Cell Surface Components Research Articles

Host Directed Autoantibodies That Fuel Tumor Progression

Autoantibodies (aAbs) have yielded productive venues for both diagnostics (eg. tumor‐associated antigens, TAAs) and therapeutics (eg. engineered drug conjugated TAAs). The paradigm for TAAs are intracellular or cell‐surface cancer cell components that are either overexpressed, expressed in a novel location, or have an altered structure or sequence that yields an immunogenic response. Another group of cancer‐relevant aAbs are directed against host proteins yet influence tumor progression and either predate the cancer and/or have arisen in response to comorbid conditions. The paradigm for this is an activating aAb against angiotensin II type 1 receptor (AT1R). This G protein coupled receptor is expressed by many cell types. In addition to a classic role in homeostatic regulation of blood pressure, AT1R activation has been implicated in altering tissue remodeling, wound repair, inflammation, energy metabolism, and thrombosis ‐ processes dysregulated in the setting of cancer. AT1RaAbs whose epitope maps to extracellular loop 2 significantly associated with levels of markers for apoptosis (sFas, sFasL), macrophage activation (neopterin, chitotriosidase) and inflammation (pro‐inflammatory cytokines) when serum from normal or lung cancer subjects was analyzed by ELISA. High serum levels of AT1RaAb were associated with poor outcomes following treatment. Lung cancer cells treated with isolated AT1RaAbs were analyzed for signaling pathways, cellular proliferation, survival (clonogenic assay following methotrexate challenge) and invasiveness (Boyden chamber assay). The data are consistent with (a) AT1RaAb binding of target receptors and constitutively activating NFkB‐dependent chronic inflammatory/remodeling state promoting tumor progression, and (b) serum AT1aAb levels being predictive of disease recurrence.

Effects of Salt Stress on Cell Surface Properties and Symbiotic Performance of Root Nodulating Bacteria

In the current study fifty root nodulating bacterial strains isolated from Cicer arietinum (Chickpea) and Vigna radiata (Mungbean) nodules were subjected to different NaCl concentrations. Out of these 20 salt tolerant isolates were selected for the production of cell surface component like β-glucan, lipopolysaccharide, exopolysaccharide and motility under salt stress condition. Symbiotic performance of Vigna radiata and Cicer arietinum were done under salt stress condition by using 4 isolates (CMR3,CMR13,MR5.MR11) which showed the tolerant behavior in all cell surface properties and one sensitive isolate (MR30) was used for comparision.All growth parameters of selected legumes were enhanced at 5% salt concentration as compare to control in CMR3,CMR13,MR5.MR11. A decline in all parameters was observed in isolate that does not show good cell surface components under salt stress. This shows the inhibition of cell surface properties under salt stress, affects the symbiotic performance of both parameters negatively. The present study concludes that under salt stress conditions, tolerant strains successfully overcome the stressful environmental conditions by maintaining the factors, essential for symbiosis in an ideal system.

Identification of genes differentially expressed in husk tomato (Physalis philadelphica) in response to whitefly (Trialeurodes vaporariorum) infestation

Plants respond to phloem-feeding whiteflies by extensive changes in gene expression. To identify differentially expressed genes in husk tomato plants (Physalis philadelphica) infested with Trialeurodes vaporariorum, young plants were challenged with adult whiteflies, and forward and reverse subtractive libraries were constructed from infested leaves at 5 and 15 days after infestation. Several genes were identified as up-regulated; these included a diversity of genes involved in plant defense responses, protein synthesis or degradation, and cell wall fortification or modification. Genes required for amino acid biosynthesis, lipid metabolism and synthesis, including cell surface components such as suberin, responses to stress, photosynthesis and other functions, were similarly induced. Down-regulated genes were also identified, most prominently kinases and aquaporin genes. Similarities in defense responses between tomato and P. philadelphica were noted regarding the expression of certain genes in response to nematode, aphid, or whitefly. A role for abscisic acid, brassinosteroids, and cytokinins in the regulated response to whitefly infestation in P. philadelphica was also implied by the expression pattern of phytohormone-associated genes, including genes coding for proteins containing F-box motifs. Differential expression of selected genes was validated by quantitative real-time PCR. The possible role played by some of these genes during whitefly infestation is discussed.

Selection of Antibodies Interfering with Cell Surface Receptor Signaling Using Embryonic Stem Cell Differentiation.

Antibodies able to bind and modify the function of cell surface signaling components in vivo are increasingly being used as therapeutic drugs. The identification of such "functional" antibodies from within large antibody pools is, therefore, the subject of intense research. Here we describe a novel cell-based expression and reporting system for the identification of functional antibodies from antigen-binding populations preselected with phage display. The system involves inducible expression of the antibody gene population from the Rosa-26 locus of embryonic stem (ES) cells, followed by secretion of the antibodies during ES cell differentiation. Target antigens are cell-surface signaling components (receptors or ligands) with a known effect on the direction of cell differentiation (FGFR1 mediating ES cell exit from self renewal in this particular protocol). Therefore, inhibition or activation of these components by functional antibodies in a few elite clones causes a shift in the differentiation outcomes of these clones, leading to their phenotypic selection. Functional antibody genes are then recovered from positive clones and used to produce the purified antibodies, which can be tested for their ability to affect cell fates exogenously. Identified functional antibody genes can be further introduced in different stem cell types. Inducible expression of functional antibodies has a temporally controlled protein-knockdown capability, which can be used to study the unknown role of the signaling pathway in different developmental contexts. Moreover, it provides a means for control of stem cell differentiation with potential in vivo applications.

The Contribution of Cell Surface Components to the Neutrophil Mechanosensitivity to Shear Stresses

This review discusses the regulation of neutrophils by fluid shear stress in the context of factors that may govern cell mechanosensitivity and its influence on cell functions. There is substantial evidence that mechanoreceptors located on the peripheral membrane contribute to the ability of shear stress to regulate cell activity. In the case of neutrophils, the formyl peptide receptor (FPR) and the CD18 integrins on the cell membrane have been shown to provide neutrophils with the ability to sense shear stresses in their local environment and alter their physiological state, accordingly. This configuration is also found for other types of cells, although they involve different cell-specific mechanoreceptors. Moreover, from an examination of the neutrophil mechanotransducing capacity, it is apparent that cellular mechanosensitivity depends on a number of factors that, if altered, contribute to dysregulation and ultimately pathophysiology. To exemplify this, we first describe the neutrophil responses to shear exposure. We then review two neutrophil mechanoreceptors, specifically FPR and CD18 integrins, which participate in controlling cell activity levels under physiological conditions. Next, we discuss the various factors that may alter neutrophil mechanosensitivity to shear stress and how these may underlie the circulatory pathobiology of two cardiovascular disease states: hypertension and hypercholesterolemia. Based on the material presented, it is conceivable that cell mechanosensitivity is a powerful global metric that permits a more efficient approach to understanding the contribution of mechanobiology to physiology and to disease processes.

Transcriptome analysis of Enterococcus faecalis during mammalian infection shows cells undergo adaptation and exist in a stringent response state.

As both a commensal and a major cause of healthcare-associated infections in humans, Enterococcus faecalis is a remarkably adaptable organism. We investigated how E. faecalis adapts in a mammalian host as a pathogen by characterizing changes in the transcriptome during infection in a rabbit model of subdermal abscess formation using transcriptional microarrays. The microarray experiments detected 222 and 291 differentially regulated genes in E. faecalis OG1RF at two and eight hours after subdermal chamber inoculation, respectively. The profile of significantly regulated genes at two hours post-inoculation included genes involved in stress response, metabolism, nutrient acquisition, and cell surface components, suggesting genome-wide adaptation to growth in an altered environment. At eight hours post-inoculation, 88% of the differentially expressed genes were down-regulated and matched a transcriptional profile consistent with a (p)ppGpp-mediated stringent response. Subsequent subdermal abscess infections with E. faecalis mutants lacking the (p)ppGpp synthetase/hydrolase RSH, the small synthetase RelQ, or both enzymes, suggest that intracellular (p)ppGpp levels, but not stringent response activation, influence persistence in the model. The ability of cells to synthesize (p)ppGpp was also found to be important for growth in human serum and whole blood. The data presented in this report provide the first genome-wide insights on E. faecalis in vivo gene expression and regulation measured by transcriptional profiling during infection in a mammalian host and show that (p)ppGpp levels affect viability of E. faecalis in multiple conditions relevant to mammalian infection. The subdermal abscess model can serve as a novel experimental system for studying the E. faecalis stringent response in the context of the mammalian immune system.

The multifaceted roles of metabolic enzymes in the Paracoccidioides species complex.

Paracoccidioides species are dimorphic fungi and are the etiologic agents of paracoccidioidomycosis, which is a serious disease that involves multiple organs. The many tissues colonized by this fungus suggest a variety of surface molecules involved in adhesion. A surprising finding is that most enzymes in the glycolytic pathway, tricarboxylic acid (TCA) cycle and glyoxylate cycle in Paracoccidioides spp. have adhesive properties that aid in interacting with the host extracellular matrix and thus act as ‘moonlighting’ proteins. Moonlighting proteins have multiple functions, which adds a dimension to cellular complexity and benefit cells in several ways. This phenomenon occurs in both eukaryotes and prokaryotes. For example, moonlighting proteins from the glycolytic pathway or TCA cycle can play a role in bacterial pathogenesis by either acting as proteins secreted in a conventional pathway and/or as cell surface components that facilitate adhesion or adherence. This review outlines the multifunctionality exhibited by many Paracoccidioides spp. enzymes, including aconitase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate lyase, malate synthase, triose phosphate isomerase, fumarase, and enolase. We discuss the roles that moonlighting activities play in the virulence characteristics of this fungus and several other human pathogens during their interactions with the host.

Live imaging provides new insights on dynamic F-actin filopodia and differential endocytosis during myoblast fusion in Drosophila.

The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the myotube.

IGFBP-2 inhibits adipogenesis and lipogenesis in human visceral, but not subcutaneous, adipocytes.

IGF-binding protein (IGFBP)-2 is the principal IGFBP produced by white adipocytes during adipogenesis, and circulating levels are reduced in obesity. Overexpression of IGFBP-2 in transgenic mice prevents obesity, but depot-specific effects of IGFBP-2 on adipo/lipogenesis are unknown. The present study aimed to investigate whether IGFBP-2 affects adipo/lipogenesis in a depot-specific manner and explore potential mechanisms. Following adipocyte characterisation, IGFBP-2 levels were measured from human subcutaneous and visceral preadipocytes, and IGFBP-2 dose-responses were then undertaken with exogenous IGFBP-2 in an in vitro IGF-I-free system to examine adipo/lipogenesis. Following this, both types of adipocytes were transfected with human siRNA IGFBP-2 to assess auto-/para-/intra-crine effects, with and without additional add-back IGFBP-2. To elucidate the potential mechanisms, visceral preadipocytes were treated with either wild-type or Heparin Binding Domain (HBD)-mutant IGFBP-2 (which is unable to bind to cell-surface components), and experiments were also undertaken using Echistatin (an integrin receptor blocker). Outcomes included gene expression profiles, protein levels and phosphorylation and lipid staining. Human visceral adipocytes produced significantly more IGFBP-2 than subcutaneous adipocytes. Subsequent dose-responses to IGFBP-2 demonstrated significant reductions in adipo/lipogenesis in visceral, but not subcutaneous, adipocytes in response to increasing IGFBP-2. Silencing IGFBP-2 resulted in exaggerated adipo/lipogenesis in visceral, but not subcutaneous, adipocytes, an effect completely inhibited by add-back IGFBP-2. These effects occurred in the absence of changes in IGF-I levels. HBD-mutant IGFBP-2 had reduced effects compared with wild-type IGFBP-2. Wild-type IGFBP-2 increased phosphorylation of focal adhesion kinase (FAK) and decreased phosphatase and tensin homolog (PTEN) levels, suggestive of integrin-mediated signalling. Blockade of this signalling, using Echistatin, completely negated the effects of IGFBP-2 on visceral adipo/lipogenesis. IGFBP-2 inhibits both adipogenesis and lipogenesis in visceral, but not subcutaneous, adipocytes. This depot-specific impairment appears to be independent of IGF-I and involves cell-surface association of IGFBP-2 and activation of integrin signalling pathways.

Cell-surface translational dynamics of nicotinic acetylcholine receptors.

Synapse efficacy heavily relies on the number of neurotransmitter receptors available at a given time. In addition to the equilibrium between the biosynthetic production, exocytic delivery and recycling of receptors on the one hand, and the endocytic internalization on the other, lateral diffusion and clustering of receptors at the cell membrane play key roles in determining the amount of active receptors at the synapse. Mobile receptors traffic between reservoir compartments and the synapse by thermally driven Brownian motion, and become immobilized at the peri-synaptic region or the synapse by: (a) clustering mediated by homotropic inter-molecular receptor–receptor associations; (b) heterotropic associations with non-receptor scaffolding proteins or the subjacent cytoskeletal meshwork, leading to diffusional “trapping,” and (c) protein-lipid interactions, particularly with the neutral lipid cholesterol. This review assesses the contribution of some of these mechanisms to the supramolecular organization and dynamics of the paradigm neurotransmitter receptor of muscle and neuronal cells -the nicotinic acetylcholine receptor (nAChR). Currently available information stemming from various complementary biophysical techniques commonly used to interrogate the dynamics of cell-surface components is critically discussed. The translational mobility of nAChRs at the cell surface differs between muscle and neuronal receptors in terms of diffusion coefficients and residence intervals at the synapse, which cover an ample range of time regimes. A peculiar feature of brain α7 nAChR is its ability to spend much of its time confined peri-synaptically, vicinal to glutamatergic (excitatory) and GABAergic (inhibitory) synapses. An important function of the α7 nAChR may thus be visiting the territories of other neurotransmitter receptors, differentially regulating the dynamic equilibrium between excitation and inhibition, depending on its residence time in each domain.

HIV's ticket to ride: Cytotoxic T-lymphocyte-activated dendritic cells exploited for virus intercellular transfer.

For HIV-1 to effectively establish infection and persistence, it must circumvent host immune detection. The capacity of HIV-1 to efficiently mutate and generate multivariant strains in response to host immune selective pressure provides the virus with a particularly effective means of evading cytotoxic T-lymphocyte (CTL) recognition and elimination.1 However, a novel concept has emerged suggesting that HIV-1 can actually benefit from the strategic “baiting” and partial activation of HIV-1-specific CTL responders.2 Driven by immune pressure, CTL epitope divergence can result in the occurrence of surviving epitope variants capable of selectively activating the helper activity of preexisting cross-reactive CTL to create a safe haven for the virus within antigen-presenting dendritic cells (DC). Instead of dampening the immune response through CTL recognition and subsequent killing of these HIV-1 antigen-expressing DC targets, a dysfunctional CTL:DC cross-talk occurs. As a result, these DC become activated and programmed to differentiate into proinflammatory cells displaying unique features that contribute to viral dissemination and persistence.2 We have found that CTL-activated DC are capable of producing high levels of proinflammatory cytokines such as interleukin (IL)-12 and IL-6, but also simultaneously express some immunosuppressive factors such as IL-10 and PD-L1, which may contribute to their resistance to direct cellular attack. They are also programmed to produce a wide range of chemokines, including CCL4, CCL5, CXCL9, and CXCL10, that can preferentially attract activated CD4+ T helper (Th) cells, the main target of HIV-1 infection. Moreover, the production of CCL19 by these DC also promotes their interaction with CCR7+/CD45RA+ naive Th cells as well as CCR7+/CD45RO+ central memory Th cells, the major cellular reservoir of latent HIV-1.3 During this secondary interaction with Th cells, these CTL-programmed DC uniquely undergo fantastic morphological changes, characterized by their rapid development of numerous, far-reaching tunneling nanotube-like membrane protrusions. This intriguing phenomenon ultimately leads to the formation of complex nanotube networks, which facilitate the direct transfer of both cell surface and cytoplasmic components between interconnected neighboring and distant cells. Importantly, these membrane bridges can also serve as highways for HIV-1 to utilize for efficient cell-to-cell transfer (Fig. 1). FIG. 1. Live-cell differential interference contrast image of eukaryotic green fluorescent protein (EGFP)-expressing HIV-1-like particles trafficking via dendritic cell (DC) membrane bridges. Our findings add a new dimension to the proposed concept of “original antigenic sin”4 in which the promiscuous nature of preexisting CTL can be exploited by an evolving virus to modulate the functional and physical characteristics of the DC, and to create an inflammatory environment suitable for viral spread and persistence within the host. These findings also highlight the need to consider the detrimental potential of selectively activating dysfunctional memory CTL responses when implementing anti-HIV-1 vaccine strategies.

Intracellular trafficking of AIP56, an NF-κB-cleaving toxin from Photobacterium damselae subsp. piscicida.

AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-κB. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-κB p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway.

Lactobacillus gasseri SBT2055 reduces infection by and colonization of Campylobacter jejuni.

Campylobacter is a normal inhabitant of the chicken gut. Pathogenic infection with this organism in humans is accompanied by severe inflammation of the intestinal mucosal surface. The aim of this study was to evaluate the ability of Lactobacillus gasseri SBT2055 (LG2055) to inhibit the adhesion and invasion of Campylobacter jejuni in vitro and to suppress C. jejuni colonization of chicks in vivo. Pretreatment with LG2055 significantly reduced adhesion to and invasion of a human epithelial cell line, Intestine 407, by C. jejuni 81–176. Methanol (MeOH)-fixed LG2055 also reduced infection by C. jejuni 81–176. However, proteinase K (ProK)-treated LG2055 eliminated the inhibitory effects. Moreover, LG2055 co-aggregated with C. jejuni 81–176. ProK treatment prevented this co-aggregation, indicating that the co-aggregation phenotype mediated by the proteinaceous cell-surface components of LG2055 is important for reducing C. jejuni 81–176 adhesion and invasion. In an in vivo assay, oral doses of LG2055 were administered to chicks daily for 14 days after oral inoculation with C. jejuni 81–176. At 14 days post-inoculation, chicks treated with LG2055 had significantly reduced cecum colonization by C. jejuni. Reduction in the number of C. jejuni 81–176 cells adhering to and internalized by human epithelial cells demonstrated that LG2055 is an organism that effectively and competitively excludes C. jejuni 81–176. In addition, the results of the chick colonization assay suggest that treatment with LG2055 could be useful in suppressing C. jejuni colonization of the chicks at early growth stages.

A rapid live-cell ELISA for characterizing antibodies against cell surface antigens of Chlamydomonas reinhardtii and its use in isolating algae from natural environments with related cell wall components.

BackgroundCell walls are essential for most bacteria, archaea, fungi, algae and land plants to provide shape, structural integrity and protection from numerous biotic and abiotic environmental factors. In the case of eukaryotic algae, relatively little is known of the composition, structure or mechanisms of assembly of cell walls in individual species or between species and how these differences enable algae to inhabit a great diversity of environments. In this paper we describe the use of camelid antibody fragments (VHHs) and a streamlined ELISA assay as powerful new tools for obtaining mono-specific reagents for detecting individual algal cell wall components and for isolating algae that share a particular cell surface component.ResultsTo develop new microalgal bioprospecting tools to aid in the search of environmental samples for algae that share similar cell wall and cell surface components, we have produced single-chain camelid antibodies raised against cell surface components of the single-cell alga, Chlamydomonas reinhardtii. We have cloned the variable-region domains (VHHs) from the camelid heavy-chain-only antibodies and overproduced tagged versions of these monoclonal-like antibodies in E. coli. Using these VHHs, we have developed an accurate, facile, low cost ELISA that uses live cells as a source of antigens in their native conformation and that requires less than 90 minutes to perform. This ELISA technique was demonstrated to be as accurate as standard ELISAs that employ proteins from cell lysates and that generally require >24 hours to complete. Among the cloned VHHs, VHH B11, exhibited the highest affinity (EC50 < 1 nM) for the C. reinhardtii cell surface. The live-cell ELISA procedure was employed to detect algae sharing cell surface components with C. reinhardtii in water samples from natural environments. In addition, mCherry-tagged VHH B11 was used along with fluorescence activated cell sorting (FACS) to select individual axenic isolates of presumed wild relatives of C. reinhardtii and other Chlorphyceae from the same environmental samples.ConclusionsCamelid antibody VHH domains provide a highly specific tool for detection of individual cell wall components of algae and for allowing the selection of algae that share a particular cell surface molecule from diverse ecosystems.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0244-0) contains supplementary material, which is available to authorized users.

Electron microscopy methods for studying plasma membranes.

Electron microscopy allows direct visualization of the underlying organization of cell surface components on a nano-scale. Immuno-gold labelling of isolated plasma membranes generates point patterns that enable mapping of protein and lipid distributions. 2D spatial statistics reveals the extent to which these distributions are clustered or dispersed and allows the extent of co-localization between different cell surface components to be precisely determined. This approach has been successfully applied to the study of signalling network organization and the consequences of physiological changes in modulating cell surface function.

Endometrial apical glycoproteomic analysis reveals roles for cadherin 6, desmoglein-2 and plexin b2 in epithelial integrity.

Implantation failure is one of the major causes of infertility and remains a major barrier to assisted reproduction success. Initial receptivity to implantation is regulated by the endometrial luminal epithelium under maternal hormonal control. Identification of epithelial cell surface components involved in embryo attachment will have translational applications in early pregnancy failure, infertility and contraception. In this study, vectorial biotinylation has been used to characterize the apical glycoproteome of Ishikawa cells, a polarized cell line that serves as a model of the implantation-receptive human endometrial luminal epithelium. Of 46 surface-associated glycoproteins detected by mass spectrometry, half are newly reported in this cell type; a subgroup of these were chosen for evaluation in tissue, and all were shown to be expressed apically in vivo in the mid-secretory (implantation) phase of the menstrual cycle, thus validating the model. Eleven adhesion molecules were detected, some already known to be involved in implantation, others novel. Cadherin 6, desmoglein 2 and plexin b2 were surprisingly found in the apical as well as the lateral membrane domain; their knock-down compromised epithelial integrity. This method of targeting glycosylated apical surface moieties in a polarized epithelial culture model shows excellent selectivity and identifies candidate cell adhesion molecules that are also present in vivo in secretory phase endometrial epithelium.

The effect of cell surface components on adhesion ability of Lactobacillus rhamnosus.

The aim of this study was to analyze the cell envelope components and surface properties of two phenotypes of Lactobacillus rhamnosus isolated from the human gastrointestinal tract. The ability of the bacteria to adhere to human intestinal cells and to aggregate with other bacteria was determined. L. rhamnosus strains E/N and PEN differed with regard to the presence of exopolysaccharides (EPS) and specific surface proteins. Transmission electron microscopy showed differences in the structure of the outer cell surface of the strains tested. Bacterial surface properties were analyzed by Fourier transform infrared spectroscopy, fatty acid methyl esters and hydrophobicity assays. Aggregation capacity and adhesion of the tested strains to the human colon adenocarcinoma cell line HT29 was determined. The results indicated a high adhesion and aggregation ability of L. rhamnosus PEN, which possessed specific surface proteins, had a unique fatty acid content, and did not synthesize EPS. Adherence of L. rhamnosus was dependent on specific interactions and was promoted by surface proteins (42–114 kDa) and specific fatty acids. Polysaccharides likely hindered bacterial adhesion and aggregation by masking protein receptors. This study provides information on the cell envelope constituents of lactobacilli that influence bacterial aggregation and adhesion to intestinal cells. This knowledge will help to understand better their specific contribution in commensal–host interactions and adaptation to this ecological niche.

Well-defined biomimetic surfaces to characterize glycosaminoglycan-mediated interactions on the molecular, supramolecular and cellular levels

Glycosaminoglycans (GAGs) are ubiquitously present at the cell surface and in extracellular matrix, and crucial for matrix assembly, cell–cell and cell-matrix interactions. The supramolecular presentation of GAG chains, along with other matrix components, is likely to be functionally important but remains challenging to control and to characterize, both in vivo and in vitro. We present a method to create well-defined biomimetic surfaces that display GAGs, either alone or together with other cell ligands, in a background that suppresses non-specific binding. Through the design of the immobilization platform – a streptavidin monolayer serves as a molecular breadboard for the attachment of various biotinylated ligands – and a set of surface-sensitive in situ analysis techniques (including quartz crystal microbalance and spectroscopic ellipsometry), the biomimetic surfaces are tailor made with tight control on biomolecular orientation, surface density and lateral mobility. Analysing the interactions between a selected GAG (heparan sulphate, HS) and the HS-binding chemokine CXCL12α (also called SDF-1α), we demonstrate that these surfaces are versatile for biomolecular and cellular interaction studies. T-lymphocytes are found to adhere specifically to surfaces presenting CXCL12α, both when reversibly bound through HS and when irreversibly immobilized on the inert surface, even in the absence of any bona fide cell adhesion ligand. Moreover, surfaces which present both HS-bound CXCL12α and the intercellular adhesion molecule 1 (ICAM-1) synergistically promote cell adhesion. Our surface biofunctionalization strategy should be broadly applicable for functional studies that require a well-defined supramolecular presentation of GAGs along with other matrix or cell-surface components.

A multi-approach study of influence of growth temperature and nutrient deprivation in a strain of Aeromonas hydrophila

In the present study we investigated the behavior of an Aeromonas hydrophila strain in prolonged nutrient deprivation condition analyzing the possible link among survival, cell morphology and adhesive characteristics and correlating them with the expression of the 43kDa outer membrane protein (OMP). The strain was inoculated in mineral and drinking chlorinated water, and in Nutrient Broth as a control with incubation at 4 and 24°C for 176days. Specimens were analyzed at different times during starvation stress. Viability was assessed by flow cytometry and growth by plate count technique; morphology and adhesivity were detected by optical and electron microscopy. The 43kDa OMP expression at different times was determined after immunoblotting assay using a polyclonal antibody produced in rabbit. The results showed a long-term viability as evidenced by cytofluorimetric analysis; however, the prolonged starvation led to the shift from the normal rod shaped cells to spherical forms in the last phases of incubation especially at 24°C. Concomitantly with the appearance of spherical cells we noted a reduction of the 43kDa OMP content and adhesive ability. Therefore, our results suggest a role of the 43kDa OMP as adhesin in A. hydrophila. In conclusion, we demonstrated that the bacterium can long survive under stress conditions, however adopting strategies which can lead to a loss of some cell surface components involved in the interactions with eukaryotic cells, therefore modifying its virulence properties.

Label-free in vitro visualization and characterization of caveolar bulbs during stimulated re-epithelialization.

Tip-enhanced Raman scattering (TERS) was paired with real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) to characterize lipid aggregates during stimulated re-epithelialization using an in vitro wound healing model. In this study, lipid fluctuations in the plasma membrane of epidermal keratinocytes were studied at multiple time points post-wounding. TERS measurements for the first time were also combined with sample analysis after initial wounding and 24 h of wound healing. This enabled simultaneous visualization and characterization of caveolar bulb distribution during wound healing stages, providing noninvasive insight into their associated lipid structure and coating protein, caveolin, in the nanometer range. The combination of Raman spectroscopy and scanning probe microscopy in TERS gives access to topographic and chemical structure information in a single experiment. It is the intrinsic specificity and sensitivity of TERS that enable this discrete detection of cell surface components on the nanometer scale. In contrast with competing biochemical methods, the applied technique does not interfere with the cellular composition, enabling lipid structure analysis without digestion or detergents, and displayed great potential for future biological in vivo studies.