Adherens junctions as molecular regulators of emergent tissue mechanics.

Tissue and organ development during embryogenesis relies on the collective and coordinated action of many cells. Recent studies have revealed that tissue material properties, including transitions between fluid and solid tissue states, are controlled in space and time to shape embryonic structures and regulate cell behaviours. Although the collective cellular flows that sculpt tissues are guided by tissue-level physical changes, these ultimately emerge from cellular-level and subcellular-level molecular mechanisms. Adherens junctions are key subcellular structures, built from clusters of classical cadherin receptors. They mediate physical interactions between cells and connect biochemical signalling to the physical characteristics of cell contacts, hence playing a fundamental role in tissue morphogenesis. In this Review, we take advantage of the results of recent, quantitative measurements of tissue mechanics to relate the molecular and cellular characteristics of adherens junctions, including adhesion strength, tension and dynamics, to the emergent physical state of embryonic tissues. We focus on systems in which cell-cell interactions are the primary contributor to morphogenesis, without significant contribution from cell-matrix interactions. We suggest that emergent tissue mechanics is an important direction for future research, bridging cell biology, developmental biology and mechanobiology to provide a holistic understanding of morphogenesis in health and disease.

Organization, dynamics and mechanoregulation of integrin-mediated cell-ECM adhesions.

The ability of animal cells to sense, adhere to and remodel their local extracellular matrix (ECM) is central to control of cell shape, mechanical responsiveness, motility and signalling, and hence to development, tissue formation, wound healing and the immune response. Cell-ECM interactions occur at various specialized, multi-protein adhesion complexes that serve to physically link the ECM to the cytoskeleton and the intracellular signalling apparatus. This occurs predominantly via clustered transmembrane receptors of the integrin family. Here we review how the interplay of mechanical forces, biochemical signalling and molecular self-organization determines the composition, organization, mechanosensitivity and dynamics of these adhesions. Progress in the identification of core multi-protein modules within the adhesions and characterization of rearrangements of their components in response to force, together with advanced imaging approaches, has improved understanding of adhesion maturation and turnover and the relationships between adhesion structures and functions. Perturbations of adhesion contribute to a broad range of diseases and to age-related dysfunction, thus an improved understanding of their molecular nature may facilitate therapeutic intervention in these conditions.

Adhesion Protein Structure, Molecular Affinities, and Principles of Cell-Cell Recognition.

The ability of cells to organize into multicellular structures in precise patterns requires that they "recognize" one another with high specificity. We discuss recent progress in understanding the molecular basis of cell-cell recognition, including unique phenomena associated with neuronal interactions. We describe structures of select adhesion receptor complexes and their assembly into larger intercellular junction structures and discuss emerging principles that relate cell-cell organization to the binding specificities and energetics of adhesion receptors. Armed with these insights, advances in protein design and gene editing should pave the way for breakthroughs toward understanding the molecular basis of cell patterning in vivo.

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.

Regulation of Collective Metastasis by Nanolumenal Signaling.

Collective metastasis is defined as the cohesive migration and metastasis of multicellular tumor cell clusters. Disrupting various cell adhesion genes markedly reduces cluster formation and colonization efficiency, yet the downstream signals transmitted by clustering remain largely unknown. Here, we use mouse and human breast cancer models to identify a collective signal generated by tumor cell clusters supporting metastatic colonization. We show that tumor cell clusters produce the growth factor epigen and concentrate it within nanolumina-intercellular compartments sealed by cell-cell junctions and lined with microvilli-like protrusions. Epigen knockdown profoundly reduces metastatic outgrowth and switches clusters from a proliferative to a collective migratory state. Tumor cell clusters from basal-like 2, but not mesenchymal-like, triple-negative breast cancer cell lines have increased epigen expression, sealed nanolumina, and impaired outgrowth upon nanolumenal junction disruption. We propose that nanolumenal signaling could offer a therapeutic target for aggressive metastatic breast cancers.

Structural Basis of Teneurin-Latrophilin Interaction in Repulsive Guidance of Migrating Neurons.

Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.

Integrins and Their Role in Immune Cell Adhesion.

One of the 2019 Canada Gairdner International Awards recognizes Timothy Springer's discovery of the first immune system adhesion molecules involved in lymphocyte homing and the translation of those discoveries into therapeutics for autoimmune disease and cancer.

The Architecture of Talin1 Reveals an Autoinhibition Mechanism.

Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting integrins in the plasma membrane with the actomyosin cytoskeleton. To understand how talin function is regulated, we determined a cryoelectron microscopy (cryo-EM) structure of full-length talin1 revealing a two-way mode of autoinhibition. The actin-binding rod domains fold into a 15-nm globular arrangement that is interlocked by the integrin-binding FERM head. In turn, the rod domains R9 and R12 shield access of the FERM domain to integrin and the phospholipid PIP2 at the membrane. This mechanism likely ensures synchronous inhibition of integrin, membrane, and cytoskeleton binding. We also demonstrate that compacted talin1 reversibly unfolds to an ∼60-nm string-like conformation, revealing interaction sites for vinculin and actin. Our data explain how fast switching between active and inactive conformations of talin could regulate FA turnover, a process critical for cell adhesion and signaling.

A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns.

Synthetic multicellular systems hold promise as models for understanding natural development of biofilms and higher organisms and as tools for engineering complex multi-component metabolic pathways and materials. However, such efforts require tools to adhere cells into defined morphologies and patterns, and these tools are currently lacking. Here, we report a 100% genetically encoded synthetic platform for modular cell-cell adhesion in Escherichia coli, which provides control over multicellular self-assembly. Adhesive selectivity is provided by a library of outer membrane-displayed nanobodies and antigens with orthogonal intra-library specificities, while affinity is controlled by intrinsic adhesin affinity, competitive inhibition, and inducible expression. We demonstrate the resulting capabilities for quantitative rational design of well-defined morphologies and patterns through homophilic and heterophilic interactions, lattice-like self-assembly, phase separation, differential adhesion, and sequential layering. Compatible with synthetic biology standards, this adhesion toolbox will enable construction of high-level multicellular designs and shed light on the evolutionary transition to multicellularity.

EBF1-deficient bone marrow stroma elicits persistent changes in HSC potential.

Crosstalk between mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) is essential for hematopoietic homeostasis and lineage output. Here, we investigate how transcriptional changes in bone marrow (BM) MSCs result in long-lasting effects on HSCs. Single-cell analysis of Cxcl12-abundant reticular (CAR) cells and PDGFRα+Sca1+ (PαS) cells revealed an extensive cellular heterogeneity but uniform expression of the transcription factor gene Ebf1. Conditional deletion of Ebf1 in these MSCs altered their cellular composition, chromatin structure and gene expression profiles, including the reduced expression of adhesion-related genes. Functionally, the stromal-specific Ebf1 inactivation results in impaired adhesion of HSCs, leading to reduced quiescence and diminished myeloid output. Most notably, HSCs residing in the Ebf1-deficient niche underwent changes in their cellular composition and chromatin structure that persist in serial transplantations. Thus, genetic alterations in the BM niche lead to long-term functional changes of HSCs.

Mechanisms of 3D cell migration.

Cell migration is essential for physiological processes as diverse as development, immune defence and wound healing. It is also a hallmark of cancer malignancy. Thousands of publications have elucidated detailed molecular and biophysical mechanisms of cultured cells migrating on flat, 2D substrates of glass and plastic. However, much less is known about how cells successfully navigate the complex 3D environments of living tissues. In these more complex, native environments, cells use multiple modes of migration, including mesenchymal, amoeboid, lobopodial and collective, and these are governed by the local extracellular microenvironment, specific modalities of Rho GTPase signalling and non-muscle myosin contractility. Migration through 3D environments is challenging because it requires the cell to squeeze through complex or dense extracellular structures. Doing so requires specific cellular adaptations to mechanical features of the extracellular matrix (ECM) or its remodelling. In addition, besides navigating through diverse ECM environments and overcoming extracellular barriers, cells often interact with neighbouring cells and tissues through physical and signalling interactions. Accordingly, cells need to call on an impressively wide diversity of mechanisms to meet these challenges. This Review examines how cells use both classical and novel mechanisms of locomotion as they traverse challenging 3D matrices and cellular environments. It focuses on principles rather than details of migratory mechanisms and draws comparisons between 1D, 2D and 3D migration.

Cadherin adhesion and mechanotransduction.

Cadherins are the principal adhesion proteins at intercellular junctions and function as the biochemical Velcro that binds cells together. Besides this mechanical function, cadherin complexes are also mechanotransducers that sense changes in tension and trigger adaptive reinforcement of intercellular junctions. The assembly and regulation of cadherin adhesions are central to their mechanical functions, and new evidence is presented for a comprehensive model of cadherin adhesion, which is surprisingly more complex than previously appreciated. Recent findings also shed new light on mechanisms that regulate cadherin junction assembly, adhesion, and mechanotransduction. We further describe recent evidence for cadherin-based mechanotransduction, and the rudiments of the molecular mechanism, which involves α-catenin and vinculin as key elements. Potential roles of a broader cast of possible force-sensitive partners are considered, as well as known and speculative biological consequences of adhesion and force transduction at cadherin-mediated junctions.

A flexible loop in the paxillin LIM3 domain mediates its direct binding to integrin ß subunits.

Integrins are fundamental for cell adhesion and the formation of focal adhesions (FA). Accordingly, these receptors guide embryonic development, tissue maintenance, and haemostasis but are also involved in cancer invasion and metastasis. A detailed understanding of the molecular interactions that drive integrin activation, FA assembly, and downstream signalling cascades is critical. Here, we reveal a direct association of paxillin, a marker protein of FA sites, with the cytoplasmic tails of the integrin ß1 and ß3 subunits. The binding interface resides in paxillin's LIM3 domain, where based on the NMR structure and functional analyses, a flexible, 7-amino acid loop engages the unstructured part of the integrin cytoplasmic tail. Genetic manipulation of the involved residues in either paxillin or integrin ß3 compromises cell adhesion and motility of murine fibroblasts. This direct interaction between paxillin and the integrin cytoplasmic domain identifies an alternative, kindlin-independent mode of integrin outside-in signalling particularly important for integrin ß3 function.

The structure and mechanics of the cell cortex depend on the location and adhesion state.

Cells exist in different phenotypes and can transition between them. A phenotype may be characterized by many different aspects. Here, we focus on the example of whether the cell is adhered or suspended and choose particular parameters related to the structure and mechanics of the actin cortex. The cortex is essential to cell mechanics, morphology, and function, such as for adhesion, migration, and division of animal cells. To predict and control cellular functions and prevent malfunctioning, it is necessary to understand the actin cortex. The structure of the cortex governs cell mechanics; however, the relationship between the architecture and mechanics of the cortex is not yet well enough understood to be able to predict one from the other. Therefore, we quantitatively measured structural and mechanical cortex parameters, including cortical thickness, cortex mesh size, actin bundling, and cortex stiffness. These measurements required developing a combination of measurement techniques in scanning electron, expansion, confocal, and atomic force microscopy. We found that the structure and mechanics of the cortex of cells in interphase are different depending on whether the cell is suspended or adhered. We deduced general correlations between structural and mechanical properties and show how these findings can be explained within the framework of semiflexible polymer network theory. We tested the model predictions by perturbing the properties of the actin within the cortex using compounds. Our work provides an important step toward predictions of cell mechanics from cortical structures and suggests how cortex remodeling between different phenotypes impacts the mechanical properties of cells.

Deposited footprints let cells switch between confined, oscillatory, and exploratory migration.

For eukaryotic cells to heal wounds, respond to immune signals, or metastasize, they must migrate, often by adhering to extracellular matrix (ECM). Cells may also deposit ECM components, leaving behind a footprint that influences their crawling. Recent experiments showed that some epithelial cell lines on micropatterned adhesive stripes move persistently in regions they have previously crawled over, where footprints have been formed, but barely advance into unexplored regions, creating an oscillatory migration of increasing amplitude. Here, we explore through mathematical modeling how footprint deposition and cell responses to footprint combine to allow cells to develop oscillation and other complex migratory motions. We simulate cell crawling with a phase field model coupled to a biochemical model of cell polarity, assuming local contact with the deposited footprint activates Rac1, a protein that establishes the cell's front. Depending on footprint deposition rate and response to the footprint, cells on micropatterned lines can display many types of motility, including confined, oscillatory, and persistent motion. On two-dimensional (2D) substrates, we predict a transition between cells undergoing circular motion and cells developing an exploratory phenotype. Small quantitative changes in a cell's interaction with its footprint can completely alter exploration, allowing cells to tightly regulate their motion, leading to different motility phenotypes (confined vs. exploratory) in different cells when deposition or sensing is variable from cell to cell. Consistent with our computational predictions, we find in earlier experimental data evidence of cells undergoing both circular and exploratory motion.

The focal adhesion protein talin is a mechanically gated A-kinase anchoring protein.

Protein kinase A (PKA) is a ubiquitous, promiscuous kinase whose activity is specified through subcellular localization mediated by A-kinase anchoring proteins (AKAPs). PKA has complex roles as both an effector and a regulator of integrin-mediated cell adhesion to extracellular matrix (ECM). Recent observations demonstrate that PKA is an active component of focal adhesions (FA), suggesting the existence of one or more FA AKAPs. Using a promiscuous biotin ligase fused to PKA type-IIα regulatory (RIIα) subunits and subcellular fractionation, we identify the archetypal FA protein talin1 as an AKAP. Talin is a large, mechanosensitive scaffold that directly links integrins to actin filaments and promotes FA assembly by recruiting additional components in a force-dependent manner. The rod region of talin1 consists of 62 α-helices bundled into 13 rod domains, R1 to R13. Direct binding assays and NMR spectroscopy identify helix41 in the R9 subdomain of talin as the PKA binding site. PKA binding to helix41 requires unfolding of the R9 domain, which requires the linker region between R9 and R10. Experiments with single molecules and in cells manipulated to alter actomyosin contractility demonstrate that the PKA-talin interaction is regulated by mechanical force across the talin molecule. Finally, talin mutations that disrupt PKA binding also decrease levels of total and phosphorylated PKA RII subunits as well as phosphorylation of VASP, a known PKA substrate, within FA. These observations identify a mechanically gated anchoring protein for PKA, a force-dependent binding partner for talin1, and a potential pathway for adhesion-associated mechanotransduction.

Nanoscale dynamics of the cadherin-catenin complex bound to vinculin revealed by neutron spin echo spectroscopy.

We report a neutron spin echo (NSE) study of the nanoscale dynamics of the cell-cell adhesion cadherin-catenin complex bound to vinculin. Our measurements and theoretical physics analyses of the NSE data reveal that the dynamics of full-length α-catenin, ß-catenin, and vinculin residing in the cadherin-catenin-vinculin complex become activated, involving nanoscale motions in this complex. The cadherin-catenin complex is the central component of the cell-cell adherens junction (AJ) and is fundamental to embryogenesis, tissue wound healing, neuronal plasticity, cancer metastasis, and cardiovascular health and disease. A highly dynamic cadherin-catenin-vinculin complex provides the molecular dynamics basis for the flexibility and elasticity that are necessary for the AJs to function as force transducers. Our theoretical physics analysis provides a way to elucidate these driving nanoscale motions within the complex without requiring large-scale numerical simulations, providing insights not accessible by other techniques. We propose a three-way "motorman" entropic spring model for the dynamic cadherin-catenin-vinculin complex, which allows the complex to function as a flexible and elastic force transducer.

Controversy in mechanotransduction - the role of endothelial cell-cell junctions in fluid shear stress sensing.

Fluid shear stress (FSS) from blood flow, sensed by the vascular endothelial cells (ECs) that line all blood vessels, regulates vascular development during embryogenesis, controls adult vascular physiology and determines the location of atherosclerotic plaque formation. Although a number of papers have reported a crucial role for cell-cell adhesions or adhesion receptors in these processes, a recent publication has challenged this paradigm, presenting evidence that ECs can very rapidly align in fluid flow as single cells without cell-cell contacts. To address this controversy, four independent laboratories assessed EC alignment in fluid flow across a range of EC cell types. These studies demonstrate a strict requirement for cell-cell contact in shear stress sensing over timescales consistent with previous literature and inconsistent with the newly published data.

Fibronectin induces a transition from amoeboid to a fan morphology and modifies migration in Entamoeba histolytica.

Cell migration modes can vary, depending on a number of environmental and intracellular factors. The high motility of the pathogenic amoeba Entamoeba histolytica is a decisive factor in its ability to cross the human colonic barrier. We used quantitative live imaging techniques to study the migration of this parasite on fibronectin, a key tissue component. Entamoeba histolytica amoebae on fibronectin contain abundant podosome-like structures. By using a laminar flow chamber, we determined that the adhesion forces generated on fibronectin were twice those on non-coated glass. When migrating on fibronectin, elongated amoeboid cells converted into fan-shaped cells characterized by the presence of a dorsal column of F-actin and a broad cytoplasmic extension at the front. The fan shape depended on the Arp2/3 complex, and the amoebae moved laterally and more slowly. Intracellular measurements of physical variables related to fluid dynamics revealed that cytoplasmic pressure gradients were weaker within fan-shaped cells; hence, actomyosin motors might be less involved in driving the cell body forward. We also found that the Rho-associated coiled-coil containing protein kinase regulated podosome dynamics. We conclude that E. histolytica spontaneously changes its migration mode as a function of the substrate composition. This adaptive ability might favour E. histolytica's invasion of human colonic tissue. By combining microfluidic experiments, mechanical modelling, and image analysis, our work also introduces a computational pipeline for the study of cell migration.