On the adhesion-velocity relation and length adaptation of motile cells on stepped fibronectin lanes.

The biphasic adhesion-velocity relation is a universal observation in mesenchymal cell motility. It has been explained by adhesion-promoted forces pushing the front and resisting motion at the rear. Yet, there is little quantitative understanding of how these forces control cell velocity. We study motion of MDA-MB-231 cells on microlanes with fields of alternating Fibronectin densities to address this topic and derive a mathematical model from the leading-edge force balance and the force-dependent polymerization rate. It reproduces quantitatively our measured adhesion-velocity relation and results with keratocytes, PtK1 cells, and CHO cells. Our results confirm that the force pushing the leading-edge membrane drives lamellipodial retrograde flow. Forces resisting motion originate along the whole cell length. All motion-related forces are controlled by adhesion and velocity, which allows motion, even with higher Fibronectin density at the rear than at the front. We find the pathway from Fibronectin density to adhesion structures to involve strong positive feedbacks. Suppressing myosin activity reduces the positive feedback. At transitions between different Fibronectin densities, steady motion is perturbed and leads to changes of cell length and front and rear velocity. Cells exhibit an intrinsic length set by adhesion strength, which, together with the length dynamics, suggests a spring-like front-rear interaction force. We provide a quantitative mechanistic picture of the adhesion-velocity relation and cell response to adhesion changes integrating force-dependent polymerization, retrograde flow, positive feedback from integrin to adhesion structures, and spring-like front-rear interaction.

On multistability and constitutive relations of cell motion on fibronectin lanes.

Cell motility on flat substrates exhibits coexisting steady and oscillatory morphodynamics, the biphasic adhesion-velocity relation, and the universal correlation between speed and persistence (UCSP) as simultaneous observations common to many cell types. Their universality and concurrency suggest a unifying mechanism causing all three of them. Stick-slip models for cells on one-dimensional lanes suggest multistability to arise from the nonlinear friction of retrograde flow. This study suggests a mechanical mechanism controlled by integrin signaling on the basis of a biophysical model and analysis of trajectories of MDA-MB-231 cells on fibronectin lanes, which additionally explains the constitutive relations. The experiments exhibit cells with steady or oscillatory morphodynamics and either spread or moving with spontaneous transitions between the dynamic regimes, spread and moving, and spontaneous direction reversals. Our biophysical model is based on the force balance at the protrusion edge, the noisy clutch of retrograde flow, and a response function of friction and membrane drag to integrin signaling. The theory reproduces the experimentally observed cell states, characteristics of oscillations, and state probabilities. Analysis of experiments with the biophysical model establishes a stick-slip oscillation mechanism, and explains multistability of cell states and the statistics of state transitions. It suggests protrusion competition to cause direction reversal events, the statistics of which explain the UCSP. The effect of integrin signaling on drag and friction explains the adhesion-velocity relation and cell behavior at fibronectin density steps. The dynamics of our mechanism are nonlinear flow mechanics driven by F-actin polymerization and shaped by the noisy clutch of retrograde flow friction, protrusion competition via membrane tension, and drag forces. Integrin signaling controls the parameters of the mechanical system.

Area and Geometry Dependence of Cell Migration in Asymmetric Two-State Micropatterns.

Microstructured surfaces provide a unique framework to probe cell migration and cytoskeletal dynamics in a standardized manner. Here, we report on the steady-state occupancy probability of cells in asymmetric two-state microstructures that consist of two fibronectin-coated adhesion sites connected by a thin guidance cue. In these dumbbell-like structures, cells transition between the two sites in a repeated and stochastic manner, and average dwell times in the respective microenvironments are determined from the cell trajectories. We study the dynamics of human breast carcinoma cells (MDA-MB-231) in these microstructures as a function of area, shape, and orientation of the adhesion sites. On square adhesive sites with different areas, we find that the occupancy probability ratio is directly proportional to the ratio of corresponding adhesion site areas. These asymmetries are well captured by a simple model for the stochastic nonlinear dynamics of the cells, which reveals generic features of the motion. Sites of equal area but different shape lead to equal occupancy if shapes are isotropic (e.g., squared or circular). In contrast, an asymmetry in the occupancy is induced by anisotropic shapes like rhombi, triangles, or rectangles that enable motion in the direction perpendicular to the transition axis. Analysis of the two-dimensional motion of cells between two rectangles with orthogonal orientation suggests that cellular transition rates depend on the cell polarization induced by anisotropic micropatterns. Taken together, our results illustrate how two-state micropatterns provide a dynamic migration assay with distinct dwell times and relative cell occupancy as readouts, which may be useful to probe cell-microenvironment interactions.

Effect of Citrate- and Gold-Stabilized Superparamagnetic Iron Oxide Nanoparticles on Head and Neck Tumor Cell Lines during Combination Therapy with Ionizing Radiation.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. They are associated with alcohol and tobacco consumption, as well as infection with human papillomaviruses (HPV). Therapeutic options include radiochemotherapy, surgery or chemotherapy. Nanoparticles are becoming more and more important in medicine. They can be used diagnostically, but also therapeutically. In order to provide therapeutic alternatives in the treatment of HNSCC, the effect of citrate-coated superparamagnetic iron oxide nanoparticles (Citrate-SPIONs) and gold-coated superparamagnetic iron oxide nanoparticles (Au-SPIONs) in combination with ionizing irradiation (IR) on two HPV positive and two HPV negative HNSCC and healthy fibroblasts and keratinocytes cell lines were tested. Effects on apoptosis and necrosis were analyzed by using flow cytometry. Cell survival studies were performed with a colony formation assay. To better understand where the SPIONs interact, light microscopy images and immunofluorescence studies were performed. The HNSCC and healthy cell lines showed different responses to the investigated SPIONs. The cytotoxic effects of SPIONs, in combination with IR, are dependent on the type of SPIONs, the dose administered and the cell type treated. They are independent of HPV status. Reasons for the different cytotoxic effect are probably the different compositions of the SPIONs and the related different interaction of the SPIONs intracellularly and paramembranously, which lead to different strong formations of double strand breaks.

Non-Markovian data-driven modeling of single-cell motility.

Trajectories of human breast cancer cells moving on one-dimensional circular tracks are modeled by the non-Markovian version of the Langevin equation that includes an arbitrary memory function. When averaged over cells, the velocity distribution exhibits spurious non-Gaussian behavior, while single cells are characterized by Gaussian velocity distributions. Accordingly, the data are described by a linear memory model which includes different random walk models that were previously used to account for various aspects of cell motility such as migratory persistence, non-Markovian effects, colored noise, and anomalous diffusion. The memory function is extracted from the trajectory data without restrictions or assumptions, thus making our approach truly data driven, and is used for unbiased single-cell comparison. The cell memory displays time-delayed single-exponential negative friction, which clearly distinguishes cell motion from the simple persistent random walk model and suggests a regulatory feedback mechanism that controls cell migration. Based on the extracted memory function we formulate a generalized exactly solvable cell migration model which indicates that negative friction generates cell persistence over long timescales. The nonequilibrium character of cell motion is investigated by mapping the non-Markovian Langevin equation with memory onto a Markovian model that involves a hidden degree of freedom and is equivalent to the underdamped active Ornstein-Uhlenbeck process.

Quasi-periodic migration of single cells on short microlanes.

Cell migration on microlanes represents a suitable and simple platform for the exploration of the molecular mechanisms underlying cell cytoskeleton dynamics. Here, we report on the quasi-periodic movement of cells confined in stripe-shaped microlanes. We observe persistent polarized cell shapes and directed pole-to-pole motion within the microlanes. Cells depolarize at one end of a given microlane, followed by delayed repolarization towards the opposite end. We analyze cell motility via the spatial velocity distribution, the velocity frequency spectrum and the reversal time as a measure for depolarization and spontaneous repolarization of cells at the microlane ends. The frequent encounters of a boundary in the stripe geometry provides a robust framework for quantitative investigations of the cytoskeleton protrusion and repolarization dynamics. In a first advance to rigorously test physical models of cell migration, we find that the statistics of the cell migration is recapitulated by a Cellular Potts model with a minimal description of cytoskeleton dynamics. Using LifeAct-GFP transfected cells and microlanes with differently shaped ends, we show that the local deformation of the leading cell edge in response to the tip geometry can locally either amplify or quench actin polymerization, while leaving the average reversal times unaffected.

Inducible microRNA-200c decreases motility of breast cancer cells and reduces filamin A.

Cancer progression and metastases are frequently related to changes of cell motility. Amongst others, the microRNA-200c (miR-200c) was shown to maintain the epithelial state of cells and to hamper migration. Here, we describe two miR-200c inducible breast cancer cell lines, derived from miR-200c knock-out MCF7 cells as well as from the miR-200c-negative MDA-MB-231 cells and report on the emerging phenotypic effects after miR-200s induction. The induction of miR-200c expression seems to effect a rapid reduction of cell motility, as determined by 1D microlane migration assays. Sustained expression of miR200c leads to a changed morphology and reveals a novel mechanism by which miR-200c interferes with cytoskeletal components. We find that filamin A expression is attenuated by miRNA-200c induced downregulation of the transcription factors c-Jun and MRTF/SRF. This potentially novel pathway that is independent of the prominent ZEB axis could lead to a broader understanding of the role that miR200c plays in cancer metastasis.

Ring-Shaped Microlanes and Chemical Barriers as a Platform for Probing Single-Cell Migration.

Quantification and discrimination of pharmaceutical and disease-related effects on cell migration requires detailed characterization of single-cell motility. In this context, micropatterned substrates that constrain cells within defined geometries facilitate quantitative readout of locomotion. Here, we study quasi-one-dimensional cell migration in ring-shaped microlanes. We observe bimodal behavior in form of alternating states of directional migration (run state) and reorientation (rest state). Both states show exponential lifetime distributions with characteristic persistence times, which, together with the cell velocity in the run state, provide a set of parameters that succinctly describe cell motion. By introducing PEGylated barriers of different widths into the lane, we extend this description by quantifying the effects of abrupt changes in substrate chemistry on migrating cells. The transit probability decreases exponentially as a function of barrier width, thus specifying a characteristic penetration depth of the leading lamellipodia. Applying this fingerprint-like characterization of cell motion, we compare different cell lines, and demonstrate that the cancer drug candidate salinomycin affects transit probability and resting time, but not run time or run velocity. Hence, the presented assay allows to assess multiple migration-related parameters, permits detailed characterization of cell motility, and has potential applications in cell biology and advanced drug screening.

Contractility as a global regulator of cellular morphology, velocity, and directionality in low-adhesive fibrillary micro-environments.

Recent reports demonstrated that migration in fibrillary environments can be mimicked by spatial confinement achieved with micro-patterning [1]. Here we investigated whether a model system based on linearly structured surfaces allows to draw conclusions about migration of endothelial cells (ECs) in fibrillary 3D environments. We found that ECs on 3 µm wide tracks (termed as 1D) migrate less efficient in comparison to ECs on broader tracks in regard to velocity and directional persistence. The frequent changes of direction in ECs on narrow tracks are accompanied by pronounced cell rounding and membrane blebbing, while cells migrating with an elongated morphology display a single lamellipodium. This behavior is contractility-dependent as both modes can be provoked by manipulating activity of myosin II (blebbistatin or calyculin A, respectively). The comparison between 1D and 3D migrating cells revealed a striking similarity in actin architecture and in switching between two morphologies. ECs move more directed but slower upon inhibition of contractility in 1D and 3D, in contrast to 2D cell culture. We conclude that micro-patterning can be used to study morphological switches in a controlled manner with a prognostic value for 3D environments. Moreover, we identified blebbing as a new aspect of EC migration.