Heterophilic Interactions Research Articles

A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns

Synthetic multicellular systems hold promise as models for understanding natural development of biofilms and higher organisms and as tools for engineering complex multi-component metabolic pathways and materials. However, such efforts require tools to adhere cells into defined morphologies and patterns, and these tools are currently lacking. Here, we report a100% genetically encoded synthetic platform for modular cell-cell adhesion in Escherichia coli, which provides control over multicellular self-assembly. Adhesive selectivity is provided by a library of outer membrane-displayed nanobodies and antigens with orthogonal intra-library specificities, while affinity iscontrolled by intrinsic adhesin affinity, competitive inhibition, and inducible expression. We demonstrate the resulting capabilities for quantitative rational design of well-defined morphologies and patterns through homophilic and heterophilic interactions, lattice-like self-assembly, phase separation, differential adhesion, and sequential layering. Compatible with synthetic biology standards, this adhesion toolbox will enable construction of high-level multicellular designs and shed light on the evolutionary transition to multicellularity.

Abstract 1321: Mutational landscape of adherens junctions in skin cutaneous melanoma

Abstract Cadherin proteins regulate cell-cell contact through calcium-dependent manner, thus play pivotal roles in morphogenetic and differentiation processes during tissue development. Reprograming in cadherin profiles contributes to epithelial-mesenchymal transition (EMT), leading to disorganized lining of epithelial tissues and cancer metastasis. To elucidate genetic impacts of classical cadherins on melanoma development, mutation profiles were analyzed using data from the TCGA databank. Among 20 classical cadherins, CDH6 showed the highest mutation frequency (11.3 %), and most of which were G to A or C to T nucleotide substitutions, a signature for UV-caused DNA damage. Two groups of unique hotspots were found on the fifth cadherin domain (D5) (S524L and S526F) and cytoplasmic cadherin domain (CDH-C) (E662K and E722K), respectively. Protein interactome and expression co-occurrence analyses placed CDH6 at the center of core cadherin network and showed strong heterophilic interactions of CDH6 with CDH4, CDH9, and CDH12. Multiple sequence alignment indicated the involvement of those hotspots in the conserved regions, which are responsible for heterophilic interaction on D5 or catenin (alpha, beta, and gamma) binding on CDH-C. Molecular modeling defined alterations in protein structures of the D5 and CDH-C domains caused by the hotspot mutations, which may influence cell-cell contact and the down-stream signaling. In sum, our data suggest CDH6 as the key player to modulate adherens junctions during the development of melanoma. Citation Format: Praveen Kumar Korla, Daniel Esguerra Gracilla, Brian Yu-Ting Kuo, Jim Jinn-Chyuan Sheu. Mutational landscape of adherens junctions in skin cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1321.

Ultrastructural changes in endometrial desmosomes of desmoglein 2 mutant mice.

The intercellular binding of desmosomal junctions is mediated by cadherins of the desmoglein (Dsg) and desmocollin (Dsc) type. Dsg2 mutant mice with deletion of a substantial segment of the extracellular EC1-EC2 domain, which is believed to participate in homo- and heterophilic desmosomal cadherin interactions, develop cardiac fibrosis and ventricular dilation. Widening of the intercellular cleft and complete intercalated disc ruptures can be observed in the hearts of these mice. Since a reduced litter size of homozygous Dsg2 mutant mice was noted and a functional correlation between desmosomes and embryo implantation has been deduced from animal studies, we looked for an alteration of desmosomes in uterine endometrial epithelium. Shape and number of desmosomes as well as the expression of Dsg2 and the desmosomal plaque protein desmoplakin (Dsp) were investigated by electron microscopy and immunohistochemistry in 12 oestrous-dated mice (7 wildtype and 5 homozygous Dsg2 mutant mice) at the age of 9-17weeks. The immunohistochemical detection of Dsg2 was diminished in the mutants and the number of desmosomes was significantly reduced as revealed by electron microscopy. In addition, the intercellular desmosomal space measured in electron micrographs was considerably widened in the Dsg2 mutants. The increased intercellular spacing can be explained by the partial deletion of the extracellular EC1-EC2 domain of Dsg2. Whether these changes explain the reduced number of offspring of homozygous Dsg2 mutant mice remains to be further investigated.

Helicobacter pylori adhesin HopQ disrupts trans dimerization in human CEACAMs.

The human gastric pathogen Helicobacter pylori is a major causative agent of gastritis, peptic ulcer disease, and gastric cancer. Aspart of its adhesive lifestyle, the bacterium targets members ofthe carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family by the conserved outer membrane adhesin HopQ. The HopQ-CEACAM1 interaction is associated with inflammatory responses and enables the intracellular delivery and phosphorylation of the CagA oncoprotein via a yet unknown mechanism. Here, we generated crystal structures of HopQ isotypes I and II bound to the N-terminal domain of human CEACAM1 (C1ND) and elucidated the structural basis of H.pylori specificity toward human CEACAM receptors. Both HopQ alleles target the β-strands G, F, and C of C1ND, which form the trans dimerization interface in homo- and heterophilic CEACAM interactions. Using SAXS, we show that the HopQ ectodomain is sufficient to induce C1ND monomerization and thus providing H.pylori a route to influence CEACAM-mediated cell adherence and signaling events.

Homophilic and Heterophilic Interactions of Type II Cadherins Identify Specificity Groups Underlying Cell-Adhesive Behavior

SUMMARYType II cadherins are cell-cell adhesion proteins critical for tissue patterning and neuronal targeting but whose molecular binding code remains poorly understood. Here, we delineate binding preferences for type II cadherin cell-adhesive regions, revealing extensive heterophilic interactions between specific pairs, in addition to homophilic interactions. Three distinct specificity groups emerge from our analysis with members that share highly similar heterophilic binding patterns and favor binding to one another. Structures of adhesive fragments from each specificity group confirm near-identical dimer topology conserved throughout the family, allowing interface residues whose conservation corresponds to specificity preferences to be identified. We show that targeted mutation of these residues converts binding preferences between specificity groups in biophysical and co-culture assays. Our results provide a detailed understanding of the type II cadherin interaction map and a basis for defining their role in tissue patterning and for the emerging importance of their heterophilic interactions in neural connectivity.

Studying Variations in CEACAM1 Nanoscale Organization, Structure, and Dynamics

CEACAMs are cell surface glycoproteins involved in homo- and hetero-philic intercellular interactions that control cellular growth, differentiation, tumourigenesis, inflammation and infection. There is growing evidence regarding the role that CEACAM1 isoforms play in regulating interactions between various cell types. In particular, given its potential as a tumour suppressor or immunomodulator, CEACAM1 is being targeted in clinical trials. However, there remain clear challenges owing to observation that different CEACAM1 isoforms display cell-type specific preferential expression patterns under different physiological conditions. Moreover, the ability of CEACAMs to form dimers and higher-order oligomers, which are thought to impact regulation of intercellular signals, adds considerable complexity to our understanding of its functional role(s). Super resolution microscopy has enabled the quantitative characterization of the nanoscale distribution of many membrane proteins, including their ability to form both micro- and nano-scale clusters. However, clustering and self-association are not necessarily the same and mapping how oligomeric state impact clustering and / or the oligomeric state of the proteins within a cluster is of particular interest to many. We report here on the development and application of a correlative STORM/homoFRET strategy to study CEACAM1 isoforms and mutants. This approach exploits eYFP for homoFRET measurements, an Alexa Fluor 647 (AF647) labeled an anti-eYFP single domain antibody (nanobody) for dSTORM, and Voronoi tessalation to perform cluster analysis of dSTORM dataset. Building on these data, we have also applied co-localization approaches to measure the CEACAM1 association to lipid-ordered regions, ezrin, and actin. We have further explored the use of single particle tracking (SPT) and mean square displacement analysis (MSD) to characterize CEACAM1 dynamics as a function of oligomeric state and location. These studies have helped develop a more fulsome perspective on the structure, association, and dynamics of this important class of membrane proteins.

ILT3.Fc-CD166 Interaction Induces Inactivation of p70 S6 Kinase and Inhibits Tumor Cell Growth.

The blockade of immune checkpoints by anti-receptor and/or anti-ligand mAb is one of the most promising approaches to cancer immunotherapy. The interaction between Ig-like transcript 3 (ILT3), a marker of tolerogenic dendritic cells, also known as LILRB4/LIR5/CD85k, and its still unidentified ligand on the surface of activated human T cells is potentially important for immune checkpoint blockade. To identify the ILT3 ligand, we generated mAb by immunizing mice with Jurkat acute T cell leukemia, which binds ILT3.Fc to its membrane. Flow cytometry, mass spectrometry, and Biacore studies demonstrated that the ILT3 ligand is a CD166/activated leukocyte cell adhesion molecule. Knockdown of CD166 in primary human T cells by nucleofection abolished the capacity of ILT3.Fc to inhibit CD4+ Th cell proliferation and to induce the generation of CD8+CD28- T suppressor cells. CD166 displays strong heterophilic interaction with CD6 and weaker homophilic CD166-CD166 cell adhesion interaction. ILT3.Fc inhibited the growth of CD166+ tumor cell lines (TCL) derived from lymphoid malignancies in vitro and in vivo. CRISPR-Cas9-based knockout of CD166 from TCL abrogated ILT3.Fc binding and its tumor-inhibitory effect. The mechanism underlying the effect of ILT3.Fc on tumor cell growth involves inhibition of the p70S6K signaling pathway. Blockade of CD166 by ILT3.Fc inhibited progression of human TCL in NOD.Cg-Prkdc Il-2rg/SzJ mice, suggesting its potential immunotherapeutic value.

Structural-Mechanical and Biochemical Functions of Classical Cadherins at Cellular Junctions: A Review and Some Hypotheses.

This article begins with a general review of cell adhesion molecules (CAMs) and narrows the focus down progressively to the cadherins (calcium binding-dependent CAMs), classifications of subfamilies of the cadherins, type I (E- and N-) cadherins, evolutionary relationships amongst cadherins, structural-mechanical and functional consequences of calcium binding to the cadherins, differential molecular interactions involving the extracellular (ecto) and intracellular (cytoplasmic) domains of the cadherins, multiple adherence-related homophilic and heterophilic interactions and associated functions of E- and N-cadherin in organismal development and disease and cadherin trafficking and membrane rafts. It ends by summarizing multiple perspectives and hypotheses concerning different aspects of cadherin structure, stability and function.

A Synthetic Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns

Synthetic multicellular systems hold promise for understanding natural development of biofilms and higher organisms, as well as for engineering complex multi-component metabolic pathways and materials. However, such efforts require tools to adhere cells into defined morphologies and patterns, and these tools are currently lacking. Here we report the first 100% genetically encoded synthetic platform for modular cell-cell adhesion in Escherichia coli, which provides control over multicellular self-assembly. Adhesive selectivity is provided by a library of outer membrane-displayed nanobody and antigen peptides with orthogonal intra-library specificities, while affinity is controlled by intrinsic adhesin affinity, competitive inhibition, and inducible expression. We demonstrate the resulting capabilities for quantitative rational design of well-defined morphologies and patterns through homophilic and heterophilic interactions, lattice-like self-assembly, phase separation, differential adhesion, and sequential layering. This adhesion toolbox, compatible with synthetic biology standards, will enable construction of high-level multicellular designs and shed light on the evolutionary transition to multicellularity.

Evaluation of Cytochalasin B-Induced Membrane Vesicles Fusion Specificity with Target Cells.

Extracellular vesicles (EV) represent a promising vector system for biomolecules and drug delivery due to their natural origin and participation in intercellular communication. As the quantity of EVs is limited, it was proposed to induce the release of membrane vesicles from the surface of human cells by treatment with cytochalasin B. Cytochalasin B-induced membrane vesicles (CIMVs) were successfully tested as a vector for delivery of dye, nanoparticles, and a chemotherapeutic. However, it remained unclear whether CIMVs possess fusion specificity with target cells and thus might be used for more targeted delivery of therapeutics. To answer this question, CIMVs were obtained from human prostate cancer PC3 cells. The diameter of obtained CIMVs was 962,13 ± 140,6 nm. We found that there is no statistically significant preference in PC3 CIMVs fusion with target cells of the same type. According to our observations, the greatest impact on CIMVs entry into target cells is by the heterophilic interaction of CIMV membrane receptors with the surface proteins of target cells.

The Impact of Adhesion Molecules on the In Vitro Culture and Differentiation of Stem Cells

The term "cell adhesion" represents cell-cell interactions and the interaction between the cell and the extracellular matrix (ECM). These interactions are crucial for the development of the stem cells niche to determine stem cell shape. The ECM is considered as a natural niche for cell residence. Cell adhesion molecules (CAMs) enable cell-cell interactions and the interactions between the cell and ECM via various mechanisms, such as trans-interaction and the heterophilic interactions. These interactions promote a broad spectrum of cell signaling that directly or indirectly modulates stem cell proliferation, self-renewal property, adhesion, and multilineage differentiation. Over many years, animal-derived feeder layers and culture components have been used for stem cell culture, which produces stem cells unsuitable for clinical applications in the regenerative medicine. This review briefly describes the stages of the development of stem cell culture toward a defined condition and the drawbacks of using animal-derived culture components. Stem cell niche-derived and ECM-derived adhesion molecules and their detailed impact on stem cell adhesion and functions will be discussed. Efficient and novel adhesion molecules for stem cell culture and differentiation are needed for further large-scale applications in tissue regeneration.

Different properties of polysialic acids synthesized by the polysialyltransferases ST8SIA2 and ST8SIA4.

Polysialic acid (polySia) is mainly found as a modification of neural cell adhesion molecule (NCAM) in whole embryonic brains, as well as restricted areas of adult vertebrate brains, including the hippocampus. PolySia shows not only repulsive effects on NCAM-involved cell-cell interactions due to its bulky and hydrated properties, but also attractive effects on the interaction with neurologically active molecules, which exerts a reservoir function. Two different polysialyltransferases, ST8SIA2 and ST8SIA4, are involved in the synthesis of polySia chains; however, to date, the differences of the properties between polySia chains synthesized by these two enzymes remain unknown. In this study, to clarify this point, we first prepared polySia-NCAMs from HEK293 cells stably expressing ST8SIA4 and ST8SIA2, or ST8SIA2 (SNP-7), a mutant ST8SIA2 derived from a schizophrenia patient. The conventional sensitive chemical and immunological characterizations showed that the quantity and quality (structural features) of polySia are not so much different between ST8SIA4- and ST8SIA2-synthesized ones, apart from those of ST8SIA2 (SNP-7). Then, we assessed the homophilic and heterophilic interactions mediated by polySia-NCAM by adopting a surface plasmon resonance measurement as an in vitro analytical method. Our novel findings are as follows: (i) the ST8SIA2- and ST8SIA4-synthesized polySia-NCAMs exhibited different attractive and repulsive effects than each other; (ii) both polySia- and oligoSia-NCAMs synthesized by ST8SIA2 were able to bind polySia-NCAMs; (iii) the polySia-NCAM synthesized by a ST8SIA2 (SNP-7) showed markedly altered attractive and repulsive properties. Collectively, polySia-NCAM is suggested to simultaneously possess both attractive and repulsive properties that are highly regulated by the two polysialyltransferases.

CD15s/CD62E Interaction Mediates the Adhesion of Non-Small Cell Lung Cancer Cells on Brain Endothelial Cells: Implications for Cerebral Metastasis.

Expression of the cell adhesion molecule (CAM), Sialyl Lewis X (CD15s) correlates with cancer metastasis, while expression of E-selectin (CD62E) is stimulated by TNF-α. CD15s/CD62E interaction plays a key role in the homing process of circulating leukocytes. We investigated the heterophilic interaction of CD15s and CD62E in brain metastasis-related cancer cell adhesion. CD15s and CD62E were characterised in human brain endothelium (hCMEC/D3), primary non-small cell lung cancer (NSCLC) (COR-L105 and A549) and metastatic NSCLC (SEBTA-001 and NCI-H1299) using immunocytochemistry, Western blotting, flow cytometry and immunohistochemistry in human brain tissue sections. TNF-α (25 pg/mL) stimulated extracellular expression of CD62E while adhesion assays, under both static and physiological flow live-cell conditions, explored the effect of CD15s-mAb immunoblocking on adhesion of cancer cell–brain endothelium. CD15s was faintly expressed on hCMEC/D3, while high levels were observed on primary NSCLC cells with expression highest on metastatic NSCLC cells (p < 0.001). CD62E was highly expressed on hCMEC/D3 cells activated with TNF-α, with lower levels on primary and metastatic NSCLC cells. CD15s and CD62E were expressed on lung metastatic brain biopsies. CD15s/CD62E interaction was localised at adhesion sites of cancer cell–brain endothelium. CD15s immunoblocking significantly decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (p < 0.001), highlighting the role of CD15s–CD62E interaction in brain metastasis.

Dynamic expression of nectins in enamel organs of mouse incisors

ObjectivesNectins are immunoglobulin-like cell–cell adhesion molecules and are of four types, namely, nectin-1, nectin-2, nectin-3, and nectin-4. Cleft lip/palate-ectodermal dysplasia is caused by a mutation in the nectin-1 gene locus. However, nectin-1-deficient (KO) mice only show mild tooth defects. This study determined the intracellular localization of nectins in mouse mandibular incisors to identify their heterophilic interactions during amelogenesis. MethodsNectin localization was determined by performing immunohistochemical analysis with confocal microscopy. Nectin gene expression was determined by performing quantitative reverse transcription-PCR. Phenotypes of nectin-2-KO mice were examined by performing micro-computed tomography, histological analysis, and organ culture. ResultsWe found that mRNA levels of nectin-1 and nectin-3 genes were higher than those of nectin-2 and nectin-4 genes in mouse enamel organs. Nectin-2 and nectin-4 were strongly expressed at the apical adherens junctions of secretory-stage ameloblasts, whereas nectin-1 and nectin-3 were distributed between the basal adherens junction of maturation-stage ameloblasts and the stratum intermedium and papillary layers. Nectin-2-KO mice showed normal mandibular tooth shape, incisor surface pigmentation, and histology. Moreover, organ cultures of the tooth organs of nectin-2-KO mice proceeded normally. ConclusionThese results indicate that nectins show graded and overlapping distribution in mouse incisors.

Mapping Ceacam 1-4L's Nanoscale Spatial Distribution, Structure, Dynamics, and Association with Lipid Ordered Domains by Super-Resolution Microscopy

Super-resolution microscopy has provided novel quantitative insights into the nanoscale spatial distribution of membrane proteins, including the observation that some systems form micro- and nano-sized clusters. However, details of the protein's self-association state (monomer / dimer / oligomer) within the different cluster types are often missing. Since it has been shown that a protein's oligomeric form might be a crucial determinant of its function, it is important to have a technique capable of measuring both spatial distribution and association state. To that end, we have applied a correlated dSTORM/homo-FRET approach to characterize CEACAM 1-4L, the progenitor isoform of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAMs) family. CEACAMs are cell surface glycoproteins involved in homo- and hetero-philic intercellular interactions that control cellular growth, differentiation, tumourigenesis, inflammation and infection. While CEACAM1-4L is known to exist as a monomer or oligomer with the former being hypothesized to be involved in activatory intercellular interactions, the basis for its spatial distribution and association state as a function of distribution remain poorly understood. Our correlated approach involved live cell homo-FRET studies of eYFP-CEACAM1-4L in HeLa cells followed by fixed cell dSTORM imaging performed using nanobody labeling with Alexa Fluor 647 (AF647) conjugated to an anti-eYFP single domain antibody. Cluster analysis of the dSTORM datasets using Voronoi tessellation revealed that the CEACAM1-4L nanoclusters were preferentially comprised of monomeric CEACAM1-4L while microclusters contained a heterogeneous distribution of CEACAM1-4L monomers and oligomers. To further characterize these clusters, super-resolution imaging using Alexa-647 labelled cholera toxin was performed to examine the co-localization of lipid ordered domains, which are known to drive membrane protein lateral distribution and clustering, with CEACAM1-4L. Finally, live-cell single particle tracking (SPT) of CEACAM1-4L was performed to investigate the putative confined, obstructed, free diffusion, and directed motion dynamics of CEACAM1-4L.

The functional roles of T-cadherin in mammalian biology

T-cadherin is a cadherin and cell adhesion molecule that is anchored to the cell surface membrane through a glycosylphosphatidylinositol moiety. T-cadherin lacks a transmembrane and cytoskeletal domain, suggesting that it must interact with other membrane-bound molecules to elicit cellular signaling to modulate normal cellular functions, and alternatively its absence can be a factor in promoting neoplasia. Moreover, apart from binding to itself it can sequester adiponectin to the cell surface. Consistent with these observations, recent research has expanded the scope of T-cadherin’s role in cancer, neuronal function, metabolism and cardiovascular disease. In this context, we highlight the experimental and genomic evidence that links T-cadherin with these diseases. In particular, we discuss how T-cadherin homophilic and heterophilic interactions impact on signaling pathways and cellular behavior.

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC'C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC'C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions.

3D Structure and Interaction of p24β and p24δ Golgi Dynamics Domains: Implication for p24 Complex Formation and Cargo Transport

The p24 family consists of four subfamilies (p24α, p24β, p24γ, and p24δ), and the proteins are thought to form hetero-oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. The proteins possess a conserved luminal Golgi dynamics (GOLD) domain, whose functions are largely unknown. Here, we present structural and biochemical studies of p24β1 and p24δ1 GOLD domains. Use of GOLD domain-deleted mutants revealed that the GOLD domain of p24δ1 is required for proper p24 hetero-oligomeric complex formation and efficient transport of GPI-anchored proteins. The p24β1 and p24δ1 GOLD domains share a common β-sandwich fold with a characteristic intrasheet disulfide bond. The GOLD domain of p24δ1 crystallized as dimers, allowing the analysis of a homophilic interaction site. Surface plasmon resonance and solution NMR analyses revealed that p24β1 and p24δ1 GOLD domains interact weakly (Kd= ~10−4M). Bi-protein titration provided interaction site maps. We propose that the heterophilic interaction of p24 GOLD domains contributes to the formation of the p24 hetero-oligomeric complex and to efficient cargo transport.

Glycosylation-dependent binding of galectin-8 to activated leukocyte cell adhesion molecule (ALCAM/CD166) promotes its surface segregation on breast cancer cells

BackgroundWe previously demonstrated that the activated leukocyte cell adhesion molecule (ALCAM/CD166) can interact with galectin-8 (Gal-8) in endothelial cells. ALCAM is a member of the immunoglobulin superfamily that promotes homophilic and heterophilic cell–cell interactions. Gal-8 is a “tandem-repeat”-type galectin, known as a matricellular protein involved in cell adhesion. Here, we analyzed the physical interaction between both molecules in breast cancer cells and the functional relevance of this phenomenon. MethodsWe performed binding assays by surface plasmon resonance to study the interaction between Gal-8 and the recombinant glycosylated ALCAM ectodomain or endogenous ALCAM from MDA-MB-231 breast cancer cells. We also analyzed the binding of ALCAM-silenced or control breast cancer cells to immobilized Gal-8 by SPR. In internalization assays, we evaluated the influence of Gal-8 on ALCAM surface localization. ResultsWe showed that recombinant glycosylated ALCAM and endogenous ALCAM from breast carcinoma cells physically interacted with Gal-8 in a glycosylation-dependent fashion displaying a differential behavior compared to non-glycosylated ALCAM. Moreover, ALCAM-silenced breast cancer cells exhibited reduced binding to Gal-8 relative to control cells. Importantly, exogenously added Gal-8 provoked ALCAM segregation, probably trapping this adhesion molecule at the surface of breast cancer cells. ConclusionsOur data indicate that Gal-8 interacts with ALCAM at the surface of breast cancer cells through glycosylation-dependent mechanisms. General significanceA novel heterophilic interaction between ALCAM and Gal-8 is demonstrated here, suggesting its physiologic relevance in the biology of breast cancer cells.

Evolutionary Dynamics of Homophily and Heterophily.

Most social interactions do not take place at random. In many situations, individuals choose their interaction partners on the basis of phenotypic cues. When this happens, individuals are often homophilic, that is, they tend to interact with individuals that are similar to them. Here we investigate the joint evolution of phenotypic cues and cue-dependent interaction strategies. By a combination of individual-based simulations and analytical arguments, we show that homophily evolves less easily than earlier studies suggest. The evolutionary interplay of cues and cue-based behaviour is intricate and has many interesting facets. For example, an interaction strategy like heterophily may stably persist in the population even if it is selected against in association with any particular cue. Homophily persisted for extensive periods of time just in those simulations where homophilic interactions provide a lower (rather than a higher) payoff than heterophilic interactions. Our results indicate that even the simplest cue-based social interactions can have rich dynamics and a surprising diversity of evolutionary outcomes.