A continuum mechanical model of cell motion driven by a biphasic traction stress.

The aim of this paper is to place the cell locomotion problem within the general framework of classical continuum mechanics, and while doing so, to account for the deformation of the actin network in the cytoskeleton; the myosin activity on the lamellum including its effect on depolymerization at the trailing edge; model the stress-dependent driving forces and kinetic laws controlling polymerization at the leading edge, depolymerization at the trailing edge and ATP hydrolysis consistently with the dissipation inequality; and, based on the observations in Gardel et al. (Gardel et al. 2008 J. Cell Biol. 183, 999-1005 (doi:10.1083/jcb.200810060)), include a biphasic velocity-dependent traction stress acting on the actin network. While we chose certain specific models for each of these, in part to allow for an analytical solution, the generality of the framework allows one to readily introduce different constitutive laws to describe these phenomena as might be needed, for example, to study some different type of cells. As described in §5, the predictions of the model compare well with observations such as the magnitude of the very different actin retrograde speeds in the lamellum and lamellipodium including their jump at the interface, the magnitude of the cell speed, and the relative lengths of the lamellipodium and lamellum.

Siliceous scales in the centrohelid heliozoan Raphidocystis contractilis facilitate settlement to the substratum.

The centrohelid heliozoan Raphidocystis contractilis has hundreds of small scales on the surface of the cell body. To understand the biological functions of the scales, comparative examinations were conducted between wild-type and scale-deficient strains that has naturally lost scales after long-term cultivation. The scale-deficient strain exhibited decreased adhesion to the substratum and had a lower sedimentation rate in water than the wild-type strain, suggesting that the scale may have the ability to attach quickly and strongly to the substratum. Percoll density gradient centrifugation showed that the scale-deficient strain had a lower density than that of the wild-type strain. In the wild-type strain, more scaled cells were observed in the higher specific gravity fractions. During the long-term culture of cells, only the cells suspended in the upper area of the flask were transferred to fresh medium. By repeating this procedure, we may have selected only cells that did not possess normal scales. In the natural environment, centrohelid heliozoans are easily flushed away if they cannot adhere strongly to the bottom. These results suggest that they use scales to ensure effective adhesion to the substratum.

Light-sheet microscopy reveals dorsoventral asymmetric membrane dynamics of Amoeba proteus during pressure-driven locomotion.

Amoebae are found all around the world and play an essential role in the carbon cycle in the environment. Therefore, the behavior of amoebae is a crucial factor when considering the global environment. Amoebae change their distribution through amoeboid locomotion, which are classified into several modes. In the pressure-driven mode, intracellular hydrostatic pressure generated by the contraction of cellular cortex actomyosin causes the pseudopod to extend. During amoeboid locomotion, the cellular surface exhibits dynamic deformation. Therefore, to understand the mechanism of amoeboid locomotion, it is important to characterize cellular membrane dynamics. Here, to clarify membrane dynamics during pressure-driven amoeboid locomotion, we developed a polkadot membrane staining method and performed light-sheet microscopy in Amoeba proteus, which exhibits typical pressure-driven amoeboid locomotion. It was observed that the whole cell membrane moved in the direction of movement, and the dorsal cell membrane in the posterior part of the cell moved more slowly than the other membrane. In addition, membrane complexity varied depending on the focused characteristic size of the membrane structure, and in general, the dorsal side was more complex than the ventral side. In summary, the membrane dynamics of Amoeba proteus during pressure-driven locomotion are asymmetric between the dorsal and ventral sides. This article has an associated interview with the co-first authors of the paper.

Recent advances in experimental design and data analysis to characterize prokaryotic motility.

Bacterial motility plays a key role in important cell processes such as chemotaxis and biofilm formation, but is challenging to quantify due to the small size of the individual microorganisms and the complex interplay of biological and physical factors that influence motility phenotypes. Swimming, the first type of motility described in bacteria, still remains largely unquantified. Light microscopy has enabled qualitative characterization of swimming patterns seen in different strains, such as run and tumble, run-reverse-flick, run and slow, stop and coil, and push and pull, which has allowed for elucidation of the underlying physics. However, quantifying these behaviors (e.g., identifying run distances and speeds, turn angles and behavior by surfaces or cell-cell interactions) remains a challenging task. A qualitative and quantitative understanding of bacterial motility is needed to bridge the gap between experimentation, omics analysis, and bacterial motility theory. In this review, we discuss the strengths and limitations of how phase contrast microscopy, fluorescence microscopy, and digital holographic microscopy have been used to quantify bacterial motility. Approaches to automated software analysis, including cell recognition, tracking, and track analysis, are also discussed with a view to providing a guide for experimenters to setting up the appropriate imaging and analysis system for their needs.

Radix Glycyrrhizae Preparata Induces Cell Cycle Arrest and Induced Caspase-Dependent Apoptosis in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is a highly aggressive and devastating brain tumor characterized by poor prognosis and high rates of recurrence. Despite advances in multidisciplinary treatment, GBM constinues to have a poor overall survival. The Radix Glycyrrhizae Preparata (RGP) has been reported to possess anti-allergic, neuroprotective, antioxidative, and anti-inflammatory activities. However, it not clear what effect it may have on tumorigenesis in GBM. This study demonstrated that RGP reduced glioma cell viability and attenuated glioma cell locomotion in GBM8401 and U87MG cells. RGP treated cells had significant increase in the percentage of apoptotic cells and rise in the percentage of caspase-3 activity. In addition, the results of study's cell cycle analysis also showed that RGP arrested glioma cells at G2/M phase and Cell failure pass the G2 checkpoint by RGP treatment in GBM8401 Cells. Based on the above results, it seems to imply that RGP activated DNA damage checkpoint system and cell cycle regulators and induce apoptosis in established GBM cells. In conclusion, RGP can inhibit proliferation, cell locomotion, cell cycle progression and induce apoptosis in GBM cells in vitro.

High Content Imaging of Barrett's-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions.

BACKGROUND & AIMS: Esophageal adenocarcinoma (EAC) develops from within Barrett's esophagus (BE) concomitant with gastroesophageal reflux disease (GERD). Wound healing processes and cellular transitions, such as epithelial-mesenchymal transitions, may contribute to the development of BE and the eventual migratory escape of metastatic cancer cells. Herein, we attempt to identify the genes underlying esophageal cellular transitions and their potential regulation by the low pH environments observed in GERD and commonly encountered by escaping cancer cells. METHODS: Small interfering RNA library screening and high-content imaging analysis outlined changes in BE high-grade dysplasia (HGD) and EAC cell morphologies after gene silencing. Gene expression microarray data and low pH exposures studies modeling GERD-associated pulses (pH 4.0, 10 min) and tumor microenvironments (pH 6.0, constant) were used. RESULTS: Statistical analysis of small interfering RNA screening data defined 207 genes (Z-score >2.0), in 12 distinct morphologic clusters, whose suppression significantly altered BE-HGD cell morphology. The most significant genes in this list included KIF11, RRM2, NUBP2, P66BETA, DUX1, UBE3A, ITGB8, GAS1, GPS1, and PRC1. Guided by gene expression microarray study data, both pulsatile and constant low pH exposures were observed to suppress the expression of GPS1 and RRM2 in a nonoverlapping temporal manner in both BE-HGD and EAC cells, with no changes observed in squamous esophageal cells. Functional studies uncovered that GPS1 and RRM2 contributed to amoeboid and mesenchymal cellular transitions, respectively, as characterized by differential rates of cell motility, pseudopodia formation, and altered expression of the mesenchymal markers vimentin and E-cadherin. CONCLUSIONS: Collectively, we have shown that low pH microenvironments associated with GERD, and tumor invasive edges, can modulate the expression of genes that triggered esophageal cellular transitions potentially critical to colonization and invasion.

Analogies in 3D molecular visualisations: development of a cell biology animation 'How cells move - a new interpretation of old data'.

Cell biology and imaging technology have vastly improved over the past decades, enabling scientists to dissect the inner workings of a cell. In addition to technical limits on spatial and temporal resolution, which obscure the picture at the molecular level, the sheer density and complexity of information impede clear understanding. 3D molecular visualisation has therefore blossomed as a way to translate molecular data in a more tangible form. Whilst the molecular machinery involved in cell locomotion has been extensively studied, existing narratives describing how cells generate the forces that drive movement remain unclear. Polymerisation of a protein called actin is clearly essential. The general belief in the cell migration field is that actin polymerisation's main role is to push the leading edge of the cell forwards, while the rest of the cell follows passively. The cell migration & chemotaxis group at the CRUK Beatson Institute propose an alternative hypothesis, in which actin filaments constitute cables. Motor proteins pull on these cables, causing them to behave like the treads of a tank and drive cell movement. This article describes the development of a 3D animation that uses analogical reasoning to contrast the 'tank' hypothesis for cell locomotion with the current dogma.

Viscoelasticity of single cells-from subcellular to cellular level.

This review deals with insights into complex cellular structures and processes obtained by measuring viscoelastic impedances of the cell envelope and the cytoplasm by colloidal bead microrheometry. I first introduce a mechanical cell model that allows us to understand their unique ability of mechanical self-stabilization by actin microtubule crosstalk. In the second part, I show how cell movements can be driven by pulsatile or propagating solitary actin gelatin waves (SAGW) that are generated on nascent adhesion domains by logistically controlled membrane recruitment of functional proteins by electrostatic-hydrophobic forces. The global polarization of cell migration is guided by actin-microtubule crosstalk that is mediated by the Ca++ and strain-sensitive supramolecular scaffolding protein IQGAP. In the third part, I introduce the traction force microscopy as a tool to measure the forces between somatic cells and the tissue ´Here I show, how absolute values of viscoelastic impedances of the composite cell envelope can be obtained by deformation field mapping techniques. In the fourth part, it is shown how the dynamic mechanical properties of the active viscoplastic cytoplasmic space can be evaluated using colloidal beads as phantom endosomes. Separate measurements of velocity distributions of directed and random motions of phantom endosomes, yield local values of transport forces, viscosities and life times of directed motion along microtubules. The last part deals with biomimetic experiments allowing us to quantitatively evaluate the mechanical properties of passive and active actin networks on the basis of the percolation theory of gelation.

Role of mechanical cues in shaping neuronal morphology and connectivity.

Neuronal circuits, the functional building blocks of the nervous system, assemble during development through a series of dynamic processes including the migration of neurons to their final position, the growth and navigation of axons and their synaptic connection with target cells. While the role of chemical cues in guiding neuronal migration and axonal development has been extensively analysed, the contribution of mechanical inputs, such as forces and stiffness, has received far less attention. In this article, we review the in vitro and more recent in vivo studies supporting the notion that mechanical signals are critical for multiple aspects of neuronal circuit assembly, from the emergence of axons to the formation of functional synapses. By combining live imaging approaches with tools designed to measure and manipulate the mechanical environment of neurons, the emerging field of neuromechanics will add a new paradigm in our understanding of neuronal development and potentially inspire novel regenerative therapies.

LRP5 associates with specific subsets of macrophages: Molecular and functional effects.

Innate and acquired immunity is involved in the progression of atherosclerosis. The molecular mechanisms ruling monocyte to macrophage (Mø) differentiation are not yet fully understood. Different subtypes of plaque macrophages that have differentiated from monocytes recruited from circulating blood, have been characterized based on surface epitopes. We have recently shown that LRP5, a member of the LDL receptor superfamily supporting Wnt signalling, has an important role in monocyte to macrophage differentiation. The aim of this study was to investigate whether the CD16- and CD16+ macrophage subsets found in human atherosclerotic plaques have a differential LRP5 expression/function and Wnt signalling potential. We show for the first time that LRP5 expression is significantly higher in human CD16+Mø derived from CD14(+)CD16(+) monocytes than in CD16-Mø macrophages derived from CD14(+)CD16(-) monocytes. LRP5 is not found in human healthy vessel or arterial intimal thickening but is found in advanced human atherosclerotic lesions co-localizing only with the CD16+Mø macrophage subset. LRP5 expressing macrophages infiltrate the deep layers of atherosclerotic plaques towards the intima-media boundaries showing increased migratory activity and higher phagocytic activity. The equivalent for human patrolling CD14(+)CD16(+) monocytes in mice, CD115(+)GR1(low) monocytes, also show an increased expression of LRP5. In summary, classical CD14(+)CD16(-)monocytes that differentiate into CD16-Mø do not express LRP5. Instead, human monocytes expressing LRP5 differentiate into CD16+Mø antiinflammatory macrophages. These antiinflammatory macrophages are found in advanced atherosclerotic human plaques. Thus LRP5 is a signature of the anti-inflammatory defensive phenotype of macrophages.

Allometry in Physarum plasmodium during free locomotion: size versus shape, speed and rhythm.

Physarum plasmodium is a giant unicellular organism whose length can vary by more than three orders of magnitude. Using plasmodia ranging in size from 100 µm to 10 cm, we investigated the size dependency of their thickness distributions and locomotion speeds during free locomotion. (1) In the longitudinal direction, the organism is thickest close to the front, and decreases exponentially in thickness towards the rear. The slenderness ratio varies with body size according to a power law, such that large plasmodia are long and flat, whereas small plasmodia are short and thick. (2) The mean locomotion speed is proportional to the mean maximum thickness of the frontal part. By conducting a dimensional analysis, possible physical models are discussed. (3) The intrinsic period of the thickness oscillation, which is related to shuttle streaming (period 1-2 min), increases logarithmically with body size. (4) Various characteristics exhibit size-independent, long-period (20±10 min) oscillations, including speed, shape and intrinsic thickness oscillation period. These variations are closely coupled to formation of the entire cell shape, including undulation of thickness along the longitudinal axis and timing of branching of the frontal tip. Based on these experimental results and those reported previously, we propose a simple mathematical model for cell locomotion.

How actin/myosin crosstalks guide the adhesion, locomotion and polarization of cells.

Cell-tissue-tissue interaction is determined by specific short range forces between cell adhesion molecules (CAMs) and ligands of the tissue, long range repulsion forces mediated by cell surface grafted macromolecules and adhesion-induced elastic stresses in the cell envelope. This interplay of forces triggers the rapid random clustering of tightly coupled linkers. By coupling of actin gel patches to the intracellular domains of the CAMs, these clusters can grow in a secondary process resulting in the formation of functional adhesion microdomains (ADs). The ADs can act as biochemical steering centers by recruiting and activating functional proteins, such as GTPases and associated regulating proteins, through electrostatic-hydrophobic forces with cationic lipid domains that act as attractive centers. First, I summarize physical concepts of cell adhesion revealed by studies of biomimetic systems. Then I describe the role of the adhesion domains as biochemical signaling platforms and force transmission centers promoting cellular protrusions, in terms of a shell string model of cells. Protrusion forces are generated by actin gelation triggered by molecular machines (focal adhesion kinase (FAK), Src-kinases and associated adaptors) which assemble around newly formed integrin clusters. They recruit and activate the GTPases Rac-1 and actin gelation promoters to charged membrane domains via electrostatic-hydrophobic forces. The cell front is pushed forward in a cyclic and stepwise manner and the step-width is determined by the dynamics antagonistic interplay between Rac-1 and RhoA. The global cell polarization in the direction of motion is mediated by the actin-microtubule (MT) crosstalk at adhesion domains. Supramolecular actin-MT assemblies at the front help to promote actin polymerization. At the rear they regulate the dismantling of the ADs through the Ca(++)-mediated activation of the protease calpain and trigger their disruption by RhoA mediated contraction via stress fibers. This article is part of a Special Issue entitled: Mechanobiology.

Coordination of contractility, adhesion and flow in migrating Physarum amoebae.

This work examines the relationship between spatio-temporal coordination of intracellular flow and traction stress and the speed of amoeboid locomotion of microplasmodia of Physarum polycephalum. We simultaneously perform particle image velocimetry and traction stress microscopy to measure the velocity of cytoplasmic flow and the stresses applied to the substrate by migrating Physarum microamoebae. In parallel, we develop a mathematical model of a motile cell which includes forces from the viscous cytosol, a poro-elastic, contractile cytoskeleton and adhesive interactions with the substrate. Our experiments show that flow and traction stress exhibit back-to-front-directed waves with a distinct phase difference. The model demonstrates that the direction and speed of locomotion are determined by this coordination between contraction, flow and adhesion. Using the model, we identify forms of coordination that generate model predictions consistent with experiments. We demonstrate that this coordination produces near optimal migration speed and is insensitive to heterogeneity in substrate adhesiveness. While it is generally thought that amoeboid motility is robust to changes in extracellular geometry and the nature of extracellular adhesion, our results demonstrate that coordination of adhesive forces is essential to producing robust migration.

A steering mechanism for phototaxis in Chlamydomonas.

Chlamydomonas shows both positive and negative phototaxis. It has a single eyespot near its equator, and as the cell rotates during the forward motion, the light signal received by the eyespot varies. We use a simple mechanical model of Chlamydomonas that couples the flagellar beat pattern to the light intensity at the eyespot to demonstrate a mechanism for phototactic steering that is consistent with observations. The direction of phototaxis is controlled by a parameter in our model, and the steering mechanism is robust to noise. Our model shows switching between directed phototaxis when the light is on and run-and-tumble behaviour in the dark.

Invadosomes in their natural habitat.

Podosomes and invadopodia (collectively known as invadosomes) are small, F-actin-rich protrusions that are located at points of cell-ECM contacts and endow cells with invasive capabilities. So far, they have been identified in human or murine immune (myelomonocytic), vascular and cancer cells. The overarching reason for studying invadosomes is their connection to human disease. For example, macrophages and osteoclasts lacking Wiskott-Aldrich syndrome protein (WASp) are not able to form podosomes, and this leads to altered macrophage chemotaxis and defective bone resorption by osteoclasts. In contrast, the ability of cancer cells to form invadopodia is associated with high invasive and metastatic potentials. While invadosome composition, dynamics and signaling cascades leading to their assembly can be followed easily in in vitro assays, studying their contribution to pathophysiological processes in situ remains challenging. A number of recent papers have started to address this issue and describe invadosomes in situ in mouse models of cancer, cardiovascular disease and angiogenesis. In addition, in vivo invadosome homologs have been reported in developmental model systems such as C. elegans, zebrafish and sea squirt. Comparative analyses among different invasion mechanisms as they happen in their natural habitats, i.e., in situ, may provide an outline of the invadosome evolutionary history, and guide our understanding of the roles of the invasion process in pathophysiology versus development.

3D arrays for high throughput assay of cell migration and electrotaxis.

Cell behaviour in 3D environments can be significantly different from those in 2D cultures. With many different 3D matrices being developed and many experimental modalities used to modulate cell behaviour in 3D, it is necessary to develop high throughput techniques to study behaviour in 3D. We report on a 3D array on slide and have adapted this to our electrotaxis chamber, thereby offering a novel approach to quantify cellular responses to electric fields (EFs) in 3D conditions, in different matrices, with different strains of cells, under various field strengths. These developments used Dictyostelium cells to illustrate possible applications and limitations.

Emergent complexity of the cytoskeleton: from single filaments to tissue.

Despite their overwhelming complexity, living cells display a high degree of internal mechanical and functional organization which can largely be attributed to the intracellular biopolymer scaffold, the cytoskeleton. Being a very complex system far from thermodynamic equilibrium, the cytoskeleton's ability to organize is at the same time challenging and fascinating. The extensive amounts of frequently interacting cellular building blocks and their inherent multifunctionality permits highly adaptive behavior and obstructs a purely reductionist approach. Nevertheless (and despite the field's relative novelty), the physics approach has already proved to be extremely successful in revealing very fundamental concepts of cytoskeleton organization and behavior. This review aims at introducing the physics of the cytoskeleton ranging from single biopolymer filaments to multicellular organisms. Throughout this wide range of phenomena, the focus is set on the intertwined nature of the different physical scales (levels of complexity) that give rise to numerous emergent properties by means of self-organization or self-assembly.

Studies on the gastrulation of amphibian embryos: Ultrastructure of the migrating cells of anurans.

In order to investigate the ultrastructure of the migrating cells in anuran gastrulae, three anurans, which belong to three different genera, were observed with transmission electron microscopy supported by light microscopy of the 1 µm sections and scanning electron microscopy. Fine filopodial cell processes, as well as cell processes probably flattened against the inner surface of the blastocoel wall, were formed by the migrating cells. Blebs and lobopodial cell processes were frequently observed inBufo, sometimes inXenopus, but not observed inRana. Microfilaments were observed in the cell processes. Focal close contacts, probably having adhesive properties, were made between the migrating cells and the inner surface of the blastocoel wall. These observations suggest that the cells migrate along the inner surface of the blastocoel wall by forming filopodia and pseudopodia flattened against the wall. The role of the blebs and lobopodial cell processes requires more investigation.