A comparative molecular force spectroscopy study of homophilic JAM-A interactions and JAM-A interactions with reovirus attachment protein sigma1

JAM-A belongs to a family of immunoglobulin-like proteins called junctional adhesion molecules (JAMs) that localize at epithelial and endothelial intercellular tight junctions. JAM-A is also expressed on dendritic cells, neutrophils, and platelets. Homophilic JAM-A interactions play an important role in regulating paracellular permeability and leukocyte transmigration across epithelial monolayers and endothelial cell junctions, respectively. In addition, JAM-A is a receptor for the reovirus attachment protein, sigma1. In this study, we used single molecular force spectroscopy to compare the kinetics of JAM-A interactions with itself and sigma1. A chimeric murine JAM-A/Fc fusion protein and the purified sigma1 head domain were used to probe murine L929 cells, which express JAM-A and are susceptible to reovirus infection. The bond half-life (t(1/2)) of homophilic JAM-A interactions was found to be shorter (k(off)(o) = 0.688 +/- 0.349 s(-1)) than that of sigma1/JAM-A interactions (k(off)(o) = 0.067 +/- 0.041 s(-1)). These results are in accordance with the physiological functions of JAM-A and sigma1. A short bond lifetime imparts a highly dynamic nature to homophilic JAM-A interactions for regulating tight junction permeability while stable interactions between sigma1 and JAM-A likely anchor the virus to the cell surface and facilitate viral entry.     

EndoCAM: a novel endothelial cell-cell adhesion molecule

Cell-cell adhesion is controlled by many molecules found on the cell surface. In addition to the constituents of well-defined junctional structures, there are the molecules that are thought to play a role in the initial interactions of cells and that appear at precise times during development. These include the cadherins and cell adhesion molecules (CAMs). Representatives of these families of adhesion molecules have been isolated from most of the major tissues. The notable exception is the vascular endothelium. Here we report the identification of a cell surface molecule designated "endoCAM" (endothelial Cell Adhesion Molecule), which may function as an endothelial cell-cell adhesion molecule. EndoCAM is a 130-kD glycoprotein expressed on the surface of endothelial cells both in culture and in situ. It is localized to the borders of contiguous endothelial cells. It is also present on platelets and white blood cells. Antibodies against endoCAM prevent the initial formation of endothelial cell-cell contacts. Despite similarities in size and intercellular location, endoCAM does not appear to be a member of the cadherin family of adhesion receptors. The serologic and protease susceptibility characteristics of endoCAM are different from those of the known cadherins, including an endogenous endothelial cadherin. Although the precise biologic function of endoCAM has not been determined, it appears to be one of the molecules responsible for regulating endothelial cell-cell adhesion processes and may be involved in platelet and white blood cell interactions with the endothelium.     

The JAM family of junctional adhesion molecules

Junctional adhesion molecules are a family of glycoproteins characterised by two immunoglobulin folds (VH- and C2-type) in the extracellular domain. Junctional adhesion molecule proteins localise to intercellular junctions of polarised endothelial and epithelial cells but can also be expressed on circulating leukocytes and platelets. In addition, they bind several ligands, in both a homophilic and heterophilic manner, and associate with several cytoplasmic partners. All these features represent the likely determinants for the role of junctional adhesion molecule proteins in processes as diverse as junction assembly, leukocyte transmigration and platelet activation.     

Evaluation of intermolecular forces in a circulating system

Intercellular interactions, which are mediated by a variety of complex intercellular molecules through the processes of formation and dissociation of molecular bonds, play a critical role in regulating cellular functions in biological systems. Various approaches are applied to evaluate intercellular or molecular bonding forces. To quantify the intermolecular interaction forces, flow chamber has become a meaningful technique as it can ultimately mimic the cellular microenvironment in vivo under physiological flow conditions. Hydrodynamic forces are usually used to predict the intercellular forces down to the single molecular level. However, results show that only using hydrodynamic force will overestimate up to 30% of the receptor-ligand strength when the non-specific forces such as Derjaguin-Landau-Verway-Overbeek (DLVO) forces become un-neglected. Due to the nature of high ion concentration in the physiological condition, electrostatic force is largely screened which will cause DLVO force unbalanced. In this study, we propose to take account of the DLVO force, including van der Waals (VDW) force and electrostatic force, to predict the intermolecular forces of a cell doublet and cell-substrate model in a circulating system. Results also show that the DLVO force has a nonlinear effect as the cell-cell or cell-substrate distance changes. In addition, we used the framework of high accuracy hydrodynamic theories proved in colloidal systems. It is concluded that DLVO force could not be ignored in quantitative studies of molecular interaction forces in circulating system. More accurate prediction of intercellular forces needs to take account of both hydrodynamic force and DLVO force.     

1H, 13C and 15N chemical shift assignments of Ninjurin1 Extracellular N-terminal Domain

Cell adhesion molecules play a crucial role in fundamental biological processes via regulating cell–cell interactions. Nerve injury induced protein1 (Ninjurin1) is a novel adhesion protein that has no significant homology with other known cell adhesion molecules. Here we present the assignment of an 81 aa construct for human Ninjurin1 Extracellular N-Terminal (ENT) domain, which comprises the critical adhesion domain.     

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH₃-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.     

Force spectroscopy studies on protein–ligand interactions: A single protein mechanics perspective

Protein–ligand interactions are ubiquitous and play important roles in almost every biological process. The direct elucidation of the thermodynamic, structural and functional consequences of protein–ligand interactions is thus of critical importance to decipher the mechanism underlying these biological processes. A toolbox containing a variety of powerful techniques has been developed to quantitatively study protein–ligand interactions in vitro as well as in living systems. The development of atomic force microscopy-based single molecule force spectroscopy techniques has expanded this toolbox and made it possible to directly probe the mechanical consequence of ligand binding on proteins. Many recent experiments have revealed how ligand binding affects the mechanical stability and mechanical unfolding dynamics of proteins, and provided mechanistic understanding on these effects. The enhancement effect of mechanical stability by ligand binding has been used to help tune the mechanical stability of proteins in a rational manner and develop novel functional binding assays for protein–ligand interactions. Single molecule force spectroscopy studies have started to shed new lights on the structural and functional consequence of ligand binding on proteins that bear force under their biological settings.     

Antibody against the Leu-CAM beta-chain (CD18) promotes both LFA-1- and CR3-dependent adhesion events.

The Leukocytic cell-adhesion molecule (beta 2 integrin) family of adhesion molecules play a key role in the intercellular adhesive interactions necessary for normal immune cell function. In this study, we report an antibody that recognizes an epitope on the Leukocytic cell-adhesion molecule common beta-chain (CD18) and promotes both lymphocyte function-associated Ag-1- and CR3-dependent adhesion events. The antibody recognizes a temperature-sensitive epitope that is not dependent on the presence of divalent cations. It is proposed that antibody binding promotes a conformational change in both lymphocyte function-associated Ag-1 and CR3, which may mimic a natural activation mechanism, resulting in increased cellular adhesion.     

Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy

Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.     

Plasma-membrane-Bound mcromoleculas are dynamically aggregated to form non-random codistribution patterns of selected functional elements. Do pattern recognition processes govern antigen presentation and intercellular interactions?

Molecular recognition processes between cell surface elements are discussed with special reference to cell surface pattern formation of membrane-bound integral proteins. The existence, as detected by flow cytometric resonance energy transfer (Appendix), and significance of cell surface patterns involving the interleukin-2 receptor, the T-cell receptor–CD3 system, the intercellular adhesion molecule ICAM-1, and the major histocompatilibilty complex class I and II molecules in the plasma membrane of lymphocytes are described. The modulation of antigen presentation by transmembrane potential changes is discussed, and a general role of transmembrane potential changes, and therefore of icon channel activities, adduced as one of the major regulatory mechanisms of cell–cell communications. A general role in the mediation and regulation of intercellular interactions is suggested for cell-surface macromolecular patterns. The dynamic pattern of protein and lipid molecules in the plasma membrane is generated by the genetic code, but has a remarkable flexibility and may be one of the major instruments of accomodation and recognition processes at the cellular level.     

UNDERSTANDING INTERCELLULAR INTERACTIONS AND CELL ADHESION: LESSONS FROM STUDIES ON PROTEIN—METAL INTERACTIONS

To understand cell—cell interactions and the interactions of cells to non-biological materials, studies on binding forces between cellular proteins and between proteins and non-biological material such as metal surfaces are essential. The adsorption of proteins to solid—water interfaces is a multifactorial and a multistep process. First steps are determined by long-range interactions where surface properties such as hydrophobicity, distribution of charged groups, ion concentrations and pH play important roles. In later steps structural rearrangements in the protein molecule and dehydration effects become more important making the adsorption process often irreversible. In the following we demonstrate that protein A and tubulin have a specific type of interaction to metal surfaces probably as an intermediate step in the adsorption process. The proteins were attached to the tip of a microfabricated cantilever in such a way that only one molecule interacts with the surface. By recording force—distance curves with an atomic force microscope the adhesion forces of single molecules binding to gold, titanium and indium—tinoxid surfaces were measured.     

Direct measurement of single-molecule visco-elasticity in atomic force microscope force-extension experiments

Measuring the visco-elastic properties of biological macromolecules constitutes an important step towards the understanding of dynamic biological processes, such as cell adhesion, muscle function, or plant cell wall stability. Force spectroscopy techniques based on the atomic force microscope (AFM) are increasingly used to study the complex visco-elastic response of (bio-)molecules on a single-molecule level. These experiments either require that the AFM cantilever is actively oscillated or that the molecule is clamped at constant force to monitor thermal cantilever motion. Here we demonstrate that the visco-elasticity of single bio-molecules can readily be extracted from the Brownian cantilever motion during conventional force-extension measurements. It is shown that the characteristics of the cantilever determine the signal-to-noise (S/N) ratio and time resolution. Using a small cantilever, the visco-elastic properties of single dextran molecules were resolved with a time resolution of 8.3 ms. The presented approach can be directly applied to probe the dynamic response of complex bio-molecular systems or proteins in force-extension experiments. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Adhesive and Signaling Functions of Cadherins and Catenins in Vertebrate Development

Properly regulated intercellular adhesion is critical for normal development of all metazoan organisms. Adherens junctions play an especially prominent role in development because they link the adhesive function of cadherin–catenin protein complexes to the dynamic forces of the actin cytoskeleton, which helps to orchestrate a spatially confined and very dynamic assembly of intercellular connections. Intriguingly, in addition to maintaining intercellular adhesion, cadherin–catenin proteins are linked to several major developmental signaling pathways crucial for normal morphogenesis. In this article we will highlight the key genetic studies that uncovered the role of cadherin–catenin proteins in vertebrate development and discuss the potential role of these proteins as molecular biosensors of external cellular microenvironment that may spatially confine signaling molecules and polarity cues to orchestrate cellular behavior throughout the complex process of normal morphogenesis.Development of any multicellular organism is impossible without a dynamic and properly regulated intercellular adhesion. Adhesive contacts between cells provide a physical anchoring system that is necessary to form highly organized tissues, and these contacts are essential for effective intercellular communication that ensures the homeostasis and survival of the entire organism. A number of unique developmental processes, including such early events as embryonic compaction and first cell fate specification, as well as later tissue morphogenesis and organogenesis, rely on a dynamic balance between cellular adhesion and migration. Cadherin–catenin protein complexes, which constitute the core of a specialized subtype of cellular adhesion structures termed adherens junctions (AJs), play a particularly important role during these processes. Apart from maintaining adhesive contacts at the cell–cell junctions, they are actively involved in epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions, which are crucial to sustain the tissue plasticity during development. Most importantly, the components of cadherin–catenin complexes are tightly linked to several major signaling networks controlling cell division, differentiation, and apoptosis and this feature is crucial for the broad roles of the AJs throughout the vertebrate development (see Cavey and Lecuit 2009).This article will focus on the role of cadherin–catenin proteins in regulating the signaling events critical for vertebrate development. Altering the expression pattern of particular cadherin–catenin complex components in the developing embryo often leads to major developmental defects, which reflect their role in both signaling and mechanical adhesion. In this article, we will highlight crucial findings suggesting that cadherin–catenin complexes provide not only the structural integrity of the tissue, but may also serve as biosensors of the external cellular microenvironment that modulate cellular behavior and make individual cells work together to ensure the fitness of the entire organism.     

Cell adhesion: More than just glue (Review)

The ability of cells to interact with each other and their surroundings in a co-ordinated manner depends on multiple adhesive interactions between neighbouring cells and their extracellular environment. These adhesive interactions are mediated by a family of cell surface proteins, termed cell adhesion molecules. Fortunately these adhesion molecules fall into distinct families with adhesive interactions varying in strength from strong binding involved in the maintenance of tissue architecture to more transient, less avid, dynamic interactions observed in leukocyte biology. Adhesion molecules are extremely versatile cell surface receptors which not only stick cells together but provide biochemical and physical signals that regulate a range of diverse functions, such as cell proliferation, gene expression, differentiation, apoptosis and migration. In addition, like many other cell surface molecules, they have been usurped as portals of entry for pathogens, including prions. How the mechanical and chemical messages generated from adhesion molecules are integrated with other signalling pathways (such as receptor tyrosine kinases and phosphatases) and the role that aberrant cell adhesion plays in developmental defects and disease pathology are currently very active areas of research. This review focuses on the biochemical features that define whether a cell surface molecule can act as an adhesion molecule, and discusses five specific examples of how cell adhesion molecules function as more than just 'sticky’ receptors. The discussion is confined to the signalling events mediated by members of the integrin, cadherin and immunoglobulin gene superfamilies. It is suggested that, by controlling the membrane organization of signalling receptors, by imposing spatial organization, and by regulating the local concentration of cytosolic adapter proteins, intercellular and cell-matrix adhesion is more than just glue holding cells together. Rather dynamic ‘conversations’ and the formation of multi-protein complexes between adhesion molecules, growth factor receptors and matrix macromolecules can now provide a molecular explanation for the long-observed but poorly understood requirement for a number of seemingly distinct cell surface molecules to be engaged for efficient cell function to occur.     

Cell adhesion: more than just glue (review)

The ability of cells to interact with each other and their surroundings in a co-ordinated manner depends on multiple adhesive interactions between neighbouring cells and their extracellular environment. These adhesive interactions are mediated by a family of cell surface proteins, termed cell adhesion molecules. Fortunately these adhesion molecules fall into distinct families with adhesive interactions varying in strength from strong binding involved in the maintenance of tissue architecture to more transient, less avid, dynamic interactions observed in leukocyte biology. Adhesion molecules are extremely versatile cell surface receptors which not only stick cells together but provide biochemical and physical signals that regulate a range of diverse functions, such as cell proliferation, gene expression, differentiation, apoptosis and migration. In addition, like many other cell surface molecules, they have been usurped as portals of entry for pathogens, including prions. How the mechanical and chemical messages generated from adhesion molecules are integrated with other signalling pathways (such as receptor tyrosine kinases and phosphatases) and the role that aberrant cell adhesion plays in developmental defects and disease pathology are currently very active areas of research. This review focuses on the biochemical features that define whether a cell surface molecule can act as an adhesion molecule, and discusses five specific examples of how cell adhesion molecules function as more than just 'sticky' receptors. The discussion is confined to the signalling events mediated by members of the integrin, cadherin and immunoglobulin gene superfamilies. It is suggested that, by controlling the membrane organization of signalling receptors, by imposing spatial organization, and by regulating the local concentration of cytosolic adapter proteins, intercellular and cell-matrix adhesion is more than just glue holding cells together. Rather dynamic 'conversations' and the formation of multi-protein complexes between adhesion molecules, growth factor receptors and matrix macromolecules can now provide a molecular explanation for the long-observed but poorly understood requirement for a number of seemingly distinct cell surface molecules to be engaged for efficient cell function to occur.     

Modulation of intercellular adherens-type junctions and tyrosine phosphorylation of their components in RSV-transformed cultured chick lens cells.

Transformation of cultured chick lens epithelial cells with a temperature-sensitive mutant of Rous sarcoma virus (tsRSV) leads to radical changes in cell shape and interactions. When cultured at the restrictive temperature (42 degrees C), the transformed cells largely retained epithelial morphology and intercellular adherens junctions (AJ), whereas on switch to the permissive temperature (37 degrees C) they rapidly became fibroblastoid, their AJ deteriorated, and cell adhesion molecules (A-CAM) (N-cadherin) largely disappeared from intercellular contact sites. The microfilament system that was primarily associated with these junctions was markedly rearranged on shift to 37 degrees C and remained associated mainly with cell-substrate focal contacts. These apparent changes in intercellular AJ were not accompanied by significant alterations in the cellular content of several junction-associated molecules, including A-CAM, vinculin, and talin. Immunolabeling with phosphotyrosine-specific antibodies indicated that both cell-substrate and intercellular AJ were the major cellular targets for the pp60v-src tyrosine-specific protein kinase. It was further shown that intercellular AJ components serve as substrates to tyrosine kinases also in nontransformed lens cells, because the addition of a combination of vanadate and H2O2--which are potent inhibitors of protein tyrosine phosphatases--leads to a remarkable accumulation of immunoreactive phosphotyrosine-containing proteins in these junctions. This finding suggests that intercellular junctions are major sites of action of protein tyrosine kinases and that protein tyrosine phosphatases play a major role in the regulation of phosphotyrosine levels in AJ of both normal and RSV-transformed cells.     

Discrete interactions in cell adhesion measured by single-molecule force spectroscopy

Cell-cell adhesion mediated by specific cell-surface molecules is essential for multicellular development. Here we quantify de-adhesion forces at the resolution of individual cell-adhesion molecules, by controlling the interactions between single cells and combining single-molecule force spectroscopy with genetic manipulation. Our measurements are focused on a glycoprotein, contact site A (csA), as a prototype of cell-adhesion proteins. csA is expressed in aggregating cells of Dictyostelium discoideum, which are engaged in development of a multicellular organism. Adhesion between two adjacent cell surfaces involves discrete interactions characterized by an unbinding force of 23 +/- 8 pN, measured at a rupture rate of 2.5 +/- 0.5 microm s-1.     

Adhesion molecules in the regulation of CNS myelination

Myelination is necessary both for rapid salutatory conduction and the long-term survival of the axon. In the CNS the myelin sheath is formed by the oligodendrocytes. Each oligodendrocyte myelinates several axons and, as the number of wraps around each axon is determined precisely by the axon diameter, this requires a close, highly regulated interaction between the axons and each of the oligodendrocyte processes. Adhesion molecules are likely to play an important role in the bi-directional signalling between axon and oligodendrocyte that underlies this interaction. Here we review the current knowledge of the function of adhesion molecules in the different phases of oligodendrocyte differentiation and myelination, and discuss how the properties of these proteins defined by other cell biological systems indicates potential roles in oligodendrocytes. We show how the function of a number of different adhesion and cell-cell interaction molecules such as polysialic acid neural cell adhesion molecule, Lingo-1, Notch, neuregulin, integrins and extracellullar matrix proteins provide negative and positive signals that coordinate the formation of the myelin membrane. Compiling this information from a number of different cell biological and genetic experiments helps us to understand the pathology of multiple sclerosis and direct new areas of research that might eventually lead to potential drug targets to increase remyelination.     

The Cas family docking protein, HEF1, promotes the formation of neurite-like membrane extensions

The Cas family proteins are a family of adhesion docking molecules that mediate protein-protein interactions and contribute to a number of signal transduction pathways. Recent studies of two family members, p130Cas and Sin, have suggested that they may play a role in neurite formation. The current study demonstrates that the third family member, HEF1, can also stimulate the formation of neurite-like processes, in the presence of Rho kinase inhibitors. The HEF1-promoted processes actively extend from the cell body and resemble neurites both in the manner of process extension and in the distribution of adhesion-associated molecules. The HEF1-promoted processes are dependent on the presence of an intact microtubule system and can be inhibited by co-expression of either constitutively active Rac or Cdc42 GTPase. Together, our data support a role for the Cas proteins in regulating cellular morphologies that contribute to tissue specialization.