The Glue that Binds Us: The Hunt for the Molecular Basis for Multicellularity.

This year's Canada Gairdner International Prize is shared by Rolf Kemler and Masatoshi Takeichi for the discovery of the cadherin family of Ca2+-dependent cell-cell adhesion proteins, which play essential roles in animal evolution, tissue development, and homeostasis, and are disrupted in human cancers.

Primary cilium loss in mammalian cells occurs predominantly by whole-cilium shedding.

The primary cilium is a central signaling hub in cell proliferation and differentiation and is built and disassembled every cell cycle in many animal cells. Disassembly is critically important, as misregulation or delay of cilia loss leads to cell cycle defects. The physical means by which cilia are lost are poorly understood but are thought to involve resorption of ciliary components into the cell body. To investigate cilium loss in mammalian cells, we used live-cell imaging to comprehensively characterize individual events. The predominant mode of cilium loss was rapid deciliation, in which the membrane and axoneme of the cilium was shed from the cell. Gradual resorption was also observed, as well as events in which a period of gradual resorption was followed by rapid deciliation. Deciliation resulted in intact shed cilia that could be recovered from culture medium and contained both membrane and axoneme proteins. We modulated levels of katanin and intracellular calcium, two putative regulators of deciliation, and found that excess katanin promotes cilia loss by deciliation, independently of calcium. Together, these results suggest that mammalian ciliary loss involves a tunable decision between deciliation and resorption.

Coordinated protein sorting, targeting and distribution in polarized cells.

The polarized distribution of functions in polarized cells requires the coordinated interaction of three machineries that modify the basic mechanisms of intracellular protein trafficking and distribution. First, intrinsic protein-sorting signals and cellular decoding machineries regulate protein trafficking to plasma membrane domains; second, intracellular signalling complexes define the plasma membrane domains to which proteins are delivered; and third, proteins that are involved in cell-cell and cell-substrate adhesion orientate the three-dimensional distribution of intracellular signalling complexes and, accordingly, the direction of membrane traffic. The integration of these mechanisms into a complex and dynamic network is crucial for normal tissue function and is often defective in disease states.

MEMS device for applying shear and tension to an epithelium combined with fluorescent live cell imaging.

Mechanical forces play important roles in the biological function of cells and tissues. While numerous studies have probed the force response of cells and measured cell-generated forces, they have primarily focused on tensile, but not shear forces. Here, we describe the design, fabrication, and application of a silicon micromachined device that is capable of independently applying and sensing both tensile and shear forces in an epithelial cell monolayer. We integrated the device with an upright microscope to enable live cell brightfield and fluorescent imaging of cells over many hours following mechanical perturbation. Using devices of increasing stiffness and the same displacement input, we demonstrate that epithelia exhibit concomitant higher maximum resistive tensile forces and quicker force relaxation. In addition, we characterized the force response of the epithelium to cyclic shear loading. While the maximum resistive forces of epithelia under cyclic shear perturbation remained unchanged between cycles, cyclic loading led to faster relaxation of the resistive forces. The device presented here can be applied to studying the force response of other monolayer-forming cell types and is compatible with pharmacological perturbation of cell structures and functions.

Changes in E-cadherin rigidity sensing regulate cell adhesion.

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell-cell adhesion.

E-cadherin and LGN align epithelial cell divisions with tissue tension independently of cell shape.

Tissue morphogenesis requires the coordinated regulation of cellular behavior, which includes the orientation of cell division that defines the position of daughter cells in the tissue. Cell division orientation is instructed by biochemical and mechanical signals from the local tissue environment, but how those signals control mitotic spindle orientation is not fully understood. Here, we tested how mechanical tension across an epithelial monolayer is sensed to orient cell divisions. Tension across Madin-Darby canine kidney cell monolayers was increased by a low level of uniaxial stretch, which oriented cell divisions with the stretch axis irrespective of the orientation of the cell long axis. We demonstrate that stretch-induced division orientation required mechanotransduction through E-cadherin cell-cell adhesions. Increased tension on the E-cadherin complex promoted the junctional recruitment of the protein LGN, a core component of the spindle orientation machinery that binds the cytosolic tail of E-cadherin. Consequently, uniaxial stretch triggered a polarized cortical distribution of LGN. Selective disruption of trans engagement of E-cadherin in an otherwise cohesive cell monolayer, or loss of LGN expression, resulted in randomly oriented cell divisions in the presence of uniaxial stretch. Our findings indicate that E-cadherin plays a key role in sensing polarized tensile forces across the tissue and transducing this information to the spindle orientation machinery to align cell divisions.

Analysis of a vinculin homolog in a sponge (phylum Porifera) reveals that vertebrate-like cell adhesions emerged early in animal evolution.

The evolution of cell-adhesion mechanisms in animals facilitated the assembly of organized multicellular tissues. Studies in traditional animal models have revealed two predominant adhesion structures, the adherens junction (AJ) and focal adhesions (FAs), which are involved in the attachment of neighboring cells to each other and to the secreted extracellular matrix (ECM), respectively. The AJ (containing cadherins and catenins) and FAs (comprising integrins, talin, and paxillin) differ in protein composition, but both junctions contain the actin-binding protein vinculin. The near ubiquity of these structures in animals suggests that AJ and FAs evolved early, possibly coincident with multicellularity. However, a challenge to this perspective is that previous studies of sponges-a divergent animal lineage-indicate that their tissues are organized primarily by an alternative, sponge-specific cell-adhesion mechanism called "aggregation factor." In this study, we examined the structure, biochemical properties, and tissue localization of a vinculin ortholog in the sponge Oscarella pearsei (Op). Our results indicate that Op vinculin localizes to both cell-cell and cell-ECM contacts and has biochemical and structural properties similar to those of vertebrate vinculin. We propose that Op vinculin played a role in cell adhesion and tissue organization in the last common ancestor of sponges and other animals. These findings provide compelling evidence that sponge tissues are indeed organized like epithelia in other animals and support the notion that AJ- and FA-like structures extend to the earliest periods of animal evolution.

Epithelial self-healing is recapitulated by a 3D biomimetic E-cadherin junction.

Epithelial monolayers undergo self-healing when wounded. During healing, cells collectively migrate into the wound site, and the converging tissue fronts collide and form a stable interface. To heal, migrating tissues must form cell-cell adhesions and reorganize from the front-rear polarity characteristic of cell migration to the apical-basal polarity of an epithelium. However, identifying the "stop signal" that induces colliding tissues to cease migrating and heal remains an open question. Epithelial cells form integrin-based adhesions to the basal extracellular matrix (ECM) and E-cadherin-mediated cell-cell adhesions on the orthogonal, lateral surfaces between cells. Current biological tools have been unable to probe this multicellular 3D interface to determine the stop signal. We addressed this problem by developing a unique biointerface that mimicked the 3D organization of epithelial cell adhesions. This "minimal tissue mimic" (MTM) comprised a basal ECM substrate and a vertical surface coated with purified extracellular domain of E-cadherin, and was designed for collision with the healing edge of an epithelial monolayer. Three-dimensional imaging showed that adhesions formed between cells, and the E-cadherin-coated MTM resembled the morphology and dynamics of native epithelial cell-cell junctions and induced the same polarity transition that occurs during epithelial self-healing. These results indicate that E-cadherin presented in the proper 3D context constitutes a minimum essential stop signal to induce self-healing. That the Ecad:Fc MTM stably integrated into an epithelial tissue and reduced migration at the interface suggests that this biointerface is a complimentary approach to existing tissue-material interfaces.

Structural and functional characterization of Caenorhabditis elegans α-catenin reveals constitutive binding to ß-catenin and F-actin.

Intercellular epithelial junctions formed by classical cadherins, ß-catenin, and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and regulation are poorly understood. Studies of cadherin-catenin assembly in a number of metazoans have revealed both similarities and unexpected differences in the biochemical properties of the cadherin·catenin complex that likely reflect the developmental and environmental requirements of different tissues and organisms. Here, we report the structural and biochemical characterization of HMP-1, the Caenorhabditis elegans α-catenin homolog, and compare it with mammalian α-catenin. HMP-1 shares overall similarity in structure and actin-binding properties, but displayed differences in conformational flexibility and allosteric regulation from mammalian α-catenin. HMP-1 bound filamentous actin with an affinity in the single micromolar range, even when complexed with the ß-catenin homolog HMP-2 or when present in a complex of HMP-2 and the cadherin homolog HMR-1, indicating that HMP-1 binding to F-actin is not allosterically regulated by the HMP-2·HMR-1 complex. The middle (i.e. M) domain of HMP-1 appeared to be less conformationally flexible than mammalian α-catenin, which may underlie the dampened effect of HMP-2 binding on HMP-1 actin-binding activity compared with that of the mammalian homolog. In conclusion, our data indicate that HMP-1 constitutively binds ß-catenin and F-actin, and although the overall structure and function of HMP-1 and related α-catenins are similar, the vertebrate proteins appear to be under more complex conformational regulation.

Mechano-transduction: from molecules to tissues.

External forces play complex roles in cell organization, fate, and homeostasis. Changes in these forces, or how cells respond to them, can result in abnormal embryonic development and diseases in adults. How cells sense and respond to these mechanical stimuli requires an understanding of the biophysical principles that underlie changes in protein conformation and result in alterations in the organization and function of cells and tissues. Here, we discuss mechano-transduction as it applies to protein conformation, cellular organization, and multi-cell (tissue) function.

Different roles of cadherins in the assembly and structural integrity of the desmosome complex.

Adhesion between cells is established by the formation of specialized intercellular junctional complexes, such as desmosomes. Desmosomes contain isoforms of two members of the cadherin superfamily of cell adhesion proteins, desmocollins (Dsc) and desmogleins (Dsg), but their combinatorial roles in desmosome assembly are not understood. To uncouple desmosome assembly from other cell-cell adhesion complexes, we used micro-patterned substrates of Dsc2aFc and/or Dsg2Fc and collagen IV; we show that Dsc2aFc, but not Dsg2Fc, was necessary and sufficient to recruit desmosome-specific desmoplakin into desmosome puncta and produce strong adhesive binding. Single-molecule force spectroscopy showed that monomeric Dsc2a, but not Dsg2, formed Ca(2+)-dependent homophilic bonds, and that Dsg2 formed Ca(2+)-independent heterophilic bonds with Dsc2a. A W2A mutation in Dsc2a inhibited Ca(2+)-dependent homophilic binding, similar to classical cadherins, and Dsc2aW2A, but not Dsg2W2A, was excluded from desmosomes in MDCK cells. These results indicate that Dsc2a, but not Dsg2, is required for desmosome assembly through homophilic Ca(2+)- and W2-dependent binding, and that Dsg2 might be involved later in regulating a switch to Ca(2+)-independent adhesion in mature desmosomes.

Sept7b is essential for pronephric function and development of left-right asymmetry in zebrafish embryogenesis.

The conserved septin family of filamentous small GTPases plays important roles in mitosis, cell migration and cell morphogenesis by forming scaffolds and diffusion barriers. Recent studies in cultured cells in vitro indicate that a septin complex of septin 2, 7 and 9 is required for ciliogenesis and cilia function, but septin function in ciliogenesis in vertebrate organs in vivo is not understood. We show that sept7b is expressed in ciliated cells in different tissues during early zebrafish development. Knockdown of sept7b by using morpholino antisense oligonucleotides caused misorientation of basal bodies and cilia, reduction of apical actin and the shortening of motile cilia in Kupffer's vesicle and pronephric tubules. This resulted in pericardial and yolk sac edema, body axis curvature and hydrocephaly. Notably, in sept7b morphants we detected strong left-right asymmetry defects in the heart and lateral plate mesoderm (situs inversus), reduced fluid flow in the kidney, the formation of kidney cysts and loss of glomerular filtration barrier function. Thus, sept7b is essential during zebrafish development for pronephric function and ciliogenesis, and loss of expression of sept7b results in defects that resemble human ciliopathies.

αE-catenin regulates cell-cell adhesion and membrane blebbing during zebrafish epiboly.

αE-catenin is an actin-binding protein associated with the E-cadherin-based adherens junction that regulates cell-cell adhesion. Recent studies identified additional E-cadherin-independent roles of αE-catenin in regulating plasma membrane dynamics and cell migration. However, little is known about the roles of αE-catenin in these different cellular processes in vivo during early vertebrate development. Here, we examined the functions of αE-catenin in cell-cell adhesion, cell migration and plasma membrane dynamics during morphogenetic processes that drive epiboly in early Danio rerio (zebrafish) development. We show that depletion of αE-catenin caused a defect in radial intercalation that was associated with decreased cell-cell adhesion, in a similar manner to E-cadherin depletion. Depletion of αE-catenin also caused deep cells to have protracted plasma membrane blebbing, and a defect in plasma membrane recruitment of ERM proteins that are involved in controlling membrane-to-cortex attachment and membrane blebbing. Significantly, depletion of both E-cadherin and αE-catenin suppressed plasma membrane blebbing. We suggest that during radial intercalation the activities of E-cadherin and αE-catenin in the maintenance of membrane-to-cortex attachment are balanced, resulting in stabilization of cell-cell adhesion and suppression of membrane blebbing, thereby enabling proper radial intercalation.

Galvanotactic control of collective cell migration in epithelial monolayers.

Many normal and pathological biological processes involve the migration of epithelial cell sheets. This arises from complex emergent behaviour resulting from the interplay between cellular signalling networks and the forces that physically couple the cells. Here, we demonstrate that collective migration of an epithelium can be interactively guided by applying electric fields that bias the underlying signalling networks. We show that complex, spatiotemporal cues are locally interpreted by the epithelium, resulting in rapid, coordinated responses such as a collective U-turn, divergent migration, and unchecked migration against an obstacle. We observed that the degree of external control depends on the size and shape of the cell population, and on the existence of physical coupling between cells. Together, our results offer design and engineering principles for the rational manipulation of the collective behaviour and material properties of a tissue.

ß-catenin at the centrosome: discrete pools of ß-catenin communicate during mitosis and may co-ordinate centrosome functions and cell cycle progression.

Beta-catenin is a multifunctional protein with critical roles in cell-cell adhesion, Wnt-signaling and the centrosome cycle. Whereas the roles of ß-catenin in cell-cell adhesion and Wnt-signaling have been studied extensively, the mechanism(s) involving ß-catenin in centrosome functions are poorly understood. ß-Catenin localizes to centrosomes and promotes mitotic progression. NIMA-related protein kinase 2 (Nek2), which stimulates centrosome separation, binds to and phosphorylates ß-catenin. ß-Catenin interacting proteins involved in Wnt signaling such as adenomatous polyposis coli, Axin, and GSK3ß, are also localized at centrosomes and play roles in promoting mitotic progression. Additionally, proteins associated with cell-cell adhesion sites, such as dynein, regulate mitotic spindle positioning. These roles of proteins at the cell cortex and Wnt signaling that involve ß-catenin indicate a cross-talk between different sub-cellular sites in the cell at mitosis, and that different pools of ß-catenin may co-ordinate centrosome functions and cell cycle progression.

E-cadherin is under constitutive actomyosin-generated tension that is increased at cell-cell contacts upon externally applied stretch.

Classical cadherins are transmembrane proteins at the core of intercellular adhesion complexes in cohesive metazoan tissues. The extracellular domain of classical cadherins forms intercellular bonds with cadherins on neighboring cells, whereas the cytoplasmic domain recruits catenins, which in turn associate with additional cytoskeleton binding and regulatory proteins. Cadherin/catenin complexes are hypothesized to play a role in the transduction of mechanical forces that shape cells and tissues during development, regeneration, and disease. Whether mechanical forces are transduced directly through cadherins is unknown. To address this question, we used a Förster resonance energy transfer (FRET)-based molecular tension sensor to test the origin and magnitude of tensile forces transmitted through the cytoplasmic domain of E-cadherin in epithelial cells. We show that the actomyosin cytoskeleton exerts pN-tensile force on E-cadherin, and that this tension requires the catenin-binding domain of E-cadherin and αE-catenin. Surprisingly, the actomyosin cytoskeleton constitutively exerts tension on E-cadherin at the plasma membrane regardless of whether or not E-cadherin is recruited to cell-cell contacts, although tension is further increased at cell-cell contacts when adhering cells are stretched. Our findings thus point to a constitutive role of E-cadherin in transducing mechanical forces between the actomyosin cytoskeleton and the plasma membrane, not only at cell-cell junctions but throughout the cell surface.

Danio rerio αE-catenin is a monomeric F-actin binding protein with distinct properties from Mus musculus αE-catenin.

It is unknown whether homologs of the cadherin·catenin complex have conserved structures and functions across the Metazoa. Mammalian αE-catenin is an allosterically regulated actin-binding protein that binds the cadherin·ß-catenin complex as a monomer and whose dimerization potentiates F-actin association. We tested whether these functional properties are conserved in another vertebrate, the zebrafish Danio rerio. Here we show, despite 90% sequence identity, that Danio rerio and Mus musculus αE-catenin have striking functional differences. We demonstrate that D. rerio αE-catenin is monomeric by size exclusion chromatography, native PAGE, and small angle x-ray scattering. D. rerio αE-catenin binds F-actin in cosedimentation assays as a monomer and as an α/ß-catenin heterodimer complex. D. rerio αE-catenin also bundles F-actin, as shown by negative stained transmission electron microscopy, and does not inhibit Arp2/3 complex-mediated actin nucleation in bulk polymerization assays. Thus, core properties of α-catenin function, F-actin and ß-catenin binding, are conserved between mouse and zebrafish. We speculate that unique regulatory properties have evolved to match specific developmental requirements.

Mitogen-activated protein kinase (MAPK/ERK) regulates adenomatous polyposis coli during growth-factor-induced cell extension.

Regulation of the microtubule- and actin-binding protein adenomatous polyposis coli (APC) is crucial for the formation of cell extensions in many cell types. This process requires inhibition of glycogen synthase kinase-3ß (GSK-3ß), which otherwise phosphorylates APC and decreases APC-mediated microtubule bundling. Although it is assumed, therefore, that APC phosphorylation is decreased during initiation of cell extensions, the phosphorylation state of APC has never been analyzed directly. We show here that NGF- and EGF-induced initial cell extensions result in APC phosphorylation by the MAPK/ERK pathway, which, in parallel with inhibition of GSK-3ß, promotes localization of APC to the tip of cell extensions. Whereas GSK-3ß inhibition promotes APC binding and stabilization of microtubules, we show that phosphorylation by ERK inhibits the interaction of APC with F-actin, and APC-mediated F-actin bundling, but not APC-mediated microtubule bundling, in vitro. These results identify a previously unknown APC regulatory pathway during growth-factor-induced cell extension, and indicate that the GSK-3ß and ERK pathways act in parallel to regulate interactions between APC and the cytoskeleton during the formation of cell extensions.

Catenins: playing both sides of the synapse.

Synapses of the central nervous system (CNS) are specialized cell-cell junctions that mediate intercellular signal transmission from one neuron to another. The directional nature of signal relay requires synaptic contacts to be morphologically asymmetric with distinct protein components, while changes in synaptic communication during neural network formation require synapses to be plastic. Synapse morphology and plasticity require a dynamic actin cytoskeleton. Classical cadherins, which are junctional proteins associated with the actin cytoskeleton, localize to synapses and regulate synaptic adhesion, stability and remodeling. The major intracellular components of cadherin junctions are the catenin proteins, and increasing evidence suggests that cadherin-catenin complexes modulate an array of synaptic processes. Here we review the role of catenins in regulating the development of pre- and postsynaptic compartments and function in synaptic plasticity, with particular focus on their role in regulating the actin cytoskeleton.

An epithelial tissue in Dictyostelium challenges the traditional origin of metazoan multicellularity.

We hypothesize that aspects of animal multicellularity originated before the divergence of metazoans from fungi and social amoebae. Polarized epithelial tissues are a defining feature of metazoans and contribute to the diversity of animal body plans. The recent finding of a polarized epithelium in the non-metazoan social amoeba Dictyostelium discoideum demonstrates that epithelial tissue is not a unique feature of metazoans, and challenges the traditional paradigm that multicellularity evolved independently in social amoebae and metazoans. An alternative view, presented here, is that the common ancestor of social amoebae, fungi, and animals spent a portion of its life cycle in a multicellular state and possessed molecular machinery necessary for forming an epithelial tissue. Some descendants of this ancestor retained multicellularity, while others reverted to unicellularity. This hypothesis makes testable predictions regarding tissue organization in close relatives of metazoans and provides a novel conceptual framework for studies of early animal evolution.