Modulation of the reaction rate of regulating protein induces large morphological and motional change of amoebic cell

Morphologies of moving amoebae are categorized into two types. One is the "neutrophil" type in which the long axis of cell roughly coincides with its moving direction. This type of cell extends a leading edge at the front and retracts a narrow tail at the rear, whose shape has been often drawn as a typical amoeba in textbooks. The other one is the "keratocyte" type with widespread lamellipodia along the front side arc. Short axis of cell in this type roughly coincides with its moving direction. In order to understand what kind of molecular feature causes conversion between two types of morphologies, and how two typical morphologies are maintained, a mathematical model of amoebic cells is developed. This model describes movement of cell and intracellular reactions of activator, inhibitor and actin filaments in a unified way. It is found that the producing rate of activator is a key factor of conversion between two types. This model also explains the observed data that the keratocyte type cells tend to rapidly move along a straight line. The neutrophil type cells move along a straight line when the moving velocity is small, but they show fluctuated motions deviating from a line when they move as fast as the keratocyte type cells. Efficient energy consumption in the neutrophil type cells is predicted.     

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.     

An Experimental and Computational Study of the Effect of ActA Polarity on the Speed of Listeria monocytogenes Actin-based Motility

Listeria monocytogenes is a pathogenic bacterium that moves within infected cells and spreads directly between cells by harnessing the cell's dendritic actin machinery. This motility is dependent on expression of a single bacterial surface protein, ActA, a constitutively active Arp2,3 activator, and has been widely studied as a biochemical and biophysical model system for actin-based motility. Dendritic actin network dynamics are important for cell processes including eukaryotic cell motility, cytokinesis, and endocytosis. Here we experimentally altered the degree of ActA polarity on a population of bacteria and made use of an ActA-RFP fusion to determine the relationship between ActA distribution and speed of bacterial motion. We found a positive linear relationship for both ActA intensity and polarity with speed. We explored the underlying mechanisms of this dependence with two distinctly different quantitative models: a detailed agent-based model in which each actin filament and branched network is explicitly simulated, and a three-state continuum model that describes a simplified relationship between bacterial speed and barbed-end actin populations. In silico bacterial motility required a cooperative restraining mechanism to reconstitute our observed speed-polarity relationship, suggesting that kinetic friction between actin filaments and the bacterial surface, a restraining force previously neglected in motility models, is important in determining the effect of ActA polarity on bacterial motility. The continuum model was less restrictive, requiring only a filament number-dependent restraining mechanism to reproduce our experimental observations. However, seemingly rational assumptions in the continuum model, e.g. an average propulsive force per filament, were invalidated by further analysis with the agent-based model. We found that the average contribution to motility from side-interacting filaments was actually a function of the ActA distribution. This ActA-dependence would be difficult to intuit but emerges naturally from the nanoscale interactions in the agent-based representation.     

Coherent Motions in Confluent Cell Monolayer Sheets

Cell migration plays a pivotal role in many physiologically important processes such as embryogenesis, wound-healing, immune defense, and cancer metastasis. Although much effort has been directed toward motility of individual cells, the mechanisms underpinning collective cell migration remain poorly understood. Here we develop a collective motility model that incorporates cell mechanics and persistent random motions of individual cells to study coherent migratory motions in epithelial-like monolayers. This model, in absence of any external chemical signals, is able to explain coordinate rotational motion seen in systems ranging from two adherent cells to multicellular assemblies. We show that the competition between the active persistent force and random polarization fluctuation is responsible for the robust rotation. Passive mechanical coupling between cells is necessary but active chemical signaling between cells is not. The predicted angular motions also depend on the geometrical shape of the underlying substrate: cells exhibit collective rotation on circular substrates, but display linear back-and-forth motion on long and narrow substrates.     

Coherent Motions in Confluent Cell Monolayer Sheets

Cell migration plays a pivotal role in many physiologically important processes such as embryogenesis, wound-healing, immune defense, and cancer metastasis. Although much effort has been directed toward motility of individual cells, the mechanisms underpinning collective cell migration remain poorly understood. Here we develop a collective motility model that incorporates cell mechanics and persistent random motions of individual cells to study coherent migratory motions in epithelial-like monolayers. This model, in absence of any external chemical signals, is able to explain coordinate rotational motion seen in systems ranging from two adherent cells to multicellular assemblies. We show that the competition between the active persistent force and random polarization fluctuation is responsible for the robust rotation. Passive mechanical coupling between cells is necessary but active chemical signaling between cells is not. The predicted angular motions also depend on the geometrical shape of the underlying substrate: cells exhibit collective rotation on circular substrates, but display linear back-and-forth motion on long and narrow substrates.     

Self-organized and highly ordered domain structures within swarms of Myxococcus xanthus

Coordinated group movement (swarming) is a key aspect of Myxococcus xanthus' social behavior. Here we report observation of domain structures formed by multiple cells within large three-dimensional swarming groups grown on amorphous glass substrates, using the atomic force microscope (AFM). Novel analyses revealed that 90% of the wild type swarms displayed some form of preferential cell alignment. In contrast, cells with mutations in the social and adventurous motility systems displayed a distinct lack of cell alignment. Video microscopy observations of domain features of in vivo swarming M. xanthus cells were also consistent with the AFM data. The results presented here reveal that unique domain formation within swarms of wild type cells is a biologically driven process requiring the social and adventurous motility systems and is not a statistical phenomenon or thermodynamic process arising from liquid crystal behavior.     

Quantification of Cell Edge Velocities and Traction Forces Reveals Distinct Motility Modules during Cell Spreading

Actin-based cell motility and force generation are central to immune response, tissue development, and cancer metastasis, and understanding actin cytoskeleton regulation is a major goal of cell biologists. Cell spreading is a commonly used model system for motility experiments – spreading fibroblasts exhibit stereotypic, spatially-isotropic edge dynamics during a reproducible sequence of functional phases: 1) During early spreading, cells form initial contacts with the surface. 2) The middle spreading phase exhibits rapidly increasing attachment area. 3) Late spreading is characterized by periodic contractions and stable adhesions formation. While differences in cytoskeletal regulation between phases are known, a global analysis of the spatial and temporal coordination of motility and force generation is missing. Implementing improved algorithms for analyzing edge dynamics over the entire cell periphery, we observed that a single domain of homogeneous cytoskeletal dynamics dominated each of the three phases of spreading. These domains exhibited a unique combination of biophysical and biochemical parameters – a motility module. Biophysical characterization of the motility modules revealed that the early phase was dominated by periodic, rapid membrane blebbing; the middle phase exhibited continuous protrusion with very low traction force generation; and the late phase was characterized by global periodic contractions and high force generation. Biochemically, each motility module exhibited a different distribution of the actin-related protein VASP, while inhibition of actin polymerization revealed different dependencies on barbed-end polymerization. In addition, our whole-cell analysis revealed that many cells exhibited heterogeneous combinations of motility modules in neighboring regions of the cell edge. Together, these observations support a model of motility in which regions of the cell edge exhibit one of a limited number of motility modules that, together, determine the overall motility function. Our data and algorithms are publicly available to encourage further exploration.     

Polarization and Movement of Keratocytes: A Multiscale Modelling Approach

Eukariotic cell motility is a complex phenomenon, in which the cytoskeleton and its major constituent, actin, play an essential role. Actin forms polymers of long, stiff filaments that are cross-linked into an anisotropic network inside a thin sheet-like cellular protrusion, the lamellipod. At the leading edge of this structure, polymerization of actin filaments creates the force that pushes out the membrane and leads to translocation of a motile cell. Dynamics of the actin network account for changes in cell shape, crawling motion and turning of the cell in response to external cues. Regulating the dynamics of the cytoskeleton, and playing a central role in signal transduction in the cell, are Cdc42, Rac and Rho (GTPases of the rho family, collectively known as the small G-proteins) and the actin nucleating complex, Arp2/3.In this paper, we use a multiscale modelling approach in a 2D model of a motile cell. We describe the mutual interactions of the small G-proteins, and their effects on capping and side-branching of actin filaments. We incorporate the pushing exerted by oriented actin filament ends on the cell edge, and a Rho-dependent contraction force. Combining these biochemical and mechanical aspects, we investigate the dynamics of a model epidermal fish keratocyte through in silico experiments. Our model gives insight into how, in response to some cue, a cell can polarize, form a leading edge, and move; concomitantly it explains how a keratocyte cell can maintain its shape and polarity, even after removal of the initial stimulus, and how it can change direction quickly in response to changes in its environment. We show that establishment of polarity stems from interactions of Cdc42, Rac and Rho, while maintenance and robustness of polarity is due to the rapid cytosolic diffusion of the inactive (GDI-bound) forms of the small G-proteins. Our model produces a cell shape that closely resembles the keratocytes and correct speeds for biologically reasonable parameter values. Movies of the simulations can be obtained from http://theory.bio.uu.nl/stan/keratocyte.     

Th17 cells exhibit a distinct calcium profile from Th1 and Th2 cells and have Th1-like motility and NF-AT nuclear localization

Helper T cell subsets have evolved to respond to different pathogens, and upon activation secrete distinct sets of cytokines. The discovery and identification of Th17 cells, which develop via a unique lineage from Th1 and Th2 cells, have provided new insights into aspects of immune regulation and host defense that were previously unclear. A key early signaling event upon Ag recognition is elevation of intracellular free Ca(2+), and cytokine expression can be differentially induced depending on the duration, amplitude, and pattern of Ca(2+) signaling. Th1 and Th2 cells can be distinguished by their Ca(2+) profiles, and we provide in this study the first report regarding Ca(2+) signaling in Th17 cells. Th17 cells have a distinct Ca(2+) signaling profile from Th1 and Th2 cells with intermediate sustained Ca(2+) levels and increased oscillations compared with Th2 cells. Elevated intracellular Ca(2+) has been shown to inhibit T cell motility, and we observed that Th17 cells, like Th1 cells, are less motile than Th2 cells. Analysis of NF-AT nuclear localization revealed that Th1 and Th17 cells have significantly higher levels at later time points compared with Th2 cells. Thus, these findings show that Th17 cells, in addition to their distinct cytokine response from Th1 and Th2 cells, display unique patterns of intracellular Ca(2+) signaling and Th1-like motility behavior and nuclear localization of NF-AT.     

Form and Function in Cell Motility: From Fibroblasts to Keratocytes

It is plain enough that a horse is made for running, but similar statements about motile cells are not so obvious. Here the basis for structure-function relations in cell motility is explored by application of a new computational technique that allows realistic three-dimensional simulations of cells migrating on flat substrata. With this approach, some cyber cells spontaneously display the classic irregular protrusion cycles and handmirror morphology of a crawling fibroblast, and others the steady gliding motility and crescent morphology of a fish keratocyte. The keratocyte motif is caused by optimal recycling of the cytoskeleton from the back to the front so that more of the periphery can be devoted to protrusion. These calculations are a step toward bridging the gap between the integrated mechanics and biophysics of whole cells and the microscopic molecular biology of cytoskeletal components.     

Contact dynamics during keratocyte motility

BACKGROUND: Keratocytes are specialised, rapidly moving cells that generate substantial contractile force perpendicular to their direction of locomotion. Potential roles for contractile force in cell motility include cell-body transport, regulation of adhesion, and retraction of the cell's trailing edge. RESULTS: To investigate contact dynamics, we used simultaneous confocal fluorescence and interference reflection microscopy to image keratocytes injected with fluorescent vinculin. We found that contacts formed behind the leading edge and grew beneath both the lamellipodium and the cell body. Contacts in the middle of the cell remained stationary relative to the substrate and began to disassemble as the cell body passed over them. In contrast, contacts in the lobes of the cell grew continuously and more rapidly, incorporated more vinculin, and slid inwards towards the sides of the cell body. Contact sliding often led to merging of contacts before their removal from the substrate. CONCLUSIONS: We suggest a synthesis of two existing, apparently conflicting models for keratocyte motility, in which network contraction progressively reorients actin filaments using the contacts as pivots, forming bundles that then generate lateral tension by a sliding-filament mechanism. Contact dynamics vary between the middle of the cell and the lobes. We propose that laterally opposed contractile forces first enhance contact growth and stability, but escalating force eventually pulls contacts from the substrate at the back of the cell, without interfering with the cell's forward progress.     

Quantitative proteomic profiling of the promastigotes and the intracellular amastigotes of Leishmania donovani isolates identifies novel proteins having a role in Leishmania differentiation and intracellular survival

Protozoan parasites of the genus Leishmania are important human pathogens that cycle between an extracellular promastigote stage residing in the sandflies and an intracellular amastigote stage colonizing the phagolysosomal compartment of the mammalian macrophages. Here, we used the isobaric tagging method to quantify the global proteomic differences between the promastigotes and the intracellular amastigotes of three different Leishmania donovani clones derived from the THP-1 human macrophage cell line. We identified a substantial number of differentially modulated proteins involved in nutrient acquisition and energy metabolism, cell motility and cytoskeleton, transport, cell signaling and stress response. Proteins involved in vesicular trafficking and endocytosis like the rab7 GTP binding protein, GTP-binding proteins of the Ras superfamily and developmentally regulated GTP-binding protein 1 revealed enhanced expression and also a putative dynein heavy chain protein was found to be up-regulated in the amastigotes and it probably has a role in cargo transport inside the vesicles. Significantly, in the amastigotes the expression of a protein involved in glucose transport was increased eight to fifteen-fold, whereas concentrations of several proteins associated with cell motility and cytoskeleton were reduced. Thus, the quantitative proteomic analysis of L. donovani isolates sheds light on some novel proteins that may have a role in Leishmania differentiation and intracellular survival.     

From molecular signal activation to locomotion: an integrated, multiscale analysis of cell motility on defined matrices

The adhesion, mechanics, and motility of eukaryotic cells are highly sensitive to the ligand density and stiffness of the extracellular matrix (ECM). This relationship bears profound implications for stem cell engineering, tumor invasion and metastasis. Yet, our quantitative understanding of how ECM biophysical properties, mechanotransductive signals, and assembly of contractile and adhesive structures collude to control these cell behaviors remains extremely limited. Here we present a novel multiscale model of cell migration on ECMs of defined biophysical properties that integrates local activation of biochemical signals with adhesion and force generation at the cell-ECM interface. We capture the mechanosensitivity of individual cellular components by dynamically coupling ECM properties to the activation of Rho and Rac GTPases in specific portions of the cell with actomyosin contractility, cell-ECM adhesion bond formation and rupture, and process extension and retraction. We show that our framework is capable of recreating key experimentally-observed features of the relationship between cell migration and ECM biophysical properties. In particular, our model predicts for the first time recently reported transitions from filopodial to "stick-slip" to gliding motility on ECMs of increasing stiffness, previously observed dependences of migration speed on ECM stiffness and ligand density, and high-resolution measurements of mechanosensitive protrusion dynamics during cell motility we newly obtained for this study. It also relates the biphasic dependence of cell migration speed on ECM stiffness to the tendency of the cell to polarize. By enabling the investigation of experimentally-inaccessible microscale relationships between mechanotransductive signaling, adhesion, and motility, our model offers new insight into how these factors interact with one another to produce complex migration patterns across a variety of ECM conditions.     

Glial‐cell‐mediated re‐induction of the blood‐brain barrier phenotype in brain capillary endothelial cells: A differential gel electrophoresis study

In the neurovascular unit, brain microvascular endothelial cells develop characteristic barrier features that control the molecular exchanges between the blood and the brain. These characteristics are partially or totally lost when the cells are isolated for use in in vitro blood‐brain barrier (BBB) models. Hence, the re‐induction of barrier properties is crucial for the relevance of BBB models. Although the role of astrocyte promiscuity is well established, the molecular mechanisms of re‐induction remain largely unknown. Here, we used a DIGE‐based proteomics approach to study endothelial cellular proteins showing significant quantitative variations after BBB re‐induction. We confirm that quantitative changes mainly concern proteins involved in cell structure and motility. Furthermore, we describe the possible involvement of the asymmetric dimethylarginine pathway in the BBB phenotype re‐induction process and we discuss asymmetric dimethylarginine's potential role in regulating endothelial function (in addition to its role as a by‐product of protein modification). Our results also suggest that the intracellular redox potential is lower in the in vitro brain capillary endothelial cells displaying re‐induced BBB functions than in cells with limited BBB functions.     

Type IV collagen stimulates an increase in intracellular calcium. Potential role in tumor cell motility.

Type IV collagen (Coll IV), a component of the extracellular matrix, stimulates motility in the A2058 human melanoma cell line, a response that is inhibited by pertussis toxin (PT). Fibronectin (FN)-induced chemotaxis in this cell line is not affected by PT. To understand the mechanism of cellular signaling, single cell intracellular Ca2+ responses to Coll IV and FN were studied using Fura-2 and digital imaging fluorescence microscopy. Coll IV, at a dose that stimulates motility (100 micrograms/ml, 185 nM), induces a significant rise in cytosolic free Ca2+ concentration ([Ca2+]i) within 100 s. This response is not inhibited by PT. Treatment of the cells with FN 30 micrograms/ml (70 nM), a dose that stimulates near-maximal chemotaxis, does not increase [Ca2+]i appreciably. Removal of extracellular Ca2+ fails to inhibit the Coll IV-stimulated rise in Ca2+ in all cells. Depletion of extracellular Ca2+ and pretreatment of cells with Ca2+ channel blockers only partially inhibits Coll IV-induced motility. Depletion of intracellular Ca2+ inhibits both chemotaxis and the Coll IV-induced increase in intracellular Ca2+. Coll IV does not stimulate membrane phosphoinositide hydrolysis. We conclude that Coll IV treatment induces an inositol 1,4,5-trisphosphate-independent release of intracellular Ca2+ stores which appears to play a necessary role in the chemotactic response of A2058 cells but is not mediated by a PT-sensitive G-protein. This response is not seen in cells exposed to FN, suggesting different intracellular signaling mechanisms for stimulated motility between these two extracellular matrix molecules.     

Interactions of the bacterial pathogenListeria monocytogenes with mammalian cells: Bacterial factors, cellular ligands, and signaling

Listeria monocytogenes is a food borne pathogen which has the very unique property of crossing three barriers during infection eliciting meningitis, meningo-encephalitis and abortions with a mortality rate of about 30%. Indeed, after crossing the intestinal barrier,Listeria disseminatesvia the lymph and the blood, to the brain and/or the placenta after crossing the brain-blood barrier and/or the placental barrier. During disease, this organism infects a variety of tissues and cell types in which it is mostly intracellular due to its capacity to induce its own phagocytosis into cells which are normally nonphagocytic. The strategies used byListeria to enter cells are different from those used by other well known invasive pathogens.Listeria thus appears as a fine model to study the molecular and cellular basis of bacterial invasion. In addition, not only during entry into cells but also during intra-and intercellular movement,Listeria exploits mammalian cell functions and is thus a novel tool for elucidating some unsolved fundamental aspects of cell biology, such as ligand receptor signaling and actin cytoskeleton rearrangements. In this review, the molecular and cellular basis of entry ofListeria into cells and of its intracellular motility will be discussed. Presented at the1st International Minisymposium on Cellular Microbiology: Cell Biology and Signalization in Host-Pathogen Interactions, Prague, October 6, 1997.