Movement Of Individual Cells Research Articles

The effect of sampling rate on observed statistics in a correlated random walk

Tracking the movement of individual cells or animals can provide important information about their motile behaviour, with key examples including migrating birds, foraging mammals and bacterial chemotaxis. In many experimental protocols, observations are recorded with a fixed sampling interval and the continuous underlying motion is approximated as a series of discrete steps. The size of the sampling interval significantly affects the tracking measurements, the statistics computed from observed trajectories, and the inferences drawn. Despite the widespread use of tracking data to investigate motile behaviour, many open questions remain about these effects. We use a correlated random walk model to study the variation with sampling interval of two key quantities of interest: apparent speed and angle change. Two variants of the model are considered, in which reorientations occur instantaneously and with a stationary pause, respectively. We employ stochastic simulations to study the effect of sampling on the distributions of apparent speeds and angle changes, and present novel mathematical analysis in the case of rapid sampling. Our investigation elucidates the complex nature of sampling effects for sampling intervals ranging over many orders of magnitude. Results show that inclusion of a stationary phase significantly alters the observed distributions of both quantities.

Cell coordination of collective migration by Rab11 and Moesin

Cell migration is an important process involved in developmental events and in pathologies such as cancer. Cell migration can be classified into two types: individual and collective cell movements. Compared with individual migration, collective cell migration is less understood and has drawn increasing attention lately because of its emerging role in cancer spreading. We have recently established that Rab11 is absolutely required for spatial control of Rac1 activity through the control of cell-cell communication during collective movements (Ramel, et al. 2013). Moreover, we demonstrated that Rab11 acts through the control of Moesin activity. Here, we discuss how Rab11 and Moesin could cooperate to transfer forces from cell to cell in order to insure coordinated collective cell migration.

Self-organization of bacterial biofilms is facilitated by extracellular DNA

Twitching motility-mediated biofilm expansion is a complex, multicellular behavior that enables the active colonization of surfaces by many species of bacteria. In this study we have explored the emergence of intricate network patterns of interconnected trails that form in actively expanding biofilms of Pseudomonas aeruginosa. We have used high-resolution, phase-contrast time-lapse microscopy and developed sophisticated computer vision algorithms to track and analyze individual cell movements during expansion of P. aeruginosa biofilms. We have also used atomic force microscopy to examine the topography of the substrate underneath the expanding biofilm. Our analyses reveal that at the leading edge of the biofilm, highly coherent groups of bacteria migrate across the surface of the semisolid media and in doing so create furrows along which following cells preferentially migrate. This leads to the emergence of a network of trails that guide mass transit toward the leading edges of the biofilm. We have also determined that extracellular DNA (eDNA) facilitates efficient traffic flow throughout the furrow network by maintaining coherent cell alignments, thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating front. Our analyses reveal that eDNA also coordinates the movements of cells in the leading edge vanguard rafts and is required for the assembly of cells into the "bulldozer" aggregates that forge the interconnecting furrows. Our observations have revealed that large-scale self-organization of cells in actively expanding biofilms of P. aeruginosa occurs through construction of an intricate network of furrows that is facilitated by eDNA.

Abstract 1478: The role of ERBIN in suppressing migration in colon cancer.

Abstract The LAP (leucine-rich repeat (LRR) and PDZ domain) family protein, ERBIN, is basolaterally localized protein that has previously been identified as a regulator of epithelial cell polarity and an ERBB2/HER2 binding protein. In addition, ERBIN inhibits RAF activation by disrupting by the RAS/RAF interaction. However, the functional importance of ERBIN in cancer remains elusive. In this study, we examined the role of ERBIN in regulating colon cancer progression and metastasis. Stable colon cancer cell lines, including SW480, LIM2405, and Caco2, were generated using lentiviral shRNA to deplete endogenous ERBIN expression. Our results showed that knockdown of ERBIN induced epithelial-mesenchymal transition (EMT)-like phenotype as marked by spindle-like morphology, a decrease in E-cadherin and an increase in vimentin expression. Moreover, the overall rate of cell migration was significantly increased in ERBIN knockdown cells as determined using Transwell assays. When tracking the movement of individual cells using live cell imaging, we found that ERBIN knockdown cells were considerably more motile with faster migrating speed when compared to control cells. Mechanistically, knockdown of ERBIN expression resulted in an increase in both Akt and ERK signaling activation. Taken together, we have identified a novel role of ERBIN in suppressing EMT and cell motility in colon cancer cells. Citation Format: Payton D. Stevens, Mike Yang, Tianyan Gao. The role of ERBIN in suppressing migration in colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1478. doi:10.1158/1538-7445.AM2013-1478

Misshapen decreases integrin levels to promote epithelial motility and planar polarity in Drosophila

Complex organ shapes arise from the coordinate actions of individual cells. The Drosophila egg chamber is an organ-like structure that lengthens along its anterior-posterior axis as it grows. This morphogenesis depends on an unusual form of planar polarity in the organ's outer epithelial layer, the follicle cells. Interestingly, this epithelium also undergoes a directed migration that causes the egg chamber to rotate around its anterior-posterior axis. However, the functional relationship between planar polarity and migration in this tissue is unknown. We have previously reported that mutations in the Misshapen kinase disrupt follicle cell planar polarity. Here we show that Misshapen's primary role in this system is to promote individual cell motility. Misshapen decreases integrin levels at the basal surface, which may facilitate detachment of each cell's trailing edge. These data provide mechanistic insight into Misshapen's conserved role in cell migration and suggest that follicle cell planar polarity may be an emergent property of individual cell migratory behaviors within the epithelium.

Semi-circular microgrooves to observe active movements of individual Navicula pavillardii cells

We performed a trajectory analysis of movements of Navicula pavillardii diatom cells that were confined to semi-circular microgrooves with several different curvature radii. Using the semi-circular micropattern, we succeeded in observing change of velocity of the same cell before and after the stimulation by N,N-dimethyl-p-toluidine (DMT). Because the looped grooves had longer contour length than straight grooves, it was effective to achieve the long term observation of the stimulated active cells. Although average velocity of 150 cells was significantly increased with DMT, the maximum velocity (19μm/s) of the cells was not increased after the DMT injection. This may suggest that existence of the mechanical limit of the velocity of the diatom cells. Secondly, trajectories of individual cell movements along the walls of the semi-circular microgrooves were analyzed in detail. As a result, the velocity of the cells was not affected by the curvature radii of the grooves although the trajectories indicated an obvious restriction of area of the cell motion. This suggests that the surface of the diatom is effective in minimizing the frictional force between the cell body and the wall of a groove. Finally, a simple model of cell motion in the semi-circular groove was proposed to clarify the relationships among the forces that determine cell movement.

Regulated tissue fluidity steers zebrafish body elongation

The tailbud is the posterior leading edge of the growing vertebrate embryo and consists of motile progenitors of the axial skeleton, musculature and spinal cord. We measure the 3D cell flow field of the zebrafish tailbud and identify changes in tissue fluidity revealed by reductions in the coherence of cell motion without alteration of cell velocities. We find a directed posterior flow wherein the polarization between individual cell motion is high, reflecting ordered collective migration. At the posterior tip of the tailbud, this flow makes sharp bilateral turns facilitated by extensive cell mixing due to increased directional variability of individual cell motions. Inhibition of Wnt or Fgf signaling or cadherin 2 function reduces the coherence of the flow but has different consequences for trunk and tail extension. Modeling and additional data analyses suggest that the balance between the coherence and rate of cell flow determines whether body elongation is linear or whether congestion forms within the flow and the body axis becomes contorted.

The cell biology of peritrichous flagella in Bacillus subtilis

Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labelling flagellar basal bodies, hooks and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by SwrA. Basal bodies are assembled rapidly (< 5 min) but the assembly of flagella capable of supporting motility is rate limited by filament polymerization (> 40 min). We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid-like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologues to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella and motility of individual cells than the motile behaviour of populations.

Silk Film Topography Directs Collective Epithelial Cell Migration

The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization.

Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos

A quantitative understanding of tissue morphogenesis requires description of the movements of individual cells in space and over time. In transparent embryos, such as C. elegans, fluorescently labeled nuclei can be imaged in three-dimensional time-lapse (4D) movies and automatically tracked through early cleavage divisions up to ~350 nuclei. A similar analysis of later stages of C. elegans development has been challenging owing to the increased error rates of automated tracking of large numbers of densely packed nuclei. We present Nucleitracker4D, a freely available software solution for tracking nuclei in complex embryos that integrates automated tracking of nuclei in local searches with manual curation. Using these methods, we have been able to track >99% of all nuclei generated in the C. elegans embryo. Our analysis reveals that ventral enclosure of the epidermis is accompanied by complex coordinated migration of the neuronal substrate. We can efficiently track large numbers of migrating nuclei in 4D movies of zebrafish cardiac morphogenesis, suggesting that this approach is generally useful in situations in which the number, packing or dynamics of nuclei present challenges for automated tracking.

Cell migration.

Cell migration is fundamental to establishing and maintaining the proper organization of multicellular organisms. Morphogenesis can be viewed as a consequence, in part, of cell locomotion, from large-scale migrations of epithelial sheets during gastrulation, to the movement of individual cells during development of the nervous system. In an adult organism, cell migration is essential for proper immune response, wound repair, and tissue homeostasis, while aberrant cell migration is found in various pathologies. Indeed, as our knowledge of migration increases, we can look forward to, for example, abating the spread of highly malignant cancer cells, retarding the invasion of white cells in the inflammatory process, or enhancing the healing of wounds. This article is organized in two main sections. The first section is devoted to the single-cell migrating in isolation such as occurs when leukocytes migrate during the immune response or when fibroblasts squeeze through connective tissue. The second section is devoted to cells collectively migrating as part of multicellular clusters or sheets. This second type of migration is prevalent in development, wound healing, and in some forms of cancer metastasis.

Molecular crowding of collagen: A pathway to produce highly-organized collagenous structures

Collagen in vertebrate animals is often arranged in alternating lamellae or in bundles of aligned fibrils which are designed to withstand in vivo mechanical loads. The formation of these organized structures is thought to result from a complex, large-area integration of individual cell motion and locally-controlled synthesis of fibrillar arrays via cell-surface fibripositors (direct matrix printing). The difficulty of reproducing such a process in vitro has prevented tissue engineers from constructing clinically useful load-bearing connective tissue directly from collagen. However, we and others have taken the view that long-range organizational information is potentially encoded into the structure of the collagen molecule itself, allowing the control of fibril organization to extend far from cell (or bounding) surfaces. We here demonstrate a simple, fast, cell-free method capable of producing highly-organized, anistropic collagen fibrillar lamellae de novo which persist over relatively long-distances (tens to hundreds of microns). Our approach to nanoscale organizational control takes advantage of the intrinsic physiochemical properties of collagen molecules by inducing collagen association through molecular crowding and geometric confinement. To mimic biological tissues which comprise planar, aligned collagen lamellae (e.g. cornea, lamellar bone or annulus fibrosus), type I collagen was confined to a thin, planar geometry, concentrated through molecular crowding and polymerized. The resulting fibrillar lamellae show a striking resemblance to native load-bearing lamellae in that the fibrils are small, generally aligned in the plane of the confining space and change direction en masse throughout the thickness of the construct. The process of organizational control is consistent with embryonic development where the bounded planar cell sheets produced by fibroblasts suggest a similar confinement/concentration strategy. Such a simple approach to nanoscale organizational control of structure not only makes de novo tissue engineering a possibility, but also suggests a clearer pathway to organization for fibroblasts than direct matrix printing.

Motility behavior of rpoS-deficient Escherichia coli analyzed by individual cell tracking

Motility is one of the most extensively studied cellular events conducted by bacteria, including Escherichia coli. A motility agar plate assay showed that deletion of the rpoS gene enhanced the apparent motility of the E. coli BW25113 strain, which inherently had negligible motility compared to wild-type E. coli strains, such as MG1655, with no effect on cell growth. This enhancement of motility was accompanied by drastic up-regulation of genes involved in the formation and rotation of flagella. Furthermore, an individual cell motility assay showed that the population of ΔrpoS cells had bimodal motility character, and that a minority of this population exhibited a much higher motility rate. These results support a view that a minority population contributes to increasing in apparent motility of the whole population of ΔrpoS cells.

Toxicity study of deflazacort on morphological development in zebrafish embryos

Deflazacort (DFZ) is a synthetic oxazoline derivative of prednisolone which comes under the classification of glucocorticoid. In the present study, toxicity of deflazacort on embryo development stage was evaluated using zebrafish embryos, in order to decrease the side effect and causalities. Zebrafish offer practical advantages as powerful in vivo model organisms for toxicity study. They develop rapidly and are optically transparent, making it convenient to observe the movement and fate of individual cells during embryonic development stages. Zebrafish embryos were exposed to DFZ from 1 to 144 h post fertilization to evaluate the developmental toxicity of DFZ. The results showed that DFZ with 2.5-μM concentration displays tail deformation with edema of heart muscles: tail deformation is related to spinal cord deformation. This shows that DFZ may have embryotoxic effect and it remains to explain the species-specific effects which draws conclusion regarding human risk assessments.

Importance of observation interval in two-dimensional video analysis of individual diatom cells

The effect of the observation interval on two-dimensional trajectory analysis of motility of individual diatom cells of Navicula sp. was studied by comparing thinned-out observation data. The trajectory of cell movement was visualized accurately even after thinning the data interval. However, the analysis of velocity fluctuations of individual cells was found to be significantly affected by the data interval. Reproducibility of the results was guaranteed by analyzing many independent cells. In addition, comparison between automatic and manual determination of cell positions proved that automatic determination was a reliable process. Our data indicated that two-dimensional trajectory analysis using a computer can be a powerful technique to study diatom locomotion.

Classifying general nonlinear force laws in cell-based models via the continuum limit

Although discrete cell-based frameworks are now commonly used to simulate a whole range of biological phenomena, it is typically not obvious how the numerous different types of model are related to one another, nor which one is most appropriate in a given context. Here we demonstrate how individual cell movement on the discrete scale modeled using nonlinear force laws can be described by nonlinear diffusion coefficients on the continuum scale. A general relationship between nonlinear force laws and their respective diffusion coefficients is derived in one spatial dimension and, subsequently, a range of particular examples is considered. For each case excellent agreement is observed between numerical solutions of the discrete and corresponding continuum models. Three case studies are considered in which we demonstrate how the derived nonlinear diffusion coefficients can be used to (a) relate different discrete models of cell behavior; (b) derive discrete, intercell force laws from previously posed diffusion coefficients, and (c) describe aggregative behavior in discrete simulations.

Collective motion of cells mediates segregation and pattern formation in co-cultures.

Pattern formation by segregation of cell types is an important process during embryonic development. We show that an experimentally yet unexplored mechanism based on collective motility of segregating cells enhances the effects of known pattern formation mechanisms such as differential adhesion, mechanochemical interactions or cell migration directed by morphogens. To study in vitro cell segregation we use time-lapse videomicroscopy and quantitative analysis of the main features of the motion of individual cells or groups. Our observations have been extensive, typically involving the investigation of the development of patterns containing up to 200,000 cells. By either comparing keratocyte types with different collective motility characteristics or increasing cells' directional persistence by the inhibition of Rac1 GTP-ase we demonstrate that enhanced collective cell motility results in faster cell segregation leading to the formation of more extensive patterns. The growth of the characteristic scale of patterns generally follows an algebraic scaling law with exponent values up to 0.74 in the presence of collective motion, compared to significantly smaller exponents in case of diffusive motion.

Multi-Cellular Logistics of Collective Cell Migration

During development, the formation of biological networks (such as organs and neuronal networks) is controlled by multicellular transportation phenomena based on cell migration. In multi-cellular systems, cellular locomotion is restricted by physical interactions with other cells in a crowded space, similar to passengers pushing others out of their way on a packed train. The motion of individual cells is intrinsically stochastic and may be viewed as a type of random walk. However, this walk takes place in a noisy environment because the cell interacts with its randomly moving neighbors. Despite this randomness and complexity, development is highly orchestrated and precisely regulated, following genetic (and even epigenetic) blueprints. Although individual cell migration has long been studied, the manner in which stochasticity affects multi-cellular transportation within the precisely controlled process of development remains largely unknown. To explore the general principles underlying multicellular migration, we focus on the migration of neural crest cells, which migrate collectively and form streams. We introduce a mechanical model of multi-cellular migration. Simulations based on the model show that the migration mode depends on the relative strengths of the noise from migratory and non-migratory cells. Strong noise from migratory cells and weak noise from surrounding cells causes “collective migration,” whereas strong noise from non-migratory cells causes “dispersive migration.” Moreover, our theoretical analyses reveal that migratory cells attract each other over long distances, even without direct mechanical contacts. This effective interaction depends on the stochasticity of the migratory and non-migratory cells. On the basis of these findings, we propose that stochastic behavior at the single-cell level works effectively and precisely to achieve collective migration in multi-cellular systems.

The Signaling Adaptor Eps8 Is an Essential Actin Capping Protein for Dendritic Cell Migration

Dendritic cells (DCs) flexibly adapt to different microenvironments by using diverse migration strategies that are ultimately dependent on the dynamics and structural organization of the actin cytoskeleton. Here, we have shown that DCs require the actin capping activity of the signaling adaptor Eps8 to polarize and to form elongated migratory protrusions. DCs from Eps8-deficient mice are impaired in directional and chemotactic migration in 3D invitro and are delayed in reaching the draining lymph node (DLN) invivo after inflammatory challenge. Hence, Eps8-deficient mice are unable to mount a contact hypersensitivity response. We have also shown that the DC migratory defect is cell autonomous and that Eps8 is required for the proper architectural organization of the actin meshwork and dynamics of cell protrusions. Yet, Eps8 is not necessary for antigen uptake, processing, and presentation. Thus, we have identified Eps8 as a unique actin capping protein specifically required for DC migration.

A contractile actomyosin network linked to adherens junctions by Canoe/afadin helps drive convergent extension.

Integrating individual cell movements to create tissue-level shape change is essential to building an animal. We explored mechanisms of adherens junction (AJ):cytoskeleton linkage and roles of the linkage regulator Canoe/afadin during Drosophila germband extension (GBE), a convergent-extension process elongating the body axis. We found surprising parallels between GBE and a quite different morphogenetic movement, mesoderm apical constriction. Germband cells have an apical actomyosin network undergoing cyclical contractions. These coincide with a novel cell shape change--cell extension along the anterior-posterior (AP) axis. In Canoe's absence, GBE is disrupted. The apical actomyosin network detaches from AJs at AP cell borders, reducing coordination of actomyosin contractility and cell shape change. Normal GBE requires planar polarization of AJs and the cytoskeleton. Canoe loss subtly enhances AJ planar polarity and dramatically increases planar polarity of the apical polarity proteins Bazooka/Par3 and atypical protein kinase C. Changes in Bazooka localization parallel retraction of the actomyosin network. Globally reducing AJ function does not mimic Canoe loss, but many effects are replicated by global actin disruption. Strong dose-sensitive genetic interactions between canoe and bazooka are consistent with them affecting a common process. We propose a model in which an actomyosin network linked at AP AJs by Canoe and coupled to apical polarity proteins regulates convergent extension.