Intercellular Bonds Research Articles

E-cadherin is under constitutive actomyosin-generated tension that is increased at cell–cell contacts upon externally applied stretch

Classical cadherins are transmembrane proteins at the core of intercellular adhesion complexes in cohesive metazoan tissues. The extracellular domain of classical cadherins forms intercellular bonds with cadherins on neighboring cells, whereas the cytoplasmic domain recruits catenins, which in turn associate with additional cytoskeleton binding and regulatory proteins. Cadherin/catenin complexes are hypothesized to play a role in the transduction of mechanical forces that shape cells and tissues during development, regeneration, and disease. Whether mechanical forces are transduced directly through cadherins is unknown. To address this question, we used a Förster resonance energy transfer (FRET)-based molecular tension sensor to test the origin and magnitude of tensile forces transmitted through the cytoplasmic domain of E-cadherin in epithelial cells. We show that the actomyosin cytoskeleton exerts pN-tensile force on E-cadherin, and that this tension requires the catenin-binding domain of E-cadherin and αE-catenin. Surprisingly, the actomyosin cytoskeleton constitutively exerts tension on E-cadherin at the plasma membrane regardless of whether or not E-cadherin is recruited to cell-cell contacts, although tension is further increased at cell-cell contacts when adhering cells are stretched. Our findings thus point to a constitutive role of E-cadherin in transducing mechanical forces between the actomyosin cytoskeleton and the plasma membrane, not only at cell-cell junctions but throughout the cell surface.

Looking After Polysialic Acid

Many aspects of brain function are modulated by neuronal activity; in some cases, the effects of activity are restricted to particular developmental stages, or critical periods. For instance, loss of visual input from one eye early in life leads to a shift in the responsiveness of neurons in the visual cortex toward the functional eye. The onset and time course of this critical period for ocular dominance plasticity is influenced by the maturation of GABAergic innervation, which is itself regulated by visual input and neuronal activity. But how? Di Cristo et al . found that polysialic acid [PSA, a homopolymer of sialic acid that attaches to neural cell adhesion molecules (NCAMs) and modulates intercellular bonds] and NCAMs were abundant in the neonatal mouse visual cortex but that PSA underwent a steep decline after eye opening. Pups reared in the dark showed increased PSA compared with pups reared under conventional light/dark cycles, and pharmacological suppression of visual signals suppressed the decline. In organotypic cortical cultures, enzymatic removal of PSA with endoneuramidase led to premature maturation of the innervation of pyramidal cell somata by GABAergic basket interneurons. Similarly, endoneuramidase injection into the visual cortex promoted both perisomatic GABAergic innervation and the frequency of miniature inhibitory postsynaptic currents. Moreover, it stimulated the early onset of the critical period for ocular dominance plasticity. Thus, the authors conclude that maturation of GABAergic inhibition--and thereby the initiation of ocular dominance plasticity--is regulated by an activity-dependent decrease in PSA. G. Di Cristo, B. Chattopadhyaya, S. J. Kuhlman, Y. Fu, M.-C. Bélanger, C. Z. Wu, U. Rutishauser, L. Maffei, Z. J. Huang, Activity-dependent PSA expression regulates inhibitory maturation and onset of critical period plasticity. Nat. Neurosci . 10 , 1569-1577 (2007). [PubMed]

Creation of intercellular bonds by anchoring protein ligands to membranes using the diphtheria toxin T domain

We describe the creation of cell adhesion mediated by cell surface engineering. The Flt3-ligand was fused to a membrane anchor made of the diphtheria toxin translocation domain. The fusion protein was attached to the surface of a cell by an acid pulse. Contact with another cell expressing the receptor Flt3 lead to its activation. This activity involved direct cell–cell contact. A mean force of 20 nN was needed to separate functionalized cells after 5 min of contact. Overall, we showed that it is possible to promote specific cell–cell adhesion by attaching protein ligands at the surface of cells.

An integrated agent-mathematical model of the effect of intercellular signalling via the epidermal growth factor receptor on cell proliferation

We have previously developed Epitheliome, a software agent representation of the growth and repair characteristics of epithelial cell populations, where cell behaviour is governed by a number of simple rules. In this paper, we describe how this model has been extended to incorporate an example of a molecular ‘mechanism’ behind a rule—in this case, how signalling by both endogenous and exogenous ligands of the epidermal growth factor receptor (EGFR) can impact on the proliferation of cell agents. We have developed a mathematical model representing release of endogenous ligand by cells, three-dimensional diffusion of the secreted molecules through a volume of cell culture medium, ligand–receptor binding, and bound receptor internalization and trafficking. Information relating to quantities of molecular species associated with each cell agent is frequently exchanged between the agent and signalling models, and the ratio of bound to free receptors determines cell cycle progression and hence the proliferative behaviour of the cell agents. We have applied this integrated model to examine the effect of plating density on tissue growth via autocrine/paracrine signalling. This predicts that cell growth is dependent on the concentration of exogenous ligand, but where this is limited, then growth becomes dependent on cell density and the availability of endogenous ligand. We have further modified the calcium concentration of the medium to modulate the formation of intercellular bonds between cells and shown that the increased propensity for cells to form colonies in physiological calcium does not result in significantly different patterns of receptor occupancy. In conclusion, our approach demonstrates that by combining agent-based and mathematical modelling paradigms, it is possible to probe the complex feedback relationship between the behaviour of individual cells and their interaction with one another and their environment.

T cell activation: Kinetic proofreading, serial engagement and cell adhesion

There is a fundamental difference in complexity between signaling initiated by ligands on the surface of one cell binding to receptors on the surface of another cell and ligands in solution binding to these receptors. The fact that two cells must approach each other and form a number of intercellular bonds of different types, all within the restricted geometry of the intercellular contact region, introduces the possibility of complex spatio-temporal dynamics of surface receptors that is not present otherwise. Mathematical modelling of these dynamics is in its early stages. However, useful tools have emerged. The purpose of this paper is to describe some of these mathematical tools, indicating their strengths and shortcomings, and suggest some directions for future theoretical studies.

Hydrodynamic Forces Applied on Intercellular Bonds, Soluble Molecules, and Cell-Surface Receptors

Cells and biomolecules exposed to blood circulation experience hydrodynamic forces that affect their function. We present a methodology to estimate fluid forces and force loading rates applied on cellular aggregates, cell-surface proteins, and soluble molecules. Low Reynolds-number hydrodynamic theory is employed. Selected results are presented for biological cases involving platelets, neutrophils, tumor cells, GpIb-like cell-surface receptors, and plasma von Willebrand factor (vWF)-like soluble proteins. Calculations reveal the following: 1), upon application of constant linear shear, cell aggregates and biomolecules experience time-varying forces due to their tumbling motion. 2), In comparison to neutrophil homotypic aggregates, the maximum force applied on neutrophil-platelet aggregates is approximately threefold lower. Thus, alterations in cell size may dramatically alter adhesion molecule requirement for efficient cell binding. Whereas peak forces on homotypic cell doublets are tensile, shear forces dominate in heterotypic doublets with radius ratio <0.3. 3), The peak forces on platelet GpIb and von Willebrand factor are of comparable magnitude. However, they are orders-of-magnitude lower than those applied on intercellular bonds. Charts are provided to rapidly evaluate the magnitude of hydrodynamic force and rotation time-period occurring in any given experiment. The calculation scheme may find application in studies of vascular biology and receptor biophysics.

Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter.

A colony morphology type is described in which cells of Salmonella typhimurium form a rigid multicellular network with expression of thin aggregative fimbriae that mediate tight intercellular bonds. Surface translocation of cells on plates and adherence to glass and polystyrene surfaces in biofilm assays are further characteristics of the morphotype. This morphotype (rdar) is normally expressed only at low temperature. However, in two unrelated S. typhimurium strains, spontaneous mutants were found forming rdar colonies independent of temperature. Allelic replacement proved a single point mutation in the promoter region of PagfD in each of the two mutants to be responsible for the constitutive phenotype of a multicellular behaviour. Transcription levels of the two divergently transcribed agf operons required for biogenesis of thin aggregative fimbriae by Northern blot analysis with gene probes for agfA and agfD as well as expression levels of AgfA by Western blotting were compared in the wild type, the constitutive mutants and their respective ompR and rpoS- derivatives. In the wild type the rdar morphotype and expression of thin aggregative fimbriae are restricted to low temperature on plates containing rich medium of low osmolarity, but biogenesis of thin aggregative fimbriae occurs upon iron starvation at 37 degrees C. In the upregulated mutants biogenesis of thin aggregative fimbriae is only abolished at high osmolarity at 37 degrees C and in the exponential phase in broth culture. Control of expression of thin aggregative fimbriae and rdar morphology takes place at the transcriptional level at the agfD promoter. A functional ompR allele is required, however an rpoS mutation abolishes transcription only in the wild type, but has no influence on expression of thin aggregative fimbriae in the constitutive mutants.

A model for the kinetics of homotypic cellular aggregation under static conditions

We present the formulation and testing of a mathematical model for the kinetics of homotypic cellular aggregation. The model considers cellular aggregation under no-flow conditions as a two-step process. Individual cells and cell aggregates 1) move on the tissue culture surface and 2) collide with other cells (or aggregates). These collisions lead to the formation of intercellular bonds. The aggregation kinetics are described by a system of coupled, nonlinear ordinary differential equations, and the collision frequency kernel is derived by extending Smoluchowski's colloidal flocculation theory to cell migration and aggregation on a two-dimensional surface. Our results indicate that aggregation rates strongly depend upon the motility of cells and cell aggregates, the frequency of cell-cell collisions, and the strength of intercellular bonds. Model predictions agree well with data from homotypic lymphocyte aggregation experiments using Jurkat cells activated by 33B6, an antibody to the beta 1 integrin. Since cell migration speeds and all the other model parameters can be independently measured, the aggregation model provides a quantitative methodology by which we can accurately evaluate the adhesivity and aggregation behavior of cells.

Coagulation of Particles in Shear Flow: Applications to Biological Cells

A simplified model is developed which makes it possible to describe the shear-induced coagulation of particles with different types of attractive interactions. In this model the trajectories of particles are calculated without taking into account any nonhydrodynamic interactions, while the hydrodynamic interaction is described in terms of the lubrication theory. Coagulation is assumed to occur if the hydrodynamic stretching force exerted on the doublet of particles is smaller than the attractive force binding the particles together. If the nonhydrodynamic interaction is reduced to the Van der Waals forces of molecular attraction, calculations of capture efficiency in terms of our simplified analysis are shown to be in good agreement with those carried out by other authors on the basis of analysis of numerically calculated trajectories. Further applications of our model deal with the coagulation of some biological cells, namely blood platelets, which are responsible for thrombous formation. In the first approximation activated platelets can be described as spherical elastic particles interacting via the superposition of Van der Waals attraction and biochemically specific interactions, which are attributed to receptor-coreceptor bonds between cells. The specific interactions are considered to result from the interplay of two subsystems, chemical and mechanical ones. The analysis of the kinetics of formation of intercellular bonds is carried out, yielding a simple analytic representation for the force of biospecific interaction. The biospecific interaction is shown to affect the capture efficiency, ϵ, at intermediate range of shear rate G , so that a plateau appears in the curve ϵ( G), while beyond this range coagulation is governed by Van der Waals attraction and ϵ is a decreasing function of G. This type of dependence is in qualitative agreement with that observed for blood platelet aggregation. Quantitative agreement is achieved with reasonable choice of parameters of interaction. Our approach also makes it possible to take into account the perturbation of particles' trajectories and the corrections to the force of Van der Waals and biospecific interactions due to elastic deformation of particles, which is shown to increase the capture efficiency at low shear rates and decrease it at high shear rates.

Interaction forces between red cells agglutinated by antibody. IV. Time and force dependence of break-up

We report on an extension of a previously described method to measure the hydrodynamic force to separate doublets of fixed, sphered and swollen red cells cross-linked by antibody (S. P. Tha, J. Shuster, and H. L. Goldsmith. 1986. Biophys. J. 50:1117-1126). With a traveling microtube apparatus, doublets are tracked and videotaped in a slowly accelerating Poiseuille flow in 150-microns-diameter tubes, and the hydrodynamic normal force at break-up, Fn, is computed from the measured doublet velocity and radial position. Previous results showed a large range of Fn, the mean of which increased with [antiserum], and an absence of clustering at discrete values of Fn. Since it was assumed that the cells separate the instant a critical force to break all crossbridges was reached, lack of clustering could have been due to the use of a polyclonal antiserum. We therefore studied the effect of monoclonal IgM or IgA antibody on the distribution of Fn. The results showed that the data are as scattered as ever, with Fn varying from 2 to 200 pN, and exhibit no evidence of clustering. However, the scatter in Fn could be due to the stochastic nature of intercellular bonds (E. Evans, D. Berk, and A. Leung. 1991a. Biophys. J. 59:838-848). We therefore studied the force dependence of the time to break-up under constant shear stress (Fn from 30 to 200 pN), both in Poiseuille and Couette flow, the latter by using a counter-rotating cone and plate rheoscope. When 280 doublets were rapidly accelerated in the traveling microtube and then allowed to coast in steady flow for up to 180 s, 91% survived into the constant force region; 16% of these broke up after time intervals, tP, of 2-30s. Of 340 doublets immediately exposed to constant shear in the rheoscope, 37% broke after time intervals, tc, from < 1 to 10 s. Thus, doublets do indeed break up under a constant shear stress, if given time. The average time to break-up decreased significantly with increasing force, while the fraction of doublets broken up increased. At a given Fn, the fraction of break-ups decreased with increasing [IgM], suggesting that the average number of bonds had also increased. Using a stochastic model of break-up (G. I. Bell. 1978. Science (Washington DC). 200:618-627; E. Evans, D. Berk,and A. Leung. 1991. Biophys. J. 59:838-848) and a Poisson distribution for the number of bonds, Nb, break-up in slowly accelerating Poiseuille flow and in immediate shear application in Couette flow was simulated. In Poiseuille flow, the observed range and scatter in Fn could be reproduced assuming (Nb) > 5. In the rheoscope, the time intervals and number of rotations to break-up, tc, were quite well reproduced assuming (Nb) = 4. The similarity of (Fn) for monoclonal IgM and IgA for doublet break-up under constant slow acceleration is compatible with the conclusion of Evans et al. (1991 a) for normal red cells and Xia et al. (manuscript submitted for publication) for sphered and swollen red cells, that the applied force extracts the antigen from the cell membrane.

Immunohistochemical localization of adherens junction components in blood-brain barrier microvessels of the rat

The morphology and molecular composition of intercellular adherens junctions have most frequently been described in epithelial cells and the fascia adhaerens of the intercalated disc. A group of cytoplasmic molecules is known to be associated with adherens junctions. The intercellular bond is mediated by cadherins which bridge the cells by homophilic binding. Recently, endothelial cells have also been shown to form intercellular junctions of the adherens-type. However, they are morphologically less distinct and little is known about their molecular components. In this study we report the localization of some adherens junction components in intact microvessels of the blood-brain barrier in the rat. We used antibodies raised against alpha-actinin, vinculin, zyxin, cadherin (antipan-cadherin antibody) and A-CAM (N-cadherin) in immunohistochemical experiments at light and electron microscopical levels. Microvessel walls reacted positively for all antigens throughout postnatal development. All antigens were localised, though not necessarily exclusively, to interendothelial junctions. At the ultrastructural level, pan-cadherin reactivity was present throughout the entire length of the cleft. These results could mean that in blood-brain barrier endothelial cells the complex tight junction is embedded in an adherens junction which occupies the entire length of the cleft.

The Ultrastructure of Transitional Cell Carcinomas of the Human Urinary Bladder Treated with Ethoglucid or Mitomycin

Electron microscopy studies of the ultrastructure of bladder carcinoma treated by intravesical instillation of ethoglucid and mitomycin are described. Ethoglucid acts mainly by disrupting cells’ intercellular bonds. Mitomycin acts on the cell nucleus. It is suggested that these differences in action form a basis for sequential therapy.

Quantitation of intercellular binding strength by disruptive centrifugation: Application to the analysis of adhesive interactions between P815 tumour cells and activated macrophages

The cell binding assay described in the present paper provides a quantitative measurement of intercellular binding strengths between P815 mastocytoma cells and macrophages activated for cytotoxicity. Conjugates were submitted to a dislodgement force generated by centrifugation tending to remove radiolabelled probe cells from macrophages adhering to a glass support. Non-specific binding occurred between P815 cells and the glass support, but was negligible on siliconed coverslips. The adhesive interactions macrophage/glass were strong enough to resist up to about 1 X 10(-9) N, the maximal dislodgement force tested. Completion of conjugate formation was reached at 37 degrees C after 45 to 60 min and was inhibited at 4 degrees C or in Ca++-free medium. The strengths of intercellular bonds between P815 cells and macrophages varied from 7 X 10(-11) N (the minimal force applied) to more than 9 X 10(-10) N. No clear-cut separation between weak and strong interactions was observed when the centrifugation step was performed at 4 degrees C. In contrast, when centrifugation was carried out at 37 degrees C, a subpopulation of loosely bound tumour targets could be distinguished from more strongly bound P815 cells.

Quantitative measurements of the specificity and kinetics of conjugate formation between cloned cytotoxic T lymphocytes and splenic target cells by dual parameter flow cytometry.

The formation of conjugates between cloned anti-H-2Kb and Dd cytotoxic T lymphocytes (CTL) and splenic target cells has been studied by dual parameter flow cytometry. By varying effector-target combinations and by blocking with anti-MHC class I monoclonal antibodies, we found that the specificity of conjugate formation, in general, paralleled that expected from cytotoxicity studies; however, a significant number of "nonspecific" conjugates was always observed. As expected from previous studies, conjugate formation did not occur below 10 degrees C and was inhibited by cytochalasin B, EDTA, and anti-Lyt-2 antibodies. Conjugate formation followed first-order kinetics. The rate of formation of conjugates increased with temperature from 24 degrees to 37 degrees C; at 37 degrees C, the half-time was 1.4 min. After a 6-min lag period, lysis of target cells could be detected at 37 degrees C but not at 30 degrees C or below. Because target cell lysis proceeded during a period of time when the number of conjugates remained constant, considerable effector cell recycling must have occurred. Comparisons of the fluorescence emissions from conjugated effector or target cells with those from unconjugated cells demonstrated that nearly all conjugates contained one effector cell and one target cell, independent of the ratio of the two cell types in the original mix. Once formed, anti-H-2Kb conjugates were stable when diluted into medium alone, but rapidly disaggregated in medium containing either anti-Lyt-2 or anti-Kb monoclonal antibodies, both of which blocked conjugate formation. This finding suggests that conjugates are normally stabilized by intercellular bonds that are constantly breaking and reforming at the cell:cell interface, and that the antibodies disrupt the conjugates by preventing the reformation of broken bonds.

T-cell-fibroblast hybridoma deformability and concanavalin A-induced agglutination.

Cell adhesion influences many important immunological functions such as phagocytosis or T-cell-mediated cytotoxicity. Previous work suggested that the ease of inducing intercellular bonds (i.e. binding efficiency) and the difficulty to separate bound cells with mechanical forces (i.e. binding strength) might be parameters of different significances. The present report describes a study made on two T-lymphocyte/polyoma virus transformed fibroblast hybrid subclones (3D1c and 3D1n) with markedly different adhesive properties: indeed, 3D1c cells were at the same time more readily agglutinated with concanavalin A and more easily disagglutinated than 3D1n. In order to understand these differences, a systematic comparison of various properties of 3D1c and 3D1n cells was undertaken. The following parameters were studied: surface density of concanavalin A binding sites, surface electrostatic charge, hydrophobicity, fluorescence polarization measured on individual cells, and ability to spread on a flat substrate in response to volume or surface forces. It is concluded that cell deformability and/or spreading ability might be an important determinant of binding strength, but the factors governing binding efficiency remained incompletely understood. It is suggested that the methods described in the present report might help understanding differences between various tumor cell lines with different malignant potential.

Calcium-dependent and calcium-independent adhesive mechanisms are present during initial binding events of neural retina cells.

The hypothesis that intercellular adhesion can be subdivided into two separable phenomena, an initial recognition event and a subsequent stabilization, is supported by the use of a new cell binding assay that provides a quantitative measure of intercellular binding strengths. Radioactive single cells are brought into contact with cell monolayers at 4 degrees C in sealed compartments. The compartments are inverted and a centrifugal force is then applied tending to dislodge the probe cells from the monolayers. By varying the speed of centrifugation, the force maintaining association between embryonic chick neural retina cells was determined to be on the order of 10(-5) dynes after incubation at 4 degrees C. Brief incubations at 37 degrees C resulted in significant strengthening of the intercellular bond. Using this cell binding assay, neural retina cells were shown to exhibit both a Ca++-independent and a Ca++-dependent mechanism in their initial binding to one another.

Use of preformed cell aggregates and layers to measure tissue-specific differences in intercellular adhesion

The binding of aggregates formed from various 7-day chick embryo tissues to cultured cell layers was analyzed 24 hr following trypsin dissociation of the tissues. The proprotion of aggregates binding is independent of the number of aggregates added, and changes with time over 60 min in a manner consistent with a first-order process. The adhesive parameter measured, the percentage of aggregates binding to cell layers per unit time, varies slightly with aggregate size but is not dependent upon the probability of collision of the aggregate with the layer. The rate of binding and the effect of modifiers of binding (temperature, inhibitors of oxidative metabolism and glutaraldehyde) are substantially different for neural retina interactions than for liver or heart interactions, suggesting that retina cells may form intercellular bonds via a mechanism distinct from that of liver or heart cells. The rate of binding between like tissue types is, with one exception, greater than between unlike types. Glutaraldehyde treatment of only one of the reactants abolishes this adhesive specificity. Aggregate binding provides a means of quantitatively assessing intercellular adhesion which has the advantage of reducing the effects of trypsinization on measurements of adhesion, and therefore lends itself to the investigation of cellular consequences of adhesion.

FIBROBLAST CELL KINETICS IN THE PERIODONTAL LIGAMENT OF THE MOUSE

ABSTRACTTwelve male mice were injected intraperitoneally with tritiated thymidine. Six were sacrificed after 1 hr and six after 7 days. The right manibular incisors were dissected, cut sagittally and dipped in liquid emulsion. In the exposed and stained slides, observation was restricted to the lingual side of the periodontal ligament. Cells were evaluated sagittally from the basal tooth end up to the distance of 5 mm and up to the depth of 100 μm in the direction of socket wall. Cell and grain count was evaluated separately in 100 × 10 μm rectangles, creating a two‐dimensional array onto which the periodontal ligament was mapped.The progenitor compartment extends up to the distance of 2400 μm from origin. Fibroblasts leaving this compartment migrate at different velocities, creating a velocity profile across the ligament. Adjacent to the socket wall cell movement is sluggish, whereas the fastest cell movement is exhibited by cells located 20–30 μm from the tooth.The existence of such a profile indicates a continuous renewal of intercellular bonds, consistent with a process of actively pulling the incisor from its socket by the migrating fibrocytes.

The adhesion of liver cells

The adhesion of cells in the intact rat liver, and the surface properties of isolated cells have been studied. The removal of divalent ions, probably calcium, from the liver results in a loosening of the intercellular bond so that it may then be broken by a suitable force, but does not allow the cells to separate spontaneously. Treatment by a number of proteolytic enzymes, including purified collagenase and elastase, lead to a breakdown or liver structure, largely through a spontaneous loosening of the cells from each other. Liver cells isolated from the organ following perfusion with a chelating agent are tough refractile bodies which appear morphologically intact although they can be shown biochemically to have leaked important cell constituents. Staining with aniline blue reveals a clear-cut surface layer. Following enzymic treatment this layer disappears and the cells become less refractile, more fragile and flatten on a surface, although still intact. It has been found that isolated liver cells and cells from other organs and from solid tumours will reaggregate quickly into firm masses if injected intraperitoncally into mice. Cells which have been treated with proteolytic enzymes do not do so, nor do the cells from ascites tumours. Suggestions are put forward concerning the mechanisms of cell adhesion and it is postulated that protein materials play an important part in the adhesion of adult liver cells. There is so far no evidence that a mucopolysaccharide is involved. These mechanisms of cell adhesion may Well differ between types of tissue, between adult and embryonic and between normal and malignant tissues.