Tissue Morphogenesis Through Dynamic Cell and Matrix Interactions.

Multicellular organisms generate tissues of diverse shapes and functions from cells and extracellular matrices. Their adhesion molecules mediate cell-cell and cell-matrix interactions, which not only play crucial roles in maintaining tissue integrity but also serve as key regulators of tissue morphogenesis. Cells constantly probe their environment to make decisions: They integrate chemical and mechanical information from the environment via diffusible ligand- or adhesion-based signaling to decide whether to release specific signaling molecules or enzymes, to divide or differentiate, to move away or stay, or even whether to live or die. These decisions in turn modify their environment, including the chemical nature and mechanical properties of the extracellular matrix. Tissue morphology is the physical manifestation of the remodeling of cells and matrices by their historical biochemical and biophysical landscapes. We review our understanding of matrix and adhesion molecules in tissue morphogenesis, with an emphasis on key physical interactions that drive morphogenesis.

Budding epithelial morphogenesis driven by cell-matrix versus cell-cell adhesion.

Many embryonic organs undergo epithelial morphogenesis to form tree-like hierarchical structures. However, it remains unclear what drives the budding and branching of stratified epithelia, such as in the embryonic salivary gland and pancreas. Here, we performed live-organ imaging of mouse embryonic salivary glands at single-cell resolution to reveal that budding morphogenesis is driven by expansion and folding of a distinct epithelial surface cell sheet characterized by strong cell-matrix adhesions and weak cell-cell adhesions. Profiling of single-cell transcriptomes of this epithelium revealed spatial patterns of transcription underlying these cell adhesion differences. We then synthetically reconstituted budding morphogenesis by experimentally suppressing E-cadherin expression and inducing basement membrane formation in 3D spheroid cultures of engineered cells, which required ß1-integrin-mediated cell-matrix adhesion for successful budding. Thus, stratified epithelial budding, the key first step of branching morphogenesis, is driven by an overall combination of strong cell-matrix adhesion and weak cell-cell adhesion by peripheral epithelial cells.

Ubiquitin ligases: guardians of mammalian development.

Mammalian development demands precision. Millions of molecules must be properly located in temporal order, and their function regulated, to orchestrate important steps in cell cycle progression, apoptosis, migration and differentiation, to shape developing embryos. Ubiquitin and its associated enzymes act as cellular guardians to ensure precise spatio-temporal control of key molecules during each of these important cellular processes. Loss of precision results in numerous examples of embryological disorders or even cancer. This Review discusses the crucial roles of E3 ubiquitin ligases during key steps of early mammalian development and their roles in human disease, and considers how new methods to manipulate and exploit the ubiquitin regulatory machinery - for example, the development of molecular glues and PROTACs - might facilitate clinical therapy.

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.

New dimensions in cell migration.

Studies of cell migration in three-dimensional (3D) cell culture systems and in vivo have revealed several differences when compared with cell migration in two dimensions, including their morphology and mechanical and signalling control. Here, researchers assess the contribution of 3D models to our understanding of cell migration, both in terms of the mechanisms used to drive single cell and collective cell migration and how migrating cells respond to a changing environment in vivo.

The extracellular matrix in development.

As the crucial non-cellular component of tissues, the extracellular matrix (ECM) provides both physical support and signaling regulation to cells. Some ECM molecules provide a fibrillar environment around cells, while others provide a sheet-like basement membrane scaffold beneath epithelial cells. In this Review, we focus on recent studies investigating the mechanical, biophysical and signaling cues provided to developing tissues by different types of ECM in a variety of developing organisms. In addition, we discuss how the ECM helps to regulate tissue morphology during embryonic development by governing key elements of cell shape, adhesion, migration and differentiation.

Cell-extracellular matrix dynamics.

The sites of interaction between a cell and its surrounding microenvironment serve as dynamic signaling hubs that regulate cellular adaptations during developmental processes, immune functions, wound healing, cell migration, cancer invasion and metastasis, as well as in many other disease states. For most cell types, these interactions are established by integrin receptors binding directly to extracellular matrix proteins, such as the numerous collagens or fibronectin. For the cell, these points of contact provide vital cues by sampling environmental conditions, both chemical and physical. The overall regulation of this dynamic interaction involves both extracellular and intracellular components and can be highly variable. In this review, we highlight recent advances and hypotheses about the mechanisms and regulation of cell-ECM interactions, from the molecular to the tissue level, with a particular focus on cell migration. We then explore how cancer cell invasion and metastasis are deeply rooted in altered regulation of this vital interaction.

Random versus directionally persistent cell migration.

Directional migration is an important component of cell motility. Although the basic mechanisms of random cell movement are well characterized, no single model explains the complex regulation of directional migration. Multiple factors operate at each step of cell migration to stabilize lamellipodia and maintain directional migration. Factors such as the topography of the extracellular matrix, the cellular polarity machinery, receptor signalling, integrin trafficking, integrin co-receptors and actomyosin contraction converge on regulation of the Rho family of GTPases and the control of lamellipodial protrusions to promote directional migration.

Btbd7 is essential for region-specific epithelial cell dynamics and branching morphogenesis in vivo.

Branching morphogenesis of developing organs requires coordinated but poorly understood changes in epithelial cell-cell adhesion and cell motility. We report that Btbd7 is a crucial regulator of branching morphogenesis in vivo. Btbd7 levels are elevated in peripheral cells of branching epithelial end buds, where it enhances cell motility and cell-cell adhesion dynamics. Genetic ablation of Btbd7 in mice disrupts branching morphogenesis of salivary gland, lung and kidney. Btbd7 knockout results in more tightly packed outer bud cells, which display stronger E-cadherin localization, reduced cell motility and decreased dynamics of transient cell separations associated with cleft formation; inner bud cells remain unaffected. Mechanistic analyses using in vitro MDCK cells to mimic outer bud cell behavior establish that Btbd7 promotes loss of E-cadherin from cell-cell adhesions with enhanced migration and transient cell separation. Btbd7 can enhance E-cadherin ubiquitination, internalization, and degradation in MDCK and peripheral bud cells for regulating cell dynamics. These studies show how a specific regulatory molecule, Btbd7, can function at a local region of developing organs to regulate dynamics of cell adhesion and motility during epithelial branching morphogenesis.

Characterization of stitch adhesions: Fibronectin-containing cell-cell contacts formed by fibroblasts.

Fibronectin is a multifunctional, extracellular matrix glycoprotein that exists either as an insoluble multimeric fibrillar component of the extracellular matrix or as a soluble monomer. Cells attach to fibronectin through transmembrane integrin receptors and form a variety of cell-matrix contacts. Here we show that primary fibroblasts can use fibronectin to organize a specific cell-cell contact - "stitch adhesions." This contact is formed by short parallel fibronectin fibrils connecting adjacent cells above the level of the focal adhesions that attach the cells to the substrate. Stitch adhesions contain integrin α5ß1 but not αVß3, align with actin filament bundles, and contain talin, tensin, α-actinin, vinculin, paxillin and a phosphorylated form of focal adhesion kinase. This combination of components differs from the described constituents of the known cell adhesions. Stitch adhesions are organized when protein synthesis and secretion are inhibited by cycloheximide and exogenous fibronectin is provided to the cells. The adhesion stitches described here provide an attractive model system for studying fibronectin fibrillogenesis and the mechanisms governing the formation of cellular adhesions.

Durotaxis by Human Cancer Cells.

Durotaxis is a type of directed cell migration in which cells respond to a gradient of extracellular stiffness. Using automated tracking of positional data for large sample sizes of single migrating cells, we investigated 1) whether cancer cells can undergo durotaxis; 2) whether cell durotactic efficiency varies depending on the regional compliance of stiffness gradients; 3) whether a specific cell migration parameter such as speed or time of migration correlates with durotaxis; and 4) whether Arp2/3, previously implicated in leading edge dynamics and migration, contributes to cancer cell durotaxis. Although durotaxis has been characterized primarily in nonmalignant mesenchymal cells, little is known about its role in cancer cell migration. Diffusible factors are known to affect cancer cell migration and metastasis. However, because many tumor microenvironments gradually stiffen, we hypothesized that durotaxis might also govern migration of cancer cells. We evaluated the durotactic potential of multiple cancer cell lines by employing substrate stiffness gradients mirroring the physiological stiffness encountered by cells in a variety of tissues. Automated cell tracking permitted rapid acquisition of positional data and robust statistical analyses for migrating cells. These durotaxis assays demonstrated that all cancer cell lines tested (two glioblastoma, metastatic breast cancer, and fibrosarcoma) migrated directionally in response to changes in extracellular stiffness. Unexpectedly, all cancer cell lines tested, as well as noninvasive human fibroblasts, displayed the strongest durotactic migratory response when migrating on the softest regions of stiffness gradients (2-7 kPa), with decreased responsiveness on stiff regions of gradients. Focusing on glioblastoma cells, durotactic forward migration index and displacement rates were relatively stable over time. Correlation analyses showed the expected correlation with displacement along the gradient but much less with persistence and none with cell speed. Finally, we found that inhibition of Arp2/3, an actin-nucleating protein necessary for lamellipodial protrusion, impaired durotactic migration.

Extracellular matrix dynamics in cell migration, invasion and tissue morphogenesis.

This review describes how direct visualization of the dynamic interactions of cells with different extracellular matrix microenvironments can provide novel insights into complex biological processes. Recent studies have moved characterization of cell migration and invasion from classical 2D culture systems into 1D and 3D model systems, revealing multiple differences in mechanisms of cell adhesion, migration and signalling-even though cells in 3D can still display prominent focal adhesions. Myosin II restrains cell migration speed in 2D culture but is often essential for effective 3D migration. 3D cell migration modes can switch between lamellipodial, lobopodial and/or amoeboid depending on the local matrix environment. For example, "nuclear piston" migration can be switched off by local proteolysis, and proteolytic invadopodia can be induced by a high density of fibrillar matrix. Particularly, complex remodelling of both extracellular matrix and tissues occurs during morphogenesis. Extracellular matrix supports self-assembly of embryonic tissues, but it must also be locally actively remodelled. For example, surprisingly focal remodelling of the basement membrane occurs during branching morphogenesis-numerous tiny perforations generated by proteolysis and actomyosin contractility produce a microscopically porous, flexible basement membrane meshwork for tissue expansion. Cells extend highly active blebs or protrusions towards the surrounding mesenchyme through these perforations. Concurrently, the entire basement membrane undergoes translocation in a direction opposite to bud expansion. Underlying this slowly moving 2D basement membrane translocation are highly dynamic individual cell movements. We conclude this review by describing a variety of exciting research opportunities for discovering novel insights into cell-matrix interactions.

Mechanosensing via cell-matrix adhesions in 3D microenvironments.

The extracellular matrix (ECM) microenvironment plays a central role in cell migration by providing physiochemical information that influences overall cell behavior. Much of this external information is accessed by direct interaction of the cell with ECM ligands and structures via integrin-based adhesions that are hypothesized to act as mechanosensors for testing the surrounding microenvironment. Our current understanding of these mechanical complexes is derived primarily from studies of cellular adhesions formed on two-dimensional (2D) substrates in vitro. Yet the rules of cell/ECM engagement and mechanosensing in three-dimensional (3D) microenvironments are invariably more complex under both in vitro and in vivo conditions. Here we review the current understanding of how cellular mechanosensing occurs through adhesion complexes within 3D microenvironments and discuss how these mechanisms can vary and differ from interactions on 2D substrates.

Local and global dynamics of the basement membrane during branching morphogenesis require protease activity and actomyosin contractility.

Many epithelial tissues expand rapidly during embryonic development while remaining surrounded by a basement membrane. Remodeling of the basement membrane is assumed to occur during branching morphogenesis to accommodate epithelial growth, but how such remodeling occurs is not yet clear. We report that the basement membrane is highly dynamic during branching of the salivary gland, exhibiting both local and global remodeling. At the tip of the epithelial end bud, the basement membrane becomes perforated by hundreds of well-defined microscopic holes at regions of rapid expansion. Locally, this results in a distensible, mesh-like basement membrane for controlled epithelial expansion while maintaining tissue integrity. Globally, the basement membrane translocates rearward as a whole, accumulating around the forming secondary ducts, helping to stabilize them during branching. Both local and global dynamics of the basement membrane require protease and myosin II activity. Our findings suggest that the basement membrane is rendered distensible by proteolytic degradation to allow it to be moved and remodeled by cells through actomyosin contractility to support branching morphogenesis.

Hemin activation of innate cellular response blocks human immunodeficiency virus type-1-induced osteoclastogenesis.

The normal skeletal developmental and homeostatic process termed osteoclastogenesis is exacerbated in numerous pathological conditions and causes excess bone loss. In cancer and HIV-1-infected patients, this disruption of homeostasis results in osteopenia and eventual osteoporesis. Counteracting the factors responsible for these metabolic disorders remains a challenge for preventing or minimizing this co-morbidity associated with these diseases. In this report, we demonstrate that a hemin-induced host protection mechanism not only suppresses HIV-1 associated osteoclastogenesis, but it also exhibits anti-osteoclastogenic activity for non-infected cells. Since the mode of action of hemin is both physiological and pharmacological through induction of heme oxygenase-1 (HO-1), an endogenous host protective response to an FDA-licensed therapeutic used to treat another disease, our study suggests an approach to developing novel, safe and effective therapeutic strategies for treating bone disorders, because hemin administration in humans has previously met required FDA safety standards.

At the leading edge of three-dimensional cell migration.

Cells migrating on flat two-dimensional (2D) surfaces use actin polymerization to extend the leading edge of the plasma membrane during lamellipodia-based migration. This mode of migration is not universal; it represents only one of several mechanisms of cell motility in three-dimensional (3D) environments. The distinct modes of 3D migration are strongly dependent on the physical properties of the extracellular matrix, and they can be distinguished by the structure of the leading edge and the degree of matrix adhesion. How are these distinct modes of cell motility in 3D environments related to each other and regulated? Recent studies show that the same type of cell migrating in 3D extracellular matrix can switch between different leading edge structures. This mode-switching behavior, or plasticity, by a single cell suggests that the apparent diversity of motility mechanisms is integrated by a common intracellular signaling pathway that governs the mode of cell migration. In this Commentary, we propose that the mode of 3D cell migration is governed by a signaling axis involving cell-matrix adhesions, RhoA signaling and actomyosin contractility, and that this might represent a universal mechanism that controls 3D cell migration.

Micro-environmental control of cell migration--myosin IIA is required for efficient migration in fibrillar environments through control of cell adhesion dynamics.

Recent evidence suggests that organization of the extracellular matrix (ECM) into aligned fibrils or fibril-like ECM topographies promotes rapid migration in fibroblasts. However, the mechanisms of cell migration that are altered by these changes in micro-environmental topography remain unknown. Here, using 1D fibrillar migration as a model system for oriented fibrillar 3D matrices, we find that fibroblast leading-edge dynamics are enhanced by 1D fibrillar micropatterns and demonstrate a dependence on the spatial positioning of cell adhesions. Although 1D, 2D and 3D matrix adhesions have similar assembly kinetics, both 1D and 3D adhesions are stabilized for prolonged periods, whereas both paxillin and vinculin show slower turnover rates in 1D adhesions. Moreover, actin in 1D adhesions undergoes slower retrograde flow than the actin that is present in 2D lamellipodia. These data suggest an increase in mechanical coupling between adhesions and protrusive machinery. Experimental reduction of contractility resulted in the loss of 1D adhesion structure and stability, with scattered small and unstable adhesions, and an uncoupling of adhesion protein-integrin stability. Genetic ablation of myosin IIA (MIIA) or myosin IIB (MIIB) isoforms revealed that MIIA is required for efficient migration in restricted environments as well as adhesion maturation, whereas MIIB helps to stabilize adhesions beneath the cell body. These data suggest that restricted cell environments, such as 1D patterns, require cellular contraction through MIIA to enhance adhesion stability and coupling to integrins behind the leading edge. This increase in mechanical coupling allows for greater leading-edge protrusion and rapid cell migration.

Non-coding RNAs and heme oxygenase-1 in vaccinia virus infection.

Small nuclear RNAs (snRNAs) are <200 nucleotide non-coding uridylate-rich RNAs. Although the functions of many snRNAs remain undetermined, a population of snRNAs is produced during the early phase of infection of cells by vaccinia virus. In the present study, we demonstrate a direct correlation between expression of the cytoprotective enzyme heme oxygenase-1 (HO-1), suppression of selective snRNA expression, and inhibition of vaccinia virus infection of macrophages. Hemin induced HO-1 expression, completely reversed virus-induced host snRNA expression, and suppressed vaccinia virus infection. This involvement of specific virus-induced snRNAs and associated gene clusters suggests a novel HO-1-dependent host-defense pathway in poxvirus infection.

Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk.

Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics.