The differential adhesion hypothesis: a direct evaluation

The differential adhesion hypothesis (DAH), advanced in the 1960s, proposed that the liquid-like tissue-spreading and cell segregation phenomena of development arise from tissue surface tensions that in turn arise from differences in intercellular adhesiveness. Our earlier measurements of liquid-like cell aggregate surface tensions have shown that, without exception, a cell aggregate of lower surface tension tends to envelop one of higher surface tension to which it adheres. We here measure the surface tensions of L cell aggregates transfected to express N-, P- or E-cadherin in varied, measured amounts. We report that in these aggregates, in which cadherins are essentially the only cell-cell adhesion molecules, the aggregate surface tensions are a direct, linear function of cadherin expression level. Taken together with our earlier results, the conclusion follows that the liquid-like morphogenetic cell and tissue rearrangements of cell sorting, tissue spreading and segregation represent self-assembly processes guided by the diminution of adhesive-free energy as cells tend to maximize their mutual binding. This conclusion relates to the physics governing these morphogenetic phenomena and applies independently of issues such as the specificities of intercellular adhesives.     

Is cell sorting caused by differences in the work of intercellular adhesion? A critique of the steinberg hypothesis

The differential adhesion hypothesis, developed by Malcolm Steinberg, proposes that the histotypic sorting out behavior of aggregated cells is mechanistically equivalent to certain aspects of liquid surface tension, specifically the spontaneous separation of immiscible liquids of differing surface tension. According to Steinberg's hypothesis, the adhesive forces between aggregated cells play essentially the same role in cell sorting as are played by intermolecular attractive forces in liquid surface tension.In this paper I discuss a number of crucial distinctions between intermolecular attraction (in liquids) and intercellular adhesion (in aggregates). First, liquid drops are closed systems thermodynamically whereas aggregates of living cells can generate an indeterminate amount of metabolic energy capable of altering cell positions and adhesions. Secondly, intercellular adhesions are more than just close range attractions since cells can be held together by forces in addition to those which originally pulled them together. Third, the breakage of intercellular adhesions need not be simply the reverse, thermodynamically, of the formation of those adhesions. And fourthly, because intercellular adhesion is generally concentrated at relatively small foci such as desmosomes, a maximization of intercellular adhesion does not necessarily require a maximization of intercellular contact area, or vice versa.In addition, several alternative hypotheses are proposed, each of which is theoretically capable of explaining cell sorting and the other surface tension-like aspects of cell aggregate behavior which Steinberg has sought to explain as consequences of differential adhesion. In particular, a differential surface contraction hypothesis is proposed, according to which cell sorting and related phenomena are the results of cell surface contractions induced to occur over those portions of the cell surface which are exposed to the surrounding culture medium. Because of the evidence that various invagination type movements of embryonic epithelia are caused by cell surface contractions, it is suggested that differential surface contraction is the most likely explanation of histotypic cell sorting. A number of experiments are suggested by which these various hypotheses might be tested.     

Computer simulations of cell sorting due to differential adhesion

The actions of cell adhesion molecules, in particular, cadherins during embryonic development and morphogenesis more generally, regulate many aspects of cellular interactions, regulation and signaling. Often, a gradient of cadherin expression levels drives collective and relative cell motions generating macroscopic cell sorting. Computer simulations of cell sorting have focused on the interactions of cells with only a few discrete adhesion levels between cells, ignoring biologically observed continuous variations in expression levels and possible nonlinearities in molecular binding. In this paper, we present three models relating the surface density of cadherins to the net intercellular adhesion and interfacial tension for both discrete and continuous levels of cadherin expression. We then use then the Glazier-Graner-Hogeweg (GGH) model to investigate how variations in the distribution of the number of cadherins per cell and in the choice of binding model affect cell sorting. We find that an aggregate with a continuous variation in the level of a single type of cadherin molecule sorts more slowly than one with two levels. The rate of sorting increases strongly with the interfacial tension, which depends both on the maximum difference in number of cadherins per cell and on the binding model. Our approach helps connect signaling at the molecular level to tissue-level morphogenesis.     

Computational Modeling Reveals that a Combination of Chemotaxis and Differential Adhesion Leads to Robust Cell Sorting during Tissue Patterning

Robust tissue patterning is crucial to many processes during development. The "French Flag" model of patterning, whereby naïve cells in a gradient of diffusible morphogen signal adopt different fates due to exposure to different amounts of morphogen concentration, has been the most widely proposed model for tissue patterning. However, recently, using time-lapse experiments, cell sorting has been found to be an alternative model for tissue patterning in the zebrafish neural tube. But it remains unclear what the sorting mechanism is. In this article, we used computational modeling to show that two mechanisms, chemotaxis and differential adhesion, are needed for robust cell sorting. We assessed the performance of each of the two mechanisms by quantifying the fraction of correct sorting, the fraction of stable clusters formed after correct sorting, the time needed to achieve correct sorting, and the size variations of the cells having different fates. We found that chemotaxis and differential adhesion confer different advantages to the sorting process. Chemotaxis leads to high fraction of correct sorting as individual cells will either migrate towards or away from the source depending on its cell type. However after the cells have sorted correctly, there is no interaction among cells of the same type to stabilize the sorted boundaries, leading to cell clusters that are unstable. On the other hand, differential adhesion results in low fraction of correct clusters that are more stable. In the absence of morphogen gradient noise, a combination of both chemotaxis and differential adhesion yields cell sorting that is both accurate and robust. However, in the presence of gradient noise, the simple combination of chemotaxis and differential adhesion is insufficient for cell sorting; instead, chemotaxis coupled with delayed differential adhesion is required to yield optimal sorting.     

Genetic control of intercellular adhesion or how cadherins shape the fruitfly Drosophila melanogaster

The beauty and diversity of cell shapes have always fascinated both biologists and physicists. In the early 1950, J. Holtfreter coined the term "tissue affinities" to describe the forces behind the spontaneous shaping of groups of cells. These tissue affinites were later on related to adhesive properties of cell membranes. In the 1960, Malcom Steinberg proposed the differential adhesion hypothesis (DAH) as a physical explanation of the liquid-like behaviour of tissues and cells during morphogenesis. However, the link between the cellular properties of adhesion molecules, such as the cadherins, and the physical rules that shape the body, has remained unclear. Recent in vitro studies have now shown that surface tensions, which drive the spontaneous liquid-like behaviour of cell rearrangements, are a direct and linear function of cadherin expression levels. Tissue surface tensions thus arise from differences in intercellular adhesiveness, which validates the DAH in vitro. The DAH was also vindicated in vivo by stunning experiments in Drosophila. The powerful genetic tools available in Drosophila allow to manipulate the levels and patterns of expression of several cadherins and to create artificially differences in intercellular adhesiveness. The results showed that simple laws of thermodynamics, as well as quantitative and qualitative differences in cadherins expression were sufficient to explain processes as complex as the establishment of the anterior-posterior axis and the formation of the compound eye in Drosophila.     

Do β-lactam antibiotics permeabilize the outer membrane of gram-negative bacteria? An electrochemical investigation

Abstract The extent of short-term adhesion of various suspension-cultured plant cell species to polymer substrates exhibiting a wide range of surface tensions was examined. Adhesion of cells with a relatively low surface tension, suspended in distilled water, to the polymers fluorinated ethylenepropylene (FEP), polystyrene (PS), polyethylene terephthalate (PET), and sulphonated polystyrene (SPS) increased with decreasing substrate surface tension following the sequence SPS < PET < PS < FEP. These results are in agreement with the predictions of a thermodynamic model of particle adhesion which considers the role of the substrate, suspending-liquid, and cellular surface tensions. In contrast, little adhesion of relatively high surface tension cells to any of the polymer substrates was observed. Electrostatic repulsive forces between these cells and the polymer surface prevent adhesion because the magnitude of the attractive van der Waals force is small. A correlation was observed between the general adhesiveness of the various cultured plant cell species, especially to the low surface tension substrates, and the cellular surface tension determined by measuring the water contact angle on smooth layers of the cells. The cellular surface tensions ranged from approximately 42 mJ/m² for Digitalis purpurea cells to approximately 70mJ/m² for Papaver somniferum cells. Adhesion of cells to the polymer substrates increased with decreasing cellular surface tension under otherwise identical conditions. These results are also consistent with thermodynamic model predictions.     

Equilibrium mechanics of monolayered epithelium

In order to fully understand the epithelial mechanics it is essential to integrate different levels of epithelial organization. In this work, we propose a theoretical approach for connecting the macroscopic mechanical properties of a monolayered epithelium to the mechanical properties at the cellular level. The analysis is based on the established mechanical models—at the macroscopic scale the epithelium is described within the mechanics of thin layers, while the cellular level is modeled in terms of the cellular surface (cortical) tension and the intercellular adhesion. The macroscopic elastic energy of the epithelium is linked to the energy of an average epithelial cell. The epithelial equilibrium state is determined by energy minimization and the macroscopic elastic moduli are calculated from deformations around the equilibrium. The results indicate that the epithelial equilibrium state is defined by the ratio between the adhesion strength and the cellular surface tension. The lower and the upper bounds for this ratio are estimated. If the ratio is small, the epithelium is cuboidal, if it is large, the epithelium becomes columnar. Importantly, it is found that the cellular cortical tension and the intercellular adhesion alone cannot produce the flattened squamous epithelium. Any difference in the surface tension between the apical and basal cellular sides bends the epithelium towards the side with the larger surface tension. Interestingly, the analysis shows that the epithelial area expansivity modulus and the shear modulus depend only on the cellular surface tension and not on the intercellular adhesion. The results are presented in a general analytical form, and are thus applicable to a variety of monolayered epithelia, without relying on the specifics of numerical finite-element methods. In addition, by using the standard theoretical tools for multi-laminar systems, the results can be applied to epithelia consisting of layers with different mechanical properties.     

Estimating intercellular surface tension by laser-induced cell fusion

Intercellular surface tension is a key variable in understanding cellular mechanics. However, conventional methods are not well suited for measuring the absolute magnitude of intercellular surface tension because these methods require determination of the effective viscosity of the whole cell, a quantity that is difficult to measure. In this study, we present a novel method for estimating the intercellular surface tension at single-cell resolution. This method exploits the cytoplasmic flow that accompanies laser-induced cell fusion when the pressure difference between cells is large. Because the cytoplasmic viscosity can be measured using well-established technology, this method can be used to estimate the absolute magnitudes of tension. We applied this method to two-cell-stage embryos of the nematode Caenorhabditis elegans and estimated the intercellular surface tension to be in the 30-90 μN m(-1) range. Our estimate was in close agreement with cell-medium surface tensions measured at single-cell resolution.     

The mechanics of cell sorting and envelopment

Aggregates of embryonic cells undergo a variety of intriguing processes including sorting by histological type and envelopment of cell masses of one type by another. It has long been held that these processes were driven by differential adhesions, as embodied in the famous differential adhesion hypothesis (DAH). Here, we use analytical mechanics to investigate the forces that are generated by various sub-cellular structures including microfilaments, cell membranes and their associated proteins, and by sources of cell-cell adhesions. We consider how these forces cause the triple junctions between cells to move, and how these motions ultimately give rise to phenomena such as cell sorting and tissue envelopment. The analyses show that, contrary to the widely accepted DAH, differential adhesions alone are unable to drive sorting and envelopment. They show, instead, that these phenomena are driven by the combined effect of several force generators, as embodied in an equivalent surface or interfacial tension. These unconventional findings follow directly from the relevant surface physics and mechanics, and are consistent with well-known cell sorting and envelopment experiments, and with recent computer simulations.     

Thermodynamic aspects of plant cell adhesion to polymer surfaces

Summary A thermodynamic model of particle adhesion from a suspension onto a solid surface is used to predict the extent of adhesion of suspension-cultured Catharanthus roseus cells to the following polymer substrates: fluorinated ethylene-propylene (FEP), polystyrene (PS), polyethylene terephthalate (PET), sulphonated polystyrene (SPS), and glass. According to this model, the extent of adhesion is determined by the surface tensions of the plant cells, the polymer substrates, and the suspending liquid medium. Experimentally, adhesion of the washed plant cells was found to decrease with increasing substrate surface tension, following the sequence FEP>PS>PET>SPS>glass, when the surface tension of the liquid was greater than that of the plant cells, in agreement with the model. However, adhesion increased with increasing substrate surface tension when the liquid surface tension was lower than the cellular surface tension, also in agreement with the model. When the liquid and cellular tensions were equal the extent of adhesion was independent of the substrate surface tension. This also agrees with model predictions and leads to a value for the surface tension of C. roseus cells of approximately 54 ergs/cm² which is in agreement with a value obtained from contact angle measurements on layers of cells and sedimentation volume analysis. The cellular surface tension determined by the sedimentation volume method showed a biphasic alteration during growth cycles of C. roseus cell cultures. These variations (between 55 and 58 ergs/cm²) agree with the pattern of adhesion previously described.     

Surface thermodynamics of leukocyte and platelet adhesion to polymer surfaces

Adhesion of leukocytes and platelets to solid substrates of different surface tensions and hence different wettability is studied from a thermodynamic point of view. A simple thermodynamic model predicts that cellular adhesion should increase with increasing surface tension of the solid substrate if the surface tension of the medium in which the cells are suspended is lower than the surface tension of the cells. If the surface tension of the suspending medium is higher than that of the cells, the opposite behavior is predicted. These predictions are borne out completely by neutrophil adhesion tests, where the surface tension of the aequeous suspending medium is varied by addition of dimethyl sulfoxide (DMSO). Platelet adhesion experiments also confirm these predictions, the only difference being that surface tensions of the suspending medium above that of the platelets cannot be realized, owing to exudation of surface active solutes from the platelets. Utilization of the thermodynamic prediction that cellular adhesion should become independent of the surface tension of the substrate when the surface tensions of the cells and that of the suspending medium are equal leads to a value of the surface tension of neutrophils of 69.0 erg/cm^2,† in excellent agreement with the value obtained from contact angles measured on layers of cells.     

Cell adhesive affinity does not dictate primitive endoderm segregation and positioning during murine embryoid body formation

The classical cell sorting experiments undertaken by Townes and Holtfreter described the intrinsic propensity of dissociated embryonic cells to self‐organize and reconcile into their original embryonic germ layers with characteristic histotypic positioning. Steinberg presented the differential adhesion hypothesis to explain these patterning phenomena. Here, we have reappraised these issues by implementing embryoid bodies to model the patterning of epiblast and primitive endoderm layers. We have used combinations of embryonic stem (ES) cells and their derivatives differentiated by retinoic acid treatment to model epiblast and endoderm cells, and wild‐type or E‐cadherin null cells to represent strongly or weakly adherent cells, respectively. One cell type was fluorescently labeled and reconstituted with another heterotypically to generate chimeric embryoid bodies, and cell sorting was tracked by time‐lapse video microscopy and confirmed by immunostaining. When undifferentiated wild‐type and E‐cadherin null ES cells were mixed, the resulting cell aggregates consisted of a core of wild‐type cells surrounded by loosely associated E‐cadherin null cells, consistent with the differential adhesion hypothesis. However, when mixed with undifferentiated ES cells, the differentiated primitive endoderm‐like cells sorted to the surface to form a primitive endoderm layer irrespective of cell‐adhesive strength, contradicting the differential adhesion hypothesis. We propose that the primitive endoderm cells reach the surface by random movement, and subsequently the cells generate an apical/basal polarity that prevents reentry. Thus, the ability to generate epithelial polarity, rather than adhesive affinity, determines the surface positioning of the primitive endoderm cells. genesis 47:579–589, 2009. © 2009 Wiley‐Liss, Inc.     

New information from cell aggregate compression tests and its implications for theories of cell sorting

In order to verify theories about the mechanics of cell sorting, tissue spreading and checkerboard pattern formation, it is necessary to measure certain cell properties such as surface tension and adhesiveness. The purpose of this work is to clarify the relationship between these two important properties and to use computer simulations and analytical calculations to extract additional information from parallel plate compression tests. This paper shows that compression tests can be used to determine not only the surface tension between the aggregate and the surrounding medium, but also the effective viscosity of the cell cytoplasm and the interfacial tension that acts between the cells that make up the aggregate. The findings reported here also support a novel, differential interfacial tension-based theory for cell sorting, tissue spreading and checkerboard pattern formation, and pose further challenges to current differential adhesion-based models.     

Mechanical stress inference for two dimensional cell arrays

Many morphogenetic processes involve mechanical rearrangements of epithelial tissues that are driven by precisely regulated cytoskeletal forces and cell adhesion. The mechanical state of the cell and intercellular adhesion are not only the targets of regulation, but are themselves the likely signals that coordinate developmental process. Yet, because it is difficult to directly measure mechanical stress in vivo on sub-cellular scale, little is understood about the role of mechanics in development. Here we present an alternative approach which takes advantage of the recent progress in live imaging of morphogenetic processes and uses computational analysis of high resolution images of epithelial tissues to infer relative magnitude of forces acting within and between cells. We model intracellular stress in terms of bulk pressure and interfacial tension, allowing these parameters to vary from cell to cell and from interface to interface. Assuming that epithelial cell layers are close to mechanical equilibrium, we use the observed geometry of the two dimensional cell array to infer interfacial tensions and intracellular pressures. Here we present the mathematical formulation of the proposed Mechanical Inverse method and apply it to the analysis of epithelial cell layers observed at the onset of ventral furrow formation in the Drosophila embryo and in the process of hair-cell determination in the avian cochlea. The analysis reveals mechanical anisotropy in the former process and mechanical heterogeneity, correlated with cell differentiation, in the latter process. The proposed method opens a way for quantitative and detailed experimental tests of models of cell and tissue mechanics.     

Differential adhesion in morphogenesis: a modern view

The spreading of one embryonic tissue over another, the sorting out of their cells when intermixed and the formation of intertissue boundaries respected by the motile border cells all have counterparts in the behavior of immiscible liquids. The 'differential adhesion hypothesis' (DAH) explains these liquid-like tissue behaviors as consequences of the generation of tissue surface and interfacial tensions arising from the adhesion energies between motile cells. The experimental verification of the DAH, the recent computational models simulating adhesion-mediated morphogenesis, and the evidence concerning the role of differential adhesion in a number of morphodynamic events, including teleost epiboly, the specification of boundaries between rhombomeres in the developing vertebrate hindbrain, epithelial-mesenchymal transitions in embryos, and malignant invasion are reviewed here.     

Surface thermodynamics of leukocyte and platelet adhesion to polymer surfaces.

Adhesion of leukocytes and platelets to solid substrates of different surface tensions and hence different wettability is studied from a thermodynamic point of view. A simple thermodynamic model predicts that a cellular adhesion should increase with increasing surface tension of the solid substrate if the surface tension of the medium in which the cells are suspended is lower than the surface tension of the cells. If the surface tension of the suspending medium is higher than that of the cells, the opposite behavior is predicted. These predictions are borne out completely by neutrophil adhesion tests, where the surface tension of the aqueous suspending medium is varied by addition of dimethyl sulfoxide (DMSO). Platelet adhesion experiments also confirm these predictions, the only difference being that surface tensions of the suspending medium above that of the platelets cannot be realized, owing to exudation of surface active solutes from the platelets. Utilization of the thermodynamic prediction that cellular adhesion should become independent of the surface tension of the substrate when the surface tensions of the cells and that of the suspending medium are equal leads to a value of the surface tension of neutrophils of 69.0 erg/cm(2), in excellent agreement with the value obtained from contact angles measured on layers of cells.     

The history of tissue tension

In recent years the phenomenon of tissue tension and its functional connection to elongation growth has regained much interest. In the present study we reconstruct older models of mechanical inhomogenities in growing plant organs, in order to establish an accurate historical background for the current discussion. We focus on the iatromechanic model developed in Stephen Hales' Vegetable Staticks, Wilhelm Hofmeister's mechanical model of negative geotropism, Julius Sachs' explanation of the development of tissue tension, and the differential-auxin-response-hypothesis by Kenneth Thimann and Charles Schneider. Each of these models is considered in the context of its respective historic and theoretical environment. In particular, the dependency of the biomechanical hypotheses on the cell theory and the hormone concept is discussed. We arrive at the conclusion that the historical development until the middle of our century is adequately described as a development towards more detailed explanations of how differential tensions are established during elongation growth in plant organs. Then we compare with the older models the structure of more recent criticism of hormonal theories of tropic curvature, and particularly the epidermal-growth-control hypothesis of Ulrich Kutschera. In contrast to the more elaborate of the older hypotheses, the recent models do not attempt an explanation of differential tensions, but instead focus on mechanical processes in organs, in which tissue tension already exists. Some conceptual implications of this discrepancy, which apparently were overlooked in the recent discussion, are briefly evaluated.     

Continuous Models for Cell Migration in Tissues and Applications to Cell Sorting via Differential Chemotaxis

Chemotaxis, the guided migration of cells in response to chemical gradients, is vital to a wide variety of biological processes, including patterning of the slime mold Dictyostelium, embryonic morphogenesis, wound healing, and tumor invasion. Continuous models of chemotaxis have been developed to describe many such systems, yet few have considered the movements within a heterogeneous tissue composed of multiple subpopulations. In this paper, a partial differential equation (PDE) model is developed to describe a tissue formed from two distinct chemotactic populations. For a “crowded” (negligible extracellular space) tissue, it is demonstrated that the model reduces to a simpler one-species system while for an “uncrowded” tissue, it captures both movement of the entire tissue (via cells attaching to/migrating within an extracellular substrate) and the within-tissue rearrangements of the separate cellular subpopulations. The model is applied to explore the sorting of a heterogeneous tissue, where it is shown that differential-chemotaxis not only generates classical sorting patterns previously seen via differential-adhesion, but also demonstrates new classes of behavior. These new phenomena include temporal dynamics consisting of a traveling wave composed of spatially sorted subpopulations reminiscent of Dictyostelium slugs.     

IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin

Remodelling of tissues depends on the coordinated regulation of multiple cellular processes, such as cell-cell communication, differential cell adhesion and programmed cell death. During pupal development, interommatidial cells (IOCs) of the Drosophila eye initially form two or three cell rows between individual ommatidia, but then rearrange into a single row of cells. The surplus cells are eliminated by programmed cell death, and the definitive hexagonal array of cells is formed, which is the basis for the regular pattern of ommatidia visible in the adult eye. Here, we show that this cell-sorting process depends on the presence of a continuous belt of the homophilic cell adhesion protein DE-cadherin at the apical end of the IOCs. Elimination of this adhesion belt by mutations in shotgun, which encodes DE-cadherin, or its disruption by overexpression of DE-cadherin, the intracellular domain of Crumbs, or by a dominant version of the monomeric GTPase Rho1 prevents localisation of the transmembrane protein IrreC-rst to the border between primary pigment cells and IOCs. As a consequence, the IOCs are not properly sorted and supernumerary cells survive. During the sorting process, Notch-mediated signalling in IOCs acts downstream of DE-cadherin to restrict IrreC-rst to this border. The data are discussed in relation to the roles of selective cell adhesion and cell signalling during tissue reorganisation.     

T cell adhesion mechanisms revealed by receptor lateral mobility

Cell surface receptors mediate the exchange of information between cells and their environment. In the case of adhesion receptors, the spatial distribution and molecular associations of the receptors are critical to their function. Therefore, understanding the mechanisms regulating the distribution and binding associations of these molecules is necessary to understand their functional regulation. Experiments characterizing the lateral mobility of adhesion receptors have revealed a set of common mechanisms that control receptor function and thus cellular behavior. The T cell provides one of the most dynamic examples of cellular adhesion. An individual T cell makes innumerable intercellular contacts with antigen presenting cells, the vascular endothelium, and many other cell types. We review here the mechanisms that regulate T cell adhesion receptor lateral mobility as a window into the molecular regulation of these systems, and we present a general framework for understanding the principles and mechanisms that are likely to be common among these and other cellular adhesion systems. We suggest that receptor lateral mobility is regulated via four major mechanisms-reorganization, recruitment, dispersion, and anchoring-and we review specific examples of T cell adhesion receptor systems that utilize one or more of these mechanisms.