Characterization of the extracellular matrix of Phaeodactylum tricornutum (Bacillariophyceae): structure,composition, and adhesive characteristics

The extracellular matrix of the ovoid and fusiform morphotypes of Phaeodactylum tricornutum (Bohlin) was characterized in detail. The structural and nanophysical properties were analyzed by microscopy. Of the two morphotypes, only the ovoid form secretes adhesive mucilage; light microscopy and scanning electron microscopy images showed that the mucilage was secreted from the girdle band region of the cell as cell‐substratum tethers, accumulating on the surface forming a biofilm. After 7 d, the secreted mucilage became entangled, forming adhesive strands that crisscrossed the substratum surface. In the initial secreted mucilage atomic force microscopy identified a high proportion of adhesive molecules without regular retraction curves and some modular‐like adhesive molecules, in the 7 d old biofilm, the adhesive molecules were longer with fewer adhesive events but greater adhesive strength. Chemical characterization was carried out on extracted proteins and polysaccharides. Differences in protein composition, monosaccharide composition, and linkage analysis are discussed in relation to the composition of the frustule and secreted adhesive mucilage. Polysaccharide analysis showed a broad range of monosaccharides and linkages across all fractions with idiosyncratic enrichment of particular monosaccharides and linkages in each fraction. 3‐linked Mannan was highly enriched in the cell frustule fractions indicating a major structural role, while Rhamnose and Fucose derivatives were enriched in the secreted fractions of the ovoid morphotype suggesting involvement in cell adhesion. Comparison of SDS‐PAGE of extracellular proteins showed two major bands for the ovoid morphotype and four for the fusiform morphotype of which only one appeared to be common to both morphotypes.     

Critical and optimal Ig domains for promotion of neurite outgrowth by L1/Ng‐CAM

Mammalian L1 and avian Ng‐CAM are homologous neural cell adhesion molecules (CAMs) that promote neurite outgrowth and cell adhesion in most neurons. Previous attempts to map these activities to discrete regions in the CAMs have suggested the involvement of a variety of different domains. However, these studies mainly used bacterially expressed proteins that were much less active on a molar basis than the native molecules. To define regions that are critical for maximal neurite outgrowth, we constructed and tested a panel of eukaryotically expressed proteins containing various extracellular segments of human L1 (hL1) or Ng‐CAM. Our results indicate that Ig domains 1–4 of hL1 are critical for homophilic binding and neurite outgrowth; however this segment is less potent than the entire extracellular region. Optimal neurite outgrowth activity was seen with proteins containing all six Ig domains of hL1 or Ng‐CAM. The adhesive properties of hL1 fragments correlated tightly with their neurite outgrowth activities, suggesting that these two processes are closely linked. These results suggest that Ig domains 1–4 form a structural cassette responsible for hL1 homophilic binding, while Ig domains 1–6 represent a functional region for optimal promotion of neurite outgrowth in vitro and possibly in vivo. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 287–302, 2000     

Adhesive modular proteins occur in the extracellular mucilage of the motile, pennate diatom Phaeodactylum tricornutum

This Letter reports on adhesive modular proteins recorded by atomic force microscopy on live cells from the extracellular mucilage secreted from, and deposited around, the motile form of the pennate diatom Phaeodactylum tricornutum. This is the first report of modular proteins and their supramolecular assemblies, called adhesive nanofibers (ANFs), to be found on diatoms that use adhesives not only for substratum adhesion, but as a conduit for cell motility. The permanent adhesive pads secreted by Toxarium undulatum, a sessile centric diatom, were previously shown to possess ANFs with a modular protein backbone. Our results reported here suggest that modular proteins may be an important component of diatom adhesives in general, and that diatoms utilize the tensile strength, toughness, and flexibility of ANFs for multiple functions. Significantly, the genome of P. tricornutum has recently been sequenced; this will allow directed searches of the genome to be made for genes with modular protein homologs, and subsequent detailed studies of their molecular structure and function.     

THE STRUCTURE AND NANOMECHANICAL PROPERTIES OF THE ADHESIVE MUCILAGE THAT MEDIATES DIATOM‐SUBSTRATUM ADHESION AND MOTILITY1

We investigated the adhesive mucilage and mechanism of cell‐substratum adhesion of two benthic raphid diatoms, the marine species Craspedostauros australis E. J. Cox and the freshwater species Pinnularia viridis (Nitzsch) Ehrenberg. SEM images of P. viridis and C. australis cells revealed the presence of multistranded tethers that appear to arise along the raphe openings and extend for a considerable distance from the cell before forming a “holdfast‐like” attachment with the substratum. We propose that the tethers result from the elongation/stretching of composite adhesive mucilage strands secreted from raphes during the onset of cell adhesion and reorientation. Atomic force microscopy (AFM) force measurements reveal that the adhesive strands originating from the nondriving raphe of live C. australis and P. viridis are highly extensible and accumulate to form tethers. During force measurements tethers can be chemically stained and are seen to extend between the cantilever tip and a cell during elongation and relaxation. In most cases, AFM force measurements recorded an interaction with a number of adhesive strands that are secreted from the raphe. The force curves of C. australis and P. viridis revealed a sawtooth pattern, suggesting the successive unbinding of modular domains when the adhesive strands were placed under stress. In addition, we applied the “fly‐fishing” technique that allowed the cantilever, suspended a distance above the cell, to interact with single adhesive strands protruding from the raphe. These force curves revealed sawtooth patterns, although the binding forces recorded were in the range for single molecule interactions.     

Dibutyryl cyclic AMP‐induced process formation in astrocytes is associated with a decrease in tyrosine phosphorylation of focal adhesion kinase and paxillin

Focal adhesion kinase (FAK or pp125FAK) is a cytosolic protein tyrosine kinase which plays an important role in integrin‐mediated signal transduction. Adhesion of cells to the substratum correlates with an increase in tyrosine phosphorylation of FAK as well as an associated protein, paxillin. In this report we show that the tyrosine phosphorylation of FAK and paxillin are decreased during dibutyryl cyclic AMP–induced (dB‐cAMP) process formation in astrocytes. When astrocytes in suspension are treated with dB‐cAMP, no alteration in morphology or tyrosine phosphorylation is observed, suggesting that both phenomena are linked and adhesion dependent. Furthermore, genistein, a tyrosine kinase inhibitor, can induce process formation in such cells, underscoring the significance of protein tyrosine kinases in maintaining the morphology of adherent cells. Finally, endothelin‐1, a vasopeptide which is known to inhibit process formation in astrocytes, inhibited the tyrosine dephosphorylation of proteins associated with dB‐cAMP treatment. These results suggest that the formation of asymmetric processes in astrocytes results from a coordinated set of alterations in the actin cytoskeleton as well as the adhesion of the cell to the substratum. Modification of the properties of such molecules is required for process formation and the dynamic modulation of astrocytic morphology in vitro and in vivo. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 407–422, 1999     

Embigin,a developmentally expressed member of the immunoglobulin super family,is also expressed during regression of prostate and mammary gland

Cell adhesion molecules (CAMs) are intimately involved in a variety of cellular processes, including development, cell growth, apoptosis, and differentiation. Interaction of CAMs with components of the extracellular matrix (ECM), growth factors, and other CAMs provides an intricate regulatory mechanism for a diverse range of cellular responses. Embigin is a developmentally expressed protein that is a member of the immunoglobulin superfamily (IgSF) class of CAMs. We have identified embigin as a gene expressed during tissue regression in rat prostate and lactating mammary gland following hormonal ablation. In the absence of the appropriate hormone, the secretory epithelial cells of these two tissues undergo successive waves of apoptotic cell death co-incident with extensive reorganization of the surviving tissue. Using Northern analysis, in situ hybridization analysis, RT-PCR, and Western analysis, we have characterized the expression of embigin mRNA and protein in both regressing prostate and mammary gland. During development of the prostate gland, increased expression of embigin is correlated with the appearance of highly organized lumenal and ductal structures. Embigin is also expressed in a variety of adult tissues including heart, liver, lung, and brain. Zoo-blot analysis with the rat embigin cDNA indicates that embigin homologs exist in species as diverse as Homo sapiens and Drosophila melanogaster, suggesting that it has been highly conserved during evolution. Embigin protein is expressed at readily detectable levels in a variety of prostate and mammary cancer cell lines, and in some cell lines the expression of embigin appears to be down-regulated in the presence of ECM. Our data have led us to propose a model in which embigin functions as a regulator of cell/ECM interactions during development and in the homeostasis of normal adult tissues. Dev. Genet. 21:268–278, 1997. © 1997 Wiley-Liss, Inc.     

Roles and modes of action of nectins in cell–cell adhesion

Nectins are Ca²⁺-independent immunoglobulin (Ig)-like cell–cell adhesion molecules (CAMs), which comprise a family consisting of four members. Each nectin homophilically and heterophilically trans-interacts and causes cell–cell adhesion. Biochemical, cell biological, and knockout mice studies have revealed that nectins play important roles in formation of many types of cell–cell junctions and cell–cell contacts, including cadherin-based adherens junctions (AJs) and synapses. Mode of action of nectins in the formation of AJs has extensively been investigated. Nectins form initial cell–cell adhesion and recruit E-cadherin to the nectin-based cell–cell adhesion sites. In addition, nectins induce activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of cadherin-based AJs through the reorganization of the actin cytoskeleton. Nectins furthermore heterophilically trans-interact with nectin-like molecules (Necls), other Ig-like CAMs, and assist or modify their various functions, such as cell adhesion, migration, and proliferation. We describe here the roles and modes of action of nectins as CAMs.     

Soluble 80-kd fragment of cell-CAM 120/80 disrupts cell-cell adhesion

Calcium-dependent cell adhesion molecules (CAMs) mediate intercellular adhesion in epithelial cells and in preimplantation mammalian embryos. One of these molecules, cell-CAM 120/80, is found on cells as a 120-kd membrane glycoprotein and as a soluble 80-kd species in conditioned culture medium [Damsky et al: Cell 34:455, 1983]. We have purified to homogeneity the soluble 80-kd fragment of cell-CAM 120/80 by using monoclonal antibody affinity chromatography. We have shown that the purified molecule can disrupt cell-cell adhesion in cultured epithelial cells, thus indicating that it is directly involved in the adhesive process. In addition, we have further characterized both the 120-kd cell-associated molecule and its 80-kd fragment, including N-terminal sequence analysis.     

Mucilage secretions of moving diatoms

Summary The raphes of a moving diatom are filled with mucilage strands which are perpendicular to the slit and protrude from the external raphe fissure. The distal ends of the strands are capable of adhering to the substratum.Navicula cuspidata moves with only the posterior half of the cell adhered; the anterior is elevated from the substratum. The species studied left no apparent mucilage trails.     

IgLON cell adhesion molecules regulate synaptogenesis in hippocampal neurons

IgLON cell adhesion molecules (CAMs) belonging to the immunoglobulin superfamily comprise of LAMP, neurotrimin (Ntm), OBCAM, and Kilon. In the present study, we performed the single and double transfection of IgLON gene constructs into hippocampal neurons in vitro and evaluated synaptic number. The quantitative analysis showed that the single over‐expression of LAMP or OBCAM increased synaptic number, while the over‐expression of Kilon reduced synaptic number and Ntm had no effects. The double over‐expression of Kilon‐Ntm, Kilon‐OBCAM, LAMP‐Ntm, and Ntm‐OBCAM decreased synaptic number and that of Kilon‐LAMP and LAMP‐OBCAM had no effect. These results suggest that IgLON CAMs participate in regulating synapse formation in hippocampal neurons. Copyright © 2009 John Wiley & Sons, Ltd.     

Dissecting focal adhesions in cells differentially expressing calreticulin: a microscopy study

Background information. Our previous studies have shown that calreticulin, a Ca²⁺‐binding chaperone located in the endoplasmic reticulum, affects cell—substratum adhesions via the induction of vinculin and N‐cadherin. Cells overexpressing calreticulin contain more vinculin than low expressers and make abundant contacts with the substratum. However, cells that express low levels of calreticulin exhibit a weak adhesive phenotype and make few, if any, focal adhesions. To date, the identity of the types of focal adhesions made by calreticulin overexpressing and low expressing cells has not been dissected. Results. The results of the present study show that calreticulin affects fibronectin matrix assembly in L fibroblast cell lines that differentially express the protein, and that these cells also differ profoundly in focal adhesion formation. Although the calreticulin overexpressing cells generate numerous interference‐reflection‐microscopy‐dark, vinculin‐ and paxillin‐containing classical focal contacts, as well as some fibrillar adhesions, the cells expressing low levels of calreticulin generate only a few weak focal adhesions. The fibronectin receptor was found to be clustered in calreticulin overexpressing cells, but diffusely distributed over the cell surface in low expressing cells. Plating L fibroblasts on fibronectin‐coated substrata induced extensive spreading in all cell lines tested. However, although calreticulin overexpressing cells were induced to form classical vinculin‐rich focal contacts, the low calreticulin expressing cells overcame their weak adhesive phenotype by induction of many tensin‐rich fibrillar adhesions, thus compensating for the low level of vinculin in these cells. Conclusions. We propose that calreticulin affects fibronectin production and, thereby, assembly, and it indirectly influences the formation and/or stability of focal contacts and fibrillar adhesions, both of which are instrumental in matrix assembly and remodelling.     

Cell-CAM 105-an adhesive cell surface glycoprotein

Summary Cell recognition and adhesion are important events in embryonic development as well as in adult physiology. In recent years several cell adhesion molecules (CAMs), that mediate adhesive interactions between vertebrate cells, have been identified and characterized. These CAMs are in general cell surface-associated high molecular weight glycoproteins. Two groups of CAMs have been classified: primary CAMs, that appear early in development; secondary CAMs, that become expressed later and with a more restricted tissue distribution. One example of a secondary CAM is cellCAM 105. This glycoprotein was originally identified in rat hepatocytes, and was shown to be involved in the reaggregation of freshly isolated hepatocytesin vitro. Physico-chemical studies on pure cellCAM 105 have demonstrated that it has adhesive properties and can bind to itself in a homophilic, calcium-independent reaction. Immunochemical and immunohistochemical investigations have shown that cellCAM 105 occurs in liver, several epithelia, vessel endothelia, platelets and polymorphonuclear leukocytes, and that it is expressed primarily in terminally differentiated cells or cells that are undergoing terminal differentiation. Available information suggests that cellCAM 105 has different functions in different cell types, and that the common functional denominator might be membrane-membrane binding. Recent data indicate that cellCAM 105 is a calmodulin-binding protein, suggesting that cellCAM-mediated cell binding could be involved in transmembrane signalling.Abbreviation CAM cell adhesion molecule     

Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans

Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and >200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion. Received: 2 December 1996 / Accepted: 6 March 1997     

Adhesion of algal cells to surfaces

This paper reports the cell–substratum interactions of planktonic (Chlorella vulgaris) and benthic (Botryococcus sudeticus) freshwater green algae with hydrophilic (glass) and hydrophobic (indium tin oxide) substrata to determine the critical parameters controlling the adhesion of algal cells to surfaces. The surface properties of the algae and substrata were quantified by measuring contact angle, electrophoretic mobility, and streaming potential. Using these data, the cell–substratum interactions were modeled using thermodynamic, DLVO, and XDLVO approaches. Finally, the rate of attachment and the strength of adhesion of the algal cells were quantified using a parallel-plate flow chamber. The results indicated that (1) acid–base interactions played a critical role in the adhesion of algae, (2) the hydrophobic alga attached at a higher density and with a higher strength of adhesion on both substrata, and (3) the XDLVO model was the most accurate in predicting the density of cells and their strength of adhesion. These results can be used to select substrata to promote/inhibit the adhesion of algal cells to surfaces.     

Microsporidia Can Acquire Lamin‐like Intermediate Filaments and Cell Adhesion Catenin‐cadherin Complexes from the Host (?)

On their spore surfaces, Microsporidia often develop a canopy of filaments with characteristics of intermediate filaments (IF), as we demonstrated in previous studies on Thelohania sp., Ameson michaelis, and Spraguea lophii. Genomic studies indicate that among invertebrates, lamins that may localize in the cytoplasm or nucleus, are the only known IF type. These IFs can bind to the substrate containing cell adhesion molecules (CAMs) cadherins, associated with β and γ catenins. The objects of this study were to determine whether microsporidia have CAMs with the attached IFs on their envelopes and to find out if these proteins are provided by the host. An examination was made for localization of lamins and CAMs on the spores of the mentioned above species and Anncaliia algerae, plus in the host animals. Then, we determined whether the spores of A. michaelis and A. algerae could bind vertebrate nuclear lamin onto the spore surface. We also tested transgenic Drosophila melanogaster stocks bearing cadherin‐GFP to see whether developing A. algerae parasites in these hosts could acquire host CAMs. The tests were positive for all these experiments. We hypothesize that microsporidia are able to acquire host lamin IFs and cell adhesion catenin–cadherin complexes from the host.     

Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125- fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.     

Physiology and Pathophysiology of Selectins,Integrins, and IgSf Cell Adhesion Molecules Focusing on Inflammation. A Paradigm Model on Infectious Endocarditis

Abstract

The development of adhesion bonds, either among cells or among cells and components of the extracellular matrix, is a crucial process. These interactions are mediated by some molecules collectively known as adhesion molecules (CAMs). CAMs are ubiquitously expressed proteins playing a central role in controlling cell migration, proliferation, survival, and apoptosis. Besides their key function in physiological maintenance of tissue integrity, CAMs play an eminent role in various pathological processes such as cardiovascular disorders, atherogenesis, atherosclerotic plaque progression and regulation of the inflammatory response. CAMs such as selectins, integrins, and immunoglobulin superfamily take part in interactions between leukocyte and vascular endothelium (leukocyte rolling, arrest, firm adhesion, migration). Experimental data and pathologic observations support the assumption that pathogenic microorganisms attach to vascular endothelial cells or sites of vascular injury initiating intravascular infections. In this review a paradigm focusing on cell adhesion molecules pathophysiology and infective endocarditis development is given.     

Mechanisms of temporary adhesion in benthic animals

Adhesive systems are ubiquitous in benthic animals and play a key role in diverse functions such as locomotion, food capture, mating, burrow building, and defence. For benthic animals that release adhesives, surface and material properties and external morphology have received little attention compared to the biochemical content of the adhesives. We address temporary adhesion of benthic animals from the following three structural levels: (a) the biochemical content of the adhesive secretions, (b) the micro‐ and mesoscopic surface geometry and material properties of the adhesive organs, and (c) the macroscopic external morphology of the adhesive organs. We show that temporary adhesion of benthic animals is affected by three structural levels: the adhesive secretions provide binding to the substratum at a molecular scale, whereas surface geometry and external morphology increase the contact area with the irregular and unpredictable profile of the substratum from micro‐ to macroscales. The biochemical content of the adhesive secretions differs between abiotic and biotic substrata. The biochemistry of the adhesives suitable for biotic substrata differentiates further according to whether adhesion must be activated quickly (e.g. as a defensive mechanism) or more slowly (e.g. during adhesion of parasites). De‐adhesion is controlled by additional secretions, enzymes, or mechanically. Due to deformability, the adhesive organs achieve intimate contact by adapting their surface profile to the roughness of the substratum. Surface projections, namely cilia, cuticular villi, papillae, and papulae increase the contact area or penetrate through the secreted adhesive to provide direct contact with the substratum. We expect that the same three structural levels investigated here will also affect the performance of artificial adhesive systems.     

On the role of cell surface associated,mucin-like glycoproteins in the pennate diatom Craspedostauros australis (Bacillariophyceae)

Diatoms are single-celled microalgae with silica-based cell walls (frustules) that are abundantly present in aquatic habitats, and form the basis of the food chain in many ecosystems. Many benthic diatoms have the remarkable ability to glide on all natural or man-made underwater surfaces using a carbohydrate- and protein-based adhesive to generate traction. Previously, three glycoproteins, termed FACs (F rustule A ssociated C omponents), have been identified from the common fouling diatom Craspedostauros australis and were implicated in surface adhesion through inhibition studies with a glycan-specific antibody. The polypeptide sequences of FACs remained unknown, and it was unresolved whether the FAC glycoproteins are indeed involved in adhesion, or whether this is achieved by different components sharing the same glycan epitope with FACs. Here we have determined the polypeptide sequences of FACs using peptide mapping by LC–MS/MS. Unexpectedly, FACs share the same polypeptide backbone (termed CaFAP1), which has a domain structure of alternating Cys-rich and Pro-Thr/Ser-rich regions reminiscent of the gel-forming mucins. By developing a genetic transformation system for C. australis, we were able to directly investigate the function of CaFAP1-based glycoproteins in vivo. GFP-tagging of CaFAP1 revealed that it constitutes a coat around all parts of the frustule and is not an integral component of the adhesive. CaFAP1-GFP producing transformants exhibited the same properties as wild type cells regarding surface adhesion and motility speed. Our results demonstrate that FAC glycoproteins are not involved in adhesion and motility, but might rather act as a lubricant to prevent fouling of the diatom surface.     

Bacteria may induce the secretion of mucin‐like proteins by the diatom Phaeodactylum tricornutum

Benthic diatoms live in photoautotrophic/heterotrophic biofilm communities embedded in a matrix of secreted extracellular polymeric substances. Closely associated bacteria influence their growth, aggregation, and secretion of exopolymers. We have studied a diatom/bacteria model community, in which a marine Roseobacter strain is able to grow with secreted diatom exopolymers as a sole source of carbon. The strain influences the aggregation of Phaeodactylum tricornutum by inducing a morphotypic transition from planktonic, fusiform cells to benthic, oval cells. Analysis of the extracellular soluble proteome of P. tricornutum in the presence and absence of bacteria revealed constitutively expressed newly identified proteins with mucin‐like domains that appear to be typical for extracellular diatom proteins. In contrast to mucins, the proline‐, serine‐, threonine‐rich (PST) domains in these proteins were also found in combination with protease‐, glucosidase‐ and leucine‐rich repeat‐domains. Bioinformatic functional predictions indicate that several of these newly identified diatom‐specific proteins may be involved in algal defense, intercellular signaling, and aggregation.