Cdc42, Rac1, and their effector IQGAP1 as molecular switches for cadherin-mediated cell-cell adhesion.

Cell-cell adhesion is a dynamic process in various cellular and developmental situations. Cadherins, well-known Ca(2+)-dependent adhesion molecules, are thought to play a major role in the regulation of cell-cell adhesion. However, the molecular mechanism underlying the rearrangement of cadherin-mediated cell-cell adhesion is largely unknown. Cdc42 and Rac1, belonging to the Rho small GTPase family, have recently been shown to be involved in the regulation of cell-cell adhesion. In addition, IQGAP1, an effector for Cdc42 and Rac1, has been shown to regulate the cadherin function through interaction with beta-catenin, a molecule associated with cadherin. In this review, we will summarize the mode of action of Cdc42 and Rac1 as well as IQGAP1 as molecular switches for the cadherin function, and then discuss physiological processes in which the Cdc42/Rac1/IQGAP1 system may be involved.     

Cadherin dimers in cell-cell adhesion

While the critical function of classic cadherin in cell-cell junctions is well established, the molecular mechanism of cadherin-based adhesion remains unclear. The elusive but principal part of this adhesion process is the cadherin-cadherin interaction maintaining the intercellular contacts. This interaction is believed to be weak, suggesting that the adhesive contacts are strengthened by the cytoskeleton-dependent clustering of numerous cadherin molecules. An examination of cadherin homodimers in living cells has shown, however, that cadherin adhesive interaction is surprisingly strong. This observation implies that the strength of the adhesive contacts is regulated by the processes disintegrating cadherin dimers. The molecular structure of these dimers and mechanisms potentially responsible for their dynamics in living cells are discussed in this review.     

Should I stay or should I go? Cadherin function and regulation in the neural crest

Our increasing comprehension of neural crest cell development has reciprocally advanced our understanding of cadherin expression, regulation, and function. As a transient population of multipotent stem cells that significantly contribute to the vertebrate body plan, neural crest cells undergo a variety of transformative processes and exhibit many cellular behaviors, including epithelial‐to‐mesenchymal transition (EMT), motility, collective cell migration, and differentiation. Multiple studies have elucidated regulatory and mechanistic details of specific cadherins during neural crest cell development in a highly contextual manner. Collectively, these results reveal that gradual changes within neural crest cells are accompanied by often times subtle, yet important, alterations in cadherin expression and function. The primary focus of this review is to coalesce recent data on cadherins in neural crest cells, from their specification to their emergence as motile cells soon after EMT, and to highlight the complexities of cadherin expression beyond our current perceptions, including the hypothesis that the neural crest EMT is a transition involving a predominantly singular cadherin switch. Further advancements in genetic approaches and molecular techniques will provide greater opportunities to integrate data from various model systems in order to distinguish unique or overlapping functions of cadherins expressed at any point throughout the ontogeny of the neural crest.     

Classical cadherin adhesion molecules: coordinating cell adhesion, signaling and the cytoskeleton

Classical cadherin adhesion molecules are fundamental determinants of tissue organization in both health and disease. Recent advances in understanding the molecular and cellular basis of cadherin function have revealed that these adhesion molecules serve as molecular couplers, linking cell surface adhesion and recognition to both the actin cytoskeleton and cell signalling pathways. We will review some of these developments, to provide an overview of progress in this rapidly-developing area of cell and developmental biology.     

Functional analysis of the structural basis of homophilic cadherin adhesion

The structures of many cell surface adhesion proteins comprise multiple tandem repeats of structurally similar domains. In many cases, the functional significance of this architecture is unknown, and there are several cases in which evidence for individual domain involvement in adhesion has been contradictory. In particular, the extracellular region of the adhesion glycoprotein cadherin consists of five tandemly arranged domains. One proposed mechanism postulated that adhesion involves only trans interactions between the outermost domains. However, subsequent investigations have generated several competing models. Here we describe direct measurements of the distance-dependent interaction potentials between cadherin mutants lacking different domains. By quantifying both the absolute distances at which opposed cadherin fragments bind and the quantized changes in the interaction potentials that result from deletions of individual domains, we demonstrate that two domains participate in homophilic cadherin binding. This finding contrasts with the current view that cadherins bind via a single, unique site on the protein surface. The potentials that result from interactions involving multiple domains generate a novel, modular binding mechanism in which opposed cadherin ectodomains can adhere in any of three antiparallel alignments.     

Role of actin filaments and cis binding in cadherin clustering and patterning

Cadherins build up clusters to maintain intercellular contact through trans and cis (lateral) bindings. Meanwhile, interactions between cadherin and the actin cytoskeleton through cadherin/F-actin linkers can affect cadherin dynamics by corralling and tethering cadherin molecules locally. Despite many experimental studies, a quantitative, mechanistic understanding of how cadherin and actin cytoskeleton interactions regulate cadherin clustering does not exist. To address this gap in knowledge, we developed a coarse-grained computational model of cadherin dynamics and their interaction with the actin cortex underlying the cell membrane. Our simulation predictions suggest that weak cis binding affinity between cadherin molecules can facilitate large cluster formation. We also found that cadherin movement inhibition by actin corralling is dependent on the concentration and length of actin filaments. This results in changes in cadherin clustering behaviors, as reflected by differences in cluster size and distribution as well as cadherin monomer trajectory. Strong cadherin/actin binding can enhance trans and cis interactions as well as cadherin clustering. By contrast, with weak cadherin/actin binding affinity, a competition between cadherin-actin binding and cis binding for a limited cadherin pool leads to temporary and unstable cadherin clusters.     

Protein interactions and disease: computational approaches to uncover the etiology of diseases

The genomic era has been characterised by vast amounts of data from diverse sources, creating a need for new tools to extract biologically meaningful information. Bioinformatics is, for the most part, responding to that need. The sparseness of the genomic data associated with diseases, however, creates a new challenge. Understanding the complex interplay between genes and proteins requires integration of data from a wide variety of sources, i.e. gene expression, genetic linkage, protein interaction, and protein structure among others. Thus, computational tools have become critical for the integration, representation and visualization of heterogeneous biomedical data. Furthermore, several bioinformatics methods have been developed to formulate predictions about the functional role of genes and proteins, including their role in diseases. After an introduction to the complex interplay between proteins and genetic diseases, this review explores recent approaches to the understanding of the mechanisms of disease at the molecular level. Finally, because most known mechanisms leading to disease involve some form of protein interaction, this review focuses on the recent methodologies for understanding diseases through their underlying protein interactions. Recent contributions from genetics, protein structure and protein interaction network analyses to the understanding of diseases are discussed here.     

Adherens junction domains are split by asymmetric division of embryonic neural stem cells

Investigating the mechanisms controlling the asymmetric division of neocortical progenitors that generate neurones in the mammalian brain is crucial for understanding the abnormalities of cortical development. Partitioning of fate determinants is a key instructive step and components of the apical junctional complex (adherens junctions), including the polarity proteins PAR3 and aPKC as well as adhesion molecules such as N‐cadherin, have been proposed to be candidate determinants. In this study, however, we found no correlation between the partitioning of N‐cadherin and fate determination. Rather, we show that adherens junctions comprise three membrane domains, and that during asymmetrical division these are split such that both daughters retain the adhesive proteins that control cell position, but only one daughter inherits the polarity proteins along with the apical membrane. This provides a molecular explanation as to how both daughters remain anchored to the ventricular surface after mitosis, while adopting different fates.     

Regulation of dendritic maintenance and growth by a mammalian 7-pass transmembrane cadherin

Drosophila Flamingo is a 7-pass transmembrane cadherin that is necessary for dendritic patterning and axon guidance. How it works at the molecular level and whether homologs of Flamingo play similar roles in mammalian neurons or not have been unanswered questions. Here, we performed loss-of-function analysis using an RNAi system and organotypic brain slice cultures to address the role of a mammalian Flamingo homolog, Celsr2. Knocking down Celsr2 resulted in prominent simplification of dendritic arbors of cortical pyramidal neurons and Purkinje neurons, and this phenotype seemed to be due to branch retraction. Cadherin domain-mediated homophilic interaction appears to be required for the maintenance of dendritic branches. Furthermore, expression of various Celsr2 forms elicited distinct responses that were dependent on an extracellular subregion outside the cadherin domains and on a portion within the carboxyl intracellular tail. Based on these findings, we discuss how Celsr2 may regulate dendritic maintenance and growth.     

Regulation of signal transduction and bacterial infection during root nodule symbiosis

Among plant-microbe interactions, root nodule symbiosis is one of the most important beneficial interactions providing legume plants with nitrogenous compounds. Over the past years a number of genes required for root nodule symbiosis has been identified but most recently great advances have been made to dissect signalling pathways and molecular interactions triggered by a set of receptor-like kinases. Genetic and biochemical approaches have not only provided evidence for the cross talk between bacterial infection of the host plant and organogenesis of a root nodule but also gained insights into dynamic regulation processes underlying successful infection events. Here, we summarise recent progress in the understanding of molecular mechanisms that regulate and trigger cellular signalling cascades during this mutualistic interaction.     

p120 serine and threonine phosphorylation is controlled by multiple ligand-receptor pathways but not cadherin ligation

p120-catenin (p120) regulates cadherin turnover and is required for cadherin stability. Extensive and dynamic phosphorylation on tyrosine, serine and threonine residues in the N-terminal regulatory domain has been postulated to regulate p120 function, possibly through modulation of the efficiency of p120/cadherin interaction. Here we have utilized novel phospho-specific monoclonal antibodies to four major p120 serine and threonine phosphorylation sites to monitor individual phosphorylation events and their consequences. Surprisingly, membrane-localization and not cadherin interaction is the main determinant in p120 serine and threonine phosphorylation and dephosphorylation. Furthermore, the phospho-status of these four residues had no obvious effect on p120's role in cadherin complex stabilization or cell-cell adhesion. Interestingly, dephosphorylation was dramatically induced by PKC activation, but PKC-independent pathways were also evident. The data suggest that p120 dephosphorylation at these sites is modulated by multiple cell surface receptors primarily through PKC-dependent pathways, but these changes do not seem to reduce p120/cadherin affinity.     

Identification of a cadherin cell adhesion recognition sequence

The molecular mechanisms by which the cadherins interact with one another to promote cell adhesion have not been elucidated. In particular, the amino acid sequences of the cadherin cell adhesion recognition sites have not been determined. Here we demonstrate that synthetic peptides containing the sequence HAV, which is common to all of the cadherins, inhibit two processes (compaction of eight-cell-stage mouse embryos and rat neurite outgrowth on astrocytes) that are known to be mediated by cadherins. The data suggest that the tripeptide HAV is a component of a cadherin cell adhesion recognition sequence.     

An alternate insulin-like growth factor I receptor signaling pathway for the progression of endothelial–mesenchymal transition

Emerging evidence suggests that endothelial-to-mesenchymal transition (EndoMT) is an important contributor to cardiovascular diseases and to vascular development and pathologies as well as in cancer progression. As in epithelial–mesenchymal transition (EMT), EndoMT may involve several regulated steps: disassembly of adherence junctions or loss of cell–cell contacts, cytoskeletal reorganization, proteases, cytokines and growth factor synthesis and secretion, extracellular matrix remodeling, membrane receptor expression, cell detachment and cell migration and differentiation. Loss of cell–cell contacts is a necessary and sufficient step in the progression of EndoMT. In endothelial cells, adherence junctions are composed of transmembrane adhesive proteins belonging to the cadherin family, with the VE-cadherin being the most important. This protein interacts with β-catenin, which links cadherin to the actin cytoskeleton. Tyrosine phosphorylation of both VE-cadherin and β-catenin is considered an important mechanism associated to the disassembly of adherence junctions or loss of cell–cell contacts. Insulin-like growth factor receptor I (IGFIR) is a transmembrane tyrosine kinase that has been involved in the alterations of cell–cell contacts and in the expression of some genes during cancer development and progression. Here, it is hypothesized that IGFIR autophosphorylation may initiate a signaling pathway that would lead to the loss of cell–cell contacts or adherence junctions, remarkable remodeling of the cytoskeleton, increased cell motility, and finally to the progressive transition towards a mesenchymal phenotype. Data supporting this hypothesis are presented here.     

Nanomechanics of the cadherin ectodomain: "canalization" by Ca2+ binding results in a new mechanical element

Cadherins form a large family of calcium-dependent cell-cell adhesion receptors involved in development, morphogenesis, synaptogenesis, differentiation, and carcinogenesis through signal mechanotransduction using an adaptor complex that connects them to the cytoskeleton. However, the molecular mechanisms underlying mechanotransduction through cadherins remain unknown, although their extracellular region (ectodomain) is thought to be critical in this process. By single molecule force spectroscopy, molecular dynamics simulations, and protein engineering, here we have directly examined the nanomechanics of the C-cadherin ectodomain and found it to be strongly dependent on the calcium concentration. In the presence of calcium, the ectodomain extends through a defined ("canalized") pathway that involves two mechanical resistance elements: a mechanical clamp from the cadherin domains and a novel mechanostable component from the interdomain calcium-binding regions ("calcium rivet") that is abolished by magnesium replacement and in a mutant intended to impede calcium coordination. By contrast, in the absence of calcium, the mechanical response of the ectodomain becomes largely "decanalized" and destabilized. The cadherin ectodomain may therefore behave as a calcium-switched "mechanical antenna" with very different mechanical responses depending on calcium concentration (which would affect its mechanical integrity and force transmission capability). The versatile mechanical design of the cadherin ectodomain and its dependence on extracellular calcium facilitate a variety of mechanical responses that, we hypothesize, could influence the various adhesive properties mediated by cadherins in tissue morphogenesis, synaptic plasticity, and disease. Our work represents the first step toward the mechanical characterization of the cadherin system, opening the door to understanding the mechanical bases of its mechanotransduction.     

Steps towards a repertoire of comprehensive maps of human protein interaction networks: the Human Proteotheque Initiative (HuPI).

Defining human protein interaction networks has become essential to develop an overall, systems-based understanding of the molecular events that sustain cell growth in normal and disease conditions. To characterize protein interaction networks from human cells, we have undertaken the development of a systematic, unbiased technology pipeline that couples experimental and computational approaches. This discovery engine is central to the Human Proteotheque Initiative (HuPI), a multidisciplinary project aimed at building a repertoire of comprehensive maps of human protein interaction networks, the Human Proteotheque. The information contained in the Proteotheque is made publicly available through an interactive web site that can be consulted to visualize some of the fundamental molecular connections formed in human cells and to determine putative functions of previously uncharacterized proteins based on guilt by association. The process governing the evolution of HuPI towards becoming a repository of accurate and complete protein interaction maps is described.     

Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins

Phage display is an in vitro method for selecting polypeptides with desired properties from a large collection of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin-receptor interaction(s). By employing phage-libraries that display single-chain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity.     

Cadherin-actin interactions at adherens junctions

The adherens junction (AJ) is a major cell-cell junction that mediates cell recognition, adhesion, morphogenesis, and tissue integrity. Although AJs transmit forces generated by actomyosin from one cell to another, AJs have long been considered as an area where signal transduction from cadherin ligation takes place through cell adhesion. Through the efforts to understand embryonic or cellular morphogenesis, dynamic interactions between the AJ and actin filaments have become crucial issues to be addressed since actin association is essential for AJ development, remodeling and function. Here, I provide an overview of cadherin-actin interaction from morphological aspects and of possible molecular mechanisms revealed by recent studies.