Quantification of Cell Movement Reveals Distinct Edge Motility Types During Cell Spreading

Actin-based motility is central to cellular processes such as migration, bacterial engulfment, and cancer metastasis, and requires precise spatial and temporal regulation of the cytoskeleton. We studied one such process, fibroblast spreading, which involves three temporal phases: early, middle, and late spreading, distinguished by differences in cell area growth. In these studies, aided by improved algorithms for analyzing edge movement, we observed that each phase was dominated by a single, kinematically and biochemically distinct cytoskeletal organization, or motility type. Specifically, early spreading was dominated by periodic blebbing; continuous protrusion occurred predominantly during middle spreading; and periodic contractions were prevalent in late spreading. Further characterization revealed that each motility type exhibited a distinct distribution of the actin-related protein VASP, while inhibition of actin polymerization by cytochalasin D treatment revealed different dependences on barbed-end polymerization. Through this detailed characterization and graded perturbation of the system, we observed that although each temporal phase of spreading was dominated by a single motility type, in general cells exhibited a variety of motility types in neighboring spatial domains of the plasma membrane edge. These observations support a model in which global signals bias local cytoskeletal biochemistry in favor of a particular motility type

Contact-controlled amoeboid motility induces dynamic cell trapping in 3D-microstructured surfaces.

On flat substrates, several cell types exhibit amoeboid migration, which is characterized by restless stochastic successions of pseudopod protrusions. The orientation and frequency of new membrane protrusions characterize efficient search modes, which can respond to external chemical stimuli as observed during chemotaxis in amoebae. To quantify the influence of mechanical stimuli induced by surface topography on the migration modes of the amoeboid model organism Dictyostelium discoideum, we apply high resolution motion analysis in microfabricated pillar arrays of defined density and geometry. Cell motion is analyzed by a two-state motility-model, distinguishing directed cellular runs from phases of isotropic migration that are characterized by randomly oriented cellular protrusions. Cells lacking myosin II or cells deprived of microtubules show significantly different behavior concerning migration velocities and migrational angle distribution, without pronounced attraction to pillars. We conclude that microtubules enhance cellular ability to react with external 3D structures. Our experiments on wild-type cells show that the switching from randomly formed pseudopods to a stabilized leading pseudopod is triggered by contact with surface structures. These alternating processes guide cells according to the available surface in their 3D environment, which we observed dynamically and in steady-state situations. As a consequence, cells perform "home-runs" in low-density pillar arrays, crawling from pillar to pillar, with a characteristic dwell time of 75 s. At the boundary between a flat surface and a 3D structured substrate, cells preferentially localize in contact with micropillars, due to the additionally available surface in the microstructured arrays. Such responses of cell motility to microstructures might open new possibilities for cell sorting in surface structured arrays

Assessment of Automated Analyses of Cell Migration on Flat and Nanostructured Surfaces

Motility studies of cells often rely on computer software that analyzes time-lapse recorded movies and establishes cell trajectories fully automatically. This raises the question of reproducibility of results, since different programs could yield significantly different results of such automated analysis. The fact that the segmentation routines of such programs are often challenged by nanostructured surfaces makes the question more pertinent. Here we illustrate how it is possible to track cells on bright field microscopy images with image analysis routines implemented in an open-source cell tracking program, PACT (Program for Automated Cell Tracking). We compare the automated motility analysis of three cell tracking programs, PACT, Autozell, and TLA, using the same movies as input for all three programs. We find that different programs track overlapping, but different subsets of cells due to different segmentation methods. Unfortunately, population averages based on such different cell populations, differ significantly in some cases. Thus, results obtained with one software package are not necessarily reproducible by other software

Systems microscopy approaches to understand cancer cell migration and metastasis

Cell migration is essential in a number of processes, including wound healing, angiogenesis and cancer metastasis. Especially, invasion of cancer cells in the surrounding tissue is a crucial step that requires increased cell motility. Cell migration is a well-orchestrated process that involves the continuous formation and disassembly of matrix adhesions. Those structural anchor points interact with the extra-cellular matrix and also participate in adhesion-dependent signalling. Although these processes are essential for cancer metastasis, little is known about the molecular mechanisms that regulate adhesion dynamics during tumour cell migration. In this review, we provide an overview of recent advanced imaging strategies together with quantitative image analysis that can be implemented to understand the dynamics of matrix adhesions and its molecular components in relation to tumour cell migration. This dynamic cell imaging together with multiparametric image analysis will help in understanding the molecular mechanisms that define cancer cell migration

Integrated Mathematical and Experimental Study of Cell Migration and Shape

Cell migration plays an essential role in many of physiological and pathological processes, including morphogenesis, inflammation, wound healing, and tumor metastasis. It is a complex process that involves multi-scale interactions between the cell and the extracellular matrix (ECM). Cells migrate through stromal ECM with native and cell-derived curvature at micron-meter scale are context-specific. How does the curvature of ECM mechanically change cell morphology and motility? Can the diverse migration behaviors from genetically identical cells be predictively using cell migrating data? We address these questions using an integrated computational and experimental approach: we developed three-dimensional biomechanical cell model and measured and analyzed a large number of cell migration images over time. Our findings suggest that 1. substrate curvature determines cell shape through contact and regulating protrusion dynamics; 2. effective cell migration is characterized with long cellular persistence time, low speed variation, spatial-temporally coordinated protrusion and contraction; 3. the cell shape variation space is low dimensional; and 4. migration behavior can be determined by a single image projected in the low dimensional cell shape variation space

Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques

Here we classify endocytic structures at the adherent (bottom) surface of many cells in culture into shorter-lived coated pits and longer-lived coated plaques which internalize by different mechanisms

Amoeboid Shape Dynamics on Flat and Topographically Modified Surfaces

I present an analysis of the shape dynamics of the amoeba Dictyostelium discoideum, a model system for the study of cellular migration. To better understand cellular migration in complicated 3-D environments, cell migration was studied on simple 3-D surfaces, such as cliffs and ridges. D. discoideum interact with surfaces without forming mature focal adhesion complexes. The cellular response to the surface topography was characterized by measuring and looking for patterns in cell shape. Dynamic cell shape is a measure of the interaction between the internal biochemical state of a cell and its external environment. For D. discoideum migrating on flat surfaces, waves of high boundary curvature were observed to travel from the cell front to the cell back. Curvature waves are also easily seen in cells that do not adhere to a surface, such as cells that are electrostatically repelled from the coverslip or cells that are extended over the edge of micro-fabricated cliffs. At the leading edge of adhered cells, these curvature waves are associated with protrusive activity, suggesting that protrusive motion can be thought of as a wave-like process. The wave-like character of protrusions provides a plausible mechanism for the ability of cells to swim in viscous fluids and to navigate complex 3-D topography. Patterning of focal adhesion complexes has previously been implicated in contact guidance (polarization or migration parallel to linear topographical structures). However, significant contact guidance is observed in D. discoideum, which lack focal adhesion complexes. Analyzing the migration of cells on nanogratings of ridges spaced various distances apart, ridges spaced about 1.5 micrometers apart were found to guide cells best. Contact guidance was modeled as an interaction between wave-like processes internal to the cell and the periodicity of the nanograting. The observed wavelength and speed of the oscillations that best couple to the surface are consistent with those of protrusive dynamics. Dynamic sensing via actin or protrusive dynamics might then play a role in contact guidance

Cellular mechanics and intracellular organization

Mechanical signals affect and regulate many aspects of the cell behaviour, including growth, differentiation, gene expression and cell death. This thesis investigates the manner by which mechanical stress perturbs the intracellular structures of the cell and induces mechanical responses. In order to correlate mechanical perturbations to cellular responses, a combined fluorescence-atomic force microscope (AFM) was used to produce well defined nanomechanical perturbations while simultaneously tracking the real-time motion of fluorescently labelled intracellular organelles in live cells. By tracking instantaneous displacements of mitochondria far from the point of indentation, insights can be gained into the long-distance propagation of forces and the role of the cytoskeleton in force transmission. Quantitative analysis and tracking of mitochondria, using several image registration and tracking techniques, revealed an increase of approximately 40% in the mean mitochondrial displacement following AFM perturbation. Furthermore, when either the actin cytoskeleton or microtubules were disrupted using anti-cytoskeletal drugs, no significant change in mitochondrial displacement was observed following indentation, revealing the crucial role of both cytoskeletal networks in the long-distance transmission of forces through the cell. In addition, the effect of retinol and conjugated linoleic acid (CLA), compounds that have diverse effects on various cellular processes, on the mechanical behaviour of the cell was examined: both compounds were found to have a significant detrimental effect on the formation of focal adhesions, which was directly correlated to the measured cell elasticity (Young’s modulus) of the cell. Furthermore, quantification of mitochondrial displacements in response to applied AFM perturbations showed force propagation through the cytoskeleton to be blunted. Treatment of the two compounds in combination showed an additive effect. These results may broaden our understanding of the interplay between cell mechanics and cellular contact with the external microenvironment, and help to shed light on the important role of retinoids and CLA in health and disease

A beta-herpesvirus with fluorescent capsids to study transport in living cells.

Fluorescent tagging of viral particles by genetic means enables the study of virus dynamics in living cells. However, the study of beta-herpesvirus entry and morphogenesis by this method is currently limited. This is due to the lack of replication competent, capsid-tagged fluorescent viruses. Here, we report on viable recombinant MCMVs carrying ectopic insertions of the small capsid protein (SCP) fused to fluorescent proteins (FPs). The FPs were inserted into an internal position which allowed the production of viable, fluorescently labeled cytomegaloviruses, which replicated with wild type kinetics in cell culture. Fluorescent particles were readily detectable by several methods. Moreover, in a spread assay, labeled capsids accumulated around the nucleus of the newly infected cells without any detectable viral gene expression suggesting normal entry and particle trafficking. These recombinants were used to record particle dynamics by live-cell microscopy during MCMV egress with high spatial as well as temporal resolution. From the resulting tracks we obtained not only mean track velocities but also their mean square displacements and diffusion coefficients. With this key information, we were able to describe particle behavior at high detail and discriminate between particle tracks exhibiting directed movement and tracks in which particles exhibited free or anomalous diffusion

Quasi-oscillatory motion of single cells on micropatterns

Zellmigration spielt eine grundlegende Rolle bei Prozessen wie Embryogenese, der Immunantwort, Wundheilung und bei der Metastasierung von Krebs. Daher ist der Mechanismus der Zellmigration, insbesondere die Dynamik des Zytoskeletts, Aktinpolymerisierung und Reaktionsdiffusionsprozesse, von großem Interesse für die Lebenswissenschaften. Zellen sind hochkomplexe dynamische Systeme, die ihren Zustand ständig verändern, wodurch sich bestimmte Morphologien und Migrationsmodi ausprägen. Die resultierenden Migrationsmuster werden durch externe Faktoren beeinflusst, die unter klassischen Kulturbedingungen nicht kontrolliert sind. Eine zentrale Herausforderung bei der Untersuchung der Zellmigration ist daher die Entwicklung spezifischer Methoden, um die Wirkung einzelner Parameter, die das Zellverhalten regulieren, untersuchen zu können. Ein möglicher Weg, die Komplexität der Umgebung zu reduzieren, besteht darin, Mikrostrukturierungstechniken zu verwenden um Zellen auf eine definierte Mikroumgebung zu beschränken. Mit solchen Strukturen kann der Freiheitsgrad der Zellbewegung reduziert werden, was es ermöglicht gezielt spezifische Eigenschaften der Zellmigration zu studieren. Darüber hinaus kann man mit Mikrostrukturierungstechnologie Felder von einer großen Anzahl identischer funktioneller Oberflächenstrukturen herstellen und so Hochdurchsatzmessungen durchführen. Im ersten Teil dieser Arbeit werden Studien zu einem neu entdeckten quasi-oszillatorischen Migrationsmodus von Einzelzellen auf kreisförmigen Mikrostrukturen vorgestellt. Wir beobachten persistente polarisierte Zellen und gerichtete Pol-zu-Pol-Bewegungen innerhalb der Strukturen. Die Zellen depolarisieren auf einer Seite der Mikrostuktur, gefolgt von einer verzögerten Repolarisierung in entgegengesetzter Richtung. Weiter wird gezeigt, dass mehrere Zelllinien (z.B. MDCK-, Huh7-, MDA-MB-231-Zellen) diesen oszillierenden Migrationsmodus auf kreis-, ellipsen- und streifenförmigen Mikrostrukturen zeigen. Im Vergleich zu kreisförmigen und elliptischen Strukturen ist das Auftreten von Oszillationen auf Streifen gehäuft feststellbar. Streifen bieten eine ideale und einfache Plattform um neue Migrationsmuster von Zellen und um den molekularen Mechanismus, der der Dynamik des Zytoskeletts zugrunde liegt, zu studieren. Im zweiten Teil dieser Arbeit analysieren wir das Zellverhalten mit Hilfe der räumlichen Geschwindigkeitsverteilung und dem Frequenzspektrum der Bewegung. Die experimentellen Daten werden mit einem zellulären Potts-Modell verglichen, das ein minimales mechanistisches Modell des dynamischen Zytoskeletts enthält. Insbesondere betrachten wir die Dauer des Umkehrprozesses als Maß für die Dauer spontaner Repolarisierung von Zellen und für die Zeit, die das führende Lamellipodium benötigt um sich am Ende des Streifens zurück zu bilden. Mit LifeAct-GFP transfizierten Zellen und Streifen mit unterschiedlich geformten Enden lassen sich Veränderungen im Verhalten an den Enden beobachten. Dies zeigt, dass die Form der Streifenenden und damit die lokale Krümmung der Zellfront Einfluss auf die Aktinpolymerisation hat. Diese Arbeit zeigt, dass Streifen für die quantitative Untersuchung von Zellmigration nützlich sind und dass erweiterte zelluläre Potts-Modelle mit einfachen mechanistischen Regeln die unterschiedlichen Migrationsphänotypen von Zellen in einer beengten Umgebung erfassen können.Cell migration plays a fundamental role in processes such as embryogenesis, immune response, wound healing and cancer metastasis. Hence the mechanisms of cell migration in particularly cytoskeleton dynamics, actin assembly, and reaction diffusion processes have received great interest in life science. Cells are highly complex dynamic systems that constantly alter their states, which leads to emerging morphologies and migratory modes. The resulting migration patterns are influenced by external cues, which are uncontrolled under classic culture conditions. Thus, a key challenge of studying cell migration is the design of specific methods to disentangle the effect of separate parameter regulating cellular behavior. A possible way to reduce the complexity of the environment is to confine cells to a defined external microenvironment by applying micropatterning techniques. Using these geometries, the degree of freedom of the cell motion can be reduced, which allows selectively studying specific characteristics of cell migration. Moreover, micropatterning technology can realize large-scale arrays of functional surface coatings, so that high throughput measurements can be obtained. In the first part of this work, studies on a newly discovered quasi-oscillatory migration mode of single cells on isotropic circular-micropatterns are presented. We observe persistent polarized cell shapes and directed pole-to-pole motion within the patterns. Cells depolarize at one side of the given micropattern, followed by delayed repolarization progressing towards the opposite side. We then show that several cell lines (e.g. MDCK, Huh7, MDA-MB-231 cells) exhibit the oscillatory migration mode on circular-shaped, ellipse-shaped, and stripe-shaped micropatterns respectively. Compared to circular and ellipse patterns, stripe-shaped microlanes enhance the occurrence of oscillations. Microlanes provide an ideal and simple platform for the exploration of emerging migration patterns of cells and the molecular mechanisms underlying cell cytoskeleton dynamics. In the second part of this work, we analyze cell motility by the spatial velocity distribution and frequency spectrum. The experimental data are compared to a Cellular Potts model that includes a minimal mechanistic model of the dynamical cytoskeleton. In particular, we evaluate the “reversal time” as a measure for spontaneous repolarization of cells as well as the time required to quench the leading lamellipodium at the microlane ends. Using LifeAct-GFP transfected cells and microlanes with differently shaped geometric ends, we found distinct scenarios at the leading edge showing that the tip geometry and hence the local deformation of the leading edge has an effect on actin polymerization. This work shows that microlanes are useful for quantitative assessment of cell migration and that extended Cellular Potts models with simple mechanistic rules capture the distinct migration phenotypes in confinement