Cost–benefit analysis of the mechanisms that enable migrating cells to sustain motility upon changes in matrix environments

Cells can move through extracellular environments with varying geometries and adhesive properties. Adaptation to these differences is achieved by switching between different modes of motility, including lamellipod-driven and blebbing motility. Further, cells can modulate their level of adhesion to the extracellular matrix (ECM) depending on both the level of force applied to the adhesions and cell intrinsic biochemical properties. We have constructed a computational model of cell motility to investigate how motile cells transition between extracellular environments with varying surface continuity, confinement and adhesion. Changes in migration strategy are an emergent property of cells as the ECM geometry and adhesion changes. The transition into confined environments with discontinuous ECM fibres is sufficient to induce shifts from lamellipod-based to blebbing motility, while changes in confinement alone within a continuous geometry are not. The geometry of the ECM facilitates plasticity, by inducing shifts where the cell has high marginal gain from a mode change, and conserving persistency where the cell can continue movement regardless of the motility mode. This regulation of cell motility is independent of global changes in cytoskeletal properties, but requires locally higher linkage between the actin network and the plasma membrane at the cell rear, and changes in internal cell pressure. In addition to matrix geometry, we consider how cells might transition between ECM of different adhesiveness. We find that this requires positive feedback between the forces cells apply on the adhesion points, and the strength of the cell–ECM adhesions on those sites. This positive feedback leads to the emergence of a small number of highly adhesive cores, similar to focal adhesions. While the range of ECM adhesion levels the cell can invade is expanded with this feedback mechanism; the velocities are lowered for conditions where the positive feedback is not vital. Thus, plasticity of cell motility sacrifices the benefits of specialization, for robustness.     

Formation of focal adhesion-stress fibre complexes coordinated by adhesive and non-adhesive surface domains

Cell motility consists of repeating cycles of protrusion of a leading edge in the direction of migration, attachment of the advancing membrane to the matrix, and pulling of the trailing edge forward. In this dynamic process there is a major role for the cytoskeleton, which drives the protrusive events via polymerisation of actin in the lamellipodium, followed by actomyosin contractility. To study the transition of the actin cytoskeleton from a 'protrusive' to 'retractive' form, we have monitored the formation of focal adhesions and stress fibres during cell migration on a micro-patterned surface. This surface consisted of parallel arrays of 2 microm-wide, fibronectin-coated gold stripes, separated by non-adhesive (poly(ethylene glycol)-coated) glass areas with variable width, ranging from 4-12 microm. Monitoring the spreading of motile cells indicated that cell spreading was equally effective along and across the adhesive stripes, as long as the non-adhesive spaces between them did not exceed 6 microm. When the width of the PEG region was 8 microm or more, cells became highly polarised upon spreading, and failed to reach the neighboring adhesive stripes. It was also noted that as soon as the protruding lamella successfully crossed the PEG-coated area and reached an adhesive region, the organisation of actin in that area was transformed from a diffuse meshwork into a bundle, oriented perpendicularly to the stripes and anchored at its ends in focal adhesions. This transition depends on actomyosin-based contractility and is apparently triggered by the adhesion to the rigid fibronectin surface.     

Simulation of the cytoskeletal response of cells on grooved or patterned substrates

We analyse the response of osteoblasts on grooved substrates via a model that accounts for the cooperative feedback between intracellular signalling, focal adhesion development and stress fibre contractility. The grooved substrate is modelled as a pattern of alternating strips on which the cell can adhere and strips on which adhesion is inhibited. The coupled modelling scheme is shown to capture some key experimental observations including (i) the observation that osteoblasts orient themselves randomly on substrates with groove pitches less than about 150 nm but they align themselves with the direction of the grooves on substrates with larger pitches and (ii) actin fibres bridge over the grooves on substrates with groove pitches less than about 150 nm but form a network of fibres aligned with the ridges, with nearly no fibres across the grooves, for substrates with groove pitches greater than about 300 nm. Using the model, we demonstrate that the degree of bridging of the stress fibres across the grooves, and consequently the cell orientation, is governed by the diffusion of signalling proteins activated at the focal adhesion sites on the ridges. For large groove pitches, the signalling proteins are dephosphorylated before they can reach the regions of the cell above the grooves and hence stress fibres cannot form in those parts of the cell. On the other hand, the stress fibre activation signal diffuses to a reasonably spatially homogeneous level on substrates with small groove pitches and hence stable stress fibres develop across the grooves in these cases. The model thus rationalizes the responsiveness of osteoblasts to the topography of substrates based on the complex feedback involving focal adhesion formation on the ridges, the triggering of signalling pathways by these adhesions and the activation of stress fibre networks by these signals.     

The simulation of stress fibre and focal adhesion development in cells on patterned substrates.

The remodelling of the cytoskeleton and focal adhesion (FA) distributions for cells on substrates with micro-patterned ligand patches is investigated using a bio-chemo-mechanical model. We investigate the effect of ligand pattern shape on the cytoskeletal arrangements and FA distributions for cells having approximately the same area. The cytoskeleton model accounts for the dynamic rearrangement of the actin/myosin stress fibres. It entails the highly nonlinear interactions between signalling, the kinetics of tension-dependent stress-fibre formation/dissolution and stress-dependent contractility. This model is coupled with another model that governs FA formation and accounts for the mechano-sensitivity of the adhesions from thermodynamic considerations. This coupled modelling scheme is shown to capture a variety of key experimental observations including: (i) the formation of high concentrations of stress fibres and FAs at the periphery of circular and triangular, convex-shaped ligand patterns; (ii) the development of high FA concentrations along the edges of the V-, T-, Y- and U-shaped concave ligand patterns; and (iii) the formation of highly aligned stress fibres along the non-adhered edges of cells on the concave ligand patterns. When appropriately calibrated, the model also accurately predicts the radii of curvature of the non-adhered edges of cells on the concave-shaped ligand patterns.     

Microfabricated Systems and Assays for Studying the Cytoskeletal Organization,Micromechanics, and Motility Patterns of Cancerous Cells

Cell motions are driven by coordinated actions of the intracellular cytoskeleton – actin, microtubules (MTs) and substrate/focal adhesions (FAs). This coordination is altered in metastatic cancer cells resulting in deregulated and increased cellular motility. Microfabrication tools, including photolithography, micromolding, microcontact printing, wet stamping and microfluidic devices have emerged as a powerful set of experimental tools with which to probe and define the differences in cytoskeleton organization/dynamics and cell motility patterns in non‐metastatic and metastatic cancer cells. In this review, we discuss four categories of microfabricated systems: (i) micropatterned substrates for studying of cell motility sub‐processes (for example, MT targeting of FAs or cell polarization); (ii) systems for studying cell mechanical properties, (iii) systems for probing overall cell motility patterns within challenging geometric confines relevant to metastasis (for example, linear and ratchet geometries), and (iv) microfluidic devices that incorporate co‐cultures of multiple cell types and chemical gradients to mimic in vivo intravasation/extravasation steps of metastasis. Together, these systems allow for creating controlled microenvironments that not only mimic complex soft tissues, but are also compatible with live cell high‐resolution imaging and quantitative analysis of single cell behavior.     

Human foetal osteoblastic cell response to polymer-demixed nanotopographic interfaces.

Nanoscale cell-substratum interactions are of significant interest in various biomedical applications. We investigated human foetal osteoblastic cell response to randomly distributed nanoisland topography with varying heights (11, 38 and 85 nm) produced by a polystyrene (PS)/polybromostyrene polymer-demixing technique. Cells displayed island-conforming lamellipodia spreading, and filopodia projections appeared to play a role in sensing the nanotopography. Cells cultured on 11 nm high islands displayed significantly enhanced cell spreading and larger cell dimensions than cells on larger nanoislands or flat PS control, on which cells often displayed a stellate shape. Development of signal transmitting structures such as focal adhesive vinculin protein and cytoskeletal actin stress fibres was more pronounced, as was their colocalization, in cells cultured on smaller nanoisland surfaces. Cell adhesion and proliferation were greater with decreasing island height. Alkaline phosphatase (AP) activity, an early stage marker of bone cell differentiation, also exhibited nanotopography dependence, i.e. higher AP activity on 11 nm islands compared with that on larger islands or flat PS. Therefore, randomly distributed island topography with varying nanoscale heights not only affect adhesion-related cell behaviour but also bone cell phenotype. Our results suggest that modulation of nanoscale topography may be exploited to control cell function at cell-biomaterial interfaces.     

Controlling fibroblast adhesion and proliferation by 1D Al2 O3 nanostructures

The fibrotic encapsulation, which is mainly accompanied by an excessive proliferation of fibroblasts, is an undesired phenomenon after the implantation of various medical devices. Beside the surface chemistry, the topography plays also a major role in the fibroblast–surface interaction. In the present study, one‐dimensional aluminium oxide (1D Al₂ O₃) nanostructures with different distribution densities were prepared to reveal the response of human fibroblasts to the surface topography. The cell size, the cell number and the ability to form well‐defined actin fibres and focal adhesions were significantly impaired with increasing distribution density of the 1D Al₂ O₃ nanostructures on the substratum.Inspec keywords: biomechanics, adhesion, surface chemistry, biomedical materials, cellular biophysics, surface topography, nanostructured materials, alumina, nanomedicineOther keywords: fibrotic encapsulation, medical devices, surface chemistry, human fibroblasts, surface topography, cell size, cell number, well‐defined actin fibres, focal adhesions, distribution density, fibroblast adhesion, 1D nanostructures, distribution densities, fibroblast proliferation, fibroblast‐surface interaction, one‐dimensional aluminium oxide nanostructures, Al₂ O₃      

Cell/surface interactions on laser micro-textured titanium-coated silicon surfaces

This paper examines the effects of nano-scale titanium coatings, and micro-groove/micro-grid patterns on cell/surface interactions on silicon surfaces. The nature of the cellular attachment and adhesion to the coated/uncoated micro-textured surfaces was elucidated by the visualization of the cells and relevant cytoskeletal & focal adhesion proteins through scanning electron microscopy and immunofluorescence staining. Increased cell spreading and proliferation rates are observed on surfaces with 50 nm thick Ti coatings. The micro-groove geometries have been shown to promote contact guidance, which leads to reduced scar tissue formation. In contrast, smooth surfaces result in random cell orientations and the increased possibility of scar tissue formation. Immunofluorescence cell staining experiments also reveal that the actin stress fibers are aligned along the groove dimensions, with discrete focal adhesions occurring along the ridges, within the grooves and at the ends of the cell extensions. The implications of the observed cell/surface interactions are discussed for possible applications of silicon in implantable biomedical systems.     

Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

Abstract

The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell–matrix interaction, using poly-ε-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.     

Focal complex maturation and bridging on 200 nm vitronectin but not fibronectin patches reveal different mechanisms of focal adhesion formation

The effects of protein type and pattern size on cell adhesion, spreading, and focal adhesion development are studied. Fibronectin and vitronectin patterns from 0.1 to 3 μm produced by colloidal lithography reveal important differences in how cells adhere to and bridge focal adhesions across protein nanopatterns versus micropatterns. Vinculin and zyxin in focal adhesions but not integrins are seen to bridge ligand gaps. Differences in protein mechanical properties are implicated as important factors in focal adhesion development.     

Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell–matrix interaction, using poly-ε-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.     

Cellular Deformations Induced by Conical Silicon Nanowire Arrays Facilitate Gene Delivery

Engineered cell–nanostructured interfaces generated by vertically aligned silicon nanowire (SiNW) arrays have become a promising platform for orchestrating cell behavior, function, and fate. However, the underlying mechanism in SiNW‐mediated intracellular access and delivery is still poorly understood. This study demonstrates the development of a gene delivery platform based on conical SiNW arrays for mechanical cell transfection, assisted by centrifugal force, for both adherent and nonadherent cells in vitro. Cells form focal adhesions on SiNWs within 6 h, and maintain high viability and motility. Such a functional and dynamic cell–SiNW interface features conformational changes in the plasma membrane and in some cases the nucleus, promoting both direct penetration and endocytosis; this synergistically facilitates SiNW‐mediated delivery of nucleic acids into immortalized cell lines, and into difficult‐to‐transfect primary immune T cells without pre‐activation. Moreover, transfected cells retrieved from SiNWs retain the capacity to proliferate—crucial to future biomedical applications. The results indicate that SiNW‐mediated intracellular delivery holds great promise for developing increasingly sophisticated investigative and therapeutic tools.     

Single-particle tracking of hepatitis B virus-like vesicle entry into cells

HBsAg, the surface antigen of the hepatitis B virus (HBV), is used as a model to study the mechanisms and dynamics of a single-enveloped virus infecting living cells by imaging and tracking at the single-particle level. By monitoring the fluorescent indicator of HBsAg particles, it is found that HBsAg enters cells via a caveolin-mediated endocytic pathway. Tracking of individual HBsAg particles in living cells reveals the anomalously actin-dependent but not microtubule-dependent motility of the internalized HBsAg particle. The motility of HBsAg particles in living cells is also analyzed quantitatively. These results may settle the long-lasting debate of whether HBV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell, and how the virus moves in the cell.     

Genetically encoded fluorescent indicators to visualize protein phosphorylation by extracellular signal-regulated kinase in single living cells

Extracellular signal-regulated kinase (ERK) is a serine/threonine protein kinase that regulates a wide variety of cell functions, such as cell growth and differentiation. To study the spatiotemporal dynamics of protein phosphor-ylation by activated ERK in living cells, we have developed genetically encoded fluorescent indicators for ERK. The present indicators are based on fluorescence resonance energy transfer (FRET) between two green fluorescent protein mutants. Phosphorylation of the indicators by activated ERK changes the FRET efficiency due to their conformational alterations. We visualized the cytosolic and nuclear activity of ERK using the present indicators. We thus found that the activation duration of ERK is considerably different between the cytosol and nucleus in living cells. The subcellular difference in the ERK activity may be fundamental to the regulation of cell functions by ERK. The present fluorescent indicators provide a powerful tool to reveal the spatiotemporal dynamics of protein phosphorylation by ERK in single living cells.     

From cytotoxicity to biocompatibility testing in vitro: cell adhesion molecule expression defines a new set of parameters

Determination of potential cytotoxicity is a central issue in current biocompatibility testing standards such as ISO and ASTM. Most of these tests do not assess biocompatibility of a biomaterial with regard to cell function. This study was aimed at screening a number of potential parameters that could be included in assessment of cell functional aspects of biocompatibility. Human umbilical vein endothelial cells (HUVEC) were seeded directly on titanium, NiCr alloy, CoCr alloy, PMMA, PE, PU, PVC, and silicone, or were exposed to the material extracts. Cytotoxicity was assessed for these materials through MTT conversion, crystal violet protein determination and Ki67 expression. In addition, expression of the cell adhesion molecules E-selectin, cadherin-5 and PECAM, as well as of the adhesion-associated proteins fibronectin and vinculin (focal adhesions), was determined by immunocytochemistry and western blotting. Cytotoxicity was not detected with the material extracts. Cells were able to adhere to bare metals, but not polymers. Fibronectin preadsorption resulted in adhesion and spreading also on the polymers. Cells were able to establish cell–cell contacts and focal adhesions. Western blotting, in combination with differential detergent extraction, indicated that linkage of cell–cell adhesion markers to the cytoskeleton may be used as an additional parameter relevant to cell function.     

Shear-induced force transmission in a multicomponent,multicell model of the endothelium

Haemodynamic forces applied at the apical surface of vascular endothelial cells (ECs) provide the mechanical signals at intracellular organelles and through the inter-connected cellular network. The objective of this study is to quantify the intracellular and intercellular stresses in a confluent vascular EC monolayer. A novel three-dimensional, multiscale and multicomponent model of focally adhered ECs is developed to account for the role of potential mechanosensors (glycocalyx layer, actin cortical layer, nucleus, cytoskeleton, focal adhesions (FAs) and adherens junctions (ADJs)) in mechanotransmission and EC deformation. The overriding issue addressed is the stress amplification in these regions, which may play a role in subcellular localization of mechanotransmission. The model predicts that the stresses are amplified 250–600-fold over apical values at ADJs and 175–200-fold at FAs for ECs exposed to a mean shear stress of 10 dyne cm⁻². Estimates of forces per molecule in the cell attachment points to the external cellular matrix and cell–cell adhesion points are of the order of 8 pN at FAs and as high as 3 pN at ADJs, suggesting that direct force-induced mechanotransmission by single molecules is possible in both. The maximum deformation of an EC in the monolayer is calculated as 400 nm in response to a mean shear stress of 1 Pa applied over the EC surface which is in accord with measurements. The model also predicts that the magnitude of the cell–cell junction inclination angle is independent of the cytoskeleton and glycocalyx. The inclination angle of the cell–cell junction is calculated to be 6.6° in an EC monolayer, which is somewhat below the measured value (9.9°) reported previously for ECs subjected to 1.6 Pa shear stress for 30 min. The present model is able, for the first time, to cross the boundaries between different length scales in order to provide a global view of potential locations of mechanotransmission.     

Exploring the formation of focal adhesions on patterned surfaces using super-resolution imaging

The formation of focal adhesions on various sizes of fibronectin patterns, ranging from 200 μm to 250 nm, was systematically investigated by total internal reflection fluorescence microscopy and super-resolution imaging. It was found that cells adhered to and spread on these micro/nanopatterns, forming focal adhesions. On a micrometer scale the shape of the focal adhesions was elongated. However, on the nanometer scale, the shape of focal adhesions became dotlike. To further explore the distribution of focal adhesion proteins formed on surfaces, a localization-based super-resolution imaging technique was employed in order to determine the position and density of vinculin proteins. A characteristic distance of 50 nm was found between vinculin molecules in the focal adhesions, which did not depend on the size of the fibronectin nanopatterns. This distance was found to be crucial for the formation of focal adhesions. In addition, the density of vinculin at the focal adhesions formed on the nanopatterns increased as the pattern size decreased. The density of the protein was found to be 425 ± 247, 584 ± 302, and 703 ± 305 proteins μm(-2) on the 600, 400, and 250 nm fibronectin patterns respectively. Whereas 226 ± 77 proteins μm(-2) was measured for the matured focal adhesions on homogeneous fibronectin coated substrates. The increase in vinculin density implies that an increase in mechanical load was applied to the focal adhesions formed on the smaller nanopatterns.     

Topographic cell instructive patterns to control cell adhesion,polarization and migration

Topographic patterns are known to affect cellular processes such as adhesion, migration and differentiation. However, the optimal way to deliver topographic signals to provide cells with precise instructions has not been defined yet. In this work, we hypothesize that topographic patterns may be able to control the sensing and adhesion machinery of cells when their interval features are tuned on the characteristic lengths of filopodial probing and focal adhesions (FAs). Features separated by distance beyond the length of filopodia cannot be readily perceived; therefore, the formation of new adhesions is discouraged. If, however, topographic features are separated by a distance within the reach of filopodia extension, cells can establish contact between adjacent topographic islands. In the latter case, cell adhesion and polarization rely upon the growth of FAs occurring on a specific length scale that depends on the chemical properties of the surface. Topographic patterns and chemical properties may interfere with the growth of FAs, thus making adhesions unstable. To test this hypothesis, we fabricated different micropatterned surfaces displaying feature dimensions and adhesive properties able to interfere with the filopodial sensing and the adhesion maturation, selectively. Our data demonstrate that it is possible to exert a potent control on cell adhesion, elongation and migration by tuning topographic features’ dimensions and surface chemistry.     

Effect of pore size of self-organized honeycomb-patterned polymer films on spreading, focal adhesion, proliferation, and function of endothelial cells

The design of nano- and microstructures based on self-organization is a key area of research in the search for new materials, and it has a variety of potential applications in tissue engineering scaffolds. We have reported a honeycomb-patterned polymer film (honeycomb film) with highly regular pores that is formed by self-organization. This study describes the behavior of vascular endothelial cells (ECs) on honeycomb films with four different pore sizes (5, 9, 12, and 16 microm) as well as on a flat film. We examined the influence of the honeycomb pattern and pore size on cell behavior. The changes in cell morphologies, actin filaments, vinculin clusters, cell proliferation, and secreted extracellular matrix (ECM) (fibronectin, laminin, type IV collagen, and elastin) production profiles were observed by using optical, fluorescence, and scanning electron microscopy. The ECs that adhered to the flat film showed an elongated morphology with random orientation; the actin filaments and focal adhesions were not conspicuous. On the other hand, the ECs on the honeycomb films exhibited greater spreading and flattening; the degree of spreading of the ECs increased with an increase in the pore size. The actin filaments and focal adhesions appeared conspicuous, and the focal adhesions localized along the edge of the honeycomb pores were distributed over the entire projected cell area. The honeycomb film with a pore size of 5 microm showed the highest cell proliferation and ECM production profiles. These results suggest that the honeycomb film is a suitable material for designing a new vascular device.     

Rear actomyosin contractility-driven directional cell migration in three-dimensional matrices: a mechano-chemical coupling mechanism

Cell migration is of vital importance in many biological processes, including organismal development, immune response and development of vascular diseases. For instance, migration of vascular smooth muscle cells from the media to intima is an essential part of the development of atherosclerosis and restenosis after stent deployment. While it is well characterized that cells use actin polymerization at the leading edge to propel themselves to move on two-dimensional substrates, the migration modes of cells in three-dimensional matrices relevant to in vivo environments remain unclear. Intracellular tension, which is created by myosin II activity, fulfils a vital role in regulating cell migration. We note that there is compelling evidence from theoretical and experimental work that myosin II accumulates at the cell rear, either isoform-dependent or -independent, leading to three-dimensional migration modes driven by posterior myosin II tension. The scenario is not limited to amoeboid migration, and it is also seen in mesenchymal migration in which a two-dimensional-like migration mode based on front protrusions is often expected, suggesting that there may exist universal underlying mechanisms. In this review, we aim to shed some light on how anisotropic myosin II localization induces cell motility in three-dimensional environments from a biomechanical view. We demonstrate an interesting mechanism where an interplay between mechanical myosin II recruitment and biochemical myosin II activation triggers directional migration in three-dimensional matrices. In the case of amoeboid three-dimensional migration, myosin II first accumulates at the cell rear to induce a slight polarization displayed as a uropod-like structure under the action of a tension-dependent mechanism. Subsequent biochemical signalling pathways initiate actomyosin contractility, producing traction forces on the adhesion system or creating prominent motile forces through blebbing activity, to drive cells to move. In mesenchymal three-dimensional migration, cells can also take advantage of the elastic properties of three-dimensional matrices to move. A minor myosin isoform, myosin IIB, is retained by relatively stiff three-dimensional matrices at the posterior side, then activated by signalling cascades, facilitating prominent cell polarization by establishing front–back polarity and creating cell rear. Myosin IIB initiates cell polarization and coordinates with the major isoform myosin IIA-assembled stress fibres, to power the directional migration of cells in the three-dimensional matrix.