A model for shape generation by strain and cell-cell adhesion in the epithelium of an arthropod leg segment

We present a model for the energetic factors determining the most stable shape of a tubular epithelium such as the hypodermis of an arthropod leg segment. The model uses the analysis by Steinberg (1963) of rearrangement of cells in aggregates under the influence of differential adhesion, combining this analysis with the assumption that the epithelium behaves as an elastic sheet. The epithelium is assumed to consist of blocks of cells with different adhesive affinities, which remain unmixed in a quilt pattern. Rearrangement of cells within each block can adjust the shape of the tube by changing the shapes of the blocks. By means of such rearrangements the tube develops that shape which minimizes a free energy. The free energy is the difference between the energy of mechanical strain due to bending of the epithelium and the work of adhesion among cells. Minimization of the free energy for a cylindrical segment yields a scaling relation involving the length and radius of the segment. Leg segments of Drosophila conformed approximately to this relation, with deviations which suggest that a whole-limb pattern of adhesive affinities modulates the shaping effects of an adhesive pattern repeated in each leg segment. The model also predicts a transient deformation in an epithelium following a grafting operation. For example, deleting a slab of tissue from a tubular segment and reuniting the cut ends should produce a constriction of the tube at the host-graft junction. We propose that patterns of strain and adhesion can provide positional information which regulates subsequent development. Local increases in strain or adhesive disparity may stimulate mitoses; the resulting changes in distribution of cells will affect morphogenesis.     

Aggregation and disaggregation of red blood cells

The aggregation of red blood cells may be analyzed as an interaction of an adhesive surface energy and the elastic stored energy which results from deformation of the cell. The adhesive surface energy is the work required to separate a unit adhered area and is the resultant of adhesive forces due to the bridging molecules that bind the cells together and the electrostatic repulsion due to surface charge. The elastic strain energy in the case of the red blood is associated with the membrane elasticity only since the interior of the cell is liquid. The membrane elasticity is due both to bending stiffness and shear. The area expansion is small and may be neglected. These assumptions allow realistic computation of red cell shapes in rouleaux. The disaggregation of rouleaux requires an external force which must overcome the adhesive energy and also supply additional elastic energy of deformation. Depending on the geometry, the initial effect of elastic energy may tend to aid disaggregation. In a shear flow, the stresses on a suspended rouleau alternately tend to compress and to disaggregate the cells if they are free to rotate. This introduces a time dependence so that viscous effects due to the viscosity of the cell membrane, the cell cytoplasm and the external fluid may play a role in determining whether disaggregation proceeds to completion or not.     

Shear-driven rolling of DNA-adhesive microspheres

Many biologically important cell binding processes, such as the rolling of leukocytes in the vasculature, are multivalent, being mediated by large numbers of weak binding ligands. Quantitative agreement between experiments and models of rolling has been elusive and often limited by the poor understanding of the binding and unbinding kinetics of the ligands involved. Here, we present a cell-free experimental model for such rolling, consisting of polymer microspheres whose adhesion to a glass surface is mediated by ligands with well-understood force-dependent binding free energy—short complementary DNA strands. We observe robust rolling activity for certain values of the shear rate and the grafted DNA strands’ binding free energy and force sensitivity. The simulation framework developed to model leukocyte rolling, adhesive dynamics, quantitatively captures the mean rolling velocity and lateral diffusivity of the experimental particles using known values of the experimental parameters. Moreover, our model captures the velocity variations seen within the trajectories of single particles. Particle-to-particle variations can be attributed to small, plausible differences in particle characteristics. Overall, our findings confirm that state-of-the-art adhesive dynamics simulations are able to capture the complex physics of particle rolling, boding well for their extension to modeling more complex systems of rolling cells.     

Adhesive Interactions and the Patterning of Nerve Connections: An Experimental and Theoretical Approach

Adhesive interactions have long been proposed to play a centralrole in the patterning of neural structures and their interconnections.Many of these ideas are based upon experiments on the projectionfrom the eye to the optic tectum (the retinotectal projection)in lowervertebrates. In order to test the feasibility of suchproposals, a detailed model, based largely on adhesive interactionsbetween cells, has been developed. Computer simulations of themodelshow that simple adhesive interactions are sufficient toexplain much of the literature on the patterning of the amphibianretinotectal projection. Aspects of the model have been experimetallytested through the use of antibodies to known adhesive molecules.The results of these experiments appear consistent with therole of adhesion in the patterning of the connections and withthe predictions of the model. Although such experiments demonstratethe power of adhesive cell interactions in the patterning ofnerve connections, additional experiments and simulations demonstratethat some other non-adhesive processes may play a role. In particular,the addition of a process that is dependent on the activityof the neurons allows the model to better fit the literature.An activity-dependent competition between neurons for adhesivesites on the target cells appears to be sufficient to play thisrole.     

Energy of adhesion of human T cells to adsorption layers of monoclonal antibodies measured by a film trapping technique.

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level.     

A continuum approach to modelling cell-cell adhesion

Cells adhere to each other through the binding of cell adhesion molecules at the cell surface. This process, known as cell-cell adhesion, is fundamental in many areas of biology, including early embryo development, tissue homeostasis and tumour growth. In this paper we develop a new continuous mathematical model of this phenomenon by considering the movement of cells in response to the adhesive forces generated through binding. We demonstrate that our model predicts the aggregation behaviour of a disassociated adhesive cell population. Further, when the model is extended to represent the interactions between multiple populations, we demonstrate that it is capable of replicating the different types of cell sorting behaviour observed experimentally. The resulting pattern formation is a direct consequence of the relative strengths of self-population and cross-population adhesive bonds in the model. While cell sorting behaviour has been captured previously with discrete approaches, it has not, until now, been observed with a fully continuous model.     

A supramolecular theory for specificity in intracellular adhesion

This paper suggests that specificity in cell-cell adhesion may result from the supramolecular conformation or organization of cell surface adhesive molecules that may be similar or identical between cells of different adhesive affinities. A model is presented and its application to results from sea urchin gamete adhesion in vitro is discussed. In this system, we have observed that species specificity can be lost without losing adhesive capability. This suggests that specificity and adhesion reside at different levels of organization and the same or similar biochemical basis exists for gamete adhesive interactions of different species of urchins.     

Neurite extension across regions of low cell-substratum adhesivity: implications for the guidepost hypothesis of axonal pathfinding

According to the adhesive "guidepost" hypothesis, pioneer axons follow pathways marked by specific nonadjacent cells (guidepost cells). The hypothesis implies that high adhesivity between extending axons and guidepost cells facilitates axon extension across low-adhesivity tissues or spaces between guidepost cells. This study investigates the ability of a high-adhesivity substratum to promote axonal extension across a low-adhesivity substratum in vitro. Dissociated chick embryo dorsal root ganglion neurons are cultured on a substratum consisting of areas of high-adhesivity substratum-bound laminin (i.e., model adhesive guideposts) separated by a low-adhesivity agarose substratum. Increasing the cell-substratum adhesivity of these guideposts results in an increase in the percentage of neurites spanning a given width of the low-adhesivity substratum. Filopodial processes at the tips of neurites can extend over the low-adhesivity substratum. Apparently, filopodial contact with high-adhesivity guideposts enables neurites to extend across intervening low-adhesivity substrata. The maximum width of low-adhesivity substratum discontinuities spanned by some neurites in vitro is comparable to the distance between some putative guidepost cells in insects. Consistent with the adhesive guidepost hypothesis, these findings demonstrate neurite extension on a substratum of discontinuous cell-substratum adhesivity.     

Dynamic interplay between adhesive and lateral E-cadherin dimers

E-cadherin, an adhesive transmembrane protein of epithelial adherens junctions, forms two types of detergent-resistant dimers: adhesive dimers consisting of cadherin molecules derived from two neighboring cells and lateral dimers incorporating cadherins of the same cell. Both dimers depend on the integrity of the same residue, Trp156. While the relative amounts of these complexes are not certain, we show here that in epithelial A-431 cells, adhesive dimers may be a prevalent form. Inactivation of the calcium-binding sites, located between successive cadherin ectodomains, drastically reduced the amount of adhesive dimers and concomitantly increased the amount of lateral dimers. A similar interdependence of adhesive and lateral dimers was observed in digitonin-permeabilized cells. In these cells, adhesive dimers immediately disassembled after lowering the Ca2+ concentration below 0.1 mM. The disappearance of adhesive dimers was counterbalanced by an increase in Trp156-dependent lateral dimers. Increasing the calcium concentration to a normal level rapidly restored the original balance between adhesive and lateral dimers. We also present evidence that E-cadherin dimers in vivo have a short lifetime. These observations suggest that cadherin-mediated adhesion is based on the dynamic cycling of E-cadherin between monomeric and adhesive dimer states.     

The adhesion of clinical isolates of Campylobacter jejuni to intestinal epithelial cells in vitro

The adhesive activity of C. jejuni isolated from feces of children with Campylobacter infection was studied with the use of a newly developed model. 47 clinical isolates were analyzed; of these, 91% were found to be enteroadhesive to a variable degree. As the result of in vitro studies, Campylobacter were found to have much greater tropism to colonic cells and epithelial cells of Peyer's patches in comparison with the epithelial cells of the small intestine. The correlation between the degree of adhesive activity and the severity of the course of Campylobacter infection in children.     

A study of the adhesive, locomotory and invasive behaviour of Walker 256 carcinosarcoma cells

We have succeeded in selecting two variant strains of the Walker 256 carcinosarcoma which display markedly different adhesive properties. Both the high (W256A) and the low (W256S) adhesive variants respond chemotactically towards 10(-8) M f-met-leu-phe (FMLP) although there is a significant difference in their locomotory ability. Nevertheless, the fact that the essentially non-adherent W256S cells can migrate in vitro argues against any simple relationship between adhesion and locomotion. We suggest that traction is important in locomotion but that it need not arise only from direct adhesive interaction. We have also tested the invasive behaviour of the W256 variants using an in vitro model system in which disruption of a cellular barrier by the invasive cells can be recorded electrophysiologically. Although leucocytes can penetrate such a barrier they do so only under chemotactic stimulation, whereas W256 tumour cells of either variant strain will do so spontaneously. The tumour variants induce cell retraction within the barrier and this may lead ultimately to cell detachment and death. The holes which arise may then be colonized by tumour cells, and in this way the invasive process could be promoted. The molecular mechanisms by which tumour cells achieve destruction of the cellular barrier are not clear, but it is likely that a number of enzymes are involved.     

Probing the Interaction Forces of Prostate Cancer Cells with Collagen I and Bone Marrow Derived Stem Cells on the Single Cell Level

Adhesion of metastasizing prostate carcinoma cells was quantified for two carcinoma model cell lines LNCaP (lymph node-specific) and PC3 (bone marrow-specific). By time-lapse microscopy and force spectroscopy we found PC3 cells to preferentially adhere to bone marrow-derived mesenchymal stem cells (SCP1 cell line). Using atomic force microscopy (AFM) based force spectroscopy, the mechanical pattern of the adhesion to SCP1 cells was characterized for both prostate cancer cell lines and compared to a substrate consisting of pure collagen type I. PC3 cells dissipated more energy (27.6 aJ) during the forced de-adhesion AFM experiments and showed significantly more adhesive and stronger bonds compared to LNCaP cells (20.1 aJ). The characteristic signatures of the detachment force traces revealed that, in contrast to the LNCaP cells, PC3 cells seem to utilize their filopodia in addition to establish adhesive bonds. Taken together, our study clearly demonstrates that PC3 cells have a superior adhesive affinity to bone marrow mesenchymal stem cells, compared to LNCaP. Semi-quantitative PCR on both prostate carcinoma cell lines revealed the expression of two Col-I binding integrin receptors, α1β1 and α2β1 in PC3 cells, suggesting their possible involvement in the specific interaction to the substrates. Further understanding of the exact mechanisms behind this phenomenon might lead to optimized therapeutic applications targeting the metastatic behavior of certain prostate cancer cells towards bone tissue.     

Affinity of red blood cell membrane for particle surfaces measured by the extent of particle encapsulation.

An experimental technique and a simple analysis are presented that can be used to quantitate the affinity of red blood cell membrane for surfaces of small beads or microsomal particles up to 3 micrometers Diam. The technique is demonstrated with an example of dextran-mediated adhesion of small spherical red cell fragments to normal red blood cells. Cells and particles are positioned for contact by manipulation with glass micropipets. The mechanical equilibrium of the adhesive contact is represented by the variational expression that the decrease in interfacial free energy due to a virtual increase in contact area is balanced by the increase in elastic energy of the membrane due to virtual deformation. The surface affinity is the reduction in free energy per unit area of the interface associated with the formation of adhesive contact. From numerical computations of equilibrium configurations, the surface affinity is derived as a function of the fractional extent of particle encapsulation. The range of surface affinities for which the results are applicable is increased over previous techniques to several times the value of the elastic shear modulus. It is shown that bending rigidity of the membrane has little effect on the analytical results for particles 1--3 micrometers Diam and that results are essentially the same for both cup- and disk-shaped red cells. A simple analytical model is shown to give a good approximation for surface affinity (normalized by the elastic shear modulus) as a function of the fractional extent of particle encapsulation. The model predicts that a particle would be almost completely vacuolized for surface affinities greater than or equal to 10 times the elastic shear modulus. Based on an elastic shear modulus of 6.6 x 10(-3) dyn/cm, the range for the red cell-particle surface affinity as measured by this technique is from approximately 7 x 10(-4) to 7 x 10(-2) erg/cm2. Also, an approximate relation is derived for the level of surface affinity necessary to produce particle vacuolization by a phospholipid bilayer surface which possesses bending rigidity and a fixed tension.     

Synergistic and Hierarchical Adhesive and Topographic Guidance of BHK Cells

Guided cell movement is a fundamental process in development and regeneration. We have used microengineered culture substrates to study the interaction between model topographic and adhesive guidance cues in steering BHK cell orientation. Grooves 0.1, 0.5, 1.0, 3.0, and 6.0 μm deep together with pitch-matched aminosilane tracks 5, 12, 25, 50, and 100 μm wide were fabricated on fused silica substrates using photolithographic and dry-etching techniques. The cues were presented to the cells individually, simultaneously in parallel and orthogonally opposed. Cells aligned most strongly to 25-μm-wide adhesive tracks and to 5-μm-wide, 6-μm-deep grooves. Stress fibers and vinculin were found to align with the adhesive tracks and to the grooves and ridges. Cell alignment was profoundly enhanced on all surfaces that presented both cues in parallel. Cells were able to switch alignment from ridges to grooves, and vice versa, depending on the location of superimposed adhesive tracks. Cells aligned preferentially to adhesive tracks superimposed orthogonally over grooves of matched pitch, traversing numerous grooves and ridges. The strength of the cues was more closely matched on narrower 3- and 6-μm-deep gratings with cells showing evidence of alignment to both cues. Confocal fluorescence microscopy revealed two groups of mutually opposed f-actin stress fibers within the same cell, one oriented with the topographic cues and the other with the adhesive cues. However, the adhesive response was consistently dominant. We conclude that cells are able to detect and respond to multiple guidance cues simultaneously. The adhesive and topographic guidance cues modeled here were capable of interacting both synergistically and hierarchically to guide cell orientation.     

Characterization of monoclonal antibodies against human red blood group cells antigens by laser backscattering

A problem in immunohematology is to define the antibody quality which is related to its affinity expressed by the equilibrium constant. The activity of an antibody can be measured by the strength of its interaction, related to the adhesive energy exchanged during RBC agglutination which depends on the antigen-antibody liaison strength. To estimate this adhesive energy, two methods are used in this paper. Firstly, the dissociation behaviour of suspended RBC agglutinates was analysed by laser backscattering intensity (r) in a Couette flow. Backscattered intensity issued from shear-induced mechanical dissociation is recorded and submitted to a numerical process to obtain the energy parameter (ED). Secondly, a modification of this technique is proposed for measuring specific binding energy. Samples were exposed to increasing shear stress, and backscattered intensity was recorded. A constant increase of this intensity with raising shear stress was observed, pointed to a progressive dissociation of RBC agglutinates into smaller ones. Considering that complete dissociation of agglutinates is only approached asymptotically it is assumed that the final break-up of doublets (two-cell agglutinates) is produced at a critical shear stress (tauC) reflecting the work done to breaking-up the molecular bridges between both adjacent cells. This shear stress is defined by the extrapolation of the linear part of the curves [r-log tau] to the backscattered signal (r0) corresponding to the complete dispersion of RBCs. These approaches permit to define the specific surface adhesive energy (Gamma) by using the Derjaguin relation and to assess the functional characterization of specific immunoglobulins. In conclusion, two parameters characterizing monoclonal antibody agglutination properties, ED and Gamma, were estimated by laser backscattering methods, which could be very useful for antibodies quality control.     

The structure-function relationships in Drosophila neurotactin show that cholinesterasic domains may have adhesive properties.

Neurotactin (Nrt), a Drosophila transmembrane glycoprotein which is expressed in neuronal and epithelial tissues during embryonic and larval stages, exhibits heterophilic adhesive properties. The extracellular domain is composed of a catalytically inactive cholinesterase-like domain. A three-dimensional model deduced from the crystal structure of Torpedo acetylcholinesterase (AChE) has been constructed for Nrt and suggests that its extracellular domain is composed of two sub-domains organized around a gorge: an N-terminal region, whose three-dimensional structure is almost identical to that of Torpedo AChE, and a less conserved C-terminal region. By using truncated Nrt molecules and a homotypic cell aggregation assay which involves a soluble ligand activity, it has been possible to show that the adhesive function is localized in the N-terminal region of the extracellular domain comprised between His347 and His482. The C-terminal region of the protein can be removed without impairing Nrt adhesive properties, suggesting that the two sub-domains are structurally independent. Chimeric molecules in which the Nrt cholinesterase-like domain has been replaced by homologous domains from Drosophila AChE, Torpedo AChE or Drosophila glutactin (Glt), share similar adhesive properties. These properties may require the presence of Nrt cytoplasmic and transmembrane domains since authentic Drosophila AChE does not behave as an adhesive molecule when transfected in S2 cells.     

Ultrastructure of the adhesive tentacles of the limnomedusa Vallentinia gabriella (Hydrozoa,Olindiadidae)

Summary The specialized adhesive exumbrellar tentacles of the limnomedusa Vallentinia gabriella were examined by light microscopy and scanning and transmission electron microscopy. The adhesive region first differentiates some distance from the tentacle tip. As differentiation proceeds the distal part is reduced and the adhesive region comes to lie at the tentacle tip. The adhesive epithelium consists of flagellated and non-flagellated glandular cells, a few nematocytes, and a nerve plexus. The glandular cells are characterized by electron-dense granules and bundles of microtubules. The microtubules, being anchored to the mesoglea, are oriented parallel to the longitudinal axis of the cell and extend up to the cell apex. It can be assumed that the microtubules are involved in the transport of secretory granules to the cell apex. Bundles of neurites run adjacent to the mesoglea between the basal processes of the glandular cells. The neurites form interneural synapses and synapses with glandular cells. It is suggested that detachment of the specialized adhesive tentacles is under nervous control.     

The attachment complex of brachiolaria larvae of the sea star <Emphasis Type="Italic">Asterias rubens</Emphasis> (Echinodermata): an ultrastructural and immunocytochemical study

The attachment complex of brachiolaria larvae of the asteroid Asterias rubens comprises three brachiolar arms and an adhesive disc located on the preoral lobe. The former are used in temporary attachment and sensory testing of the substratum, whereas the latter is used for permanent fixation to the substratum at the onset of metamorphosis. Brachiolar arms are hollow structures consisting of an extensible stem tipped by a crown of dome-like ciliated papillae. The papilla epidermis is composed of secretory cells (type A, B and C cells), non-secretory ciliated cells, neurosecretory-like cells and support cells. Type A and B secretory cells fill a large part of the papilla epidermis and are always closely associated. They presumably form a duo-gland adhesive system in which type A and B cells are respectively adhesive and de-adhesive in function. The adhesive disc is an epidermal structure mainly composed of secretory cells and support cells. Secretory cells produce the cement, which anchor the metamorphic larva to the substratum until the podia are developed. The relatedness between the composition of the adhesive material in the brachiolaria attachment complex and in the podia of adults was investigated by immunocytochemistry using antibodies raised against podial adhesive secretions of A. rubens. Type A secretory cells were the only immunolabelled cells indicating that their temporary adhesive shares common epitopes with the one of podia. The attachment pattern displayed by the individuals of A. rubens during the perimetamorphic period—temporary, permanent, temporary—is unique among marine non-vertebrate Metazoa.     

Dynamic adhesion and separation of cells in vitro. I. Mathematical analysis of the experimental system

The contact interaction between cells and thewalls of microchambers through which the cells are passing are quantitated in terms of parameters which are estimated from macroscopic experimental data. For this purpose, an analysis is made of the internal geometry of glass bead-columns. A mathematical model is presented which describes the passage of cells down the column. This model relates the temporal pattern of the number of cells entering and leaving the bead-column to the “dynamic adhesive” properties of a cell as it passes through a microscopic section of the bead-column.     

Functional morphology of coronal and peristomeal podia in Sphaerechinus granularis (Echinodermata,Echinoida)

Summary Coronal podia of Sphaerechinus granularis are anchoring (adhering) appendages involved in either locomotion or capture of drift materials. Adhesion is not due to the presumed sucker action of the disc but relies entirely on secretions of the disc epidermis. Peristomeal podia function in wrapping together food particles or food fragments in an adhesive material thus facilitating their capture by the Aristotle's lantern. In both types of podia, the disc epidermis is made up of four cell types: non-ciliated secretory cells (NCS cells) that contain graules whose content is at least partly mucopolysaccharidic in nature, ciliated secretory cells (CS cells) containing granules of unknown nature, ciliated non-secretory cells (CNS cells) and support cells. The cilia of CS cells are subeuticular whereas those of CNS cells, although also short and rigid, traverse the cuticle and protrude in the outer medium. All these cells are presumably involved in an adhesive/de-adhesive process functioning as a duogland adhesive system. Adhesive secretion would be produced by NCS cells and de-adhesive secretion by CS cells. These secretions would be controlled through stimulations by the two types of ciliated cells (receptor cells) which presumably interact with the secretory cells by way of the nerve plexus. This model of adhesion/de-adhesion fits well with the activities of both coronal and peristomeal podia. The secretion of NCS cells would make up a bridge of adhesive material between a podium and the substratum (coronal podia) or would coat and gather food particles (peristomeal podia), respectively. The de-adhesive material enclosed in the granules of CS cells would allow the podia (either coronal or peristomeal) to easily become detached from the substratum and to always remain clear of any particles.Research Assistant, National Fund for Scientific Research (Belgium)