in growth curves, proliferation kinetics, and viability between the groups compared. Moreover, no alterations were observed in the cell morphology. Interestingly, TEM images indicated that the nanoparticles are uptaken by the cells and accumulate in cytoplasmic vesicles. These results suggest promising future medical applications for these nanoparticles.

Successful implantation is associated with a unique spatial pattern of vascular remodeling, characterized by profound peripheral neo-vascularization surrounding a peri-embryo avascular niche. We hypothesized that hyaluronan controls the formation of the unique vascular pattern encompassing the embryo. This hypothesis was evaluated by genetic modification of hyaluronan metabolism specifically targeted to embryonic trophoblast cells. The outcome of altered hyaluronan deposition on uterine vascular remodeling and post-implantation development were analyzed by MRI, detailed histological examinations, and RNA-sequencing of uterine NK cells. Our experiments revealed that eliminating the anti-angiogenic hyaluronan, led to elevated expression of MMP-9, VEGF-A and its receptor VEGFR-2, accompanied by reduced recruitment of uterine NK cells. Further local decrease in VEGFR-3 resulted in impaired formation of vascular sinuous folds, ectopic angiogenesis and dysfunctional uterine NK cells. Conversely, enhanced deposition of hyaluronan caused the expansion of the maternal embryo barrier, leading to an increased diffusion distance and aborted implantation. These results demonstrate a pivotal role for hyaluronan in successful pregnancy by fine tuning the peri-embryo avascular niche and maternal vascular morphogenesis.

The bell-shaped members of Cnidaria typically move around by swimming whereas the Hydra polyp can perform locomotion on solid substrates in aquatic environment. To address the biomechanics of locomotion on rigid substrates, we studied the 'somersaulting' locomotion in Hydra. We applied atomic force microscopy on the whole animal to measure the local mechanical properties of body column and identified the existence of differential Young's modulus between the shoulder region versus rest of the body column at 3:1 ratio. We show that this movement depends primarily on graded variations in tissue stiffness of the body column and is explained by computational models that accurately recapitulate the mechanics involved in this process. We demonstrate that perturbation of the observed stiffness gradient in hydra body column by modulating the extracellular matrix (ECM) polymerization impairs the 'somersault' movement. These results provide mechanistic basis for the evolutionary significance of differential ECM properties and tissue stiffness.

In nature, bacteria reside in biofilms - multicellular differentiated communities held together by extracellular matrix. In this work, we identified a novel subpopulation essential for biofilm formation – mineral-forming cells. This subpopulation contains an intracellular calcium-accumulating niche, in which the formation of a calcium carbonate mineral is initiated. As the biofilm colony develops, this mineral grows in a controlled manner, forming a functional macrostructure that serves the entire community. The molecular mechanisms promoting calcite scaffold formation were conserved between three distant phyla – the Gram-positive Bacillus subtilis, Gram-negative Pseudomonas aeruginosa and the actinobacterium Mycobacterium abscessus. Biofilm development of all three species was similarly impaired by inhibition of calcium uptake and carbonate accumulation. Moreover, chemical inhibition and mutations targeting mineralization both significantly reduced the attachment of P. aeruginosa to the lung, as well as the subsequent damage inflicted by biofilms to lung tissues, and restored their sensitivity to antibiotics. The evolutionary conserved cellular pathway controlling the fundamental feature of biofilm development uncovered in this work offers novel druggable targets for antibiotics to combat otherwise untreatable biofilm infections.