Targeted migration of pherophorin‐S indicates extensive extracellular matrix dynamics in Volvox carteri

Hydroxyproline‐rich glycoproteins (HRGPs) constitute a major group of proteins of the extracellular matrix (ECM). The multicellular green alga Volvox carteri is a suitable model organism in which to study the evolutionary transition to multicellularity, including the basic principles and characteristics of an ECM. In Volvox, the ECM is dominated by a single HRGP family: the pherophorins. Our inventory amounts to 117 pherophorin‐related genes in V. carteri. We focused on a pherophorin with an unexpected characteristic: pherophorin‐S is a soluble, non‐cross‐linked ECM protein. Using transformants expressing a YFP‐tagged pherophorin‐S we observed the synthesis and secretion of pherophorin‐S by somatic cells in vivo, and we then traced the protein during its conspicuous migration to the ECM around prehatching juveniles and its localized concentration there. Our results provide insights into how an ECM zone surrounding the progeny is remotely affected by distantly located parental somatic cells. In view of the properties and migration of pherophorin‐S, we conclude that pherophorin‐S is likely to act as an ECM plasticizer to allow for dynamic ECM remodeling.     

The extracellular matrix of Volvox carteri: molecular structure of the cellular compartment

The extracellular matrix (ECM) of Volvox contains insoluble fibrous layers that surround individual cells at a distance to form contiguous cellular compartments. Using immunological techniques, we identified a sulfated surface glycoprotein (SSG 185) as the monomeric precursor of this substructure within the ECM. The primary structure of the SSG 185 poly-peptide chain has been derived from cDNA and genomic DNA. A central domain of the protein, 80 amino acid residues long, consists almost exclusively of hydroxyproline residues. The chemical structure of the highly sulfated polysaccharide covalently attached to SSG 185 has been determined by permethylation analysis. As revealed by EM, SSG 185 is a rod-shaped molecule with a 21-nm-long polysaccharide strand protruding from its central region. The chemical nature of the cross- links between SSG 185 monomers is discussed.     

Self-assembly and cross-linking of Volvox extracellular matrix glycoproteins are specifically inhibited by Ellman's reagent.

A major impediment to the biochemical characterization of extracellular matrices from algae (as well as higher plants) is the extensive covalent cross-linking that exists in the matrix, rendering most components insoluble and resistant to conventional extraction procedures. In the multicellular green alga Volvox, biogenesis of the extracellular matrix (ECM) is initiated immediately after the process of embryonic inversion. At this stage of development, the sulfhydryl reagent 5, 5'-dithio-bis(2-nitrobenzoic acid), known as Ellman's reagent, interferes in a highly specific manner with ECM biogenesis. Treated post-inversion embryos are no longer able to assemble an intact ECM and consequently dissociate into a suspension of single cells. Dissociated cells remain viable and continue to secrete ECM proteins into the growth medium, as documented by the identification of several members of the pherophorin family. Cross-linked ECM polymers such as sulfated surface glycoprotein 185 remain in a soluble state. Thus, treatment with Ellman's reagent opens a simple approach for the isolation and characterization of otherwise inaccessible monomeric precursors.     

Evidence for autocatalytic cross-linking of hydroxyproline-rich glycoproteins during extracellular matrix assembly in Volvox

The alga Volvox carteri is one of the simplest multicellular organisms, yet it has a surprisingly complex extracellular matrix (ECM), making Volvox suitable as a model system in which to study ECM self-assembly. Here, we analyze the primary structures and post-translational modifications of two main ECM components synthesized in response to sexual induction as well as wounding. These proteins are members of the pherophorin family with as yet unknown properties. They contain polyhydroxyproline spacers as long as 500 and 2750 residues. Even the highly purified proteins retain the capacity to self-assemble and cross-link, producing an insoluble fibrous network in an apparently autocatalytic reaction. This pherophorin-based network is located within the deep zone of the ECM. A molecular genetic search for additional members of the pherophorin family indicates that at least nine different pherophorin species can be expected to serve as precursors for ECM substructures. Therefore, the highly diversified members of the pherophorin family represent region-specific morphological building blocks for ECM assembly and cross-linking.     

Development-dependent modification of the extracellular matrix by a sulphated glycoprotein in Volvox carteri

We report the chemical characterization of the highly sulphated glycoprotein SSG 185 from Volvox carteri. SSG 185 is a hydroxyproline-containing, extracellular glycoprotein. The sulphate residues are clustered within the parent saccharide structure of SSG 185, since on mercaptolysis all the sulphate residues are recovered in a small saccharide fragment containing mannose, arabinose and sulphate (in a molar ratio of 2). SSG 185 is a short-lived molecule, serving as a precursor for a high mol. wt. component of the extracellular matrix. Synthesis of SSG 185 is developmentally controlled. Different SSG 185 variants, with unknown modifications in the sulphated saccharide fragment, are synthesized at different developmental stages or under the influence of the sexual inducer. These modifications remain conserved in the aggregated state of SSG 185, indicating the development-dependent modification of the extracellular matrix.     

Developmental SEM observations on an extracellular matrix in embryogenic calli ofDrosera rotundifolia andZea mays

Summary Primary embryogenic callus ofDrosera rotundifolia and long-term cultured embryogenic callus ofZea mays possess a conspicuous extracellular matrix (ECM) around and between embryogenic cells. The structural arrangement of ECM depends on the developmental stage of the embryogenic cells. Single embryoid cells were covered with, and connected by net-like material. However, surface cells of young globular embryoids were covered with a coherent layer of ECM which forms bridges with net-like material between the cells which was gradually reduced to coarse strands. When protodermis was formed on the surface of globular embryoids, the ECM disappeared completely. The ECM network was never observed on the surface of heart- and torpedo-shaped embryoids. Safranine (especially 0.1%) stabilized the structure of ECM. Digestion with pronase E and proteinase K indicated that the ECM contains proteinaceous components. Similar developmental patterns of ECM were observed in dicotyledonous and monocotyledonous examples. The ECM represents a stable morphological structure even during long-term embryogenic culture in maize.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - Dicamba 3,6-dichloro-o-anisic acid - ECM extracellular matrix - KIN kinetin - SEM scanning electron microscopy - TEM transmission electron microscopy     

Sexual pheromone induces diffusion of the pheromone-homologous polypeptide in the extracellular matrix of Volvox carteri

The C-terminal domain of pherophorin II is homologous to the sexual pheromone of Volvox carteri and is released from other domains during sexual induction. Green fluorescent protein fused to the C terminus of pherophorin II was located at the extracellular matrix directly surrounding the gonidium, the final target of the sexual-induction signal.     

A phosphodiester bridge between two arabinose residues as a structural element of an extracellular glycoprotein of Volvox carteri

The sulphated glycoprotein SSG 185 is the monomeric precursor of a highly aggregated structural element in the extracellular matrix of the multicellular green alga Volvox carteri. A phosphodiester of arabinose was isolated from a saccharide fragment of SSG 185. The structure of this phosphodiester was investigated by methylation analysis, 13C-NMR, photometric methods and enzymatic assays and identified as D-Araiota-5-phospho-5-D-Araiota. The function of this phosphodiester bridge as a crosslink of different carbohydrate chains in SSG 185 is discussed.     

Effects of tunicamycin on protein glycosylation and development inVolvox carteri

Summary The involvement of protein glycosylation in regulation of the development of the multicellular green alga,Volvox carteri, was studied using the antibiotic, tunicamycin. Three specific developmental processes were found to be affected by the antibiotic: reproductive cell maturation; establishment of polar cellular organization during embryogenesis and release of progeny spheroids from the parental spheroids. Tunicamycin inhibited the transfer of GlcNAc-1-phosphate to dolichyl phosphate which is catalyzed byVolvox membrane preparations. Changes in the glycosylation of several secreted and cellular glycoproteins were observed when proteins were labelled with radioactive amino acids and sugars in the absence and presence of tunicamycin and then electrophoresed on sodium dodecylsulfate-polyacrylamide slab gels. The levels of a few secreted proteins were reduced in tunicamycin treated cultures and one protein band appeared exclusively in the treated cells. Tunicamycin treatment also altered the electrophoretic mobility of radio-iodinated surface macromolecules. Binding of concanavalin A by tunicamycin treatedVolvox spheroids was drastically reduced. It is there-fore likely that the aberrant development results from inhibition of protein glycosylation and the consequent changes in the structure of the cellular, secreted and surface glycoproteins.     

Effect of fibroblast activation protein and alpha2-antiplasmin cleaving enzyme on collagen types I, III, and IV

The circulating enzyme, α₂-antiplasmin cleaving enzyme (APCE), has very similar sequence homology and proteolytic specificity as fibroblast activation protein (FAP), a membrane-bound proteinase. FAP is expressed on activated fibroblasts associated with rapid tissue growth as in embryogenesis, wound healing, and epithelial-derived malignancies, but not in normal tissues. Its presence on stroma suggests that FAP functions to remodel extracellular matrix (ECM) during neoplastic growth. Precise biologic substrates have not been defined for FAP, although like APCE, it cleaves α₂-antiplasmin to a derivative more easily cross-linked to fibrin. While FAP has been shown to cleave gelatin, evidence for cleavage of native collagen, the major ECM component, remains indistinct. We examined the potential proteolytic effects of FAP or APCE alone and in concert with selected matrix metalloproteinases (MMPs) on collagens I, III, and IV. SDS-PAGE analyses demonstrated that neither FAP nor APCE cleaves collagen I. Following collagen I cleavage by MMP-1, however, FAP or APCE digested collagen I into smaller peptides. These peptides were analogous to, yet different from, those produced by MMP-9 following MMP-1 cleavage. Amino-terminal sequencing and mass spectrometry analyses of digestion mixtures identified several peptide fragments within the sequences of the two collagen chains. The proteolytic synergy of APCE in the cleavage of collagen I and III was not observed with collagen IV. We conclude that FAP works in synchrony with other proteinases to cleave partially degraded or denatured collagen I and III as ECM is excavated, and that derivative peptides might function to regulate malignant cell growth and motility.     

Biofilms formed by Scedosporium and Lomentospora species: focus on the extracellular matrix

Abstract

In the present study, the composition of the extracellular matrix (ECM) of the biofilm formed by Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans on a polystyrene surface was investigated. Confocal laser scanning microscopy revealed a dense mycelial mass, with an ECM covering/interspersing the fungal cells and containing carbohydrate-rich molecules (e.g. glycoproteins) and extracellular DNA. The ECMs that were chemically extracted from mature biofilms formed by each of these fungi was predominantly composed of polysaccharides, followed by proteins, nucleic acids and sterols. In general, the amount of biofilm ECM was significantly greater in S. minutisporum and S. aurantiacum than in S. apiospermum and L. prolificans. Corroborating these results, the disarticulation of mature biofilms with enzymes, sodium metaperiodate and chelating agents occurred mainly in S. minutisporum and S. aurantiacum. Collectively, these results have revealed for the first time the composition of the ECM of the biofilms formed by Scedosporium/Lomentospora species and the role it plays in their architecture.     

Modulation of tissue transglutaminase in tubular epithelial cells alters extracellular matrix levels: A potential mechanism of tissue scarring

The up-regulation and trafficking of tissue transglutaminase (TG2) by tubular epithelial cells (TEC) has been implicated in the development of kidney scarring. TG2 catalyses the crosslinking of proteins via the formation of highly stable ε(γ-glutamyl) lysine bonds. We have proposed that TG2 may contribute to kidney scarring by accelerating extracellular matrix (ECM) deposition and by stabilising the ECM against proteolytic decay.To investigate this, we have studied ECM metabolism in Opossum kidney (OK) TEC induced to over-express TG2 by stable transfection and in tubular cells isolated from TG2 knockout mice.Increasing the expression of TG2 led to increased extracellular TG2 activity (p < 0.05), elevated ε(γ-glutamyl) lysine crosslinking in the ECM and higher levels of ECM collagen per cell by ³H-proline labelling. Immunofluorescence demonstrated that this was attributable to increased collagen III and IV levels. Higher TG2 levels were associated with an accelerated collagen deposition rate and a reduced ECM breakdown by matrix metalloproteinases (MMPs).In contrast, a lack of TG2 was associated with reduced ε(γ-glutamyl) lysine crosslinking in the ECM, causing reduced ECM collagen levels and lower ECM per cell.We report that TG2 contributes to ECM accumulation primarily by accelerating collagen deposition, but also by altering the susceptibility of the tubular ECM to decay. These findings support a role for TG2 in the expansion of the ECM associated with kidney scarring.     

Granulated metrial gland cells in the pregnant uterus of mice expressing the collagen mutation tight-skin (Tsk/+)

Summary Influences of the extracellular matrix (ECM) on the differentiation and distribution of granulated metrial gland (GMG) cells, a uterine natural killer (NK)-like cell subset, were studied by histological examination of implantation sites in the mouse mutant Tsk/+. Tsk/+ mice overproduce collagens I and III. GMG cell differentiation appeared to progress normally in Tsk/+ mice between days 6.5 and 12.5 of gestation. The distribution of GMG cells, however, was abnormal. Significant numbers of GMG cells were found in the antimesometrial and lateral decidual regions at day 8.5 of gestation and in the regions between implantation sites until day 10.5 of gestation. Loss of GMG cells from these regions normally occurs by day 6.5 of gestation. These data suggest that alterations to the ECM change the migration properties or life span of GMG cells.     

Characterization of the protease activity that cleaves the extracellular domain of beta-dystroglycan

Dystroglycan (DG) complex, composed of alphaDG and betaDG, provides a link between the extracellular matrix (ECM) and cortical cytoskeleton. Although the proteolytic processing of betaDG was reported in various physiological and pathological conditions, its exact mechanism remains unknown. In this study, we addressed this issue using the cell culture system of rat schwannoma cell line RT4. We found that the culture medium of RT4 cells was enriched with the protease activity that degrades the fusion protein construct of the extracellular domain of betaDG specifically. This activity was suppressed by the inhibitor of matrix metalloproteinase-2 (MMP-2) and MMP-9, but not by the inhibitors of MMP-1, MMP-3, MMP-8, and MMP-13. Zymography and RT-PCR analysis showed that RT4 cells secreted MMP-2 and MMP-9 into the culture medium. Finally, active MMP-2 and MMP-9 enzymes degraded the fusion protein construct of the extracellular domain of betaDG. These results indicate (1) that RT4 cells secrete the protease activity that degrades the extracellular domain of betaDG specifically and (2) that MMP-2 and MMP-9 may be involved in this process.     

Extracellular matrix protein 1 interacts with the domain III of fibulin-1C and 1D variants through its central tandem repeat 2

Extracellular matrix protein 1 (ECM1), a widely expressed glycoprotein, has been shown to harbor mutations in lipoid proteinosis (LP), an autosomal recessive disorder characterized by profound alterations in the extracellular matrix of connective tissue. The biological function of ECM1 and its role in the pathomechanisms of LP are unknown. Fibulins comprise a family of extracellular matrix components, and the prototype of this family, fibulin-1, is expressed in various connective tissues and plays a role in developmental and pathologic processes. In this study, we demonstrate that ECM1, and specifically the second tandem repeat domain which is alternatively spliced, interacts with the C-terminal segments of fibulins 1C and 1D splice variants which differ in their C-terminal domain III. The interactions were detected by yeast two-hybrid genetic system and confirmed by co-immunoprecipitations. Kinetics of the binding between ECM1 and fibulin-1D, measured by biosensor assay, revealed a K(d) of 5.71 x 10(-8) M, indicating a strong protein-protein interaction. Since distinct splice variants of ECM1 and fibulin-1 have been shown to be co-expressed in tissues affected in LP, we propose that altered ECM1/fibulin-1 interactions may play a role in the pathogenesis of this disease as well as in a number of processes involving the extracellular matrix of connective tissues.     

MT1-MMP releases latent TGF-beta1 from endothelial cell extracellular matrix via proteolytic processing of LTBP-1

Targeting of transforming growth factor beta (TGF-β) to the extracellular matrix (ECM) by latent TGF-β binding proteins (LTBPs) regulates the availability of TGF-β for interactions with endothelial cells during their quiescence and activation. However, the mechanisms which release TGF-β complexes from the ECM need elucidation. We find here that morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) resulted in membrane-type 1 matrix metalloproteinase (MT1-MMP) -mediated solubilization of latent TGF-β complexes from the ECM by proteolytic processing of LTBP-1. These processes required the activities of PKC and ERK1/2 signaling pathways and were coupled with markedly increased MT1-MMP expression. The functional role of MT1-MMP in LTBP-1 release was demonstrated by gene silencing using lentiviral short-hairpin RNA as well as by the inhibition with tissue inhibitors of metalloproteinases, TIMP-2 and TIMP-3. Negligible effects of TIMP-1 and uPA/plasmin system inhibitors indicated that secreted MMPs or uPA/plasmin system did not contribute to the release of LTBP-1. Current results identify MT1-MMP-mediated proteolytic processing of ECM-bound LTBP-1 as a mechanism to release latent TGF-β from the subendothelial matrix.     

Selective Decline of Synaptic Protein Levels in the Frontal Cortex of Female Mice Deficient in the Extracellular Metalloproteinase ADAMTS1

The chondroitin sulfate-bearing proteoglycans, also known as lecticans, are a major component of the extracellular matrix (ECM) in the central nervous system and regulate neural plasticity. Growing evidence indicates that endogenous, extracellular metalloproteinases that cleave lecticans mediate neural plasticity by altering the structure of ECM aggregates. The bulk of this in vivo data examined the matrix metalloproteinases, but another metalloproteinase family that cleaves lecticans, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), modulates structural plasticity in vitro, although few in vivo studies have tested this concept. Thus, the purpose of this study was to examine the neurological phenotype of a mouse deficient in ADAMTS1. Adamts1 mRNA was absent in the ADAMTS1 null mouse frontal cortex, but there was no change in the abundance or proteolytic processing of the prominent lecticans brevican and versican V2. However, there was a marked increase in the perinatal lectican neurocan in juvenile ADAMTS1 null female frontal cortex. More prominently, there were declines in synaptic protein levels in the ADAMTS1 null female, but not male, frontal cortex beginning at postnatal day 28. These synaptic marker declines did not affect learning or memory in the adult female ADAMTS1 null mice when tested with the radial-arm water maze. These results indicate that in vivo Adamts1 knockout leads to sexual dimorphism in frontal cortex synaptic protein levels. Since changes in lectican abundance and proteolytic processing did not accompany the synaptic protein declines, ADAMTS1 may play a nonproteolytic role in regulating neural plasticity.     

Dynamics of extracellular matrix production and turnover in tissue engineered cardiovascular structures

Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial requirements for their long-term patency and function. This complex process requires the deposition and accumulation of extracellular matrix molecules as well as the remodeling of this extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). In this study, we have investigated the dynamics of ECM production and the activity of MMPs and TIMPs in long-term tissue-engineered vascular conduits using quantitative ECM analysis, substrate gel electrophoresis, radiometric enzyme assays and Western blot analyses. Over a time period of 169 days in vivo, levels of elastin and proteoglycans/glycosaminoglycans in tissue-engineered constructs came to approximate those of their native tissue counter parts. The kinetics of collagen deposition and remodeling, however, apparently require a much longer time period. Through the use of substrate gel electrophoresis, proteolytic bands whose molecular weight was consistent with their identification as the active form of MMP-2 (approximately 64--66 kDa) were detected in all native and tissue-engineered samples. Additional proteolytic bands migrating at approximately 72 kDa representing the latent form of MMP-2 were detected in tissue-engineered samples at time points from 5 throughout 55 days. Radiometric assays of MMP-1 activity demonstrated no significant differences between the native and tissue-engineered samples. This study determines the dynamics of ECM production and turnover in a long-term tissue-engineered vascular tissue and highlights the importance of ECM remodeling in the development of successful tissue-engineered vascular structures.     

Enhancement of the adhesion of fibroblasts by peptide containing an Arg-Gly-Asp sequence with poly(ethylene glycol) into a thermo-reversible hydrogel as a synthetic extracellular matrix

In an effort to regulate the behavior of mammalian cell entrapped in a gel, the gels were functionalized with the putative cell-binding (-Arg-Gly-Asp-) (RGD) domain. The adhesion molecules composed of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides and the cell recognition ligands were inculcated into the thermo-reversible hydrogel composed of N-isopropylacrylamide, with a small amount of succinyl poly(ethylene glycol) (PEG) acrylate (MW 2000) used as the biomimetic extracellular matrix (ECM). The GRGDS-containing p(NiPAAm-co-PEG) copolymer gel was examined in vitro for its ability to promote cell spreading and to increase the viability of the cells by introducing PEG spacers. ECM poorly adhered to hydrogel lacking adhesion molecules permitting only a 20% spread of the seeded cells after 10 days. When the PEG spacer arms, which were immobilized by a peptide linkage, had been integrated into the hydrogel, the conjugation of RGD improved cell spreading by 600% in a 10-day trial.     

Composition and organisation of extracellular matrices around germ tubes and appressoria ofColletotrichum lindemuthianum

Summary The ultrastructure and composition of the extracellular matrices (ECMs) associated with germ tubes and appressoria ofColletotrichum lindemuthianum have been examined. Flexuous fibres (fimbriae), up to 6 m long and 4–30 nm in diameter, protruded from the surface of germ tubes and appressoria. Anionic colloidal gold and lectin cytochemistry showed that ECMs of germ tubes and appressoria contain basic proteins, -D-mannose and -D-galactose residues. A monoclonal antibody, UB26, was raised to infection structures isolated from leaves ofPhaseolus vulgaris infected withC. lindemuthianum. UB26 recognised a protein epitope on two glycoproteins (M_r 133,000 and 146,000). Reductions in the M_r of these proteins after treatment with peptide-N-glycosidase and trifluoromethane sulphonic acid suggest that they carry N- and O-linked side-chains. Immunofluorescence and EM-immunogold labelling showed that glycoproteins recognised by UB26 were restricted to the ECMs around germ tubes and appressoria but fimbriae were not labelled. Unlike appressorial germ tubes formed in vitro, intracellular infection hyphae were not labelled, suggesting that the glycoproteins recognised by UB26 are not present on fungal structures formed within host cells. In liquid culture, these glycoproteins were not released into the medium, suggesting they are physically linked to the cell wall. Also, the glycoproteins were not removed from glass surfaces by ultrasonication. These results suggest that glycoproteins recognised by UB26 may be involved in the adhesion of germ tubes and appressoria to substrata. Our results show that the ECMs of germ tubes and appressoria differ markedly in structure and composition from those of conidia and intracellular hyphae, and that extracellular glycoproteins are associated with specific regions of the fungal cell surface.Abbreviations ECM extracellular matrix - BPA Bauhinia purpurea agglutinin - BSA bovine serum albumin - DIC differential interference contrast - FITC fluorescein isothiocyanate - GNL Galanthus nivalis lectin - GSI-B₄ Griffonia simplicifolia isolectin B₄ - HEPES (N-(2-hydroxyethyl)piperazine-N-(2-ethanesulphonic acid) - IIF indirect immunofluorescence - IPC isopycnic centrifugation - MAb monoclonal antibody - PEG polyethylene glycol - PBS phosphate buffered saline - PNGase peptideN-glycosidase - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TCS tissue culture supernatant - TEM transmission electron microscopy - TFMS trifluoromethane sulphonic acid