The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins providing both biophysical and biochemical cues that are important regulators of cell proliferation, survival, differentiation, and migration. We present here a proteomic strategy developed to characterize the in vivo ECM composition of normal tissues and tumors using enrichment of protein extracts for ECM components and subsequent analysis by mass spectrometry. In parallel, we have developed a bioinformatic approach to predict the in silico "matrisome" defined as the ensemble of ECM proteins and associated factors. We report the characterization of the extracellular matrices of murine lung and colon, each comprising more than 100 ECM proteins and each presenting a characteristic signature. Moreover, using human tumor xenografts in mice, we show that both tumor cells and stromal cells contribute to the production of the tumor matrix and that tumors of differing metastatic potential differ in both the tumor- and the stroma-derived ECM components. The strategy we describe and illustrate here can be broadly applied and, to facilitate application of these methods by others, we provide resources including laboratory protocols, inventories of ECM domains and proteins, and instructions for bioinformatically deriving the human and mouse matrisome.     

Comparative proteomic profiling of human osteoblast‐derived extracellular matrices identifies proteins involved in mesenchymal stromal cell osteogenic differentiation and mineralization

The extracellular matrix (ECM) is a dynamic component of tissue architecture that physically supports cells and actively influences their behavior. In the context of bone regeneration, cell‐secreted ECMs have become of interest as they reproduce tissue‐architecture and modulate the promising properties of mesenchymal stem cells (MSCs). We have previously created an in vitro model of human osteoblast‐derived devitalized ECM that was osteopromotive for MSCs. The aim of this study was to identify ECM regulatory proteins able to modulate MSC differentiation to broaden the spectrum of MSC clinical applications. To this end, we created two additional models of devitalized ECMs with different mineralization phenotypes. Our results showed that the ECM derived from osteoblast‐differentiated MSCs had increased osteogenic potential compared to ECM derived from undifferentiated MSCs and non‐ECM cultures. Proteomic analysis revealed that structural ECM proteins and ribosomal proteins were upregulated in the ECM from undifferentiated MSCs. A similar response profile was obtained by treating osteoblast‐differentiating MSCs with Activin‐A. Extracellular proteins were upregulated in Activin‐A ECM, whereas mitochondrial and membrane proteins were downregulated. In summary, this study illustrates that the composition of different MSC‐secreted ECMs is important to regulate the osteogenic differentiation of MSCs. These models of devitalized ECMs could be used to modulate MSC properties to regulate bone quality.     

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.     

A refined technique for extraction of extracellular matrices from bacterial biofilms and its applicability

Biofilm‐forming bacteria embedded in polymeric extracellular matrices (ECMs) that consist of polysaccharides, proteins and/or extracellular DNAs (eDNAs) acquire high resistance to antimicrobial agents and host immune systems. To understand molecular mechanisms of biofilm formation and maintenance and to develop therapeutic countermeasures against chronic biofilm‐associated infections, reliable methods to isolate ECMs are inevitable. In this study, we refined the ECM extraction method recently reported and evaluated its applicability. Using three Staphylococcus aureus biofilms in which proteins, polysaccharides or eDNAs are major contributors to their integrity, ECMs were extracted using salts and detergents. We found that extraction with 1.5 M sodium chloride (NaCl) could be optimum for not only ECM proteins but also polysaccharides and eDNAs. In addition, long‐time incubation was not necessary for efficient ECM isolation. Lithium chloride (LiCl) was comparative to NaCl but is more expensive. In contrast to SDS, NaCl hardly caused leakage of intracellular proteins and did not affect viability of bacterial cells within biofilms. Furthermore, this method is applicable to other bacteria such as Gram‐positive Staphylococcus epidermidis and Gram‐negative Escherichia coli and Pseudomonas aeruginosa. Thus, this refined method is very simple, rapid, low cost and non‐invasive and could be used for a broad range of applications.     

Differential growth factor adsorption to calvarial osteoblast-secreted extracellular matrices instructs osteoblastic behavior

Craniosynostosis (CS), the premature ossification of cranial sutures, is attributed to increased osteogenic potential of resident osteoblasts, yet the contribution of the surrounding extracellular matrix (ECM) on osteogenic differentiation is unclear. The osteoblast-secreted ECM provides binding sites for cellular adhesion and regulates the transport and signaling of osteoinductive factors secreted by the underlying dura mater. The binding affinity of each osteoinductive factor for the ECM may amplify or mute its relative effect, thus contributing to the rate of suture fusion. The purpose of this paper was to examine the role of ECM composition derived from calvarial osteoblasts on protein binding and its resultant effect on cell phenotype. We hypothesized that potent osteoinductive proteins present during sutural fusion (e.g., bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta-1 (TGF-β1)) would exhibit distinct differences in binding when exposed to ECMs generated by human calvarial osteoblasts from unaffected control individuals (CI) or CS patients. Decellularized ECMs produced by osteoblasts from CI or CS patients were incubated in the presence of BMP-2 or TGF-β1, and the affinity of each protein was analyzed. The contribution of ECM composition to protein binding was interrogated by enzymatically modulating proteoglycan content within the ECM. BMP-2 had a similar binding affinity for each ECM, while TGF-β1 had a greater affinity for ECMs produced by osteoblasts from CI compared to CS patients. Enzymatic treatment of ECMs reduced protein binding. CS osteoblasts cultured on enzymatically-treated ECMs secreted by osteoblasts from CI patients in the presence of BMP-2 exhibited impaired osteogenic differentiation compared to cells on untreated ECMs. These data demonstrate the importance of protein binding to cell-secreted ECMs and confirm that protein-ECM interactions have an important role in directing osteoblastic differentiation of calvarial osteoblasts.     

Caprine articular,meniscus and intervertebral disc cartilage: An integral analysis of collagen network and chondrocytes

Cartilage is a tissue with only limited reparative capacities. A small part of its volume is composed of cells, the remaining part being the hydrated extracellular matrix (ECM) with collagens and proteoglycans as its main constituents. The functioning of cartilage depends heavily on its ECM. Although it is known that the various (fibro)cartilaginous tissues (articular cartilage, annulus fibrosus, nucleus pulposus, and meniscus) differ from one each other with respect to their molecular make-up, remarkable little quantitative information is available with respect to its biochemical constituents, such as collagen content, or the various posttranslational modifications of collagen. Furthermore, we have noticed that tissue-engineering strategies to replace cartilaginous tissues pay in general little attention to the biochemical differences of the tissues or the phenotypical differences of the (fibro)chondrocytes under consideration. The goal of this paper is therefore to provide quantitative biochemical data from these tissues as a reference for further studies. We have chosen the goat as the source of these tissues, as this animal is widely accepted as an animal model in orthopaedic studies, e.g. in the field of cartilage degeneration and tissue engineering. Furthermore, we provide data on mRNA levels (from genes encoding proteins/enzymes involved in the synthesis and degradation of the ECM) from (fibro)chondrocytes that are freshly isolated from these tissues and from the same (fibro)chondrocytes that are cultured for 18 days in alginate beads. Expression levels of genes involved in the cross-linking of collagen were different between cells isolated from various cartilaginous tissues. This opens the possibility to include more markers than the commonly used chondrogenic markers type II collagen and aggrecan for cartilage tissue-engineering applications.     

Proteomics Characterization of Extracellular Space Components in the Human Aorta

The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and also serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation. Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the vascular ECM and its associated proteins. Thus, its detailed composition remains to be characterized. In this study, we describe a methodology for the extraction of extracellular proteins from human aortas and their identification by proteomics. The approach is based on (a) effective decellularization to enrich for scarce extracellular proteins, (b) successful solubilization and deglycosylation of ECM proteins, and (c) relative estimation of protein abundance using spectral counting. Our three-step extraction approach resulted in the identification of 103 extracellular proteins of which one-third have never been reported in the proteomics literature of vascular tissues. In particular, three glycoproteins (podocan, sclerostin, and agrin) were identified for the first time in human aortas at the protein level. We also identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin. Moreover, our methodology allowed us to screen for proteolysis in the aortic samples based on the identification of proteolytic enzymes and their corresponding degradation products. For instance, we were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis.Vascular cells, in particular vascular smooth muscle cells, produce and maintain a complex meshwork of ECM.¹ The ECM is not only the scaffold for the anchorage and mobility of residing cells but also absorbs and transduces the shear and strain forces of the blood flow. It is primarily composed of elastin, collagen, proteoglycans, and glycoproteins. The elastin fibers and type I and III fibrillar collagens form a rigid network of highly cross-linked interstitial matrix. They offer elasticity (elastin) and tensile strength (collagens). Proteoglycans, because of their negative charge, attract water and confer resistance to compression. Finally, glycoproteins participate in matrix organization and are essential for cell attachment.The vascular ECM also serves as a substrate for the binding and retention of secreted, soluble proteins of vascular cells as well as molecules coming from the circulation, including lipoproteins, growth factors, cytokines, proteases, and protease inhibitors. These components are invariably associated with ECM proteins, especially proteoglycans. Together they comprise the vascular extracellular environment and are pivotal for disease processes, such as atherosclerosis and aneurysm formation (1).Although proteomics has been previously applied to vascular tissues, only one study has specifically targeted the extracellular vascular environment (2). This study was focused on the isolation of intimal proteoglycans from human carotid arteries. Moreover, most proteomics studies use whole tissue lysates, which are rich in cellular proteins that inevitably mask the identification of the less abundant proteins of the vascular extracellular environment (3–5). Thus, the composition of the vascular ECM and its associated proteins remains poorly defined. In the present study, we used morphologically normal human aortic samples to develop a method for the extraction of proteins present in the extracellular environment, including ECM proteins and proteins attached to the ECM. We had three specific aims: first, to reduce the contamination with cellular proteins, thereby increasing the chance of identifying scarce extracellular proteins; second, to efficiently solubilize and deglycosylate ECM proteins to improve their analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS); and third, to interface the nanoflow LC system to a recently developed injection device, which splits the flow from the analytical column, to allow the reanalysis of the same sample during a single LC-MS/MS run (RePlay, Advion).Our methodology provides a detailed overview of the aortic ECM and its associated proteins, many reported for the first time in proteomics analysis of the vasculature. Most importantly, this method could be adapted for use with other tissues to further our understanding of the composition of extracellular environment and ECM turnover under various disease conditions.     

Cell adhesion to extracellular matrix is different in marine hydrozoans compared with vertebrates

Extracellular matrices (ECMs) of phylogenetically very distant organisms were tested for their ability to support cell adhesion, spreading and DNA replication in reciprocal xenograft adhesion tests. Mechanically dissociated cells of the medusa Podocoryne carnea (Cnidaria, Hydrozoa) were seeded on ECMs of polyps and medusa, and on several ECM glycoproteins or entire ECMs from vertebrates. In reciprocal experiments, cells from different vertebrate cell-lines were seeded on ECMs of polyps, medusae and also on electrophoresed and blotted extracts of both types of ECMs. The results demonstrate that medusa cells adhere and spread on polyp and medusa ECMs but do not recognize vertebrate ECMs or purified ECM glycoproteins. Vertebrate cells in contrast adhere, spread and proliferate on ECMs of polyps and medusae. The number of attached cells depends on the cell type, the type of ECM and, in certain cases, on the stage of the cell cycle. Cell adhesion experiments with pretreated ECMs of polyps and medusae, e.g. oxidation of carbohydrate residues with sodium-metaperiodate, or blocking of certain carbohydrate moieties with the lectin wheat germ agglutinin or a carbohydrate-specific monoclonal antibody, demonstrate that ECM carbohydrates are more important for cell-ECM interactions of medusa cells than for vertebrate cells. Furthermore, the experiments indicate that polyp and medusa ECMs contain different components which strongly modulate adhesion, spreading and DNA replication of vertebrate cells.     

Fungi in Roots of Nursery Grown Pinus sylvestris: Ectomycorrhizal Colonisation, Genetic Diversity and Spatial Distribution

The aims of this study were to investigate patterns of ectomycorrhizal (ECM) colonisation and community structure on nursery grown seedlings of Pinus sylvestris, spatial distribution of ECMs in the nursery plot and genetic diversity of commonly isolated ECM basidiomycete Hebeloma cavipes. One hundred seedlings were sampled in 225?m² area using a systematic grid design. For each seedling, 20 individual root tips were randomly collected, morphotyped, and surface sterilised for fungal isolation in pure culture. Results showed that ECM community was comprised of nine distinct morphotypes among which Thelephora terrestris (39.7%), Hebeloma sp. (17.8%) and Suillus luteus (6.1%) were the most abundant. Spatial distribution of ECMs in the nursery plot was determined by their relative abundance: even in common ECMs and random in rare ones. Fungal isolation yielded 606 pure cultures, representing 71 distinct taxa. The most commonly isolated fungi were the ascomycetes Neonectria macrodidyma (20.3%), Phialocephala fortinii (13.5%), Neonectria radicicola (6.3%) and the ECM basidiomycete H. cavipes (4.5%). Intraspecific genetic diversity within 27 H. cavipes isolates was studied using two methods: restriction digestion of the amplified intergenic spacer of nuclear ribosomal DNA and genealogical concordance of five genetic markers. Five and eight genotypes were revealed by each respective method, but both of those were largely consistent, in particular, in determining the largest genotype (A) composed of 18 isolates. Mapping positions for each H. cavipes isolate and genotype in the field showed that isolates of the A genotype covered a large part of the nursery plot. This suggests that H. cavipes is largely disseminated by vegetative means of local genotypes and that nursery cultivation practices are likely to contribute to the dissemination of this species in the forest nursery soils.     

Mycorrhiza formation is not needed for early growth induction and growth-related changes in polyamines in Scots pine seedlings in vitro

Ectomycorrhizal (ECM) fungi have been shown to improve growth of the host plant before the formation of physical ECM structures, i.e. during the so-called pre-mycorrhizal phase. In the present study, changes in growth and the concentrations of individual polyamines (PAs) were followed during the mycorrhiza formation in Scots pine (Pinus sylvestris) seedlings in the presence of two ECM fungi, Pisolithus tinctorius and Paxillus involutus. The two fungus stains were chosen because they differed in infection characteristics as well as in PA and auxin production. The results were compared to our earlier study with two Suillus variegatus strains forming ECMs with Scots pine seedlings in vitro. Paxillus was not able to form ECMs whereas Pisolithus formed ECM association with Scots pine seedlings within two weeks. However, Paxillus enhanced the growth of the seedlings more than Pisolithus. Paxillus also increased putrescine (Put) concentrations of the seedlings in the pre-mycorrhizal phase much more than Pisolithus. A similar trend was observed in the free spermidine (Spd) in stems, whereas in the needles Paxillus decreased free Spd concentration. Pisolithus caused a threefold greater increase in root free Spd than Paxillus. Effects of Paxillus on the growth and PA fluctuation, excluding root free Spd, of the host plant resembled that observed in our previous in vitro study on S. variegatus–Scots pine interaction. Therefore, changes in specific PA concentrations in the pre-mycorrhizal phase seem to be related to growth induction by the ECM fungus rather than to mycorrhiza formation. Moreover, we suggest that growth induction in host plants is not necessarily followed by ECM formation.     

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.     

Proteomic analysis of extracellular matrix from the hepatic stellate cell line LX-2 identifies CYR61 and Wnt-5a as novel constituents of fibrotic liver

Activation of hepatic stellate cells (HSCs) and subsequent uncontrolled accumulation of altered extracellular matrix (ECM) underpin liver fibrosis, a wound healing response to chronic injury, which can lead to organ failure and death. We sought to catalogue the components of fibrotic liver ECM to obtain insights into disease etiology and aid identification of new biomarkers. Cell-derived ECM was isolated from the HSC line LX-2, an in vitro model of liver fibrosis, and compared to ECM from human foreskin fibroblasts (HFFs) as a control. Mass spectrometry analyses of cell-derived ECMs identified, with ≥99% confidence, 61 structural ECM or secreted proteins (48 and 31 proteins for LX-2 and HFF, respectively). Gene ontology enrichment analysis confirmed the enrichment of ECM proteins, and hierarchical clustering coupled with protein-protein interaction network analysis revealed a subset of proteins enriched to fibrotic ECM, highlighting the existence of cell type-specific ECM niches. Thirty-six proteins were enriched to LX-2 ECM as compared to HFF ECM, of which Wnt-5a and CYR61 were validated by immunohistochemistry in human and murine fibrotic liver tissue. Future studies will determine if these and other components may play a role in the etiology of hepatic fibrosis, serve as novel disease biomarkers, or open up new avenues for drug discovery.     

From molecular signal activation to locomotion: an integrated, multiscale analysis of cell motility on defined matrices

The adhesion, mechanics, and motility of eukaryotic cells are highly sensitive to the ligand density and stiffness of the extracellular matrix (ECM). This relationship bears profound implications for stem cell engineering, tumor invasion and metastasis. Yet, our quantitative understanding of how ECM biophysical properties, mechanotransductive signals, and assembly of contractile and adhesive structures collude to control these cell behaviors remains extremely limited. Here we present a novel multiscale model of cell migration on ECMs of defined biophysical properties that integrates local activation of biochemical signals with adhesion and force generation at the cell-ECM interface. We capture the mechanosensitivity of individual cellular components by dynamically coupling ECM properties to the activation of Rho and Rac GTPases in specific portions of the cell with actomyosin contractility, cell-ECM adhesion bond formation and rupture, and process extension and retraction. We show that our framework is capable of recreating key experimentally-observed features of the relationship between cell migration and ECM biophysical properties. In particular, our model predicts for the first time recently reported transitions from filopodial to "stick-slip" to gliding motility on ECMs of increasing stiffness, previously observed dependences of migration speed on ECM stiffness and ligand density, and high-resolution measurements of mechanosensitive protrusion dynamics during cell motility we newly obtained for this study. It also relates the biphasic dependence of cell migration speed on ECM stiffness to the tendency of the cell to polarize. By enabling the investigation of experimentally-inaccessible microscale relationships between mechanotransductive signaling, adhesion, and motility, our model offers new insight into how these factors interact with one another to produce complex migration patterns across a variety of ECM conditions.     

Syndecan-1-Induced ECM Fiber Alignment Requires Integrin αvβ3 and Syndecan-1 Ectodomain and Heparan Sulfate Chains

Expression of the cell surface proteoglycan syndecan-1 (Sdc1) is frequently induced in stromal fibroblasts of invasive breast carcinomas. We have recently identified a correlation between stromal Sdc1 expression and extracellular matrix (ECM) fiber alignment, both in vitro and in vivo. ECMs derived from Sdc1-positive human mammary fibroblasts (HMF) showed an aligned fiber architecture, which contrasted markedly with the more random fiber arrangement in the ECM produced by Sdc1-negative HMFs. We further demonstrated that aligned fiber architecture promotes the directional migration and invasion of breast carcinoma cells. To decipher the molecular mechanisms governing the formation of an aligned, invasion-permissive ECM, a series of Sdc1 mutants was introduced into HMF. We found that both the ectodomain and heparan sulfate chains of Sdc1 were required for full activity of Sdc1 in regulating ECM alignment, while transmembrane and cytoplasmic domains were dispensable. Sdc1 regulates the activities of several integrins via its ectodomain. Integrins are key players in the assembly of fibronectin-rich ECM. In addition, integrins are capable of regulating cell morphology and cell shape and orientation may affect ECM architecture. Therefore, we investigated the role of integrins in Sdc1-mediated ECM fiber alignment. Sdc1-overexpressing HMF gained an enhanced spindle-shaped morphology when cultured in an overconfluent state under conditions permissive for ECM production, which was partially reversed by siRNA-mediated silencing of β3 integrin expression. Moreover, suppression of αvβ3 integrin activity by a function-blocking antibody or β3 knockdown largely abolished the aligned ECM fiber architecture and consequently the invasion-permissive properties of the ECM induced by Sdc1. The results suggest that Sdc1 may modulate fibronectin fibrillogenesis and/or alter cell morphology during ECM production through αvβ3 integrin, thereby mediating ECM fiber alignment. Understanding the mechanisms governing ECM organization may lead to the development of novel stroma-targeted therapy for breast cancer, aiming at converting an invasion-permissive to an invasion-restrictive microenvironment.     

Rheological properties of cross-linked hyaluronan-gelatin hydrogels for tissue engineering

Hydrogels that mimic the natural extracellular matrix (ECM) are used in three-dimensional cell culture, cell therapy, and tissue engineering. A semi-synthetic ECM based on cross-linked hyaluronana offers experimental control of both composition and gel stiffness. The mechanical properties of the ECM in part determine the ultimate cell phenotype. We now describe a rheological study of synthetic ECM hydrogels with storage shear moduli that span three orders of magnitude, from 11 to 3 500 Pa, a range important for engineering of soft tissues. The concentration of the chemically modified HA and the cross-linking density were the main determinants of gel stiffness. Increase in the ratio of thiol-modified gelatin reduced gel stiffness by diluting the effective concentration of the HA component.     

Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis

During angiogenesis, cytoskeletal dynamics that mediate endothelial cell branching morphogenesis during vascular guidance are thought to be regulated by physical attributes of the extracellular matrix (ECM) in a process termed mechanosensing. Here, we tested the involvement of microtubules in linking mechanosensing to endothelial cell branching morphogenesis. We used a recently developed microtubule plus end-tracking program to show that specific parameters of microtubule assembly dynamics, growth speed and growth persistence, are globally and regionally modified by, and contribute to, ECM mechanosensing. We demonstrated that engagement of compliant two-dimensional or three-dimensional ECMs induces local differences in microtubule growth speed that require myosin II contractility. Finally, we found that microtubule growth persistence is modulated by myosin II-mediated compliance mechanosensing when cells are cultured on two-dimensional ECMs, whereas three-dimensional ECM engagement makes microtubule growth persistence insensitive to changes in ECM compliance. Thus, compliance and dimensionality ECM mechanosensing pathways independently regulate specific and distinct microtubule dynamics parameters in endothelial cells to guide branching morphogenesis in physically complex ECMs.     

Colony Organization in the Green Alga Botryococcus braunii (Race B) Is Specified by a Complex Extracellular Matrix

Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous β-1, 4- and/or β-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form “drapes” between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.     

The identification of six novel proteins with fibronectin or collagen type I binding activity from Streptococcus suis serotype 2

Streptococcus suis, a major swine pathogen, is an emerging zoonotic agent that causes meningitis and septic shock. Bacterial cell wall and secreted proteins are often involved in interactions with extracellular matrix proteins (ECMs), which play important roles in the initial steps of pathogenesis. In this study, 2D SDS-PAGE, western blotting-based binding affinity measurements, and microtiter plate binding assays were used to identify cell wall and secreted proteins from S. suis that interact with fibronectin and collagen type I. We identified six proteins from S. suis, including three proteins (translation elongation factor G, oligopeptide-binding protein OppA precursor, and phosphoglycerate mutase) that show both fibronectin and collagen type I binding activity. To the best of our knowledge, these three newly identified proteins had no previously reported fibronectin or collagen type I binding activity. Overall, the aim in this study was to identify proteins with ECM binding activity from S. suis and it represents the first report of six new proteins from S. suis that interact with fibronectin or collagen type I.     

雌蕊胞外基质在花粉管生长中的作用

雌蕊胞外基质对雌蕊与花粉的识别以及花粉管的定向生长有着重要的作用,是近年来植物生殖生物学的研究热点之一。与花粉萌发和花粉管生长相关的雌蕊胞外基质种类主要包括阿拉伯半乳糖蛋白、类伸展素糖蛋白、富含脯氨酸糖蛋白、钙调素、S—糖蛋白、果胶以及子房的特异性物质等。本文着重介绍这些雌蕊胞外基质的生理功能及其研究进展。     

雌蕊胞外基质在花粉管生长中的作用

雌蕊胞外基质对雌蕊与花粉的识别以及花粉管的定向生长有着重要的作用,是近年来植物生殖生物学的研究热点之一。与花粉萌发和花粉管生长相关的雌蕊胞外基质种类主要包括阿拉伯半乳糖蛋白、类伸展素糖蛋白、富含脯氨酸糖蛋白、钙调素、S-糖蛋白、果胶以及子房的特异性物质等。本文着重介绍这些雌蕊胞外基质的生理功能及其研究进展。