Changes in mesenchymal cell-basal lamina relationships preceding palatal shelf reorientation in the mouse

Two specific regions of the future nasal and oral epithelial surfaces of the secondary palatal shelves increase in cell density during shelf reorientation. The relationships of mesenchymal cells to the basal lamina underlying these regions were examined and compared to those of cells underlying adjacent regions which did not change in cell density. CD-1 mouse fetuses were obtained on day 13.5 of gestation. Some palatal shelves were excised immediately and fixed for electron microscopy; other heads were partially dissected and incubated for 4 hr prior to fixation. Although shelf movement is detected only after 6 hr incubation, the shorter time period was selected in order to detect events which precede reorientation. Electron micrographs were taken of the epithelial-mesenchymal interface of nasal and oral regions known to increase in epithelial cell density (active segments) and of nasal and oral regions which did not increase (inactive segments). Several measurements were made in a 500-nm-wide zone delimited on photographic prints. Distinct differences in mesenchymal cell configuration were found between nasal and oral regions. Active and inactive segments of each region also differed. A filamentous layer attached to the undersurface of the lamina densa was observed to vary in thickness and character between regions as well. After 4 hr incubation, differences in mesenchymal cell configuration and ultrastructure of the sublaminar zone were apparent between regions. These results suggest that local epithelial-mesenchymal interactions, possibly mediated by the extracellular matrix, precede shelf reorientation. Whether these changes in mesenchymal cell configuration actually reflect mesenchymal cell activities that are necessary for shelf reorientation remains to be elucidated.     

DOCK10-mediated Cdc42 activation is necessary for amoeboid invasion of melanoma cells

BACKGROUND: Tumor cells can move in a three-dimensional (3D) environment in either mesenchymal-type or amoeboid modes. In mesenchymal-type movement, cells have an elongated morphology with Rac-induced protrusions at the leading edge. Amoeboid cells have high levels of actomyosin contractility, and movement is associated with deformation of the cell body through the matrix without proteolysis. Because signaling pathways that control the activation of GTPases for amoeboid movement are poorly understood, we sought to identify regulators of amoeboid movement by screening an siRNA library targeting guanine nucleotide exchange factors (GEFs) for Rho-family GTPases. RESULTS: We identified DOCK10, a Cdc42 GEF, as a key player in amoeboid migration; accordingly, we find that expression of activated Cdc42 induces a mesenchymal-amoeboid transition and increases cell invasion. Silencing DOCK10 expression promotes conversion to mesenchymal migration and is associated with decreased MLC2 phosphorylation and increased Rac1 activation. Consequently, abrogating DOCK10 and Rac1 expression suppresses both amoeboid and mesenchymal migration and results in decreased invasion. We show that the Cdc42 effectors N-WASP and Pak2 are required for the maintenance of the rounded-amoeboid phenotype. Blocking Cdc42 results in loss of mesenchymal morphology, arguing that Cdc42 is also involved in mesenchymal morphology through different activation and effector pathways. CONCLUSIONS: Previous work has identified roles of Rho and Rac signaling in tumor cell movement, and we now elucidate novel roles of Cdc42 signaling in amoeboid and mesenchymal movement and tumor cell invasion.     

Mapping local matrix remodeling induced by a migrating tumor cell using three-dimensional multiple-particle tracking

Mesenchymal cell migration through a three-dimensional (3D) matrix typically involves major matrix remodeling. The direction of matrix deformation occurs locally in all three dimensions, which cannot be measured by current techniques. To probe the local, 3D, real-time deformation of a collagen matrix during tumor cell migration, we developed an assay whereby matrix-embedded beads are tracked simultaneously in all three directions with high resolution. To establish a proof of principle, we investigated patterns of collagen I matrix deformation near fibrosarcoma cells in the absence and presence of inhibitors of matrix metalloproteinases and acto-myosin contractility. Our results indicate that migrating cells show patterns of local matrix deformation toward the cell that are symmetric in magnitude with respect to the axis of cell movement. In contrast, patterns of matrix release from the cell are asymmetric: the matrix is typically relaxed first at the back of the cell, allowing forward motion, and then at the cell's leading edge. Matrix deformation in regions of the matrix near the cell's leading edge is elastic and mostly reversible, but induces irreversible matrix rupture events near the trailing edge. Our results also indicate that matrix remodeling spatially correlates with protrusive activity. This correlation is mediated by myosin II and Rac1, and eliminated after inhibition of pericellular proteolysis or ROCK. We have developed an assay based on high-resolution 3D multiple-particle tracking that allows us to probe local matrix remodeling during mesenchymal cell migration through a 3D matrix and simultaneously monitor protrusion dynamics.     

A fibre reorientation model for orthotropic multiplicative growth

The main goal of this contribution consists in the development of a remodelling framework for orthotropic continua whereby the underlying symmetry group is incorporated via two fibre families. Special emphasis is placed on the modelling of biological tissues at finite deformations. Besides the incorporation of a referential mass source, anisotropic growth is addressed by means of a multiplicative decomposition of the overall deformation gradient into an elastic and a growth distortion. Projected quantities of a configurational growth stress tensor are advocated as driving forces for time-dependent saturation–type evolution of the principal values of the growth distortion. Moreover, the reorientation of both fibre families, which directly affects the strain energy as well as the growth distortion itself, is guided by analyzing critical energy points. In particular, a time-dependent formulation is developed which aligns the fibre directions according to the principal stretch directions. Finally, the proposed framework is embedded into a finite element context so that representative numerical examples, examining growth and resorption in volume and density together with fibre reorientation, close this study.     

Emerging role for nuclear rotation and orientation in cell migration

Nucleus movement, positioning, and orientation is precisely specified and actively regulated within cells, and it plays a critical role in many cellular and developmental processes. Mutation of proteins that regulate the nucleus anchoring and movement lead to diverse pathologies, laminopathies in particular, suggesting that the nucleus correct positioning and movement is essential for proper cellular function. In motile cells that polarize toward the direction of migration, the nucleus undergoes controlled rotation promoting the alignment of the nucleus with the axis of migration. Such spatial organization of the cell appears to be optimal for the cell migration. Nuclear reorientation requires the cytoskeleton to be anchored to the nuclear envelope, which exerts pulling or pushing torque on the nucleus. Here we discuss the possible molecular mechanisms regulating the nuclear rotation and reorientation and the significance of this type of nuclear movement for cell migration.     

Emerging role for nuclear rotation and orientation in cell migration

Nucleus movement, positioning, and orientation is precisely specified and actively regulated within cells, and it plays a critical role in many cellular and developmental processes. Mutation of proteins that regulate the nucleus anchoring and movement lead to diverse pathologies, laminopathies in particular, suggesting that the nucleus correct positioning and movement is essential for proper cellular function. In motile cells that polarize toward the direction of migration, the nucleus undergoes controlled rotation promoting the alignment of the nucleus with the axis of migration. Such spatial organization of the cell appears to be optimal for the cell migration. Nuclear reorientation requires the cytoskeleton to be anchored to the nuclear envelope, which exerts pulling or pushing torque on the nucleus. Here we discuss the possible molecular mechanisms regulating the nuclear rotation and reorientation and the significance of this type of nuclear movement for cell migration.     

A hybrid computational model for collective cell durotaxis

Collective cell migration is regulated by a complex set of mechanical interactions and cellular mechanisms. Collective migration emerges from mechanisms occurring at single cell level, involving processes like contraction, polymerization and depolymerization, of cell–cell interactions and of cell–substrate adhesion. Here, we present a computational framework which simulates the dynamics of this emergent behavior conditioned by substrates with stiffness gradients. The computational model reproduces the cell’s ability to move toward the stiffer part of the substrate, process known as durotaxis. It combines the continuous formulation of truss elements and a particle-based approach to simulate the dynamics of cell–matrix adhesions and cell–cell interactions. Using this hybrid approach, researchers can quickly create a quantitative model to understand the regulatory role of different mechanical conditions on the dynamics of collective cell migration. Our model shows that durotaxis occurs due to the ability of cells to deform the substrate more in the part of lower stiffness than in the stiffer part. This effect explains why cell collective movement is more effective than single cell movement in stiffness gradient conditions. In addition, we numerically evaluate how gradient stiffness properties, cell monolayer size and force transmission between cells and extracellular matrix are crucial in regulating durotaxis.     

Actomyosin contractility spatiotemporally regulates actin network dynamics in migrating cells

Coupling interactions among mechanical and biochemical factors are important for the realization of various cellular processes that determine cell migration. Although F-actin network dynamics has been the focus of many studies, it is not yet clear how mechanical forces generated by actomyosin contractility spatiotemporally regulate this fundamental aspect of cell migration. In this study, using a combination of fluorescent speckle microscopy and particle imaging velocimetry techniques, we perturbed the actomyosin system and examined quantitatively the consequence of actomyosin contractility on F-actin network flow and deformation in the lamellipodia of actively migrating fish keratocytes. F-actin flow fields were characterized by retrograde flow at the front and anterograde flow at the back of the lamellipodia, and the two flows merged to form a convergence zone of reduced flow intensity. Interestingly, activating or inhibiting actomyosin contractility altered network flow intensity and convergence, suggesting that network dynamics is directly regulated by actomyosin contractility. Moreover, quantitative analysis of F-actin network deformation revealed that the deformation was significantly negative and predominant in the direction of cell migration. Furthermore, perturbation experiments revealed that the deformation was a function of actomyosin contractility. Based on these results, we suggest that the actin cytoskeletal structure is a mechanically self-regulating system, and we propose an elaborate pathway for the spatiotemporal self-regulation of the actin cytoskeletal structure during cell migration. In the proposed pathway, mechanical forces generated by actomyosin interactions are considered central to the realization of the various mechanochemical processes that determine cell motility.     

Short-lived, transitory cell-cell interactions foster migration-dependent aggregation

During embryonic development, motile cells aggregate into cohesive groups, which give rise to tissues and organs. The role of cell migration in regulating aggregation is unclear. The current paradigm for aggregation is based on an equilibrium model of differential cell adhesivity to neighboring cells versus the underlying substratum. In many biological contexts, however, dynamics is critical. Here, we provide evidence that multicellular aggregation dynamics involves both local adhesive interactions and transport by cell migration. Using time-lapse video microscopy, we quantified the duration of cell-cell contacts among migrating cells that collided and adhered to another cell. This lifetime of cell-cell interactions exhibited a monotonic decreasing dependence on substratum adhesivity. Parallel quantitative measurements of cell migration speed revealed that across the tested range of adhesive substrata, the mean time needed for cells to migrate and encounter another cell was greater than the mean adhesion lifetime, suggesting that aggregation dynamics may depend on cell motility instead of the local differential adhesivity of cells. Consistent with this hypothesis, aggregate size exhibited a biphasic dependence on substratum adhesivity, matching the trend we observed for cell migration speed. Our findings suggest a new role for cell motility, alongside differential adhesion, in regulating developmental aggregation events and motivate new design principles for tuning aggregation dynamics in tissue engineering applications.     

M5 mesoscopic and macroscopic models for mesenchymal motion

In this paper mesoscopic (individual based) and macroscopic (population based) models for mesenchymal motion of cells in fibre networks are developed. Mesenchymal motion is a form of cellular movement that occurs in three-dimensions through tissues formed from fibre networks, for example the invasion of tumor metastases through collagen networks. The movement of cells is guided by the directionality of the network and in addition, the network is degraded by proteases. The main results of this paper are derivations of mesoscopic and macroscopic models for mesenchymal motion in a timely varying network tissue. The mesoscopic model is based on a transport equation for correlated random walk and the macroscopic model has the form of a drift-diffusion equation where the mean drift velocity is given by the mean orientation of the tissue and the diffusion tensor is given by the variance-covariance matrix of the tissue orientations. The transport equation as well as the drift-diffusion limit are coupled to a differential equation that describes the tissue changes explicitly, where we distinguish the cases of directed and undirected tissues. As a result the drift velocity and the diffusion tensor are timely varying. We discuss relations to existing models and possible applications.Dedicated to K.P. Hadeler, a great scientist, teacher, and friend.     

CompuCell3D Simulations Reproduce Mesenchymal Cell Migration on Flat Substrates

Mesenchymal cell crawling is a critical process in normal development, in tissue function, and in many diseases. Quantitatively predictive numerical simulations of cell crawling thus have multiple scientific, medical, and technological applications. However, we still lack a low-computational-cost approach to simulate mesenchymal three-dimensional (3D) cell crawling. Here, we develop a computationally tractable 3D model (implemented as a simulation in the CompuCell3D simulation environment) of mesenchymal cells crawling on a two-dimensional substrate. The Fürth equation, the usual characterization of mean-squared displacement (MSD) curves for migrating cells, describes a motion in which, for increasing time intervals, cell movement transitions from a ballistic to a diffusive regime. Recent experiments have shown that for very short time intervals, cells exhibit an additional fast diffusive regime. Our simulations’ MSD curves reproduce the three experimentally observed temporal regimes, with fast diffusion for short time intervals, slow diffusion for long time intervals, and intermediate time -interval-ballistic motion. The resulting parameterization of the trajectories for both experiments and simulations allows the definition of time- and length scales that translate between computational and laboratory units. Rescaling by these scales allows direct quantitative comparisons among MSD curves and between velocity autocorrelation functions from experiments and simulations. Although our simulations replicate experimentally observed spontaneous symmetry breaking, short-timescale diffusive motion, and spontaneous cell-motion reorientation, their computational cost is low, allowing their use in multiscale virtual-tissue simulations. Comparisons between experimental and simulated cell motion support the hypothesis that short-time actomyosin dynamics affects longer-time cell motility. The success of the base cell-migration simulation model suggests its future application in more complex situations, including chemotaxis, migration through complex 3D matrices, and collective cell motion.     

Collective Epithelial and Mesenchymal Cell Migration During Gastrulation

Gastrulation, the process that puts the three major germlayers, the ectoderm, mesoderm and endoderm in their correct topological position in the developing embryo, is characterised by extensive highly organised collective cell migration of epithelial and mesenchymal cells. We discuss current knowledge and insights in the mechanisms controlling these cell behaviours during gastrulation in the chick embryo. We discuss several ideas that have been proposed to explain the observed large scale vortex movements of epithelial cells in the epiblast during formation of the primitive streak. We review current insights in the control and execution of the epithelial to mesenchymal transition (EMT) underlying the formation of the hypoblast and the ingression of the mesendoderm cells through the streak. We discuss the mechanisms by which the mesendoderm cells move, the nature and dynamics of the signals that guide these movements, as well as the interplay between signalling and movement that result in tissue patterning and morphogenesis. We argue that instructive cell-cell signaling and directed chemotactic movement responses to these signals are instrumental in the execution of all phases of gastrulation.     

Mathematical model for the effects of adhesion and mechanics on cell migration speed.

Migration of mammalian blood and tissue cells over adhesive surfaces is apparently mediated by specific reversible reactions between cell membrane adhesion receptors and complementary ligands attached to the substratum. Although in a number of systems these receptors and ligand molecules have been isolated and identified, a theory capable of predicting the effects of their properties on cell migration behavior currently does not exist. We present a simple mathematical model for elucidating the dependence of cell speed on adhesion-receptor/ligand binding and cell mechanical properties. Our model can be applied to propose answers to questions such as: does an optimal adhesiveness exist for cell movement? How might changes in receptor and ligand density and/or affinity affect the rate of migration? Can cell rheological properties influence movement speed? This model incorporates cytoskeletal force generation, cell polarization, and dynamic adhesion as requirements for persistent cell movement. A critical feature is the proposed existence of an asymmetry in some cell adhesion-receptor property, correlated with cell polarity. We consider two major alternative mechanisms underlying this asymmetry: (a) a spatial distribution of adhesion-receptor number due to polarized endocytic trafficking and (b) a spatial variation in adhesion-receptor/ligand bond strength. Applying a viscoelastic-solid model for cell mechanics allows us to represent one-dimensional locomotion with a system of differential equations describing cell deformation and displacement along with adhesion-receptor dynamics. In this paper, we solve these equations under the simplifying assumption that receptor dynamics are at a quasi-steady state relative to cell locomotion. Thus, our results are strictly valid for sufficiently slow cell movement, as typically observed for tissue cells such as fibroblasts. Numerical examples relevant to experimental systems are provided. Our results predict how cell speed might vary with intracellular contractile force, cell rheology, receptor/ligand kinetics, and receptor/ligand number densities. A biphasic dependence is shown to be possible with respect to some of the system parameters, with position of the maxima essentially governed by a balance between transmitted contractile force and adhesiveness. We demonstrate that predictions for the two alternative asymmetry mechanisms can be distinguished and could be experimentally tested using cell populations possessing different adhesion-receptor numbers.     

A multiscale whole-cell theory for mechanosensitive migration on viscoelastic substrates

Increasing experimental evidence validates that both the elastic stiffness and viscosity of the extracellular matrix regulate mesenchymal cell behavior, such as the rational switch between durotaxis (cell migration to stiffer regions), anti-durotaxis (migration to softer regions), and adurotaxis (stiffness-insensitive migration). To reveal the mechanisms underlying the crossover between these motility regimes, we have developed a multiscale chemomechanical whole-cell theory for mesenchymal migration. Our framework couples the subcellular focal adhesion dynamics at the cell-substrate interface with the cellular cytoskeletal mechanics and the chemical signaling pathways involving Rho GTPase proteins. Upon polarization by the Rho GTPase gradients, our simulated cell migrates by concerted peripheral protrusions and contractions, a hallmark of the mesenchymal mode. The resulting cell dynamics quantitatively reproduces the experimental migration speed as a function of the uniform substrate stiffness and explains the influence of viscosity on the migration efficiency. In the presence of stiffness gradients and absence of chemical polarization, our simulated cell can exhibit durotaxis, anti-durotaxis, and adurotaxis respectively with increasing substrate stiffness or viscosity. The cell moves toward an optimally stiff region from softer regions during durotaxis and from stiffer regions during anti-durotaxis. We show that cell polarization through steep Rho GTPase gradients can reverse the migration direction dictated by the mechanical cues. Overall, our theory demonstrates that opposing durotactic behaviors emerge via the interplay between intracellular signaling and cell-medium mechanical interactions in agreement with experiments, thereby elucidating complex mechanosensing at the single-cell level.     

Proteolysis of cortactin by calpain regulates membrane protrusion during cell migration

Calpain 2 regulates membrane protrusion during cell migration. However, relevant substrates that mediate the effects of calpain on protrusion have not been identified. One potential candidate substrate is the actin binding protein cortactin. Cortactin is a Src substrate that drives actin polymerization by activating the Arp2/3 complex and also stabilizes the cortical actin network. We now provide evidence that proteolysis of cortactin by calpain 2 regulates membrane protrusion dynamics during cell migration. We show that cortactin is a calpain 2 substrate in fibroblasts and that the preferred cleavage site occurs in a region between the actin binding repeats and the alpha-helical domain. We have generated a mutant cortactin that is resistant to calpain proteolysis but retains other biochemical properties of cortactin. Expression of the calpain-resistant cortactin, but not wild-type cortactin, impairs cell migration and increases transient membrane protrusion, suggesting that calpain proteolysis of cortactin limits membrane protrusions and regulates migration in fibroblasts. Furthermore, the enhanced protrusion observed with the calpain-resistant cortactin requires both the Arp2/3 binding site and the Src homology 3 domain of cortactin. Together, these findings suggest a novel role for calpain-mediated proteolysis of cortactin in regulating membrane protrusion dynamics during cell migration.     

Role of vitronectin in embryonic rat endocardial cell migration in vitro

Summary Vitronectin is one of the extracellular matrices that mediate cell spreading and attachment in vitro. In the present paper, we demonstrate the involvement of vitronectin in the migration of cushion mesenchymal cells of the embryonic rat heart. Immunohistochemistry established the localization of vitronectin in the myocardial cells and in some of the cushion mesenchymal cells of the truncus arteriosus and atrioventricular canal. In vitro, vitronectin, fibronectin, and collagen type-I revcaled significant stimulating activity for cushion mesenchymal cell migration. The distance migrated by cushion mesenchymal cells cultured on vitronectin, collagen type-I, or both vitronectin and fibronectin was similar, but that on fibronectin was significantly shorter. Following the addition of anti-vitronectin IgG to the medium, the migration distance of cushion mesenchymal cells on fibronectin was remarkably increased. Most explants on vitronectin or on both vitronectin and fibronectin became detached from dishes after the addition of the antivitronectin antibody. Immunostaining revealed that cushion mesenchymal cells cultured on substrata other than vitronectin synthesized vitronectin. From these results, it is suggested that vitronectin is synthesized by myocardial cells and some cushion mesenchymal cells, and that vitronectin inhibits cell movement on fibronectin. This feature of vitronectin may be important in the regulation of the migration of cushion mesenchymal cell in vivo.     

Rac activation and inactivation control plasticity of tumor cell movement

Tumor cells exhibit two different modes of individual cell movement. Mesenchymal-type movement is characterized by an elongated cellular morphology and requires extracellular proteolysis. In amoeboid movement, cells have a rounded morphology, are less dependent on proteases, and require high Rho-kinase signaling to drive elevated levels of actomyosin contractility. These two modes of cell movement are interconvertible. We show that mesenchymal-type movement in melanoma cells is driven by activation of the GTPase Rac through a complex containing NEDD9, a recently identified melanoma metastasis gene, and DOCK3, a Rac guanine nucleotide exchange factor. Rac signals through WAVE2 to direct mesenchymal movement and suppress amoeboid movement through decreasing actomyosin contractility. Conversely, in amoeboid movement, Rho-kinase signaling activates a Rac GAP, ARHGAP22, that suppresses mesenchymal movement by inactivating Rac. We demonstrate tight interplay between Rho and Rac in determining different modes of tumor cell movement, revealing how tumor cells switch between different modes of movement.     

Integrin-cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated

We have used laser optical trapping and nanometer-level motion analysis to investigate the cytoskeletal associations and surface dynamics of beta 1 integrin, a cell-substrate adhesion molecule, on the dorsal surfaces of migrating fibroblast cells. A single-beam optical gradient trap (laser tweezers) was used to restrain polystyrene beads conjugated with anti-beta 1 integrin mAbs and place them at desired locations on the cell exterior. This technique was used to demonstrate a spatial difference in integrin-cytoskeleton interactions in migrating cells. We found a distinct increase in the stable attachment of beads, and subsequent rearward flow, on the lamellipodia of locomoting cells compared with the retracting portions. Complementary to the enhanced linkage of integrin at the cell lamellipodium, the membrane was more deformable at the rear versus the front of moving cells while nonmotile cells did not exhibit this asymmetry in membrane architecture. Video microscopy and nanometer-precision tracking routines were used to study the surface dynamics of integrin on the lamellipodia of migrating cells by monitoring the displacements of colloidal gold particles coated with anti-beta 1 integrin mAbs. Small gold aggregates were rapidly transported preferentially to the leading edge of the lamellipod where they resumed diffusion restricted along the edge. This fast transport was characterized by brief periods of directed movement ("jumps") having an instantaneous velocity of 37 +/- 15 microns/min (SD), separated by periods of diffusion. In contrast, larger aggregates of gold particles and the large latex beads underwent slow, steady rearward movement (0.85 +/- 0.44 micron/min) (SD) at a rate similar to that reported for other capping events and for migration of these cells. Cell lines containing mutated beta 1 integrins were used to show that the cytoplasmic domain is essential for an asymmetry in attachment of integrin to the underlying cytoskeletal network and is also necessary for rapid, intermittent transport. However, enhanced membrane deformability at the cell rear does not require integrin-cytoskeletal interactions. We also demonstrated that posttranslational modifications of integrin could potentially play a role in these phenomena. These results suggest a scheme for the role of dynamic integrin-mediated adhesive interactions in cell migration. Integrins are transported preferentially to the cell front where they form nascent adhesions. These adhesive structures grow in size and associate with the cytoskeleton that exerts a rearward force on them. Dorsal aggregates more rearward while those on the ventral side remain fixed to the substrate allowing the cell body to move forward. Detachment of the cell rear occurs by at least two modes: (a) weakened integrin- cytoskeleton interactions, potentially mediated by local modifications of linkage proteins, which lead to weakened cell-substratum interactions and (b) ripping of integrins and the highly deformable membrane from the cell body.