Physical influences of the extracellular environment on cell migration.

The way in which a cell migrates is influenced by the physical properties of its surroundings, in particular the properties of the extracellular matrix. How the physical aspects of the cell's environment affect cell migration poses a considerable challenge when trying to understand migration in complex tissue environments and hinders the extrapolation of in vitro analyses to in vivo situations. A comprehensive understanding of these problems requires an integrated biochemical and biophysical approach. In this Review, we outline the findings that have emerged from approaches that span these disciplines, with a focus on actin-based cell migration in environments with different stiffness, dimensionality and geometry.

Alignment of the cell long axis by unidirectional tension acts cooperatively with Wnt signalling to establish planar cell polarity.

Planar cell polarity (PCP) is the aligned cell polarity within a tissue plane. Mechanical signals are known to act as a global cue for PCP, yet their exact role is still unclear. In this study, we focused on PCP in the posterior neuroectoderm of Xenopus laevis and investigated how mechanical signals regulate polarity. We reveal that the neuroectoderm is under a greater tension in the anterior-posterior direction and that perturbation of this tension causes PCP disappearance. We show that application of uniaxial stretch to explant tissues can control the orientation of PCP and that cells sense the tissue stretch indirectly through a change in their shape, rather than directly through detection of anisotropic tension. Furthermore, we reveal that PCP is most strongly established when the orientation of tissue stretch coincides with that of diffusion of locally expressed Wnt ligands, suggesting a cooperative relationship between these two PCP regulators.

Spindle reorientation in response to mechanical stress is an emergent property of the spindle positioning mechanisms.

Proper orientation of the mitotic spindle plays a crucial role in embryos, during tissue development, and in adults, where it functions to dissipate mechanical stress to maintain tissue integrity and homeostasis. While mitotic spindles have been shown to reorient in response to external mechanical stresses, the subcellular cues that mediate spindle reorientation remain unclear. Here, we used a combination of optogenetics and computational modeling to investigate how mitotic spindles respond to inhomogeneous tension within the actomyosin cortex. Strikingly, we found that the optogenetic activation of RhoA only influences spindle orientation when it is induced at both poles of the cell. Under these conditions, the sudden local increase in cortical tension induced by RhoA activation reduces pulling forces exerted by cortical regulators on astral microtubules. This leads to a perturbation of the balance of torques exerted on the spindle, which causes it to rotate. Thus, spindle rotation in response to mechanical stress is an emergent phenomenon arising from the interaction between the spindle positioning machinery and the cell cortex.

Tension at intercellular junctions is necessary for accurate orientation of cell division in the epithelium plane.

The direction in which a cell divides is set by the orientation of its mitotic spindle and is important for determining cell fate, controlling tissue shape, and maintaining tissue architecture. Divisions parallel to the epithelial plane sustain tissue expansion. By contrast, divisions perpendicular to the plane promote tissue stratification and lead to the loss of epithelial cells from the tissue-an event that has been suggested to promote metastasis. Much is known about the molecular machinery involved in orienting the spindle, but less is known about the contribution of mechanical factors, such as tissue tension, in ensuring spindle orientation in the plane of the epithelium. This is important as epithelia are continuously subjected to mechanical stresses. To explore this further, we subjected suspended epithelial monolayers devoid of extracellular matrix to varying levels of tissue tension to study the orientation of cell divisions relative to the tissue plane. This analysis revealed that lowering tissue tension by compressing epithelial monolayers or by inhibiting myosin contractility increased the frequency of out-of-plane divisions. Reciprocally, increasing tissue tension by elevating cell contractility or by tissue stretching restored accurate in-plane cell divisions. Moreover, a characterization of the geometry of cells within these epithelia suggested that spindles can sense tissue tension through its impact on tension at subcellular surfaces, independently of their shape. Overall, these data suggest that accurate spindle orientation in the plane of the epithelium relies on a threshold level of tension at intercellular junctions.

Tissue stiffening coordinates morphogenesis by triggering collective cell migration in vivo.

Collective cell migration is essential for morphogenesis, tissue remodelling and cancer invasion. In vivo, groups of cells move in an orchestrated way through tissues. This movement involves mechanical as well as molecular interactions between cells and their environment. While the role of molecular signals in collective cell migration is comparatively well understood, how tissue mechanics influence collective cell migration in vivo remains unknown. Here we investigated the importance of mechanical cues in the collective migration of the Xenopus laevis neural crest cells, an embryonic cell population whose migratory behaviour has been likened to cancer invasion. We found that, during morphogenesis, the head mesoderm underlying the cephalic neural crest stiffens. This stiffening initiates an epithelial-to-mesenchymal transition in neural crest cells and triggers their collective migration. To detect changes in their mechanical environment, neural crest cells use mechanosensation mediated by the integrin-vinculin-talin complex. By performing mechanical and molecular manipulations, we show that mesoderm stiffening is necessary and sufficient to trigger neural crest migration. Finally, we demonstrate that convergent extension of the mesoderm, which starts during gastrulation, leads to increased mesoderm stiffness by increasing the cell density underneath the neural crest. These results show that convergent extension of the mesoderm has a role as a mechanical coordinator of morphogenesis, and reveal a link between two apparently unconnected processes-gastrulation and neural crest migration-via changes in tissue mechanics. Overall, we demonstrate that changes in substrate stiffness can trigger collective cell migration by promoting epithelial-to-mesenchymal transition in vivo. More broadly, our results raise the idea that tissue mechanics combines with molecular effectors to coordinate morphogenesis.

L-selectin regulates human neutrophil transendothelial migration.

The migration of circulating neutrophils towards damaged or infected tissue is absolutely critical to the inflammatory response. L-selectin is a cell adhesion molecule abundantly expressed on circulating neutrophils. For over two decades, neutrophil L-selectin has been assigned the exclusive role of supporting tethering and rolling - the initial stages of the multi-step adhesion cascade. Here, we provide direct evidence for L-selectin contributing to neutrophil transendothelial migration (TEM). We show that L-selectin co-clusters with PECAM-1 - a well-characterised cell adhesion molecule involved in regulating neutrophil TEM. This co-clustering behaviour occurs specifically during TEM, which serves to augment ectodomain shedding of L-selectin and expedite the time taken for TEM (TTT) to complete. Blocking PECAM-1 signalling (through mutation of its cytoplasmic tail), PECAM-1-dependent adhesion or L-selectin shedding, leads to a significant delay in the TTT. Finally, we show that co-clustering of L-selectin with PECAM-1 occurs specifically across TNF- but not IL-1ß-activated endothelial monolayers - implying unique adhesion interactomes forming in a cytokine-specific manner. To our knowledge, this is the first report to implicate a non-canonical role for L-selectin in regulating neutrophil TEM.

Curling of epithelial monolayers reveals coupling between active bending and tissue tension.

Epithelial monolayers are two-dimensional cell sheets which compartmentalize the body and organs of multicellular organisms. Their morphogenesis during development or pathology results from patterned endogenous and exogenous forces and their interplay with tissue mechanical properties. In particular, bending of epithelia is thought to result from active torques generated by the polarization of myosin motors along their apicobasal axis. However, the contribution of these out-of-plane forces to morphogenesis remains challenging to evaluate because of the lack of direct mechanical measurement. Here we use epithelial curling to characterize the out-of-plane mechanics of epithelial monolayers. We find that curls of high curvature form spontaneously at the free edge of epithelial monolayers devoid of substrate in vivo and in vitro. Curling originates from an enrichment of myosin in the basal domain that generates an active spontaneous curvature. By measuring the force necessary to flatten curls, we can then estimate the active torques and the bending modulus of the tissue. Finally, we show that the extent of curling is controlled by the interplay between in-plane and out-of-plane stresses in the monolayer. Such mechanical coupling emphasizes a possible role for in-plane stresses in shaping epithelia during morphogenesis.

Single-cell approaches to cell competition: High-throughput imaging, machine learning and simulations.

Cell competition is a quality control mechanism in tissues that results in the elimination of less fit cells. Over the past decade, the phenomenon of cell competition has been identified in many physiological and pathological contexts, driven either by biochemical signaling or by mechanical forces within the tissue. In both cases, competition has generally been characterized based on the elimination of loser cells at the population level, but significantly less attention has been focused on determining how single-cell dynamics and interactions regulate population-wide changes. In this review, we describe quantitative strategies and outline the outstanding challenges in understanding the single cell rules governing tissue-scale competition dynamics. We propose quantitative metrics to characterize single cell behaviors in competition and use them to distinguish the types and outcomes of competition. We describe how such metrics can be measured experimentally using a novel combination of high-throughput imaging and machine learning algorithms. We outline the experimental challenges to quantify cell fate dynamics with high-statistical precision, and describe the utility of computational modeling in testing hypotheses not easily accessible in experiments. In particular, cell-based modeling approaches that combine mechanical interaction of cells with decision-making rules for cell fate choices provide a powerful framework to understand and reverse-engineer the diverse rules of cell competition.

Cortical cell stiffness is independent of substrate mechanics.

Cortical stiffness is an important cellular property that changes during migration, adhesion and growth. Previous atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates have suggested that cells adapt their stiffness to that of their surroundings. Here we show that the force applied by AFM to a cell results in a significant deformation of the underlying substrate if this substrate is softer than the cell. This 'soft substrate effect' leads to an underestimation of a cell's elastic modulus when analysing data using a standard Hertz model, as confirmed by finite element modelling and AFM measurements of calibrated polyacrylamide beads, microglial cells and fibroblasts. To account for this substrate deformation, we developed a 'composite cell-substrate model'. Correcting for the substrate indentation revealed that cortical cell stiffness is largely independent of substrate mechanics, which has major implications for our interpretation of many physiological and pathological processes.

Actomyosin controls planarity and folding of epithelia in response to compression.

Throughout embryonic development and adult life, epithelia are subjected to compressive deformations. While these have been shown to trigger mechanosensitive responses such as cell extrusion and differentiation, which span tens of minutes, little is known about how epithelia adapt to compression over shorter timescales. Here, using suspended epithelia, we uncover the immediate response of epithelial tissues to the application of in-plane compressive strains (5-80%). We show that fast compression induces tissue buckling followed by actomyosin-dependent tissue flattening that erases the buckle within tens of seconds, in both mono- and multi-layered epithelia. Strikingly, we identify a well-defined limit to this response, so that stable folds form in the tissue when compressive strains exceed a 'buckling threshold' of ~35%. A combination of experiment and modelling shows that this behaviour is orchestrated by adaptation of the actomyosin cytoskeleton as it re-establishes tissue tension following compression. Thus, tissue pre-tension allows epithelia to both buffer against deformation and sets their ability to form and retain folds during morphogenesis.

BRAF Modulates Stretch-Induced Intercellular Gap Formation through Localized Actin Reorganization.

Mechanical forces acting on cell-cell adhesion modulate the barrier function of endothelial cells. The actively remodeled actin cytoskeleton impinges on cell-cell adhesion to counteract external forces. We applied stress on endothelial monolayers by mechanical stretch to uncover the role of BRAF in the stress-induced response. Control cells responded to external forces by organizing and stabilizing actin cables in the stretched cell junctions. This was accompanied by an increase in intercellular gap formation, which was prevented in BRAF knockdown monolayers. In the absence of BRAF, there was excess stress fiber formation due to the enhanced reorganization of actin fibers. Our findings suggest that stretch-induced intercellular gap formation, leading to a decrease in barrier function of blood vessels, can be reverted by BRAF RNAi. This is important when the endothelium experiences changes in external stresses caused by high blood pressure, leading to edema, or by immune or cancer cells in inflammation or metastasis.

Blebs lead the way: how to migrate without lamellipodia.

Blebs are spherical membrane protrusions that are produced by contractions of the actomyosin cortex. Blebs are often considered to be a hallmark of apoptosis; however, blebs are also frequently observed during cytokinesis and during migration in three-dimensional cultures and in vivo. For tumour cells and a number of embryonic cells, blebbing migration seems to be a common alternative to the more extensively studied lamellipodium-based motility. We argue that blebs should be promoted to a more prominent place in the world of cellular protrusions.

A role for actomyosin contractility in Notch signaling.

BACKGROUND: Notch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition. Unlike many other signaling systems, however, since both the ligand and receptor are transmembrane proteins, the activation of Notch by Delta depends strictly on cell-cell contact. Furthermore, the binding of the ligand to the receptor may not be sufficient to induce signaling, since recent work in cell culture suggests that ligand-induced Notch signaling also requires a mechanical pulling force. This tension exposes a cleavage site in Notch that, when cut, activates signaling. Although it is not known if mechanical tension contributes to signaling in vivo, others have suggested that this is how endocytosis of the receptor-ligand complex contributes to the cleavage and activation of Notch. In a similar way, since Notch-mediated lateral inhibition at a distance in the dorsal thorax of the pupal fly is mediated via actin-rich protrusions, it is possible that cytoskeletal forces generated by networks of filamentous actin and non-muscle myosin during cycles of protrusion extension and retraction also contribute to Notch signaling. RESULTS: To test this hypothesis, we carried out a detailed analysis of the role of myosin II-dependent tension in Notch signaling in the developing fly and in cell culture. Using dynamic fluorescence-based reporters of Notch, we found that myosin II is important for signaling in signal sending and receiving cells in both systems-as expected if myosin II-dependent tension across the Notch-Delta complex contributes to Notch activation. While myosin II was found to contribute most to signaling at a distance, it was also required for maximal signaling between adjacent cells that share lateral contacts and for signaling between cells in culture. CONCLUSIONS: Together these results reveal a previously unappreciated role for non-muscle myosin II contractility in Notch signaling, providing further support for the idea that force contributes to the cleavage and activation of Notch in the context of ligand-dependent signaling, and a new paradigm for actomyosin-based mechanosensation.

Coordinated control of Notch/Delta signalling and cell cycle progression drives lateral inhibition-mediated tissue patterning.

Coordinating cell differentiation with cell growth and division is crucial for the successful development, homeostasis and regeneration of multicellular tissues. Here, we use bristle patterning in the fly notum as a model system to explore the regulatory and functional coupling of cell cycle progression and cell fate decision-making. The pattern of bristles and intervening epithelial cells (ECs) becomes established through Notch-mediated lateral inhibition during G2 phase of the cell cycle, as neighbouring cells physically interact with each other via lateral contacts and/or basal protrusions. Since Notch signalling controls cell division timing downstream of Cdc25, ECs in lateral contact with a Delta-expressing cell experience higher levels of Notch signalling and divide first, followed by more distant neighbours, and lastly Delta-expressing cells. Conversely, mitotic entry and cell division makes ECs refractory to lateral inhibition signalling, fixing their fate. Using a combination of experiments and computational modelling, we show that this reciprocal relationship between Notch signalling and cell cycle progression acts like a developmental clock, providing a delimited window of time during which cells decide their fate, ensuring efficient and orderly bristle patterning.

Directional memory arises from long-lived cytoskeletal asymmetries in polarized chemotactic cells.

Chemotaxis, the directional migration of cells in a chemical gradient, is robust to fluctuations associated with low chemical concentrations and dynamically changing gradients as well as high saturating chemical concentrations. Although a number of reports have identified cellular behavior consistent with a directional memory that could account for behavior in these complex environments, the quantitative and molecular details of such a memory process remain unknown. Using microfluidics to confine cellular motion to a 1D channel and control chemoattractant exposure, we observed directional memory in chemotactic neutrophil-like cells. We modeled this directional memory as a long-lived intracellular asymmetry that decays slower than observed membrane phospholipid signaling. Measurements of intracellular dynamics revealed that moesin at the cell rear is a long-lived element that when inhibited, results in a reduction of memory. Inhibition of ROCK (Rho-associated protein kinase), downstream of RhoA (Ras homolog gene family, member A), stabilized moesin and directional memory while depolymerization of microtubules (MTs) disoriented moesin deposition and also reduced directional memory. Our study reveals that long-lived polarized cytoskeletal structures, specifically moesin, actomyosin, and MTs, provide a directional memory in neutrophil-like cells even as they respond on short time scales to external chemical cues.

Emergence of homeostatic epithelial packing and stress dissipation through divisions oriented along the long cell axis.

Cell division plays an important role in animal tissue morphogenesis, which depends, critically, on the orientation of divisions. In isolated adherent cells, the orientation of mitotic spindles is sensitive to interphase cell shape and the direction of extrinsic mechanical forces. In epithelia, the relative importance of these two factors is challenging to assess. To do this, we used suspended monolayers devoid of ECM, where divisions become oriented following a stretch, allowing the regulation and function of epithelial division orientation in stress relaxation to be characterized. Using this system, we found that divisions align better with the long, interphase cell axis than with the monolayer stress axis. Nevertheless, because the application of stretch induces a global realignment of interphase long axes along the direction of extension, this is sufficient to bias the orientation of divisions in the direction of stretch. Each division redistributes the mother cell mass along the axis of division. Thus, the global bias in division orientation enables cells to act collectively to redistribute mass along the axis of stretch, helping to return the monolayer to its resting state. Further, this behavior could be quantitatively reproduced using a model designed to assess the impact of autonomous changes in mitotic cell mechanics within a stretched monolayer. In summary, the propensity of cells to divide along their long axis preserves epithelial homeostasis by facilitating both stress relaxation and isotropic growth without the need for cells to read or transduce mechanical signals.

L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro.

L-selectin is a cell adhesion molecule that tethers free-flowing leukocytes from the blood to luminal vessel walls, facilitating the initial stages of their emigration from the circulation toward an extravascular inflammatory insult. Following shear-resistant adhesion to the vessel wall, L-selectin has frequently been reported to be rapidly cleaved from the plasma membrane (known as ectodomain shedding), with little knowledge of the timing or functional consequence of this event. Using advanced imaging techniques, we observe L-selectin shedding occurring exclusively as primary human monocytes actively engage in transendothelial migration (TEM). Moreover, the shedding was localized to transmigrating pseudopods within the subendothelial space. By capturing monocytes in midtransmigration, we could monitor the subcellular distribution of L-selectin and better understand how ectodomain shedding might contribute to TEM. Mechanistically, L-selectin loses association with calmodulin (CaM; a negative regulator of shedding) specifically within transmigrating pseudopods. In contrast, L-selectin/CaM interaction remained intact in nontransmigrated regions of monocytes. We show phosphorylation of L-selectin at Ser 364 is critical for CaM dissociation, which is also restricted to the transmigrating pseudopod. Pharmacological or genetic inhibition of L-selectin shedding significantly increased pseudopodial extensions in transmigrating monocytes, which potentiated invasive behavior during TEM and prevented the establishment of front/back polarity for directional migration persistence once TEM was complete. We conclude that L-selectin shedding directly regulates polarity in transmigrated monocytes, which affirms an active role for this molecule in driving later stages of the multistep adhesion cascade.