Anchor Cell Research Articles

Vinculin head-tail interaction defines multiple early mechanisms for cell substrate rigidity sensing.

Rigidity sensing is a critical determinant of cell fate and behavior but its molecular mechanisms are poorly understood. Focal adhesions (FAs) are complexes that anchor cells to the matrix. Among their components, vinculin undergoes an auto-inhibitory head-tail interaction that regulates the recruitment of, and interactions with its partners in a force-dependent manner. It is unknown, however, whether this mechanism is involved in substrate rigidity sensing. Here, we use a range of quantitative fluorescence microscopies on live human Mesenchymal Stem Cells to address this question. We identify two distinct rigidity-sensing molecular modules in FAs, one of which involves vinculin and talin, is regulated by vinculin head-tail interaction, and targets cell morphology. Vinculin and talin are recruited independently in a rigidity-dependent manner to FAs where they directly interact in a rigidity-independent stoichiometry at a site proximal to talin head. Vinculin head-tail interaction is required on soft substrates to destabilize vinculin and talin in FAs, and to allow hMSCs branching. Another module involves paxillin and FAK, which soft substrates also destabilize, but independently of vinculin head-tail interaction. This multi-modularity may be key to allow a versatile response to complex biomechanical cues.

Collective migration in tissues.

The Minisymposium on “Cell Migration in Tissues” focused on analysis of dynamic collective cell migration in several model systems and was cochaired by Alissa M. Weaver (Vanderbilt University Medical Center) and Celeste M. Nelson (Princeton University). The program highlighted both the physical forces exerted during migration and the signaling pathways involved in the process. Celeste Nelson (Princeton University) presented results suggesting that cells migrate collectively through fibrous extracellular matrix (ECM) by exerting tensile forces at the leading edge of the cohort. Three-dimensional traction force microscopy was used to quantify the physical forces exerted by normal and malignant mammary epithelial cells undergoing collective migration through collagen gels (Gjorevski et al., 2015 ). The forces and displacements were surprisingly dynamic, with the cohort periodically relaxing as it migrated forward. Jessica Konen (Marcus laboratory, Emory University) described a new technique called spatiotemporal genomic analysis, which was used to select rare populations of leader cells from three-dimensional (3D) lung cancer spheroids and compare their gene expression patterns to that of follower cells. Leader cells were found to express higher levels of genes related to cell adhesion and vascular sprouting. These gene expression changes persisted in purified cultures. Eliah Shamir (Ewald laboratory, Johns Hopkins University) showed that in 3D culture of mammary epithelial organoids, the myoepithelial layer was required to prevent invasion and dissemination of Twist1-expressing luminal epithelial cells. Surprisingly, Twist1-induced invasion required the expression of E-cadherin, and knocking down expression of this cell–cell adhesion protein prevented dissemination downstream of Twist1 (Shamir et al., 2014 ). Joshua Bloomekatz (Yelon laboratory, University of California, San Diego) showed a critical role for platelet-derived growth factor (PDGF) in the movement of cardiomyocytes during cardiac fusion. A forward genetic screen was used to identify a mutation called refuse-to-fuse, which causes inhibition of cardiac fusion and the subsequent formation of a bifurcated ventricle. This mutation was found to disrupt splicing of pdgfra, leading to expression of a truncated form of the PDGF receptor. Expression of PDGF ligands in relatively medial locations suggested a model in which PDGF signaling coordinates the movement of cardiomyocytes toward the midline during cardiac fusion. Bong Hwan Sung (Weaver laboratory, Vanderbilt University) showed that directionally persistent migration of cancer cells is promoted by autocrine secretion of exosomes, late endosome–derived extracellular vesicles (Sung et al., 2015 ). Exosomes were shown to contain ECM proteins as cargo and to be secreted at sites of nascent adhesions. Accordingly, exosome secretion promoted adhesion assembly, migrational polarity, and the stability of lamellipodia. David Matus (Stony Brook University) used an RNA interference screen to identify a transcriptional program that maintains invasive anchor cells in a cell cycle–arrested state during uterine-vulval attachment in Caenorhabditis elegans. Nuclear hormone receptor nhr-67 (TLX) expression leads to up-regulation of cyclin-dependent kinase inhibitor cki-1, causing cell cycle arrest in G1 (Matus et al., 2015 ). Cells that lack nhr-67 are proliferative and noninvasive, failing to express matrix or form invadopodia. The Minisymposium concluded with three lightning talks. Micah Webster (Fan laboratory, Carnegie Institution for Science) showed a role for “ghost fibers,” ECM remnants of damaged muscle fibers, in organizing skeletal muscle progenitor migration, division, and fusion during regeneration in mice (Webster et al., 2016 ). Rebecca Green (Oegema laboratory, University of California, San Diego) developed a four-dimensional high-content imaging approach to screen for developmentally important genes within C. elegans embryos. Yutaka Matsubayashi (Stramer laboratory, King’s College London) showed that formation of the basement membrane in the Drosophila embryo requires rapid anterior–posterior dispersal of macrophages (hemocytes), which first appear in the head of the embryo.

SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane.

Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells.

Fabrication, Characterization, and Biocompatibility of Polymer Cored Reduced Graphene Oxide Nanofibers.

Graphene nanofibers have shown a promising potential across a wide spectrum of areas, including biology, energy, and the environment. However, fabrication of graphene nanofibers remains a challenging issue due to the broad size distribution and extremely poor solubility of graphene. Herein, we report a facile yet efficient approach for fabricating a novel class of polymer core-reduced graphene oxide shell nanofiber mat (RGO-CSNFM) by direct heat-driven self-assembly of graphene oxide sheets onto the surface of electrospun polymeric nanofibers without any requirement of surface treatment. Thus-prepared RGO-CSNFM demonstrated excellent mechanical, electrical, and biocompatible properties. RGO-CSNFM also promoted a higher cell anchorage and proliferation of human bone marrow mesenchymal stem cells (hMSCs) compared to the free-standing RGO film without the nanoscale fibrous structure. Further, cell viability of hMSCs was comparable to that on the tissue culture plates (TCPs) with a distinctive healthy morphology, indicating that the nanoscale fibrous architecture plays a critically constructive role in supporting cellular activities. In addition, the RGO-CSNFM exhibited excellent electrical conductivity, making them an ideal candidate for conductive cell culture, biosensing, and tissue engineering applications. These findings could provide a new benchmark for preparing well-defined graphene-based nanomaterial configurations and interfaces for biomedical applications.

Vaccinia-related kinase 1 is required for early uterine development in Caenorhabditis elegans

Protein kinases regulate a multitude of processes by reversible phosphorylation of target molecules. Induction of cell proliferation and differentiation are fundamental to development and rely on tightly controlled kinase activities. Vaccinia-Related Kinases (VRKs) have emerged as a multifunctional family of kinases with essential functions conserved, from nematodes and fruit flies, to humans. VRK substrates include chromatin and transcription factors, whereas deregulation of VRKs is implicated in sterility, cancer and neurological defects. In contrast to previous observations, we describe here that Caenorhabditis elegans VRK-1 is expressed in all cell types, including proliferating and post-mitotic cells. Despite the ubiquitous expression pattern, we find that vrk-1 mutants are particularly impaired in uterine development. Our data show that VRK-1 is required for uterine cell proliferation and differentiation. Moreover, the anchor cell, a specialized uterine cell, fails to fuse with neighboring cells to form the utse syncytium in vrk-1 mutants, thus providing further insight on the role of VRKs in organogenesis.

3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction.

BackgroundCellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC) is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility.Methodology/Principal FindingsWe have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum.Conclusions/SignificanceThe FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life cycle of this human parasite.

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation.

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.

1D26 2.5次元細胞外基質上における細胞-基質間接着の動態

Focal adhesions are force-sensitive, multiprotein complexes that anchor cells to the extracellular substrate. Previous studies have revealed the composition, ultrastructure, and dynamics of focal adhesions, and how they change upon intracellular and/or extracellular forces. However, little is known regarding how focal adhesions sense the geometry of the extracellular substrate. Here we develop a novel traction microscopy that allows for simultaneous evaluation of the cellular forces and the effect of local substrate curvature, i.e. how the substrate is curved out of the plane to have a 2.5D surface. While it is well known that force-induced elongation of focal adhesions occurs on a flat surface, here we interestingly found that the dynamics of individual focal adhesions is abrogated by a certain level of local substrate curvature, or 2.5D substrates. Thus, further development of focal adhesions is allowed only when they retain a straight shape on a planar surface.

Influences of LIN-12/Notch and POP-1/TCF on the Robustness of Ventral Uterine Cell Fate Specification in Caenorhabditis elegans Gonadogenesis.

The prospective ventral uterus of the hermaphrodite gonad primordium consists of two pairs of sister cells, with each pair consisting of a proximal “α” cell and a distal “β” cell. All four cells initially are competent to become the anchor cell (AC), a unique cell type that acts as the organizer of subsequent uterine and vulval development. However, the β cells soon lose this competence and always become ventral uterine precursor cells (VUs), whereas the α cells maintain their AC competence longer, until lin-12/Notch-mediated interactions between them specify one as the AC and the other as a VU. Here, we investigate this asymmetry in developmental potential and VU fate specification between the α and β sister cells. We find evidence that lin-12 activity contributes to the robustness of βVU fate at elevated temperature, that the Caenorhabditis elegans Notch paralog glp-1 is not functionally redundant with lin-12 in specifying βVU fate, and that the activity of POP-1, the sole C. elegans TCF ortholog, influences βVU fate. We propose a model for how Wnt and LIN-12/Notch signaling together lead to robust specification of the βVU fate.

Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression

Despite critical roles in development and cancer, the mechanisms that specify invasive cellular behavior are poorly understood. Through a screen of transcription factors in Caenorhabditis elegans, we identified G1 cell-cycle arrest as a precisely regulated requirement of the anchor cell (AC) invasion program. We show that the nuclear receptor nhr-67/tlx directs the AC into G1 arrest in part through regulation of the cyclin-dependent kinase inhibitor cki-1. Loss of nhr-67 resulted in non-invasive, mitotic ACs that failed to express matrix metalloproteinases or actin regulators and lack invadopodia, F-actin-rich membrane protrusions that facilitate invasion. We further show that G1 arrest is necessary for the histone deacetylase HDA-1, a key regulator of differentiation, to promote pro-invasive gene expression and invadopodia formation. Together, these results suggest that invasive cell fate requires G1 arrest and that strategies targeting both G1-arrested and actively cycling cells may be needed to halt metastatic cancer.

An Arresting Story about Basement Membrane Invasion

In this issue of Developmental Cell, Matus et al. (2015) reveal that to invade past basement membrane, the C. elegans anchor cell must cease dividing before differentiating and expressing pro-invasion genes. This demonstration of invasion and proliferation as mutually incompatible cell states has implications for our understanding of cancer metastasis.

Calpain-Mediated Positional Information Directs Cell Wall Orientation to Sustain Plant Stem Cell Activity, Growth and Development.

Eukaryotic development and stem cell control depend on the integration of cell positional sensing with cell cycle control and cell wall positioning, yet few factors that directly link these events are known. The DEFECTIVE KERNEL1 (DEK1) gene encoding the unique plant calpain protein is fundamental for development and growth, being essential to confer and maintain epidermal cell identity that allows development beyond the globular embryo stage. We show that DEK1 expression is highest in the actively dividing cells of seeds, meristems and vasculature. We further show that eliminating Arabidopsis DEK1 function leads to changes in developmental cues from the first zygotic division onward, altered microtubule patterns and misshapen cells, resulting in early embryo abortion. Expression of the embryonic marker genes WOX2, ATML1, PIN4, WUS and STM, related to axis organization, cell identity and meristem functions, is also altered in dek1 embryos. By monitoring cell layer-specific DEK1 down-regulation, we show that L1- and 35S-induced down-regulation mainly affects stem cell functions, causing severe shoot apical meristem phenotypes. These results are consistent with a requirement for DEK1 to direct layer-specific cellular activities and set downstream developmental cues. Our data suggest that DEK1 may anchor cell wall positions and control cell division and differentiation, thereby balancing the plant's requirement to maintain totipotent stem cell reservoirs while simultaneously directing growth and organ formation. A role for DEK1 in regulating microtubule-orchestrated cell wall orientation during cell division can explain its effects on embryonic development, and suggests a more general function for calpains in microtubule organization in eukaryotic cells.

Analysis of Wave Excitation of the Phase-Control ICRF Antennas with Three-Dimensional Full Wave Code on GAMMA 10

On the GAMMA 10 tandem mirror, divertor simulation experiments progress with high-temperature plasmas produced by waves in ion-cyclotron range of frequency (ICRF) and open magnetic fields. In these experiments, high-temperature and high-density plasmas are required to be produced and controlled. In order to enhance the ion heating in the anchor cell, phase-control experiments, which use two ICRF antennas installed in the central cell and the anchor cell, are introduced. In these experiments, ICRF waves in the same frequency (10 MHz) are excited. Ion heating in the anchor cell is performed more effectively with phase control. In order to analyze wave propagation in the phase-control experiments, a three-dimensional full wave code (TASK/WF), in which parallel processing has been implemented recently, is introduced. In this technical note, we discuss ICRF wave excitation and propagation in the phase-control experiments. It is clearly observed by the experiments and wave analysis that the enhancement of ion heating in the anchor cell is performed by phase-control experiments.

High-Density Plasma Production in the GAMMA 10 Central Cell with ICRF Heating on Both Anchor Cells

In the GAMMA 10 tandem mirror, divertor simulation experiments that utilize particle flux toward the west end region (called end-loss flux) have been implemented. Since a positive correlation has been reported between the end-loss flux and the central-cell density, an increase of the central-cell density is important for obtaining a higher end-loss flux on the divertor simulation experiments. By arranging the ion cyclotron range of frequency (ICRF) systems so as to excite strong ICRF waves in both anchor cells simultaneously, we have succeeded in producing high-density plasmas (line density of 1.2 × 1014 cm−2) in both anchor cells. As a result, a higher central-cell density of 4.4 × 1012 cm−3 and a higher end-loss flux of more than 1023 m−2s−1 have been obtained. One of the possible mechanisms of the high density production is a formation of positive potentials on both anchor cells. Plasmas in the central cell are confined due to those potentials.

Dimerization-driven degradation of C. elegans and human E proteins.

E proteins are conserved regulators of growth and development. We show that the Caenorhabditis elegans E-protein helix-loop-helix-2 (HLH-2) functions as a homodimer in directing development and function of the anchor cell (AC) of the gonad, the critical organizer of uterine and vulval development. Our structure-function analysis of HLH-2 indicates that dimerization drives its degradation in other uterine cells (ventral uterine precursor cells [VUs]) that initially have potential to be the AC. We also provide evidence that this mode of dimerization-driven down-regulation can target other basic HLH (bHLH) dimers as well. Remarkably, human E proteins can functionally substitute for C. elegans HLH-2 in regulating AC development and also display dimerization-dependent degradation in VUs. Our results suggest that dimerization-driven regulation of bHLH protein stability may be a conserved mechanism for differential regulation in specific cell contexts.

An Atlas of Network Topologies Reveals Design Principles for Caenorhabditis elegans Vulval Precursor Cell Fate Patterning.

The vulval precursor cell (VPC) fate patterning in Caenorhabditis elegans is a classic model experimental system for cell fate determination and patterning in development. Despite its apparent simplicity (six neighboring cells arranged in one dimension) and many experimental and computational efforts, the patterning strategy and mechanism remain controversial due to incomplete knowledge of the complex biology. Here, we carry out a comprehensive computational analysis and obtain a reservoir of all possible network topologies that are capable of VPC fate patterning under the simulation of various biological environments and regulatory rules. We identify three patterning strategies: sequential induction, morphogen gradient and lateral antagonism, depending on the features of the signal secreted from the anchor cell. The strategy of lateral antagonism, which has not been reported in previous studies of VPC patterning, employs a mutual inhibition of the 2° cell fate in neighboring cells. Robust topologies are built upon minimal topologies with basic patterning strategies and have more flexible and redundant implementations of modular functions. By simulated mutation, we find that all three strategies can reproduce experimental error patterns of mutants. We show that the topology derived by mapping currently known biochemical pathways to our model matches one of our identified functional topologies. Furthermore, our robustness analysis predicts a possible missing link related to the lateral antagonism strategy. Overall, we provide a theoretical atlas of all possible functional networks in varying environments, which may guide novel discoveries of the biological interactions in vulval development of Caenorhabditis elegans and related species.

Mad1 at the Golgi apparatus: a story beyond kinetochores

Mad1 at the Golgi apparatus: a story beyond kinetochores

Extracellular matrix biomimicry for the creation of investigational and therapeutic devices.

The extracellular matrix (ECM) is a web of fibrous proteins that serves as a scaffold for tissues and organs, and is important for maintaining homeostasis and facilitating cellular adhesion. Integrin transmembrane receptors are the primary adhesion molecules that anchor cells to the ECM, thus integrating cells with their microenvironments. Integrins play a critical role in facilitating cell-matrix interactions and promoting signal transduction, both from the cell to the ECM and vice versa, ultimately mediating cell behavior. For this reason, many advanced biomaterials employ biomimicry by replicating the form and function of fibrous ECM proteins. The ECM also acts as a reservoir for small molecules and growth factors, wherein fibrous proteins directly bind and present these bioactive moieties that facilitate cell activity. Therefore biomimicry can be enhanced by incorporating small molecules into ECM-like substrates. Biomimetic ECM materials have served as invaluable research tools for studying interactions between cells and the surrounding ECM, revealing that cell-matrix signaling is driven by mechanical forces, integrin engagement, and small molecules. Mimicking pathological ECMs has also elucidated disease specific cell behaviors. For example, biomimetic tumor microenvironments have been used to induce metastatic cell behaviors, and have thereby shown promise for in vitro cancer drug testing and targeting. Further, ECM-like substrates have been successfully employed for autologous cell recolonization for tissue engineering and wound healing. As we continue to learn more about the mechanical and biochemical characteristics of the ECM, these properties can be harnessed to develop new biomaterials, biomedical devices, and therapeutics.

Combination of X-ray crystallography, SAXS and DEER to obtain the structure of the FnIII-3,4 domains of integrin α6β4.

Integrin α6β4 is a major component of hemidesmosomes that mediate the stable anchorage of epithelial cells to the underlying basement membrane. Integrin α6β4 has also been implicated in cell proliferation and migration and in carcinoma progression. The third and fourth fibronectin type III domains (FnIII-3,4) of integrin β4 mediate binding to the hemidesmosomal proteins BPAG1e and BPAG2, and participate in signalling. Here, it is demonstrated that X-ray crystallography, small-angle X-ray scattering and double electron-electron resonance (DEER) complement each other to solve the structure of the FnIII-3,4 region. The crystal structures of the individual FnIII-3 and FnIII-4 domains were solved and the relative arrangement of the FnIII domains was elucidated by combining DEER with site-directed spin labelling. Multiple structures of the interdomain linker were modelled by Monte Carlo methods complying with DEER constraints, and the final structures were selected against experimental scattering data. FnIII-3,4 has a compact and cambered flat structure with an evolutionary conserved surface that is likely to correspond to a protein-interaction site. Finally, this hybrid method is of general application for the study of other macromolecules and complexes.

Lateral assembly of N-cadherin drives tissue integrity by stabilizing adherens junctions.

Cadherin interactions ensure the correct registry and anchorage of cells during tissue formation. Along the plasma membrane, cadherins form inter-junctional lattices via cis- and trans-dimerization. While structural studies have provided models for cadherin interactions, the molecular nature of cadherin binding in vivo remains unexplored. We undertook a multi-disciplinary approach combining live cell imaging of three-dimensional cell assemblies (spheroids) with a computational model to study the dynamics of N-cadherin interactions. Using a loss-of-function strategy, we demonstrate that each N-cadherin interface plays a distinct role in spheroid formation. We found that cis-dimerization is not a prerequisite for trans-interactions, but rather modulates trans-interfaces to ensure tissue stability. Using a model of N-cadherin junction dynamics, we show that the absence of cis-interactions results in low junction stability and loss of tissue integrity. By quantifying the binding and unbinding dynamics of the N-cadherin binding interfaces, we determined that mutating either interface results in a 10-fold increase in the dissociation constant. These findings provide new quantitative information on the steps driving cadherin intercellular adhesion and demonstrate the role of cis-interactions in junction stability.