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 force measurements of insulin monomer-monomer interactions

Direct measurement of the forces involved in protein-protein and protein-receptor interactions can, in principle, provide insight necessary for the advancement of structural biology, molecular biology, and the development of therapeutic proteins. The protein insulin is illustrative in this respect as the mechanisms of insulin dimer dissociation and insulin-insulin receptor binding are crucial to the efficacy of insulin medications for the control of diabetes. Insulin molecules, modified with a photochemically active azido functionality on specific residues, were attached to force microscope tips and opposing mica surfaces in configurations that would either favor or disfavor dimer formation. Force curve measurements performed in buffer solution revealed the complexity of the insulin monomer-monomer interaction with multiple unbinding events occurring upon tip retraction, suggesting disruption of discrete molecular bonds at the monomer-monomer interface. Furthermore, the force curves exhibit long-range unbinding events, consistent with considerable elongation of the insulin molecule prior to dissociation. The unbinding forces observed in this study are the result of a combination of molecular disentanglement and dimer dissociation processes.     

Interactions between trophoblast and uterine epithelium: monitoring of adhesive forces

At embryo implantation, it is postulated that the initial contact between blastocyst and maternal tissues is by adhesion of the trophoblast to the uterine epithelium. This cell-to-cell interaction is thought to be critical for implantation, although the actual adhesive forces have never been determined. In the present study, the atomic force microscope (AFM) was used to study the adhesion between human uterine epithelial cell lines (HEC-1-A; RL95-2) and human trophoblast-type cells (JAR). Specific interaction forces of these epithelia via their apical cell poles were determined on the basis of approach-and-separation cycles. For this purpose, the AFM tip was functionalized with JAR cells, then brought to the surface of uterine epithelial monolayers and was kept in contact for different periods of time (ms, 1, 10, 20, 40 min). The approach force curves displayed repulsive interactions for both HEC-1-A and RL95-2 cells. However, RL95-2 cells (with a smooth surface structure and a thin glycocalyx) showed lower values of the repulsive regime than HEC-1-A cells (with a rough surface structure and a thick glycocalyx). After having overcome repulsive interactions, the initial contact was followed by adhesive interactions. For contact times of 20 and 40 min, RL95-2 cells, but not HEC-1-A cells, showed specific JAR binding, i.e. the separation force curves displayed repeated rupture events in the range of 1-3 nN with a distance between 7-15 microm and, thereafter, a final rupture event at a distance of up to 45 microm. These features point to the formation of strong cell-to-cell bonds. Collectively, these studies provide the first definition of interaction forces between the trophoblast and the uterine epithelium, and are consistent with the hypothesis that an RL95-2-like architecture of uterine epithelial cells, i.e. an non-polarized phenotype, is essential for apical adhesiveness for the human trophoblast.     

Structural activity of a cloned potassium channel (ROMK1) monitored with the atomic force microscope: the "molecular-sandwich" technique

The atomic force microscope (AFM) was used to continuously follow height changes of individual protein molecules exposed to physiological stimuli. A AFM tip was coated with ROMK1 (a cloned renal epithelial potassium channel known to be highly pH sensitive) and lowered onto atomically flat mica surface until the protein was sandwiched between AFM tip and mica. Because the AFM tip was an integral part of a highly flexible cantilever, any structural alterations of the sandwiched molecule were transmitted to the cantilever. This resulted in a distortion of the cantilever that was monitored by means of a laser beam. With this system it was possible to resolve vertical height changes in the ROMK1 protein of >/=0.2 nm (approximately 5% of the molecule's height) with a time resolution of >/=1 msec. When bathed in electrolyte solution that contained the catalytic subunit of protein kinase A and 0.1 mM ATP (conditions that activate the native ion channel), we found stochastically occurring height fluctuations in the ROMK1 molecule. These changes in height were pH-dependent, being greatest at pH 7.6, and lowering the pH (either by titration or by the application of CO2) reduced their magnitude. The data show that overall changes in shape of proteins occur stochastically and increase in size and frequency when the proteins are active. This AFM "molecular-sandwich" technique, called MOST, measures structural activity of proteins in real time and could prove useful for studies on the relationship between structure and function of proteins at the molecular level.     

Simultaneous height and adhesion imaging of antibody-antigen interactions by atomic force microscopy

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.     

The deformation of polycrystalline zirconium

The deformation of polycrystalline zirconium has been examined in terms of the power-law and the thermally activated rate equation approach to deformation. It is shown that the thermal component of the flow stress is independent of strain and that the athermal component is the main contributor to work hardening. The deformation behavior is in general agreement with Fleischer’s force-distance diagram for tetragonal defect-dislocation interaction. Electron microscopy however, showed that the majority of slip activity occurred on the prism planes and interstitials in octahedral positions do not interact strongly with dislocations on the prism planes. It is therefore suggested that deformation is controlled by solute atoms interacting with the dislocation core and that this interaction has a force-distance relationship equivalent to that suggested by Fleischer.     

Molecular forces between membranes displaying neutral glycosphingolipids: evidence for carbohydrate attraction

The surface force apparatus was used to determine the fundamental forces governing the adhesion between mixed bilayer membranes comprising lactosyl ceramide (LacCer) and di-tridecanoyl-phosphatidyl choline. Forces between membranes were quantified as a function of the glycolipid surface densities, which ranged from 0 to 30 mol %. Control measurements of the forces between pure phosphatidylcholine membranes and mixed bilayers of lactosyl ceramide with phosphocholine showed that the steric thickness of the carbohydrate headgroups increased from 19 to 25 A when the glycolipid density increased from 10 to 20 mol %. The layer compressibility also decreased with increasing carbohydrate coverage, but the corresponding adhesion between lactosyl ceramide-containing membranes increased with increasing amounts of glycosphingolipid in them. The nonspecific van der Waals forces accounted for the attraction measured in the control experiments and that between identical 10 mol % LacCer bilayers. However, the increase in the adhesion with increasing glycolipid density was 2-4 times greater than predicted by Lifschitz theory. Additionally, the forces measured during separation of membranes containing 20 and 30 mol % glycosphingolipid indicated that the headgroups bind and rearrange during bilayer detachment. The interactions between the carbohydrates are weak and apparently dynamic, and they generate an additional density-dependent intermembrane attraction that is on the order of the van der Waals force.     

EFFECTS OF MATRIX MOLECULAR WEIGHT ON STRUCTURE AND REINFORCEMENT OF HIGH DENSITY POLYETHYLENEfMICA COMPOSITES

Three types of high-density polyethylene (HDPE) with different molecular weights (high,medium and low) were adopted to evaluate the influence of matrix molecular weight on the structure-propcrty relation of injection-molded HDPE/mica composites through a combination of SEM,2d-WAXS,DSC,DMA and tensile testing. Various structural factors including orientation,filler dispersion,interfacial interaction between HDPE and mica,etc.,which can impact the macroscopic mechanics,were compared in detail among the three HDPE/mica composites. The transcrystallization of HDPE on the mica surface was observed and it exhibited strong matrix molecular weight dependence. Obvious transcrystalline structure was found in the composite with low molecular weight HDPE,whereas it was hard to be detected in the composites with increased HDPE molecular weight. The best reinforcement effect in the composite with low molecular weight HDPE can be understood as mainly due to substantially improved interfacial adhesion between matrix and mica filler,which arises from the transcrystallization mechanism.     

The atomic force microscope detects ATP-sensitive protein clusters in the plasma membrane of transformed MDCK cells

Plasma membrane proteins are supposed to form clusters that allow 'functional cross-talk' between individual molecules within nanometre distance. However, such hypothetical protein clusters have not yet been shown directly in native plasma membranes. Therefore, we developed a technique to get access to the inner face of the plasma membrane of cultured transformed kidney (MDCK) cells. The authors applied atomic force microscopy (AFM) to visualize clusters of native proteins protruding from the cytoplasmic membrane surface. We used the K+ channel blocker iberiotoxin (IBTX), a positively charged toxin molecule, that binds with high affinity to plasma membrane potassium channels and to atomically flat mica. Thus, apical plasma membranes could be 'glued' with IBTX to the mica surface with the cytosolic side of the membrane accessible to the scanning AFM tip. The topography of these native inside-out membrane patches was imaged with AFM in electrolyte solution mimicking the cytosol. The plasma membrane could be clearly identified as a lipid bilayer with the characteristic height of 4.9 +/- 0.02 nm. Multiple proteins protruded from the lipid bilayer into the cytosolic space with molecule heights between 1 and 20 nm. Large protrusions were most likely protein clusters. Addition of the proteolytic enzyme pronase to the bath solution led to the disappearance of the proteins within minutes. The metabolic substrate ATP induced a shape-change of the protein clusters and smaller subunits became visible. ADP or the non-hydrolysable ATP analogue, ATP-gamma-S, could not exert similar effects. It is concluded that plasma membrane proteins (and/or membrane associated proteins) form 'functional clusters' in their native environment. The 'physiological' arrangement of the protein molecules within a cluster requires ATP.     

Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope

To achieve high-resolution topographs of native biological macromolecules in aqueous solution with the atomic force microscope (AFM) interactions between AFM tip and sample need to be considered. Short-range forces produce the submolecular information of high-resolution topographs. In contrast, no significant high-resolution information is provided by the long-range electrostatic double-layer force. However, this force can be adjusted by pH and electrolytes to distribute the force applied to the AFM tip over a large sample area. As demonstrated on fragile biological samples, adjustment of the electrolyte solution results in a local reduction of both vertical and lateral forces between the AFM tip and proteinous substructures. Under such electrostatically balanced conditions, the deformation of the native protein is minimized and the sample surface can be reproducibly contoured at a lateral resolution of 0.6 nm.     

Modifying the mechanical property and shear threshold of L-selectin adhesion independently of equilibrium properties

Interactions between adhesion molecules on two different cells differ from interactions between receptors and soluble ligands in that the adhesion molecule interaction (bond) is often subjected to force. It is widely assumed by cell biologists that the 'strength' of a bond is a simple function of the affinity of one adhesion molecule for the other, whereas biophysicists suggest that bonds have 'mechanical properties' that affect their strength. Mechanical properties are a function of the shape of the energy landscape related to bond formation and dissociation, whereas affinity is related only to the net energy change. Mechanical properties determine the amount by which the kinetics and affinity of bonds are altered by applied force. To date there has been no experimental manipulation of an adhesion molecule that has been shown to affect mechanical properties. L-selectin is an adhesion molecule that mediates lymphocyte binding to, and rolling on, high endothelial venules; these are prerequisites for the emigration of lymphocytes from the bloodstream into lymph nodes. Here we report a selective and reversible chemical modification of a mucin-like ligand that alters the mechanical properties of its bond with L-selectin. The effect of force on the rate of bond dissociation, that is, on a mechanical property, is altered, whereas there is little or no effect of the modification on the rate of bond dissociation in the absence of force. Moreover, the puzzling requirement for hydrodynamic shear flow above a threshold level for L-selectin interactions is dramatically altered.     

Atomic force microscopy for characterization of the biomaterial interface

The molecular processes that occur at the interface of an implanted biomaterial determines the host response, including phenomena such as protein adsorption, conformational changes, and subsequent interactions with cellular components. Until recently, such processes could not be observed directly. Over the past decade, atomic force microscopy (AFM) has provided mechanistic insights into the molecular level interactions that occur at the biomaterial interface. Several unique operational modes have been developed which utilize intermittent contact with the sample and decrease applied shear forces. These dynamic modes also can be used to study the role of different structural components on biomaterial micromechanical properties. Force detection techniques allow molecular level studies of individual receptor-ligand binding events, and force mapping for determining structure/function relationships. Advancements in tip manufacturing, image processing techniques, the use of model surfaces and labeling all have contributed to the advancement of the AFM as a state-of-the-art research instrument. In this report, we examine the applicability of the AFM to the study of biomaterials and cell/molecular interactions.     

Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins

The structure of the Escherichia coli chaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid. High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top. Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiae with a destabilizing Gly-->Ala mutation and RTEM beta-lactamase from Escherichia coli with two Cys-->Ala mutations, could be studied by force spectroscopy under different conditions. The results show that the interaction force decreases in the presence of ATP (but not of ATPgammaS) and that the force is smaller for native-like proteins than for the fully denatured ones. It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight. By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding.     

Experimental study of the interaction range and association rate of surface-attached cadherin 11

We describe a method allowing quantitative determination of the interaction range and association rate of individual surface-attached molecules. Spherical beads (1.4 micro(m) radius) were coated with recombinant outer domains of the newly described classical type II cadherin 11, a cell adhesion molecule. Beads were driven along cadherin-coated surfaces with a hydrodynamic force of approximately 1 pN, i.e., much less than the mechanical strength of many ligand-receptor bonds. Spheres displayed periods of slow motion interspersed with arrests of various duration. Particle position was monitored with 50 Hz frequency and 0.025 micro(m) accuracy. Nearly 1 million positions were recorded and processed. Comparison between experimental and computer-simulated trajectories suggested that velocity fluctuations might be related quantitatively to Brownian motion perpendicular to the surface. The expected amplitude of this motion was of order of 100 nm. Theoretical analysis of the relationship between sphere acceleration and velocity allowed simultaneous determination of the wall shear rate and van der Waals attraction between spheres and surface. The Hamaker constant was estimated at 2.9 x 10(-23) J. The frequency of bond formation was then determined as a function of sphere velocity. Experimental data were consistent with the view that the rate of association between a pair of adhesion molecules was approximately 1.2 x 10(-3) s-1 and the interaction range was approximately 10 nm. It is concluded that the presented methodology allows sensitive measurement of sphere-to-surface interactions (with approximately 10 fN sensitivity) as well as the effective range and rate of bond formation between individual adhesion molecules.     

Optimalization of southwestern technique for detection of binding of Epstein-Barr virus nuclear antigen 1 to origin of replication

An optimized protocol of Southwestern analysis for detection of Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) binding to the origin of viral replication (oriP) was described. The unique feature of this optimized protocol includes the restoration of the denatured proteins to native conformation after SDS-polyacrylamide gel electrophoresis and blocking of nonspecific and low-affinity binding sites prior to DNA binding. The parameters and conditions that may affect the specific interaction of EBNA-1 to oriP DNA were then determined. The specific binding was affected by divalent cations (MgCl2) and ionic strength; the optimal concentrations of MgCl2 and NaCl were observed at 10 and 300 mM, respectively. The various buffer systems and pH values tested had no apparent effect on EBNA-1 binding. Under the optimal conditions, a single protein of 68 Kd was detected and the bindings of other nonspecific, low-affinity DNA-binding proteins were abolished. The authenticity and specificity of 68 Kd protein as EBNA-1 were determined by reaction with antibody specific for EBNA-1 and competition assay with specific DNA sequence. After optimization this technique can be a powerful and yet simple means of studying protein-DNA interactions and their roles in gene expression.     

Reaction and total cross sections for low energy pi + and pi - on isospin zero nuclei

We calculated the electrostatic force between a planar interface, such as a planar-supported lipid bilayer membrane, and the tip of a stylus on which another lipid bilayer or some other biomacromolecular system might be deposited. We considered styli with rounded tips as well as conical tips. To take into account the effect of dynamical hydrogen-bonded structures in the aqueous phase, we used a theory of nonlocal electrostatics. We used the Derjaguin approximation and identified the systems for which its use is valid. We pointed out where our approach differs from previous calculations and to what extent the latter are inadequate. We found that 1) the nonlocal interactions have significant effects over distances of 10-15 A from the polar zone and that, at the surface of this zone, the effect on the calculated force can be some orders of magnitude; 2) the lipid dipoles and charges are located a distance L from the hydrophobic layer in the aqueous medium and this can have consequences that may not be appreciated if it is ignored; 3) dipoles, located in the aqueous region, can give rise to forces even though the polar layer is unchanged, and if this is ignored the interpretation of force data can be erroneous if an attempt is made to rationalize an observed force with a knowledge of an uncharged surface; 4) the shape of the stylus tip can be very important, and a failure to take this into account can result in incorrect conclusions, a point made by other workers; and 5) when L is nonzero, the presence of charges and dipoles can yield a force that can be nonmonotonic as a function of ionic concentration.     

Growth and configurational stability of circular,buckling-driven film delaminations

A study is presented of delamination at the interface between a thin elastic film bonded to a substrate under conditions in which the film is subject to equi-biaxial compression. The focus is on initially circular delaminations. When the initial delamination is sufficiently large it buckles away from the substrate producing a blister which in turn induces a driving force on the interface crack tip. A two-part theoretical analysis of the coupled buckling/fracture problem is conducted: the axisymmetric growth of the circular blister, and the stability of the circular blister to nonaxisymmetric perturbations of the interface crack front. A simple criterion is identified which excludes the possibility of wide-spread delamination. Experiments are reported for a model film/substrate system (mica bonded to aluminum) chosen to allow visualization of the interface and to permit compressive stresses in the film to be generated over the full range of interest by exploiting the large thermal expansion mismatch of the system. The experiments bear out the theoretical prediction of a regime of axisymmetric growth which gives way to nonaxisymmetric blisters after a blister becomes sufficiently large. The study suggests that the wavy-circular and worm-like blister morphologies which are usually observed for delaminated films are a manifestation of the configurational instability of the interface crack front.     

Physician-patient interactions: a comparison of analysis systems

OBJECTIVE: The medical interview has important diagnostic and therapeutic functions and requires the integration of doctor-centred and patient-centred interviewing techniques to collect accurate and complete biopsychosocial data from the patient. Analysis of the interaction between patient and doctors which occur during the medical interview allow to evaluate physicians' interview techniques and to eventually improve them. OBJECTIVE: 1. To review different Interaction Analysis Systems (IAS) used to describe doctor-patient communication in terms of clinical relevance, observational strategy, reliability and behavioural and verbal contents. 2. To critically evaluate these IASs on the basis of their relevant research outcomes. METHOD: Previous reviews on interaction and keywords for Medline research (HealthGate) listed above were utilised to collect the relevant literature. RESULTS: Seventeen classification systems were identified and ten were discussed in a chronological order. Starting from a general sociological or psycholinguistic approach, the IASs over the years have became more specific and detailed, focusing more on the medical interview and on specific topics, such as cancer or hospital medical consultations. CONCLUSIONS: When studying interactions in general practice medicine, it is important to define the significant units of interaction which allow to identify a "patient-centred approach", since this is relevant not only for obtaining reliable and complete medical and social data, but also for the recognition of patients with emotional disorders and their correct diagnosis. Listening to the patient and facilitating the expression of emotions is an important aspect of patient education too, as patients learn that talking about psychological problems to their physician is appropriate and may be therapeutic.     

Influence of tablet hardness and hydrophobicity on the adhesive properties of an acrylic resin copolymer

The adhesive properties--including the force of adhesion, elongation at adhesive failure, the modulus of adhesion, and the adhesive toughness--of an acrylic resin copolymer were determined using the butt adhesion technique. Flat-faced tablets containing up to 30% hydrogenated castor oil were coated with an aqueous dispersion of Eudragit L30D-55. Using data obtained from a Chatillon digital force gauge attached to a motorized test stand, force-deflection profiles, similar to stress-strain curves generated in the tensile testing of free films, were constructed. The surface characteristics of the tablets significantly influenced polymer-substrate interaction. The force of adhesion, the elongation at adhesive failure, and the adhesive toughness decreased as the surface of the tablet became more hydrophobic through the addition of wax to the tablet formulation. Lower adhesive properties were found with increasing tablet hardness, due to a decrease in the effective area of contact between the film coating and the tablet surface. Increased polymer loading resulted in stronger adhesion, indicating a relationship between the mechanical and adhesive properties of the polymer. The present study demonstrated that the area under the force-deflection profile in conjunction with the force of adhesion was more representative of the adhesive properties of the polymer.     

Thrombin promotes platelet-mediated melanoma cell adhesion to endothelial cells under flow conditions: role of platelet glycoproteins P-selectin and GPIIb-IIIA

We investigated the role of platelets in human melanoma cell (line 397) interaction with vascular endothelial cells (ECs) under flow conditions. The ability of the tumour cells to adhere to the EC monolayer was significantly reduced by application of flow at a shear rate of 250 s(-1). A 2.2-fold increase in tumour cell adhesion to ECs under flow was observed upon addition of thrombin receptor agonist peptide (TRAP)-activated platelets but not resting platelets. A similar increase (2.5-fold) in tumour cell adhesion to ECs under flow was observed when the tumour cells were incubated with resting platelets on thrombin-treated ECs. However, thrombin treatment of the ECs alone had no effect on tumour cell adhesion in the absence of platelets. The enhancement of tumour cell adhesion to ECs by TRAP-activated platelets was virtually abolished by blockade of the platelet glycoproteins P-selectin and GPIIb-IIIa by monoclonal antibodies. Blockade of P-selectin also inhibited the direct adhesion of TRAP-activated platelets to ECs, but did not affect the interaction of the tumour cells with platelets immobilized on subendothelial extracellular matrix (ECM). Blockade of GPIIb-IIIa inhibited both platelet-EC and platelet-tumor cell interactions. Our results indicate that tumour cell adhesion to the endothelium under flow is enhanced by platelets under conditions that allow platelet adhesion to ECs. Inhibition studies suggest that activated platelet adhesion to ECs is mediated by P-selectin and GPIIb-IIIA, and tumour cell adhesion to EC-bound platelets--mainly by GPIIb-IIIa.