Traction-regulated persistence governs durotaxis across cell types.

Collective cell migration underlies morphogenesis, wound healing and cancer invasion1,2. Most directed migration in vivo has been attributed to chemotaxis, whereby cells follow a chemical gradient3-5. Cells can also follow a stiffness gradient in vitro, a process called durotaxis3,4,6-8, but evidence for durotaxis in vivo is lacking6. Here we show that in Xenopus laevisthe neural crest-an embryonic cell population-self-generates a stiffness gradient in the adjacent placodal tissue, and follows this gradient by durotaxis. The gradient moves with the neural crest, which is continually pursuing a retreating region of high substrate stiffness. Mechanistically, the neural crest induces the gradient due to N-cadherin interactions with the placodes and senses the gradient through cell-matrix adhesions, resulting in polarized Rac activity and actomyosin contractility, which coordinates durotaxis. Durotaxis synergizes with chemotaxis, cooperatively polarizing actomyosin machinery of the cell group to prompt efficient directional collective cell migration in vivo. These results show that durotaxis and dynamic stiffness gradients exist in vivo, and gradients of chemical and mechanical signals cooperate to achieve efficient directional cell migration.

Directional cell migration due to mechanosensing for invivo microenvironment, such as microgrooved surfaces, is an essential process in tissue growth and repair in both normal and pathological states. Cell migration responses on the microgrooved surfaces might be reflected by the cell type difference, which is deeply involved in cellular physiological functions. Although the responses are implicated in focal adhesions (FAs) of cells, limited information is available about cell migration behavior on the microgrooved surfaces whose dimensions are comparable with the size of FAs. In the present study, we investigated the cell orientation and migration behavior of normal vascular smooth muscle cells (VSMCs) and cervical cancer HeLa cells on the microgrooved surface. The cells were cultured on the PDMS substrate comprising shallow grooves with 2-µm width and approximately 150-nm depth, which indicates the same order of magnitude as that of the horizontal and vertical size of FAs, respectively. The cell migration and intracellular structures were analyzed by live cell imaging and confocal fluorescence microscopy. The intracellular tension was also assessed using atomic force microscopy (AFM). VSMCs presenting well-aligned actin stress fibers with mature FAs revealed marked cell elongation and directional migration on the grooves; however, HeLa cells with nonoriented F-actin with smaller FAs did not. The internal force of the actin fibers was significantly higher in VSMCs than that in HeLa cells, and the increase or decrease in the cytoskeletal forces improved or diminished the sensing ability for shallow grooves, respectively. The results strongly indicated that directional cell migration should be modulated by cell type-specific cytoskeletal arrangements and intracellular traction forces. The differences in cell type-specific orientation and migration responses can be emphasized on the microgrooves as large as the horizontal and vertical size of FAs. The microgoove structure in the size range of the FA protein complex is a powerful tool to clarify subtle differences in the intracellular force-dependent substrate mechanosensing.

Anisotropic stiffness gradient-regulated mechanical guidance drives directional migration of cancer cells

Continuous development of cell traction force can regulate cell migration on various extracellular matrixes in vivo. However, the topographical effect on traction force is still not fully understood. Micropost sensors with parallel guiding gratings were fabricated in polydimethylsiloxane to track the cell traction force during topographical guidance in real time. The force distributions along MC3T3-E1 mouse osteoblasts were captured every minute. The traction force in the leading, middle, and trailing regions was monitored during forward and reversed cell migration. The traction force showed periodic changes during cell migration when the cell changed from elongated to contracted shape. For cell migration without guiding pattern, the leading region showed the largest traction force among the three regions, typically 5.8±0.8 nanonewton (nN) when the cell contracted and 7.1±0.5 nN when it elongated. During guided cell migration, a lower traction force was obtained. When a cell contracted, the trailing traction force was 4.1±0.4 for non-guided migration and 2.2±0.2 nN for guided migration. As a cell became elongated, the trailing traction force was 6.0±0.5 nN during non-guided migration and 4.8±0.3 nN under guidance. When a cell reversed its migration direction, the magnitudes of the traction force from the leading to the trailing regions also flipped. The cell traction force is continuously influenced by topographical guidance, which determines cell migration speed and direction. These results of cell traction force development on various topographies could lead to better cell migration control using topotaxis.

Directed cell migration is critical across biological processes spanning healing to cancer invasion, yet no existing tools allow real-time interactive guidance over such migration. We present a new bioreactor that harnesses electrotaxis-directed cell migration along electric field gradients-by integrating four independent electrodes under computer control to dynamically program electric field patterns, and hence steer cell migration. Using this platform, we programmed and characterized multiple precise, two-dimensional collective migration maneuvers in renal epithelia and primary skin keratinocyte ensembles. First, we demonstrated on-demand, 90-degree collective turning. Next, we developed a universal electrical stimulation scheme capable of programming arbitrary 2D migration maneuvers such as precise angular turns and migration in a complete circle. Our stimulation scheme proves that cells effectively time-average electric field cues, helping to elucidate the transduction timescales in electrotaxis. Together, this work represents an enabling platform for controlling cell migration with broad utility across many cell types.

During skeletal muscle regeneration caused by injury, muscle satellite cells proliferate and migrate toward the site of muscle injury. This migration is mainly induced by hepatocyte growth factor (HGF) secreted by intact myofibers and also released from injured muscle. However, the intracellular machinery for the satellite cell migration has not been elucidated. To examine the mechanisms of satellite cell migration, we utilized satellite cell-derived mouse C2C12 skeletal muscle cells. HGF induced reorganization of actin cytoskeleton to form lamellipodia in C2C12 myoblasts. HGF treatment facilitated both nondirectional migration of the myoblasts in phagokinetic track assay and directional chemotactic migration toward HGF in a three-dimensional migration chamber assay. Endogenous N-WASP and WAVE2 were concentrated in the lamellipodia at the leading edge of the migrating cells. Moreover, exogenous expression of wild-type N-WASP or WAVE2 promoted lamellipodial formation and migration. By contrast, expression of the dominant-negative mutant of N-WASP or WAVE2 and knockdown of N-WASP or WAVE2 expression by the RNA interference prevented the HGF-induced lamellipodial formation and migration. When the cells were treated with LY294002, an inhibitor of phosphatidylinositol 3-kinase, the HGF-induced lamellipodial formation and migration were abrogated. These results imply that both N-WASP and WAVE2, which are activated downstream of phosphati-dylinositol 3-kinase, are required for the migration through the lamellipodial formation of C2C12 cells induced by HGF.

The control of cell migration has an important role in processes ranging from developmental morphogenesis to the pathogenesis. In this study, we describe a novel approach to develop a micro-checkerboard patterned polymeric flat surface with discrete surface stiffness. This platform as a culture substrate allows us to explore the mechanism of durotaxis, referred to as the directed cell movement via the gradient of surface stiffness. The flat surface with different rigidity was achieved in two stages of fabrication. First, polydimethylsiloxane (PDMS) was pressed and cured on a glass substrate with trenches of varying depths in a checkerboard arrangement, and then, a thin PDMS layer was spin coated on the previous pattern to make the flat surface. The stiff region is defined by a thin layer (2.5 µm) of PDMS and the soft region is defined by a thick one (7.5 µm). To investigate the migratory cell behavior, the NIH 3T3 cell was cultured. The result demonstrates that a single cell showed clearly a migratory cell behavior toward the stiffer regions driven by the difference of effective surface stiffness. At high cell density, the effect of cell migration on effective surface stiffness decreased with increasing cell–cell interactions. However, cell migration was still dominated by difference of effective surface stiffness while fluctuating at the boundary between the stiff and soft regions. This approach enables us to control the mechanical and topological properties of surface. The developed platform will also offer a useful tool to study cell–substrate interaction mediated by surface stiffness (e.g. mechanotransduction).

Traction Forces Control Cell-Edge Dynamics and Mediate Distance Sensitivity during Cell Polarization.

Direct current electric fields (DCEFs) can induce directional migration for many cell types through activation of intracellular signaling pathways. However, the mechanisms that bridge extracellular electrical stimulation with intracellular signaling remain largely unknown. In the current study, we found that a DCEF can induce the directional migration of U87, C6 and U251 glioma cells to the cathode and stimulate the production of hydrogen peroxide and superoxide. Subsequent studies demonstrated that the electrotaxis of glioma cells were abolished by the superoxide inhibitor N-acetyl-l-cysteine (NAC) or overexpression of mitochondrial superoxide dismutase (MnSOD), but was not affected by inhibition of hydrogen peroxide through the overexpression of catalase. Furthermore, we found that the presence of NAC, as well as the overexpression of MnSOD, could almost completely abolish the activation of Akt, extracellular-signal-regulated kinase (Erk)1/2, c-Jun N-terminal kinase (JNK), and p38, although only JNK and p38 were affected by overexpression of catalase. The presenting of specific inhibitors can decrease the activation of Erk1/2 or Akt as well as the directional migration of glioma cells. Collectively, our data demonstrate that superoxide may play a critical role in DCEF-induced directional migration of glioma cells through the regulation of Akt and Erk1/2 activation. This study provides novel evidence that the superoxide is at least one of the “bridges” coupling the extracellular electric stimulation to the intracellular signals during DCEF-mediated cell directional migration.

Phosphatidylinositol 4,5 bisphosphate (PIP₂) is a key lipid messenger for regulation of cell migration. PIP₂ modulates many effectors, but the specificity of PIP₂ signalling can be defined by interactions of PIP₂-generating enzymes with PIP₂ effectors. Here, we show that type Iγ phosphatidylinositol 4-phosphate 5-kinase (PIPKIγ) interacts with the cytoskeleton regulator, IQGAP1, and modulates IQGAP1 function in migration. We reveal that PIPKIγ is required for IQGAP1 recruitment to the leading edge membrane in response to integrin or growth factor receptor activation. Moreover, IQGAP1 is a PIP₂ effector that directly binds PIP₂ through a polybasic motif and PIP₂ binding activates IQGAP1, facilitating actin polymerization. IQGAP1 mutants that lack PIPKIγ or PIP₂ binding lose the ability to control directional cell migration. Collectively, these data reveal a synergy between PIPKIγ and IQGAP1 in the control of cell migration.

Deformation Gradients Imprint the Direction and Speed of En Masse Fibroblast Migration for Fast Healing

General cellular durotaxis induced with cell-scale heterogeneity of matrix-elasticity

BackgroundSuccessful nerve regeneration depends upon directed migration of morphologically specialized repair state Schwann cells across a nerve defect. Although several groups have studied directed migration of Schwann cells in response to chemical or topographic cues, the current understanding of how the mechanical environment influences migration remains largely understudied and incomplete. Therefore, the focus of this study was to evaluate Schwann cell migration and morphodynamics in the presence of stiffness gradients, which revealed that Schwann cells can follow extracellular gradients of increasing stiffness, in a form of directed migration termed durotaxis.MethodsPolyacrylamide substrates were fabricated to mimic the range of stiffness found in peripheral nerve tissue. We assessed Schwann cell response to substrates that were either mechanically uniform or embedded with a shallow or steep stiffness gradient, respectively corresponding to the mechanical niche present during either the fluid phase or subsequent matrix phase of the peripheral nerve regeneration process. We examined cell migration (velocity and directionality) and morphology (elongation, spread area, nuclear aspect ratio, and cell process dynamics). We also characterized the surface morphology of Schwann cells by scanning electron microscopy.ResultsOn laminin-coated polyacrylamide substrates embedded with either a shallow (∼0.04 kPa/mm) or steep (∼0.95 kPa/mm) stiffness gradient, Schwann cells displayed durotaxis, increasing both their speed and directionality along the gradient materials, fabricated with elastic moduli in the range found in peripheral nerve tissue. Uniquely and unlike cell behavior reported in other cell types, the durotactic response of Schwann cells was not dependent upon the slope of the gradient. When we examined whether durotaxis behavior was accompanied by a pro-regenerative Schwann cell phenotype, we observed altered cell morphology, including increases in spread area and the number, elongation, and branching of the cellular processes, on the steep but not the shallow gradient materials. This phenotype emerged within hours of the cells adhering to the materials and was sustained throughout the 24 hour duration of the experiment. Control experiments also showed that unlike most adherent cells, Schwann cells did not alter their morphology in response to uniform substrates of different stiffnesses.ConclusionThis study is notable in its report of durotaxis of cells in response to a stiffness gradient slope, which is greater than an order of magnitude less than reported elsewhere in the literature, suggesting Schwann cells are highly sensitive detectors of mechanical heterogeneity. Altogether, this work identifies durotaxis as a new migratory modality in Schwann cells, and further shows that the presence of a steep stiffness gradient can support a pro-regenerative cell morphology.

Tug of War at the Cell-Matrix Interface

Cell migration is a crucial process during development, the immune response and wound healing. 
\nAs a consequence, aberrant cell migration can lead to tumor metastasis or autoimmune disorders. In order to migrate directionally, cells have to orchestrate a complex machinery of cytoskeletal, adhesion and signaling components to reach their place of destination. During directional migration, cells maintain a polarized state, which means that cell front and back have to be co-ordinated in a robust way. This complex process is not yet fully understood and gaining mechanistic information in model cell migration systems might help to explain regulation of cell migration in vivo.
\nAn important question is how local phenomena such as fine leading edge dynamics, Rho-GTPase signaling or cytoskeletal organization impact on establishment and maintenance of cell polarization to co-ordinate front and back activities during directional cell migration. Current models of global front and back co-ordination mostly originate from highly polarized, rapidly moving cells such as neutrophils or Dictiostelium[OP1]. However, due to the small size of these cells, it is not possible to study local phenomena such as fine leading edge dynamics, happening within time scales of seconds and spatial scales of single micrometers. In order to address questions concerning these fine dynamics, fibroblasts are a widely used model system due to their large and flat morphology. The limitation of this system is that fibroblasts are not highly polarized, precluding the study of front/back co-ordination. An integrated, multi-scale view of directional cell migration view is therefore missing.
\nTo overcome this limitation we engineered a system, which enabled us to study polarized and persistent cell migration on multiple time and length scales. In this experimental set up we allowed rat embryonic fibroblasts (REF52) to migrate on fibronectin coated line substrates. Unstimulated cells (referred to as hapto cells) or platelet derived growth factor (PDGF) treated cells (referred to as chemo cells) were studied in a variety of static or live cell imaging experiments using different spatio-temporal resolution. Hapto cells were found to undergo transient episodes of polarization and therefore characterized by low migration persistence as well as low migration velocity. In contrast, chemo cells showed a drastic increase of migration persistence, enabling them to migrate in one specific direction for hours. At the same time migration velocity was elevated by more than five times. This provides an excellent model system to study polarized cell migration.
\nIn order to explain these drastic changes in migration persistence and velocity as global migration parameters, we examined cytoskeletal, adhesion and signaling dynamics at high spatio-temporal resolution. We found that hapto cells displayed classic features previously observed during mesenchymal cell migration. A protrusive lamellipodium led to the formation of initial adhesions, called focal complexes. Directly behind the lamellipodium, these adhesions then matured in mechanosensitive adhesions, called focal adhesions, through interaction with the contractile lamella. Front adhesions connected to back focal adhesions through stress fibers. Thus, as previously proposed, this front/back linkage coupled with stress fiber tension allows the front to pull the back, leading to tail retraction.
\nSurprisingly, we observed different actin and adhesion dynamics in persistently migrating chemo cells. These cells remodeled their cytoskeleton and developed two distinct front and back functional modules which are mechanically uncoupled. Specifically, a non-contractile front module, containing a constant sized zone of podosome-like structures (PLSs) replaced the lamella and precluded contractile, retrograde F-actin flow at this subcellular localization. This allowed the PLS zone to function as mechanical insulator, leading to loss of maturation of focal complexes to focal adhesions. Tail retraction was then mediated by a 2nd contractile module that consists of a myosin cluster positioned directly at the back of the PLS zone. Thus, a front module pushes in direction of cell migration, and a contractile back module directly follows the PLS zone and pulls the cell back allowing for tail retraction.
\nBy evaluating front/back motion co-ordination, and using drug perturbations, we formally showed, that the protrusive front is mechanically uncoupled from the contractile back module. From a signaling point of view, we found that the PLS zone acts by locally inhibiting RhoA mediated contractility at the leading edge, allowing uncoupling of cell front and back.
\n
\nWe propose, that mechanical uncoupling of cell front and back by establishment of the PLS zone enables highly efficient and persistent fibroblast cell migration during exposure to a uniform concentration growth factor. The finding that cells do not necessarily require a chemokine gradient to migrate uni-directionally, but can polarize efficiently by simple exposure of an uniform concentration of growth factor in combination with topological confinement of the ECM, might have significance in vivo too. For example, neural crest cells can migrate directionally during collective cell migration in absence of a gradient. During cancer metastasis, a macrophage-tumor cell paracrine loop allows for collective cell streaming in one specific direction on collagen fibrils. While this was suggested to involve chemotaxis, it is conceivable that chemokinesis might therefore be sufficient to induce directional cell migration on the collagen fibril.
\nOur finding of mechanical uncoupling of cell front and back during chemokinesis on line substrates might provide a mean for generation of directional cell migration.
\n[OP1]Took away time and length scales
\n