Ronen Zaidel-Bar

Hierarchical assembly of cell-matrix adhesion complexes

The adhesion of cells to the extracellular matrix is a dynamic process, mediated by a series of cell-surface and matrix-associated molecules that interact with each other in a spatially and temporally regulated manner. These interactions play a major role in tissue formation, cellular migration and the induction of adhesion-mediated transmembrane signals. In this paper, we show that the formation of matrix adhesions is a hierarchical process, consisting of several sequential molecular events. One of the earliest steps in surface recognition is mediated, in some cells, by a 1 microm-thick cell-surface hyaluronan coat, which precedes the establishment of stable, cytoskeleton-associated adhesions. The earliest forms of these integrin-mediated contacts are dot-shaped FXs (focal complexes), which are formed under the protrusive lamellipodium of migrating cells. These adhesions recruit, sequentially, different anchor proteins that are involved in binding the actin cytoskeleton to the membrane. Conspicuous in its absence from FXs is zyxin, which is recruited to these sites only on retraction of the leading edge and the transformation of the FXs into a focal adhesion. Continuing application of force to focal adhesions results in the formation of fibrillar adhesions and reorganization of the extracellular matrix. The formation of these adhesions depends on actomyosin contractility and matrix pliability.

A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions

Diverse cellular processes are carried out by distinct integrin-mediated adhesions. Cell spreading and migration are driven by focal complexes; robust adhesion to the extracellular matrix by focal adhesions; and matrix remodeling by fibrillar adhesions. The mechanism(s) regulating the spatio-temporal distribution and dynamics of the three types of adhesion are unknown. Here, we combine live-cell imaging, labeling with phosphospecific-antibodies and overexpression of a novel tyrosine phosphomimetic mutant of paxillin, to demonstrate that the modulation of tyrosine phosphorylation of paxillin regulates both the assembly and turnover of adhesion sites. Moreover, phosphorylated paxillin enhanced lamellipodial protrusions, whereas non-phosphorylated paxillin was essential for fibrillar adhesion formation and for fibronectin fibrillogenesis. We further show that focal adhesion kinase preferentially interacted with the tyrosine phosphomimetic paxillin and its recruitment is implicated in high turnover of focal complexes and translocation of focal adhesions. We created a mathematical model that recapitulates the salient features of the measured dynamics, and conclude that tyrosine phosphorylation of the adaptor protein paxillin functions as a major switch, regulating the adhesive phenotype of cells.

The switchable integrin adhesome

Adhesion sites, which interconnect cells with their neighbors or with the extracellular matrix (ECM), are large multiprotein complexes that provide mechanical coupling as well as a means for cells to sense the chemical and physical properties of their environment ([Bershadsky et al., 2003][1]; [Chen

Actin-Delimited Adhesion-Independent Clustering of E-Cadherin Forms the Nanoscale Building Blocks of Adherens Junctions

E-cadherin is the major adhesion receptor in epithelial adherens junctions, which connect cells to form tissues and are essential for morphogenesis and homeostasis. The mechanism by which E-cadherin monomers cluster and become organized in adherens junctions remains poorly understood. Here, using superresolution microscopy techniques in combination with structure-informed functional mutations, we found that loosely organized clusters of approximately five E-cadherin molecules that form independently of cis or trans interactions, and that are delimited by the cortical F-actin meshwork, are the precursors of trans-ligated adhesive clusters that make up the adherens junction. The density of E-cadherin clusters was wide ranged, and notably, we could detect densities consistent with the crystal lattice structure at the core of adhesive clusters, which were dependent on extracellular domain interactions. Thus, our results elucidate the nanoscale architecture of adherens junctions, as well as the molecular mechanisms driving its assembly.

Opening the floodgates: proteomics and the integrin adhesome

Cell biologists studying cell adhesion have already figured out that cell-extracellular matrix connections, mediated by integrin receptors, are diverse and extremely complex structures. Dozens of adaptors-linking integrins with the cytoskeleton, and numerous enzymes and signaling proteins-regulating adhesion site dynamics, collectively referred to as the integrin adhesome, cooperate in mediating adhesion and activating specific signaling networks. Recent proteomic studies indicate that the known adhesome complexity is just the tip of the iceberg. In each existing category of molecular function the number of candidate components more than double the known components and several new categories are suggested. Proteomic analysis of different integrin heterodimers points to integrin-specific variations in composition and analysis of adhesion complexes under varying tension regimes highlights the force-dependent recruitment of different components, most notably LIM domain proteins.

Two Isoforms of the Drosophila RNA Binding Protein, How, Act in Opposing Directions to Regulate Tendon Cell Differentiation

Differential RNA metabolism regulates a wide array of developmental processes. Here, we describe a mechanism that controls the transition from premature Drosophila tendon precursors into mature muscle-bound tendon cells. This mechanism is based on the opposing activities of two isoforms of the RNA binding protein How. While the isoform How(L) is a negative regulator of Stripe, the key modulator of tendon cell differentiation, How(S) isoform elevates Stripe levels, thereby releasing the differentiation arrest induced by How(L). The opposing activities of the How isoforms are manifested by differential rates of mRNA degradation of the target stripe mRNA. This mechanism is conserved, as the mammalian RNA binding Quaking proteins may similarly affect the levels of Krox20, a regulator of Schwann cell maturation.

E-cadherin interactome complexity and robustness resolved by quantitative proteomics

The protein interaction network associated with E-cadherin is robust to changes in cell-cell adhesion. E-cadherin Sticks with the Program The transmembrane cell adhesion protein E-cadherin is responsible for the integrity of most epithelial tissues, forming contacts between neighboring cells in cellular structures called adherens junctions. Decreases in E-cadherin–mediated cell adhesion are associated with migratory cell behavior that occurs during normal development and cancer metastasis. Guo et al. used a mass spectrometry–based approach to comprehensively identify E-cadherin–interacting proteins in human gastric cancer cells. Using multiple approaches, they characterized the localization of many of these interacting proteins. Furthermore, analysis of cells exposed to a small molecule that prevented the ability of E-cadherin to bind to E-cadherin on other cells revealed that most of intracellular proteins that interact with E-cadherin do not require cell-cell adhesion, suggesting that the E-cadherin intracellular interaction network is robust to perturbation, which may poise cells to form strong adherens junctions when conditions are right. E-cadherin–mediated cell-cell adhesion and signaling plays an essential role in development and maintenance of healthy epithelial tissues. Adhesiveness mediated by E-cadherin is conferred by its extracellular cadherin domains and is regulated by an assembly of intracellular adaptors and enzymes associated with its cytoplasmic tail. We used proximity biotinylation and quantitative proteomics to identify 561 proteins in the vicinity of the cytoplasmic tail of E-cadherin. In addition, we used proteomics to identify proteins associated with E-cadherin–containing adhesion plaques from a cell-glass interface, which enabled the assignment of cellular localization to putative E-cadherin–interacting proteins. Moreover, by tagging identified proteins with GFP (green fluorescent protein), we determined the subcellular localization of 83 putative E-cadherin–proximal proteins and identified 24 proteins that were previously uncharacterized as part of adherens junctions. We constructed and characterized a comprehensive E-cadherin interaction network of 79 published and 394 previously uncharacterized proteins using a structure-informed database of protein-protein interactions. Finally, we found that calcium chelation, which disrupts the interaction of the extracellular E-cadherin domains, did not disrupt most intracellular protein interactions with E-cadherin, suggesting that the E-cadherin intracellular interactome is predominantly independent of cell-cell adhesion.

Cadherin adhesome at a glance

This article is part of a Minifocus on Adhesion. For further reading, please see related articles: ‘Cycling around cell–cell adhesion with Rho GTPase regulators’ by Jessica McCormack et al. ( J. Cell Sci. 126 , 379-391). ‘E-cadherin–integrin crosstalk in cancer invasion and metastasis’

E-cadherin junction formation involves an active kinetic nucleation process

Significance Epithelial (E)-cadherin-based adherens junctions are the basis of epithelial tissue integrity in Metazoans. They are composed of E-cadherin molecules interacting with each other from apposed cells. Using artificial supported lipid bilayers functionalized with the full-length extracellular domain of E-cadherin and live cells, we show that E-cadherin junction formation involves a nucleation process mediated by active filopodia retraction and requiring reduced mobility of E-cadherin on supported lipid bilayers. These results underscore the importance of controlling physical aspects of the cellular microenvironment with synthetic materials for in vitro live cell applications. In this case, tuning the mobility of a viscous fluid display surface enabled functional reconstitution of a cadherin-mediated adhesion junction. Epithelial (E)-cadherin-mediated cell−cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.

Long-range self-organization of cytoskeletal myosin II filament stacks

Although myosin II filaments are known to exist in non-muscle cells, their dynamics and organization are incompletely understood. Here, we combined structured illumination microscopy with pharmacological and genetic perturbations, to study the process of actomyosin cytoskeleton self-organization into arcs and stress fibres. A striking feature of the myosin II filament organization was their ‘registered’ alignment into stacks, spanning up to several micrometres in the direction orthogonal to the parallel actin bundles. While turnover of individual myosin II filaments was fast (characteristic half-life time 60 s) and independent of actin filament turnover, the process of stack formation lasted a longer time (in the range of several minutes) and required myosin II contractility, as well as actin filament assembly/disassembly and crosslinking (dependent on formin Fmnl3, cofilin1 and α-actinin-4). Furthermore, myosin filament stack formation involved long-range movements of individual myosin filaments towards each other suggesting the existence of attractive forces between myosin II filaments. These forces, possibly transmitted via mechanical deformations of the intervening actin filament network, may in turn remodel the actomyosin cytoskeleton and drive its self-organization.