Biochemical perturbations of the mitotic spindle in Xenopus extracts using a diffusion-based microfluidic assay

A microfluidic device is a powerful tool to manipulate in a controlled manner at spatiotemporal scales for biological systems. Here, we describe a simple diffusion-based assay to generate and measure the effect of biochemical perturbations within the cytoplasm of cell-free extracts from Xenopus eggs. Our approach comprises a microliter reservoir and a model cytoplasm that are separated by a synthetic membrane containing sub-micrometric pores through which small molecules and recombinant proteins can diffuse. We have used this system to examine the perturbation of elements of the mitotic spindle, which is a microtubule-based bipolar structure involved in the segregation of the replicated genome to daughter cells during cell division. First, we used the small molecule inhibitor monastrol to target kinesin-5, a molecular motor that maintains the microtubule spindle bipolarity. Next, we explored the dynamics of the mitotic spindle by monitoring the exchange between unpolymerized and polymerized tubulin within microtubule fibers. These results show that a simple diffusion-based system can generate biochemical perturbations directly within a cell-free cytoplasm based on Xenopus egg extracts at the time scale of minutes. Our assay is therefore suitable for monitoring the dynamics of supramolecular assemblies within cell-free extracts in response to perturbations. This strategy opens up broad perspectives including phenotype screening or mechanistic studies of biological assembly processes and could be applied to other cell-free extracts such as those derived from mammalian or bacterial cells

Adhesion of soft objects on wet substrates

International audienceWe study the dynamics of contact of a soft object (rubber bead, soft shell, vesicles, living cells) on a wet substrate by removal of the intercalated liquid film. The profiles of the contact zone are observed by reflection interference contrast microscopy. The adhesion forces (either hydrophobic, electrostatic or specific) are measured by micropipettes, flow cells or `microkarcher' techniques. For vesicles, the adhesion induces a tension of the membrane, which relaxes by the formation of transient macroscopic pores. We study the dynamics of opening and closing of pores

Un substrat de micropiliers pour étudier la migration cellulaire

Les propriétés mécaniques des cellules jouent un rôle prépondérant dans de nombreux événements de la vie cellulaire comme le développement embryonnaire, la formation des tissus ou encore le développement des métastases. La migration cellulaire est en partie caractérisée par des interactions mécaniques. Ainsi, les forces de traction qu’exercent les cellules sur leur environnement impliquent, en parallèle, une réorganisation dynamique des processus d’adhérence et du cytosquelette interne de la cellule. Pour évaluer ces forces, un substrat a été développé, constitué d’un réseau forte densité de micro-piliers déformables sur lequel se déplacent les cellules. Cette surface est fabriquée par des méthodes de lithographie empruntées à la micro-électronique. Les piliers mesurent environ un micromètre et sont en caoutchouc, donc suffisamment déformables pour fléchir sous l’effet des forces exercées par les cellules. L’analyse au microscope des déflexions individuelles de chaque pilier a permis de quantifier en temps réel les forces locales que des cellules exercent sur leur substrat lors de leurs processus d’adhérence et de dissociation.Mechanical forces play an important role in various cellular functions, such as tumor metastasis, embryonic development or tissue formation. Cell migration involves dynamics of adhesive processes and cytoskeleton remodelling, leading to traction forces between the cells and their surrounding extracellular medium. To study these mechanical forces, a number of methods have been developed to calculate tractions at the interface between the cell and the substrate by tracking the displacements of beads or microfabricated markers embedded in continuous deformable gels. These studies have provided the first reliable estimation of the traction forces under individual migrating cells. We have developed a new force sensor made of a dense array of soft micron-size pillars microfabricated using microelectronics techniques. This approach uses elastomeric substrates that are micropatterned by using a combination of hard and soft lithography. Traction forces are determined in real time by analyzing the deflections of each micropillar with an optical microscope. Indeed, the deflection is directly proportional to the force in the linear regime of small deformations. Epithelial cells are cultured on our substrates coated with extracellular matrix protein. First, we have characterized temporal and spatial distributions of traction forces of a cellular assembly. Forces are found to depend on their relative position in the monolayer : the strongest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. Consequently, these forces are quantified and correlated with the adhesion/scattering processes of the cells

Mathematical description of bacterial traveling pulses

The Keller-Segel system has been widely proposed as a model for bacterial waves driven by chemotactic processes. Current experiments on E. coli have shown precise structure of traveling pulses. We present here an alternative mathematical description of traveling pulses at a macroscopic scale. This modeling task is complemented with numerical simulations in accordance with the experimental observations. Our model is derived from an accurate kinetic description of the mesoscopic run-and-tumble process performed by bacteria. This model can account for recent experimental observations with E. coli. Qualitative agreements include the asymmetry of the pulse and transition in the collective behaviour (clustered motion versus dispersion). In addition we can capture quantitatively the main characteristics of the pulse such as the speed and the relative size of tails. This work opens several experimental and theoretical perspectives. Coefficients at the macroscopic level are derived from considerations at the cellular scale. For instance the stiffness of the signal integration process turns out to have a strong effect on collective motion. Furthermore the bottom-up scaling allows to perform preliminary mathematical analysis and write efficient numerical schemes. This model is intended as a predictive tool for the investigation of bacterial collective motion

Mathematical description of bacterial traveling pulses

The Keller-Segel system has been widely proposed as a model for bacterial waves driven by chemotactic processes. Current experiments on {\em E. coli} have shown precise structure of traveling pulses. We present here an alternative mathematical description of traveling pulses at a macroscopic scale. This modeling task is complemented with numerical simulations in accordance with the experimental observations. Our model is derived from an accurate kinetic description of the mesoscopic run-and-tumble process performed by bacteria. This model can account for recent experimental observations with {\em E. coli}. Qualitative agreements include the asymmetry of the pulse and transition in the collective behaviour (clustered motion versus dispersion). In addition we can capture quantitatively the main characteristics of the pulse such as the speed and the relative size of tails. This work opens several experimental and theoretical perspectives. Coefficients at the macroscopic level are derived from considerations at the cellular scale. For instance the stiffness of the signal integration process turns out to have a strong effect on collective motion. Furthermore the bottom-up scaling allows to perform preliminary mathematical analysis and write efficient numerical schemes. This model is intended as a predictive tool for the investigation of bacterial collective motion

Modeling E. coli Tumbles by Rotational Diffusion. Implications for Chemotaxis

The bacterium Escherichia coli in suspension in a liquid medium swims by a succession of runs and tumbles, effectively describing a random walk. The tumbles randomize incompletely, i.e. with a directional persistence, the orientation taken by the bacterium. Here, we model these tumbles by an active rotational diffusion process characterized by a diffusion coefficient and a diffusion time. In homogeneous media, this description accounts well for the experimental reorientations. In shallow gradients of nutrients, tumbles are still described by a unique rotational diffusion coefficient. Together with an increase in the run length, these tumbles significantly contribute to the net chemotactic drift via a modulation of their duration as a function of the direction of the preceding run. Finally, we discuss the limits of this model in propagating concentration waves characterized by steep gradients. In that case, the effective rotational diffusion coefficient itself varies with the direction of the preceding run. We propose that this effect is related to the number of flagella involved in the reorientation process

Comportements collectifs de bactéries en géométrie contrôlée et sous l'effet de la centrifugation

Escherichia coli est une bactérie dotée de flagelles lui permettant de se mouvoir dans un liquide. Les trajectoires de ce mouvement sont analogues à celles d'une marche aléatoire à 3 dimensions. Capable de puiser l'énergie de son environnement pour maintenir son mouvement, elle est un paradigme active autopropulsée. Les systèmes de particules actives autopropulsées ont déjà fait l'objet de nombreuses études mais le comportement de ces systèmes soumis à un champ de force homogène est encore mal compris. Ce travail a premièrement consisté à développer un dispositif exprimental afin de pouvoir visualiser l'influence de la centrifugation sur une population de bactéries. Les résultats reproductibles obtenus sur les profils d'équilibre de sédimentation nous ont permis de proposer un modèle de sphère solide afin de décrire le comportement de ce système. Par ailleurs, dans des géométries confinées, les bactéries E. coli sont capables grâce à leur chimioactisme de se déplacer collectivement. Elles peuvent alors former des ondes de concentration. Ce travail a permis de déterminer comment de telles ondes de concentration réagissaient à des perturbations provoquées par des variations géométriques de l'environnement. L'ensemble des résultats expérimentaux obtenus peuvent être interprétés avec des arguments qualitatifs simples. Finalement nous confrontons ces résultats expérimentaux à ceux obtenus par des simulations issues de modèles cinétiques.Escherichia coli is a flagellated bacterium. It swims in liquids following trajectories that are all well described by a 3D random walk. E. coli uptakes energy from its environment in order to maintain its movement, therefore it's an example of self-propelled particle (SPP). Many systems of SPP have been studied but it's still not clear how these systems react under a homogeneous force field. In this work we have designed an exprimental setup to study the effect of centrifugation on a bacteria population. We have obtained reproducible results of sedimentation profiles at equilibrium. These results are well described in a hard sphere model framework. In confined geometries, E. coli bacteria are able to move collectively using their chemotaxis. It can lead to the formation of concentration waves. In the second part of this work, we have determined how these bacterial waves react to perturbations induced by geometrical changes of their environment. All experimental results can be interpreted with simple qualitative arguments. Finally we compare these with those obtained by experimental simulation results from kinetic models .PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Ecoulements et adhésion (rôle des microstructurations)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Efficiency of a self-aminoacylating ribozyme: Effect of the length and base-composition of its 3′ extension

Variants of a previously described small self-aminoacylating ribozyme are tested in order to uncover the potentialities of a 3′ extension responsible for the esterification. The base-composition and the length of this specific part of the ribozyme are investigated. Very short extensions can still reach the active site, reflecting the small persistence length of RNA. The yield of aminoacylation is particularly high for ribozymes with extensions made up of a poly-U, for which a maximum of efficiency is observed for a total length of about 10 nucleotides. A simple model describing the behavior of this region of the ribozyme can account for the data