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.

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.

Trends in adsorption of electrocatalytic water splitting intermediates on cubic ABO3 oxides.

The reactivity of solid oxide surfaces towards adsorption of oxygen and hydrogen is a key metric for the design of new catalysts for electrochemical water splitting. In this paper, we report on trends in the adsorption energy of different adsorbed intermediates derived from the oxidation and reduction of water for ternary ABO3 oxides in the cubic perovskite structure. Our findings support a previously reported trend that rationalizes the observed lower bound in oxygen evolution (OER) overpotentials from correlations in OH* and OOH* adsorption energies. In addition, we report hydrogen adsorption energies that may be used to estimate hydrogen evolution (HER) overpotentials along with potential metrics for electrochemical metastability in reducing environments. We also report and discuss trends between atom-projected density of states and adsorption energies, which may enable a design criteria from the local electronic structure of the active site.

Inverted formin 2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly.

Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts. High-resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF-FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FAs during maturation rather than during their initial generation, and once there, acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately 3D matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SFs and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM.

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.

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.

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.

Regulation of cell adhesion and migration by cell-derived matrices.

Three-dimensional in vitro extracellular matrix models provide a physiological alternative to regular two-dimensional cell culture, though they lack the full diversity of molecular composition and physical properties of whole-animal systems. Cell-derived matrices are extracellular matrices that are the product of matrix secretion and assembly by cells cultured at high density in vitro. After the removal of the cells that produced the matrix, an assembled matrix scaffold is left that closely mimics native stromal fiber organization and molecular content. Cell-derived matrices have been shown to impart in vivo-like responses to cells cultured in these matrices. In this review, we focus on mechanisms through which the distinct molecular and topographical composition of cell-derived matrices directs cellular behavior, specifically through regulation of cell-matrix adhesions and subsequent contributions to the process of cell migration.

Region-specific epithelial cell dynamics during branching morphogenesis.

BACKGROUND: Epithelial cells of developing embryonic organs, such as salivary glands, can display substantial motility during branching morphogenesis. Their dynamic movements and molecules involved in their migration are not fully characterized. RESULTS: We generated transgenic mice expressing photo-convertible KikGR and tracked the movements of individual cells highlighted by red fluorescence in different regions of developing salivary glands. Motility was highest for outer bud epithelial cells adjacent to the basement membrane, lower in inner bud cells, and lowest in duct cells. The highly motile outer cells contacting the basement membrane were pleomorphic, whereas inner cells were rounded. Peripheral cell motility was disrupted by antibodies inhibiting α6+ß1 integrins and the nonmuscle myosin II inhibitor blebbistatin. Inner bud cell migration was unaffected by these inhibitors, but their rate of migration was stimulated by inhibiting E-cadherin. CONCLUSIONS: Cell motility in developing salivary glands was highest in cells in contact with the basement membrane. The basement membrane-associated motility of these outer bud cells depended on integrins and myosin II, but not E-cadherin. In contrast, motility of inner bud cells was restrained by E-cadherin. These findings identify the importance of integrin-dependent basement membrane association for the morphology, tissue organization, and lateral motility of morphogenetic epithelial cells.

Teneurin-4 is a novel regulator of oligodendrocyte differentiation and myelination of small-diameter axons in the CNS.

Myelination is essential for proper functioning of the CNS. In this study, we have identified a mouse mutation, designated furue, which causes tremors and hypomyelination in the CNS, particularly in the spinal cord, but not in the sciatic nerve of the PNS. In the spinal cord of the furue mice, myelination of small-diameter axons was dramatically reduced, and differentiation of oligodendrocytes, the myelin-forming cells in the CNS, was inhibited. We subsequently found that the furue mutation was associated with a transgene insertion into the teneurin-4 (Ten-4, Ten-m4/Odz4) gene, encoding a transmembrane protein of unknown function. Ten-4 was strongly expressed in the spinal cord of wild-type mice and was induced during normal oligodendrocyte differentiation. In contrast, in the furue mice, the expression of Ten-4 was absent. Differentiation and cellular process formation of oligodendrocytes were inhibited in primary cell culture from the furue mice. Cell differentiation and process formation were also inhibited in the oligodendrocyte progenitor cell line CG-4 after suppression of Ten-4 expression by shRNA. Furthermore, Ten-4 positively regulated focal adhesion kinase, an essential signaling molecule for oligodendrocyte process formation and myelination of small-diameter axons. These findings suggest that Ten-4 is a novel regulator of oligodendrocyte differentiation and that it plays a critical role in the myelination of small-diameter axons in the CNS.

Long Prehensile Protrusions Can Facilitate Cancer Cell Invasion through the Basement Membrane.

A basic process in cancer is the breaching of basement-membrane barriers to permit tissue invasion. Cancer cells can use proteases and physical mechanisms to produce initial holes in basement membranes, but how cells squeeze through this barrier into matrix environments is not well understood. We used a 3D invasion model consisting of cancer-cell spheroids encapsulated by a basement membrane and embedded in collagen to characterize the dynamic early steps in cancer-cell invasion across this barrier. We demonstrate that certain cancer cells extend exceptionally long (~30-100 µm) protrusions through basement membranes via actin and microtubule cytoskeletal function. These long protrusions use integrin adhesion and myosin II-based contractility to pull cells through the basement membrane for initial invasion. Concurrently, these long, organelle-rich protrusions pull surrounding collagen inward while propelling cancer cells outward through perforations in the basement-membrane barrier. These exceptionally long, contractile cellular protrusions can facilitate the breaching of the basement-membrane barrier as a first step in cancer metastasis.

Maintenance of pluripotency in the entire ectoderm enables neural crest formation.

The ability of the pluripotent epiblast to contribute progeny to all three germ layers is thought to be lost after gastrulation. The later-forming neural crest (NC) rises from ectoderm and it remains poorly understood how its exceptionally high stem-cell potential to generate mesodermal- and endodermal-like cells is obtained. We monitored transcriptional changes from gastrulation to neurulation using single-cell-Multiplex-Spatial-Transcriptomics (scMST) complemented with RNA-sequencing. Unexpectedly, we find maintenance of undecided Nanog/Oct4-PouV/Klf4-positive pluripotent-like pan-ectodermal stem-cells spanning the entire ectoderm late in the neurulation process with ectodermal patterning completed only at the end of neurulation when pluripotency becomes restricted to NC, challenging our understanding of gastrulation. Furthermore, broad ectodermal pluripotency is found at all axial levels unrelated to the NC lineage the cells later commit to, suggesting a general role in stemness enhancement and proposing a mechanism by which the NC acquires its ability to form derivatives beyond "ectodermal-capacity" in chick and mouse embryos.

Maintenance of pluripotency-like signature in the entire ectoderm leads to neural crest stem cell potential.

The ability of the pluripotent epiblast to contribute progeny to all three germ layers is thought to be lost after gastrulation. The later-forming neural crest (NC) rises from ectoderm and it remains poorly understood how its exceptionally high stem-cell potential to generate mesodermal- and endodermal-like derivatives is obtained. Here, we monitor transcriptional changes from gastrulation to neurulation using single-cell-Multiplex-Spatial-Transcriptomics (scMST) complemented with RNA-sequencing. We show maintenance of pluripotency-like signature (Nanog, Oct4/PouV, Klf4-positive) in undecided pan-ectodermal stem-cells spanning the entire ectoderm late during neurulation with ectodermal patterning completed only at the end of neurulation when the pluripotency-like signature becomes restricted to NC, challenging our understanding of gastrulation. Furthermore, broad ectodermal pluripotency-like signature is found at multiple axial levels unrelated to the NC lineage the cells later commit to, suggesting a general role in stemness enhancement and proposing a mechanism by which the NC acquires its ability to form derivatives beyond "ectodermal-capacity" in chick and mouse embryos.

Biglycan regulates bone development and regeneration.

Endochondral bone development and regeneration relies on activation and proliferation of periosteum derived-cells (PDCs). Biglycan (Bgn), a small proteoglycan found in extracellular matrix, is known to be expressed in bone and cartilage, however little is known about its influence during bone development. Here we link biglycan with osteoblast maturation starting during embryonic development that later affects bone integrity and strength. Biglycan gene deletion reduced the inflammatory response after fracture, leading to impaired periosteal expansion and callus formation. Using a novel 3D scaffold with PDCs, we found that biglycan could be important for the cartilage phase preceding bone formation. The absence of biglycan led to accelerated bone development with high levels of osteopontin, which appeared to be detrimental to the structural integrity of the bone. Collectively, our study identifies biglycan as an influencing factor in PDCs activation during bone development and bone regeneration after fracture.

A ubiquitin-based effector-to-inhibitor switch coordinates early brain, craniofacial, and skin development.

The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and neural crest-derived craniofacial structures are unclear. Here, biochemical disease-variant profiling reveals a posttranslational pathway that drives early ectodermal differentiation in the vertebrate head. The anteriorly expressed ubiquitin ligase CRL3-KLHL4 restricts signaling of the ubiquitous cytoskeletal regulator CDC42. This regulation relies on the CDC42-activating complex GIT1-ßPIX, which CRL3-KLHL4 exploits as a substrate-specific co-adaptor to recognize and monoubiquitylate PAK1. Surprisingly, we find that ubiquitylation converts the canonical CDC42 effector PAK1 into a CDC42 inhibitor. Loss of CRL3-KLHL4 or a disease-associated KLHL4 variant reduce PAK1 ubiquitylation causing overactivation of CDC42 signaling and defective ectodermal patterning and neurulation. Thus, tissue-specific restriction of CDC42 signaling by a ubiquitin-based effector-to-inhibitor is essential for early face, brain, and skin formation, revealing how cell-fate and morphometric changes are coordinated to ensure faithful organ development.