Fibronectin–collagen binding and requirement during cellular adhesion

Fibronectin isolated from human plasma and from the extracellular matrices of cell monolayers mediates the attachment in vitro and spreading of trypsin-treated cells on a collagen substratum. Fibronectin-dependent kinetics of cellular attachment to collagen were studied for several adherent cell types. It was shown that trypsin-treated human umbilical-cord cells, mouse sarcoma CMT81 cells, endothelial cells, and human fibroblasts from a patient with Glanzmann's disease were completely dependent on fibronectin for their attachment to collagen, whereas guinea-pig and monkey smooth-muscle cells and chick-embryo secondary fibroblasts displayed varying degrees of dependence on fibronectin for their attachment. Radiolabelled human plasma fibronectin possessed similar affinity for collagen types I, II and III from a variety of sources. The fibronectin bound equally well to the collagens with or without prior urea treatment. However, in the fibronectin-mediated adhesion assay using PyBHK fibroblasts, a greater number of cells adhered and more spreading was observed on urea-treated collagen. Fibronectin extracted from the extracellular matrix of chick-embryo fibroblasts and that purified from human plasma demonstrated very similar kinetics of complexing to collagencoated tissue-culture dishes. Fibronectin from both sources bound to collagen in the presence of 0.05–4.0m-NaCl and over the pH range 2.6–10.6. The binding was inhibited when fibronectin was incubated with 40–80% ethylene glycol, the ionic detergents sodium dodecyl sulphate and deoxycholate, and the non-ionic detergents Nonidet P-40, Tween 80 and Triton X-100, all at a concentration of 0.1%. From these results we proposed that fibronectin–collagen complexing is mainly attributable to hydrophobic interactions.     

Endothelial cell–cell adhesion during zebrafish vascular development

The vertebrate vasculature is an essential organ network with major roles in health and disease. The establishment of balanced cell–cell adhesion in the endothelium is crucial for the functionality of the vascular system. Furthermore, the correct patterning and integration of vascular endothelial cell–cell adhesion drives the morphogenesis of new vessels, and is thought to couple physical forces with signaling outcomes during development. Here, we review insights into this process that have come from studies in zebrafish. First, we describe mutants in which endothelial adhesion is perturbed, second we describe recent progress using in vivo cell biological approaches that allow the visualization of endothelial cell–cell junctions. These studies underline the profound potential of this model system to dissect in great detail the function of both known and novel regulators of endothelial cell–cell adhesion.     

Fibronectin fibril pattern displays the force balance of cell–matrix adhesion

Formation of fibrillar patterns of fibronectin on polymer substrates with gradated physicochemical surface properties was analysed during early stages of endothelial cell adhesion. Fibronectin was pre-adsorbed onto three maleic anhydride copolymer thin films with distinct differences in the protein adsorption strength as verified by heteroexchange experiments. The evolved micrometer scale fibrillar patterns of fibronectin on the compared polymer surfaces were characterized after 50 min of cellular reorganization by an auto-correlation analysis using fluorescence microscopy data. Statistical analysis revealed a decrease of the typical spacings of the fibronectin fibrils from 2.6 to 1.8 m with decreasing fibronectin adsorption strength to the substrate. Size and density of focal adhesions correlated with this dependence of the fibronectin fibril pattern. From these data a model was developed relating the fibronectin fibril pattern to the fibronectin-substrate adsorption strength through the cytoskeletal force regulation mechanism of the cell.     

Modeling of cell adhesion and deformation mediated by receptor–ligand interactions

The current work is devoted to studying adhesion and deformation of biological cells mediated by receptors and ligands in order to enhance the existing models. Due to the sufficient in-plane continuity and fluidity of the phospholipid molecules, an isotropic continuum fluid membrane is proposed for modeling the cell membrane. The developed constitutive model accounts for the influence of the presence of receptors on the deformation and adhesion of the cell membrane through the introduction of spontaneous area dilation. Motivated by physics, a nonlinear receptor–ligand binding force is introduced based on charge-induced dipole interaction. Diffusion of the receptors on the membrane is governed by the receptor–ligand interaction via Fick’s Law and receptor-ligand interaction. The developed model is then applied to study the deformation and adhesion of a biological cell. The proposed model is used to study the role of the material, binding, spontaneous area dilation and environmental properties on the deformation and adhesion of the cell.     

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion

Background

Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated proline–glycine–proline (N-ac-PGP). In the current study, we investigate whether N-ac-PGP influences β₂-integrin activation and function in neutrophilic firm adhesion to endothelium.

Methods

Human polymorphonuclear leukocytes (PMNs) were isolated from fresh human blood. Subsequently, a transmigration assay was performed to evaluate the active migration of PMNs towards N-ac-PGP. Furthermore, the effect of the tripeptide on β₂-integrin activation was assessed by performing the adhesion assay using fibrinogen as a ligand. To determine whether this effect was due to conformational change of β₂-integrins, antibodies against CD11b and CD18 were used in the adhesion assay and the expression pattern of CD11b was determined.

Results

Human neutrophils transmigrated through an endothelial cell layer in response to basolateral N-ac-PGP. N-ac-PGP induced also a neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that CD11b/CD18 (Mac-1) was responsible for the N-ac-PGP-induced firm adhesion of neutrophils to fibrinogen. Pertussis toxin decreased the Mac-1 activation indicating the involvement of G-proteins. N-ac-PGP most likely activated Mac-1 by initiating a conformational change, since the expression pattern of Mac-1 on the cell surface did not change significantly.

Conclusions

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion.

General significance

This is the first study to describe that the chemo-attractant N-ac-PGP also activates Mac-1 on the surface of neutrophils, which can additionally contribute to neutrophilic transmigration into the lung tissue during lung inflammation.     

The frequency and duration of Salmonella–macrophage adhesion events determines infection efficiency

Salmonella enterica causes a range of important diseases in humans and a in a variety of animal species. The ability of bacteria to adhere to, invade and survive within host cells plays an important role in the pathogenesis of Salmonella infections. In systemic salmonellosis, macrophages constitute a niche for the proliferation of bacteria within the host organism. Salmonella enterica serovar Typhimurium is flagellated and the frequency with which this bacterium collides with a cell is important for infection efficiency. We investigated how bacterial motility affects infection efficiency, using a combination of population-level macrophage infection experiments and direct imaging of single-cell infection events, comparing wild-type and motility mutants. Non-motile and aflagellate bacterial strains, in contrast to wild-type bacteria, collide less frequently with macrophages, are in contact with the cell for less time and infect less frequently. Run-biased Salmonella also collide less frequently with macrophages but maintain contact with macrophages for a longer period of time than wild-type strains and infect the cells more readily. Our results suggest that uptake of S. Typhimurium by macrophages is dependent upon the duration of contact time of the bacterium with the cell, in addition to the frequency with which the bacteria collide with the cell.     

Effects of curvature and cell–cell interaction on cell adhesion in microvessels

It has been found that both circulating blood cells and tumor cells are more easily adherent to curved microvessels than straight ones. This motivated us to investigate numerically the effect of the curvature of the curved vessel on cell adhesion. In this study, the fluid dynamics was carried out by the lattice Boltzmann method (LBM), and the cell dynamics was governed by the Newton’s law of translation and rotation. The adhesive dynamics model involved the effect of receptor-ligand bonds between circulating cells and endothelial cells (ECs). It is found that the curved vessel would increase the simultaneous bond number, and the probability of cell adhesion is increased consequently. The interaction between traveling cells would also affect the cell adhesion significantly. For two-cell case, the simultaneous bond number of the rear cell is increased significantly, and the curvature of microvessel further enhances the probability of cell adhesion.     

Host–pathogen interactions: Impenetrable barriers or entry portals? The role of cell–cell adhesion during infection

Cell-cell adhesion plays a fundamental role in cell polarity and organogenesis. It also contributes to the formation and establishment of physical barriers against microbial infections. However, a large number of pathogens, from viruses to bacteria and parasites, have developed countless strategies to specifically target cell adhesion molecules in order to adhere to and invade epithelial cells, disrupt epithelial integrity, and access deeper tissues for dissemination. The study of all these processes has contributed to the characterization of molecular machineries at the junctions of eukaryotic cells that have been better understood by using pathogens as probes.     

An Elmo–Dock complex locally controls Rho GTPases and actin remodeling during cadherin-mediated adhesion

Cell–cell contact formation is a dynamic process requiring the coordination of cadherin-based cell–cell adhesion and integrin-based cell migration. A genome-wide RNA interference screen for proteins required specifically for cadherin-dependent cell–cell adhesion identified an Elmo–Dock complex. This was unexpected as Elmo–Dock complexes act downstream of integrin signaling as Rac guanine-nucleotide exchange factors. In this paper, we show that Elmo2 recruits Dock1 to initial cell–cell contacts in Madin–Darby canine kidney cells. At cell–cell contacts, both Elmo2 and Dock1 are essential for the rapid recruitment and spreading of E-cadherin, actin reorganization, localized Rac and Rho GTPase activities, and the development of strong cell–cell adhesion. Upon completion of cell–cell adhesion, Elmo2 and Dock1 no longer localize to cell–cell contacts and are not required subsequently for the maintenance of cell–cell adhesion. These studies show that Elmo–Dock complexes are involved in both integrin- and cadherin-based adhesions, which may help to coordinate the transition of cells from migration to strong cell–cell adhesion.     

A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell–cell adhesion

Cadherins and associated catenins provide an important structural interface between neighboring cells, the actin cytoskeleton, and intracellular signaling pathways in a variety of cell types throughout the Metazoa. However, the full inventory of the proteins and pathways required for cadherin-mediated adhesion has not been established. To this end, we completed a genome-wide (∼14,000 genes) ribonucleic acid interference (RNAi) screen that targeted Ca²⁺-dependent adhesion in DE-cadherin–expressing Drosophila melanogaster S2 cells in suspension culture. This novel screen eliminated Ca²⁺-independent cell–cell adhesion, integrin-based adhesion, cell spreading, and cell migration. We identified 17 interconnected regulatory hubs, based on protein functions and protein–protein interactions that regulate the levels of the core cadherin–catenin complex and coordinate cadherin-mediated cell–cell adhesion. Representative proteins from these hubs were analyzed further in Drosophila oogenesis, using targeted germline RNAi, and adhesion was analyzed in Madin–Darby canine kidney mammalian epithelial cell–cell adhesion. These experiments reveal roles for a diversity of cellular pathways that are required for cadherin function in Metazoa, including cytoskeleton organization, cell–substrate interactions, and nuclear and cytoplasmic signaling.     

Down-regulation of Pkd2 by siRNAs suppresses cell–cell adhesion in the mouse melanoma cells

The Pkd2 gene encodes an integral protein (~130 kDa), named polycystin-2 (PC-2). PC-2 is mainly involved in autosomal dominant polycystic kidney disease. Recently, polycystin-1/polycystin-2 complex has been shown to act as an adhesion complex mediating or regulating cell–cell or cell–matrix adhesion, suggesting that PC-2 may play a role in cell–cell/cell–matrix interactions. Here, we knocked down the expression of Pkd2 gene with small interfering RNAs (siRNAs) in the mouse melanoma cells (B16 cells), indicating that the cells transfected with the targeted siRNAs significantly suppressed cell–cell adhesion, but not cell–matrix adhesion, compared to the cells transfected with non-targeted control (NC) siRNA. This study provides the first directly functional evidence that PC-2 mediates cell–cell adhesion. Furthermore, we demonstrated that PC-2 modulated cell–cell adhesion may be, at least partially, associated with E-cadherin. Collectively, these findings for the first time showed that PC-2 may mediate cell–cell adhesion, at least partially, through E-cadherin.     

Bioactivity of nitrolinoleate: effects on adhesion molecules and CD40–CD40L system

The vascular effects of nitrolinoleate (LNO₂), an endogenous product of linoleic acid (LA) nitration by nitric oxide-derived species and a potential nitrosating agent, were investigated on rat endothelial-leukocyte interactions. Confocal microscopy analysis demonstrated that LNO₂ was capable to deliver free radical nitric oxide (^·NO) into cells, 5 min after its administration to cultured cells, with a peak of liberation at 30 min. THP-1 monocytes incubated with LNO₂ for 5 min presented nitrosation of CD40, leading to its inactivation. Other anti-inflammatory actions of LNO₂ were observed in vivo by intravital microscopy assays. LNO₂ decreased the number of adhered leukocytes in postcapillary venules of the mesentery network. In addition to this, LNO₂ reduced mRNA and protein expression of β2-integrin in circulating leukocytes, as well as VCAM-1 in endothelial cells isolated from postcapillary venules, confirming its antiadhesive effects on both cell types. Moreover, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, partially abolished the inhibitory action of LNO₂ on leukocyte-endothelium interaction, suggesting that the antiadhesion effects of LNO₂ involve a dual role in leukocyte adhesion, acting as a nitric oxide donor as well as through nitric oxide-independent mechanisms. In conclusion, LNO₂ inhibited adhesion molecules expression and promoted ^·NO inactivation of the CD40–CD40L system, both important processes of the inflammatory response.     

XLMR protein related to neurite extension (Xpn/KIAA2022) regulates cell–cell and cell–matrix adhesion and migration

X-linked mental retardation (XLMR) is a common cause of moderate to severe intellectual disability in males. XLMR protein related to neurite extension (Xpn, also known as KIAA2022) has been implicated as a gene responsible for XLMR in humans. Although Xpn is highly expressed in the developing brain and is involved in neurite outgrowth in PC12 cells and neurons, little is known about the functional role of Xpn. Here, we show that Xpn regulates cell–cell and cell–matrix adhesion and migration in PC12 cells. Xpn knockdown enhanced cell–cell and cell–matrix adhesion mediated by N-cadherin and β1-integrin, respectively. N-Cadherin and β1-integrin expression at the mRNA and protein levels was significantly increased in Xpn knockdown PC12 cells. Furthermore, overexpressed Xpn protein was strongly expressed in the nuclei of PC12 and 293T cells. Finally, depletion of Xpn perturbed cellular migration by enhancing N-cadherin and β1-integrin expression in a PC12 cell wound healing assay. We conclude that Xpn regulates cell–cell and cell–matrix adhesion and cellular migration by regulating the expression of adhesion molecules.     

Interleukin-1β enhances cell adhesion in human endothelial cells via microRNA-1914–5p suppression

Atherosclerosis is a chronic inflammatory disease and the underlying cause of most cardiovascular diseases. Interleukin (IL)-1β facilitates early atherogenic lesion formation by increasing monocyte adhesion to endothelial cells via upregulation of adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1). MicroRNAs (miRNAs) have been shown to be associated with inflammatory conditions in the vascular system. The expression of circulating miR-1914–5p is reportedly downregulated in patients with cardiovascular diseases. However, the role of miR-1914–5p downregulation in IL-1β–induced endothelial cell dysfunction and the effect of miR-1914–5p on lesion formation remain unclear. Therefore, we investigated whether miR-1914–5p is associated with monocyte adhesion in human endothelial cells. IL-1β decreased miR-1914–5p expression in EA.hy926 cells. In addition, miR-1914–5p depletion enhanced ICAM-1 expression and monocyte adhesion in EA.hy926 cells. Moreover, miR-1914–5p mimic suppressed monocyte adhesion and ICAM-1 expression induced by IL-1β in endothelial cells. These results suggest that suppression of miR-1914–5p expression by IL-1β may be an important regulator in mediating monocyte adhesion in endothelial cells. Further investigation of miR-1914–5p may lead to the development of novel therapeutic strategies for atherosclerosis.     

α-Tubulin K40 acetylation is required for contact inhibition of proliferation and cell–substrate adhesion

Acetylation of α-tubulin on lysine 40 marks long-lived microtubules in structures such as axons and cilia, and yet the physiological role of α-tubulin K40 acetylation is elusive. Although genetic ablation of the α-tubulin K40 acetyltransferase αTat1 in mice did not lead to detectable phenotypes in the developing animals, contact inhibition of proliferation and cell–substrate adhesion were significantly compromised in cultured αTat1^−/− fibroblasts. First, αTat1^−/− fibroblasts kept proliferating beyond the confluent monolayer stage. Congruently, αTat1^−/− cells failed to activate Hippo signaling in response to increased cell density, and the microtubule association of the Hippo regulator Merlin was disrupted. Second, αTat1^−/− cells contained very few focal adhesions, and their ability to adhere to growth surfaces was greatly impaired. Whereas the catalytic activity of αTAT1 was dispensable for monolayer formation, it was necessary for cell adhesion and restrained cell proliferation and activation of the Hippo pathway at elevated cell density. Because α-tubulin K40 acetylation is largely eliminated by deletion of αTAT1, we propose that acetylated microtubules regulate contact inhibition of proliferation through the Hippo pathway.     

Candida biofilms: is adhesion sexy?

The development of Candida albicans biofilms requires two types of adhesion molecule - the Als proteins and Hwp1. Mutational analyses have recently revealed that these molecules play complementary roles, and their characteristics suggest that they may have evolved from primitive mating agglutinins.