Soichiro Yamada

Deconstructing the Cadherin-Catenin-Actin Complex

Spatial and functional organization of cells in tissues is determined by cell-cell adhesion, thought to be initiated through trans-interactions between extracellular domains of the cadherin family of adhesion proteins, and strengthened by linkage to the actin cytoskeleton. Prevailing dogma is that cadherins are linked to the actin cytoskeleton through beta-catenin and alpha-catenin, although the quaternary complex has never been demonstrated. We test this hypothesis and find that alpha-catenin does not interact with actin filaments and the E-cadherin-beta-catenin complex simultaneously, even in the presence of the actin binding proteins vinculin and alpha-actinin, either in solution or on isolated cadherin-containing membranes. Direct analysis in polarized cells shows that mobilities of E-cadherin, beta-catenin, and alpha-catenin are similar, regardless of the dynamic state of actin assembly, whereas actin and several actin binding proteins have higher mobilities. These results suggest that the linkage between the cadherin-catenin complex and actin filaments is more dynamic than previously appreciated.

α-Catenin Is a Molecular Switch that Binds E-Cadherin-β-Catenin and Regulates Actin-Filament Assembly

Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that α-catenin links the adhesion protein E-cadherin through β-catenin to the actin cytoskeleton, in the accompanying paper we report that α-catenin does not bind simultaneously to both E-cadherin-β-catenin and actin filaments. Here we demonstrate that α-catenin exists as a monomer or a homodimer with different binding properties. Monomeric α-catenin binds more strongly to E-cadherin-β-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that α-catenin is an allosteric protein. Significantly, α-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for α-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion.

Mechanics of living cells measured by laser tracking microrheology

To establish laser-tracking microrheology (LTM) as a new technique for quantifying cytoskeletal mechanics, we measure viscoelastic moduli with wide bandwidth (5 decades) within living cells. With the first subcellular measurements of viscoelastic phase angles, LTM provides estimates of solid versus liquid behavior at different frequencies. In LTM, the viscoelastic shear moduli are inferred from the Brownian motion of particles embedded in the cytoskeletal network. Custom laser optoelectronics provide sub-nanometer and near-microsecond resolution of particle trajectories. The kidney epithelial cell line, COS7, has numerous spherical lipid-storage granules that are ideal probes for noninvasive LTM. Although most granules are percolating through perinuclear spaces, a subset of perinuclear granules is embedded in dense viscoelastic cytoplasm. Over all time scales embedded particles exhibit subdiffusive behavior and are not merely tethered by molecular motors. At low frequencies, lamellar regions (820 +/- 520 dyne/cm(2)) are more rigid than viscoelastic perinuclear regions (330 +/- 250 dyne/cm(2), p < 0.0001), but spectra converge at high frequencies. Although the actin-disrupting agent, latrunculin A, softens and liquefies lamellae, physiological levels of F-actin, alone (11 +/- 1.2 dyne/cm(2)) are approximately 70-fold softer than lamellae. Therefore, F-actin is necessary for lamellae mechanics, but not sufficient. Furthermore, in time-lapse of apparently quiescent cells, individual lamellar granules can show approximately 4-fold changes in moduli that last >10 s. Over a broad range of frequencies (0.1-30, 000 rad/s), LTM provides a unique ability to noninvasively quantify dynamic, local changes in cell viscoelasticity.

Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell–cell adhesion

Spatiotemporal coordination of cell–cell adhesion involving lamellipodial interactions, cadherin engagement, and the lateral expansion of the contact is poorly understood. Using high-resolution live-cell imaging, biosensors, and small molecule inhibitors, we investigate how Rac1 and RhoA regulate actin dynamics during de novo contact formation between pairs of epithelial cells. Active Rac1, the Arp2/3 complex, and lamellipodia are initially localized to de novo contacts but rapidly diminish as E-cadherin accumulates; further rounds of activation and down-regulation of Rac1 and Arp2/3 occur at the contacting membrane periphery, and this cycle repeats as a restricted membrane zone that moves outward with the expanding contact. The cortical bundle of actin filaments dissolves beneath the expanding contacts, leaving actin bundles at the contact edges. RhoA and actomyosin contractility are activated at the contact edges and are required to drive expansion and completion of cell–cell adhesion. We show that zones of Rac1 and lamellipodia activity and of RhoA and actomyosin contractility are restricted to the periphery of contacting membranes and together drive initiation, expansion, and completion of cell–cell adhesion.

The 'ins' and 'outs' of intermediate filament organization.

A major function shared by several types of cytoplasmic intermediate filaments (IFs) is to stabilize cellular architecture against the mechanical forces it is subjected to. As for other fibrous cytoskeletal arrays, a crucial determinant of this function is the spatial organization of IFs in the cytoplasm. However, very few crossbridging proteins are specific for IFs - most IF-associated proteins known to exert a structural role act to tether IFs to other major cytoskeletal elements, such as F-actin, microtubules or adhesion complexes. In addition, IFs are endowed with the ability to participate in their own organization. This intriguing property is probably connected to the unusual degree of sequence diversity and sequence-specific regulation that characterize IF genes and their proteins. This dependence upon a combination of extrinsic and intrinsic determinants contributes to distinguish IFs from other fibrous cytoskeletal polymers and is key to their function.

Biochemical and structural analysis of α-catenin in cell–cell contacts

Cadherins are transmembrane adhesion molecules that mediate homotypic cell-cell contact. In adherens junctions, the cytoplasmic domain of cadherins is functionally linked to the actin cytoskeleton through a series of proteins known as catenins. E-cadherin binds to beta-catenin, which in turn binds to alpha-catenin to form a ternary complex. alpha-Catenin also binds to actin, and it was assumed previously that alpha-catenin links the cadherin-catenin complex to actin. However, biochemical, structural and live-cell imaging studies of the cadherin-catenin complex and its interaction with actin show that binding of beta-catenin to alpha-catenin prevents the latter from binding to actin. Biochemical and structural data indicate that alpha-catenin acts as an allosteric protein whose conformation and activity changes depending on whether or not it is bound to beta-catenin. Initial contacts between cells occur on dynamic lamellipodia formed by polymerization of branched actin networks, a process controlled by the Arp2/3 (actin-related protein 2/3) complex. alpha-Catenin can suppress the activity of Arp2/3 by competing for actin filaments. These findings lead to a model for adherens junction formation in which clustering of the cadherin-beta-catenin complex recruits high levels of alpha-catenin that can suppress the Arp2/3 complex, leading to cessation of lamellipodial movement and formation of a stable contact. Thus alpha-catenin appears to play a central role in cell-cell contact formation.

E-cadherin plays an essential role in collective directional migration of large epithelial sheets

In wound healing and development, large epithelial sheets migrate collectively, in defined directions, and maintain tight cell–cell adhesion. This type of movement ensures an essential function of epithelia, a barrier, which is lost when cells lose connection and move in isolation. Unless wounded, epithelial sheets in cultures normally do not have overall directional migration. Cell migration is mostly studied when cells are in isolation and in the absence of mature cell–cell adhesion; the mechanisms of the migration of epithelial sheets are less well understood. We used small electric fields (EFs) as a directional cue to instigate and guide migration of epithelial sheets. Significantly, cells in monolayer migrated far more efficiently and directionally than cells in isolation or smaller cell clusters. We demonstrated for the first time the group size-dependent directional migratory response in several types of epithelial cells. Gap junctions made a minimal contribution to the directional collective migration. Breaking down calcium-dependent cell–cell adhesion significantly reduced directional sheet migration. Furthermore, E-cadherin blocking antibodies abolished migration of cell sheets. Traction force analysis revealed an important role of forces that cells in the leading rows exert on the substratum. With EF, the traction forces of the leading edge cells coordinated in directional re-orientation. Our study thus identifies a novel mechanism—E-cadherin dependence and coordinated traction forces of leading cells in collective directional migration of large epithelial sheets.

N-cadherin-mediated cell-cell adhesion promotes cell migration in a three-dimensional matrix

Summary Cancer cells that originate from epithelial tissues typically lose epithelial specific cell–cell junctions, but these transformed cells are not devoid of cell–cell adhesion proteins. Using hepatocyte-growth-factor-treated MDCK cells that underwent a complete epithelial-to-mesenchymal transition, we analyzed cell–cell adhesion between these highly invasive transformed epithelial cells in a three-dimensional (3D) collagen matrix. In a 3D matrix, these transformed cells formed elongated multicellular chains, and migrated faster and more persistently than single cells in isolation. In addition, the cell clusters were enriched with stress-fiber-like actin bundles that provided contractile forces. N-cadherin-knockdown cells failed to form cell–cell junctions or migrate, and the expression of the N-cadherin cytoplasmic or extracellular domain partially rescued the knockdown phenotype. By contrast, the expression of N-cadherin–&agr;-catenin chimera rescued the knockdown phenotype, but individual cells within the cell clusters were less mobile. Together, our findings suggest that a dynamic N-cadherin and actin linkage is required for efficient 3D collective migration.

The nonhelical tail domain of keratin 14 promotes filament bundling and enhances the mechanical properties of keratin intermediate filaments in vitro

Keratin filaments arise from the copolymerization of type I and II sequences, and form a pancytoplasmic network that provides vital mechanical support to epithelial cells. Keratins 5 and 14 are expressed as a pair in basal cells of stratified epithelia, where they occur as bundled arrays of filaments. In vitro, bundles of K5–K14 filaments can be induced in the absence of cross-linkers, and exhibit enhanced resistance to mechanical strain. This property is not exhibited by copolymers of K5 and tailless K14, in which the nonhelical tail domain has been removed, or copolymers of K5 and K19, a type I keratin featuring a short tail domain. The purified K14 tail domain binds keratin filaments in vitro with specificity (kD ∼2 μM). When transiently expressed in cultured cells, the K14 tail domain associates with endogenous keratin filaments. Utilization of the K14 tail domain as a bait in a yeast two-hybrid screen pulls out type I keratin sequences from a skin cDNA library. These data suggest that the tail domain of K14 contributes to the ability of K5–K14 filaments to self-organize into large bundles showing enhanced mechanical resilience in vitro.

Self-organization of an acentrosomal microtubule network at the basal cortex of polarized epithelial cells

Mechanisms underlying the organization of centrosome-derived microtubule arrays are well understood, but less is known about how acentrosomal microtubule networks are formed. The basal cortex of polarized epithelial cells contains a microtubule network of mixed polarity. We examined how this network is organized by imaging microtubule dynamics in acentrosomal basal cytoplasts derived from these cells. We show that the steady-state microtubule network appears to form by a combination of microtubule–microtubule and microtubule–cortex interactions, both of which increase microtubule stability. We used computational modeling to determine whether these microtubule parameters are sufficient to generate a steady-state acentrosomal microtubule network. Microtubules undergoing dynamic instability without any stabilization points continuously remodel their organization without reaching a steady-state network. However, the addition of increased microtubule stabilization at microtubule–microtubule and microtubule–cortex interactions results in the rapid assembly of a steady-state microtubule network in silico that is remarkably similar to networks formed in situ. These results define minimal parameters for the self-organization of an acentrosomal microtubule network.