Pleurotaenium trabecula,a desmid of wetland biofilms: the extracellular matrix and adhesion mechanisms1

Pleurotaenium trabecula (Ehren.) Nägeli is a placoderm desmid that commonly occurs in wetland biofilms of the southeastern Adirondacks (NY, USA). It often displays a distinctive habit whereby the cell remains attached to the substrate via the polar end of one semicell, while the remainder of the cell is suspended in the water column. In this study, we examined the extracellular matrix (ECM) of this alga to elucidate its adhesion mechanisms and postadhesion behavior. The ECM consists of the following: (i) an extracellular polymeric substance (EPS), which includes polyanionic and sulfated polysaccharides; (ii) a thin pectin‐containing primary cell wall, which is quickly sloughed off after postcytokinetic semicell expansion; and (iii) a thick secondary cell wall that is perforated with a distinct pore complex. Each pore of this complex possesses an external network of densely aggregated fibrils. Selective solubilization and immunolabeling studies suggest that these fibrillar aggregates or “adhesion centers” (i.e., ACs) contain arabinogalactan protein and are involved in initial adhesion of the cell to a substrate. We propose that postinitial adhesion behavior entails localized secretion of EPS derived from a large pool of EPS‐containing vesicles situated in the peripheral cytoplasm. As the EPS absorbs water, hygroscopic pressure breaks the connections between the ACs on the cell wall and substrate and allows a portion of a cell to lift up into the water column.     

Integrin–ECM interactions and membrane-associated Catalase cooperate to promote resilience of the Drosophila intestinal epithelium

Balancing cellular demise and survival constitutes a key feature of resilience mechanisms that underlie the control of epithelial tissue damage. These resilience mechanisms often limit the burden of adaptive cellular stress responses to internal or external threats. We recently identified Diedel, a secreted protein/cytokine, as a potent antagonist of apoptosis-induced regulated cell death in the Drosophila intestinal midgut epithelium during aging. Here, we show that Diedel is a ligand for RGD-binding Integrins and is thus required for maintaining midgut epithelial cell attachment to the extracellular matrix (ECM)-derived basement membrane. Exploiting this function of Diedel, we uncovered a resilience mechanism of epithelial tissues, mediated by Integrin–ECM interactions, which shapes cell death spreading through the regulation of cell detachment and thus cell survival. Moreover, we found that resilient epithelial cells, enriched for Diedel–Integrin–ECM interactions, are characterized by membrane association of Catalase, thus preserving extracellular reactive oxygen species (ROS) balance to maintain epithelial integrity. Intracellular Catalase can relocalize to the extracellular membrane to limit cell death spreading and repair Integrin–ECM interactions induced by the amplification of extracellular ROS, which is a critical adaptive stress response. Membrane-associated Catalase, synergized with Integrin–ECM interactions, likely constitutes a resilience mechanism that helps balance cellular demise and survival within epithelial tissues.

A key feature of the resilience mechanisms that underlie the control of epithelial tissue damage is the balance between cell death and survival. This study shows that the anti-oxidant enzyme catalase can relocate to membranes in order to promote the resilience of the Drosophila midgut epithelium, synergizing with integrin-ECM interactions to prevent the spread of cell death.     

Role and impact of the extracellular matrix on integrin‐mediated pancreatic β‐cell functions

Understanding the organisation and role of the extracellular matrix (ECM) in islets of Langerhans is critical for maintaining pancreatic β‐cells, and to recognise and revert the physiopathology of diabetes. Indeed, integrin‐mediated adhesion signalling in response to the pancreatic ECM plays crucial roles in β‐cell survival and insulin secretion, two major functions, which are affected in diabetes. Here, we would like to present an update on the major components of the pancreatic ECM, their role during integrin‐mediated cell‐matrix adhesions and how they are affected during diabetes. To treat diabetes, a promising approach consists in replacing β‐cells by transplantation. However, efficiency is low, because β‐cells suffer of anoikis, due to enzymatic digestion of the pancreatic ECM, which affects the survival of insulin‐secreting β‐cells. The strategy of adding ECM components during transplantation, to reproduce the pancreatic microenvironment, is a challenging task, as many of the regulatory mechanisms that control ECM deposition and turnover are not sufficiently understood. A better comprehension of the impact of the ECM on the adhesion and integrin‐dependent signalling in β‐cells is primordial to improve the healthy state of islets to prevent the onset of diabetes as well as for enhancing the efficiency of the islet transplantation therapy.     

The Matrix Reloaded: How Sensing the Extracellular Matrix Synchronizes Bacterial Communities

In response to chemical communication, bacterial cells often organize themselves into complex multicellular communities that carry out specialized tasks. These communities are frequently referred to as biofilms, which involve the collective behavior of different cell types. Like cells of multicellular eukaryotes, the biofilm cells are surrounded by self-produced polymers that constitute the extracellular matrix (ECM), which binds them to each other and to the surface. In multicellular eukaryotes, it has been evident for decades that cell-ECM interactions control multiple cellular processes during development. While cells both in biofilms and in multicellular eukaryotes are surrounded by ECM and activate various genetic programs, until recently it has been unclear whether cell-ECM interactions are recruited in bacterial communicative behaviors. In this review, we describe the examples reported thus far for ECM involvement in control of cell behavior throughout the different stages of biofilm formation. The studies presented in this review have provided a newly emerging perspective of the bacterial ECM as an active player in regulation of biofilm development.     

The ABC transporter Snu and the extracellular protein Snsl cooperate in the formation of the lipid-based inward and outward barrier in the skin of Drosophila

Lipids in extracellular matrices (ECM) contribute to barrier function and stability of epithelial tissues such as the pulmonary alveoli and the skin. In insects, skin waterproofness depends on the outermost layer of the extracellular cuticle termed envelope that contains cuticulin, an unidentified water-repellent complex molecule composed of proteins, lipids and catecholamines. Based on live-imaging analyses of fruit fly larvae, we find that initially envelope units are assembled within putative vesicles harbouring the ABC transporter Snu and the extracellular protein Snsl. In a second step, the content of these vesicles is distributed to cuticular lipid-transporting nanotubes named pore canals and to the cuticle surface in dependence of Snu function. Consistently, the surface of snu and snsl mutant larvae is depleted from lipids and cuticulin. By consequence, these animals suffer uncontrolled water loss and penetration of xenobiotics. Our data allude to a two-step model of envelope i.e. barrier formation. The proposed mechanism in principle parallels the events occurring during differentiation of the lipid-based ECM by keratinocytes in the vertebrate skin suggesting establishment of analogous mechanisms of skin barrier formation in vertebrates and invertebrates.     

Trafficking mechanisms of extracellular matrix macromolecules: Insights from vertebrate development and human diseases

Cellular life depends on protein transport and membrane traffic. In multicellular organisms, membrane traffic is required for extracellular matrix deposition, cell adhesion, growth factor release, and receptor signaling, which are collectively required to integrate the development and physiology of tissues and organs. Understanding the regulatory mechanisms that govern cargo and membrane flow presents a prime challenge in cell biology. Extracellular matrix (ECM) secretion remains poorly understood, although given its essential roles in the regulation of cell migration, differentiation, and survival, ECM secretion mechanisms are likely to be tightly controlled.Recent studies in vertebrate model systems, from fishes to mammals and in human patients, have revealed complex and diverse loss-of-function phenotypes associated with mutations in components of the secretory machinery. A broad spectrum of diseases from skeletal and cardiovascular to neurological deficits have been linked to ECM trafficking. These discoveries have directly challenged the prevailing view of secretion as an essential but monolithic process. Here, we will discuss the latest findings on mechanisms of ECM trafficking in vertebrates.     

DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides

One intriguing discovery in modern microbiology is the extensive presence of extracellular DNA (eDNA) within biofilms of various bacterial species. Although several biological functions have been suggested for eDNA, including involvement in biofilm formation, the detailed mechanism of eDNA integration into biofilm architecture is still poorly understood. In the biofilms formed by Myxococcus xanthus, a Gram-negative soil bacterium with complex morphogenesis and social behaviors, DNA was found within both extracted and native extracellular matrices (ECM). Further examination revealed that these eDNA molecules formed well organized structures that were similar in appearance to the organization of exopolysaccharides (EPS) in ECM. Biochemical and image analyses confirmed that eDNA bound to and colocalized with EPS within the ECM of starvation biofilms and fruiting bodies. In addition, ECM containing eDNA exhibited greater physical strength and biological stress resistance compared to DNase I treated ECM. Taken together, these findings demonstrate that DNA interacts with EPS and strengthens biofilm structures in M. xanthus.     

Integrins and EGFR coordinately regulate the pro-apoptotic protein Bim to prevent anoikis

Epithelial cells must adhere to the extracellular matrix (ECM) for survival, as detachment from matrix triggers apoptosis or anoikis. Integrins are major mediators of adhesion between cells and ECM proteins, and transduce signals required for cell survival. Recent evidence suggests that integrin receptors are coupled to growth factor receptors in the regulation of multiple biological functions; however, mechanisms involved in coordinate regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we identify the pro-apoptotic protein Bim as a critical mediator of anoikis in epithelial cells. Bim is strongly induced after cell detachment and downregulation of Bim expression by RNA interference (RNAi) inhibits anoikis. Detachment-induced expression of Bim requires a lack of beta(1)-integrin engagement, downregulation of EGF receptor (EGFR) expression and inhibition of Erk signalling. Overexpressed EGFR was uncoupled from integrin regulation, resulting in the maintenance of Erk activation in suspension, and a block in Bim expression and anoikis. Thus, Bim functions as a key sensor of integrin and growth factor signals to the Erk pathway, and loss of such coordinate regulation may contribute to tumour progression.     

Distribution of bacterial proteins in biofilms formed by non-typeable Haemophilus influenzae.

The ability to preserve the fragile ultrastructural organization of bacterial biofilms using cryo-preparation methods for electron microscopy has enabled us to probe sections through non-typeable Haemophilus influenzae (NTHi) biofilms and determine the localization of NTHi-specific lipooligosaccharide (LOS) and proteins within these structures. Some of the proteins we examined are currently being considered as candidates for vaccine development, so it is important that their distribution and accessibility within the biofilms formed by NTHi be determined. We have localized LOS to the extracellular matrix (ECM) of the biofilm and the P6 outer membrane protein to the membrane of what appear to be viable bacteria within the biofilm. The Hap and HWM1/HMW2 adhesive proteins were associated with bacteria within the biofilm and were present in the biofilm ECM. The IgA1 protease is a secreted protein that was also associated with NTHi in the biofilm and was in the ECM, but was more concentrated in the top region of the biofilm, suggesting a role in protecting biofilm bacteria from antibody attack.     

Identification of Ata, a multifunctional trimeric autotransporter of Acinetobacter baumannii

Acinetobacter baumannii has recently emerged as a highly troublesome nosocomial pathogen, especially in patients in intensive care units and in those undergoing mechanical ventilation. We have identified a surface protein adhesin of A. baumannii, designated the Acinetobacter trimeric autotransporter (Ata), that contains all of the typical features of trimeric autotransporters (TA), including a long signal peptide followed by an N-terminal, surface-exposed passenger domain and a C-terminal domain encoding 4 β-strands. To demonstrate that Ata encoded a TA, we created a fusion protein in which we replaced the entire passenger domain of Ata with the epitope tag V5, which can be tracked with specific monoclonal antibodies, and demonstrated that the C-terminal 101 amino acids of Ata were capable of exporting the heterologous V5 tag to the surface of A. baumannii in a trimeric form. We found that Ata played a role in biofilm formation and bound to various extracellular matrix/basal membrane (ECM/BM) components, including collagen types I, III, IV, and V and laminin. Moreover, Ata mediated the adhesion of whole A. baumannii cells to immobilized collagen type IV and played a role in the survival of A. baumannii in a lethal model of systemic infection in immunocompetent mice. Taken together, these results reveal that Ata is a TA of A. baumannii involved in virulence, including biofilm formation, binding to ECM/BM proteins, mediating the adhesion of A. baumannii cells to collagen type IV, and contributing to the survival of A. baumannii in a mouse model of lethal infection.     

Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells

Background  

Interactions of cells with the extracellular matrix (ECM) are critical for the establishment and maintenance of stem cell self-renewal and differentiation. However, the ECM is a complex mixture of matrix molecules; little is known about the role of ECM components in human embryonic stem cell (hESC) differentiation into neural progenitors and neurons.     

A mitochondrial protein, Bit1, mediates apoptosis regulated by integrins and Groucho/TLE corepressors

A delicate balance of signals regulates cell survival. One set of these signals is derived from integrin-mediated cell adhesion to the extracellular matrix (ECM). Loss of cell attachment to the ECM causes apoptosis, a process known as anoikis. In searching for proteins involved in cell adhesion-dependent regulation of anoikis, we identified Bit1, a mitochondrial protein that is released into the cytoplasm during apoptosis. Cytoplasmic Bit1 forms a complex with AES, a small Groucho/transducin-like enhancer of split (TLE) protein, and induces cell death with characteristics of caspase-independent apoptosis. Cell attachment to fibronectin counteracts the apoptotic effect of Bit1 and AES. Increasing Bit1 expression enhances anoikis, while suppressing the expression reduces it. Thus, we have elucidated an integrin-controlled pathway that is, at least in part, responsible for the cell survival effects of cell-ECM interactions.     

Structure of a WW domain containing fragment of dystrophin in complex with beta-dystroglycan

Dystrophin and beta-dystroglycan are components of the dystrophin-glycoprotein complex (DGC), a multimolecular assembly that spans the cell membrane and links the actin cytoskeleton to the extracellular basal lamina. Defects in the dystrophin gene are the cause of Duchenne and Becker muscular dystrophies. The C-terminal region of dystrophin binds the cytoplasmic tail of beta-dystroglycan, in part through the interaction of its WW domain with a proline-rich motif in the tail of beta-dystroglycan. Here we report the crystal structure of this portion of dystrophin in complex with the proline-rich binding site in beta-dystroglycan. The structure shows that the dystrophin WW domain is embedded in an adjacent helical region that contains two EF-hand-like domains. The beta-dystroglycan peptide binds a composite surface formed by the WW domain and one of these EF-hands. Additionally, the structure reveals striking similarities in the mechanisms of proline recognition employed by WW domains and SH3 domains.     

Variation in the ratio of curli and phosphoethanolamine cellulose associated with biofilm architecture and properties

Bacterial biofilms are communities of bacteria entangled in a self‐produced extracellular matrix (ECM). Escherichia coli direct the assembly of two insoluble biopolymers, curli amyloid fibers, and phosphoethanolamine (pEtN) cellulose, to build remarkable biofilm architectures. Intense curiosity surrounds how bacteria harness these amyloid‐polysaccharide composites to build biofilms, and how these biopolymers function to benefit bacterial communities. Defining ECM composition involving insoluble polymeric assemblies poses unique challenges to analysis and, thus, to comparing strains with quantitative ECM molecular correlates. In this work, we present results from a sum‐of‐the‐parts ¹³C solid‐state nuclear magnetic resonance (NMR) analysis to define the curli‐to‐pEtN cellulose ratio in the isolated ECM of the E. coli laboratory K12 strain, AR3110. We compare and contrast the compositional analysis and comprehensive biofilm phenotypes for AR3110 and a well‐studied clinical isolate, UTI89. The ECM isolated from AR3110 contains approximately twice the amount of pEtN cellulose relative to curli content as UTI89, revealing plasticity in matrix assembly principles among strains. The two parent strains and a panel of relevant gene mutants were investigated in three biofilm models, examining: (a) macrocolonies on agar, (b) pellicles at the liquid‐air interface, and (c) biomass accumulation on plastic. We describe the influence of curli, cellulose, and the pEtN modification on biofilm phenotypes with power in the direct comparison of these strains. The results suggest that curli more strongly influence adhesion, while pEtN cellulose drives cohesion. Their individual and combined influence depends on both the biofilm modality (agar, pellicle, or plastic‐associated) and the strain itself.     

A three-dimensional collagen-fiber network model of the extracellular matrix for the simulation of the mechanical behaviors and micro structures

The extracellular matrix (ECM) provides structural and biochemical support to cells and tissues, which is a critical factor for modulating cell dynamic behavior and intercellular communication. In order to further understand the mechanisms of the interactive relationship between cell and the ECM, we developed a three-dimensional (3D) collagen-fiber network model to simulate the micro structure and mechanical behaviors of the ECM and studied the stress–strain relationship as well as the deformation of the ECM under tension. In the model, the collagen-fiber network consists of abundant random distributed collagen fibers and some crosslinks, in which each fiber is modeled as an elastic beam and a crosslink is modeled as a linear spring with tensile limit, it means crosslinks will fail while the tensile forces exceed the limit of spring. With the given parameters of the beam and the spring, the simulated tensile stress–strain relation of the ECM highly matches the experimental results including damaged and failed behaviors. Moreover, by applying the maximal inscribed sphere method, we measured the size distribution of pores in the fiber network and learned the variation of the distribution with deformation. We also defined the alignment of the collagen-fibers to depict the orientation of fibers in the ECM quantitatively. By the study of changes of the alignment and the damaged crosslinks against the tensile strain, this paper reveals the comprehensive mechanisms of four stages of ‘toe’, ‘linear’, ‘damage’ and ‘failure’ in the tensile stress–strain relation of the ECM which can provide further insight in the study of cell-ECM interaction.     

Engineering strategies to recapitulate epithelial morphogenesis within synthetic three-dimensional extracellular matrix with tunable mechanical properties

The mechanical properties (e.g. stiffness) of the extracellular matrix (ECM) influence cell fate and tissue morphogenesis and contribute to disease progression. Nevertheless, our understanding of the mechanisms by which ECM rigidity modulates cell behavior and fate remains rudimentary. To address this issue, a number of two and three-dimensional (3D) hydrogel systems have been used to explore the effects of the mechanical properties of the ECM on cell behavior. Unfortunately, many of these systems have limited application because fiber architecture, adhesiveness and/or pore size often change in parallel when gel elasticity is varied. Here we describe the use of ECM-adsorbed, synthetic, self-assembling peptide (SAP) gels that are able to recapitulate normal epithelial acini morphogenesis and gene expression in a 3D context. By exploiting the range of viscoelasticity attainable with these SAP gels, and their ability to recreate native-like ECM fibril topology with minimal variability in ligand density and pore size, we were able to reconstitute normal and tumor-like phenotypes and gene expression patterns in nonmalignant mammary epithelial cells. Accordingly, this SAP hydrogel system presents the first tunable system capable of independently assessing the interplay between ECM stiffness and multi-cellular epithelial phenotype in a 3D context.     

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.     

Influence of anodic biofilm growth on bioelectricity production in single chambered mediatorless microbial fuel cell using mixed anaerobic consortia

The effect of anodic biofilm growth and extent of its coverage on the anodic surface of a single chambered mediatorless microbial fuel cell (MFC) was evaluated for bioelectricity generation using designed synthetic wastewater (DSW) and chemical wastewater (CW) as substrates and anaerobic mixed consortia as biocatalyst. Three MFCs (plain graphite electrodes, air cathode, Nafion membrane) were operated separately with variable biofilm coverage [control; anode surface coverage (ASC), 0%], partially developed biofilm [PDB; ASC approximately 44%; 90 days] and fully developed biofilm [FDB; ASC approximately 96%; 180 days] under acidophilic conditions (pH 6) at room temperature. The study depicted the effectiveness of anodic biofilm formation in enhancing the extracellular electron transfer in the absence of mediators. Higher specific power production [29mW/kg COD(R) (CW and DSW)], specific energy yield [100.46J/kg VSS (CW)], specific power yield [0.245W/kg VSS (DSW); 0.282W/kg VSS (CW)] and substrate removal efficiency of 66.07% (substrate degradation rate, 0.903kgCOD/m(3)-day) along with effective functioning fuel cell at relatively higher resistance [4.5kOmega (DSW); 14.9kOmega (CW)] correspond to sustainable power [0.008mW (DSW); 0.021mW (CW)] and effective electron discharge (at higher resistance) and recovery (Coulomb efficiency; 27.03%) were observed especially with FDB operation. Cyclic voltammetry analysis documented six-fold increment in energy output from control (1.812mJ) to PDB (10.666mJ) operations and about eight-fold increment in energy from PDB to FDB (86.856mJ). Biofilm configured MFC was shown to have the potential to selectively support the growth of electrogenic bacteria with robust characteristics, capable of generating higher power yields along with substrate degradation especially operated with characteristically complex wastewaters as substrates.