Effets collectifs dans la matière vivante : des anneaux de cytokinèse aux monocouches épithéliales

The emergence of collective behavior from the interaction of individual units is not clear. In this thesis, we address this question in two different systems at different scales. At the micrometer scale during cytokinetic ring constriction, we show that acto-myosin self-organizes into rotating and static clusters in fission yeast and mammalian cells. These self-organizations arise from common interaction rules, but to serve distinct functions, transport and stress generation respectively. At 100 micrometers scale, we report correlated pulsations of cells in an epithelial monolayer. We show the key roles of substrate friction, and the tight coupling between cell area, cell height and contractility. We also present two other studies: synthetic polyamines for studying actin polymerization in vivo, and direction reversal in single cell migration during ratchetaxis. Altogether, this PhD illustrates the importance of physical phenomena in cellular dynamics.L’émergence de comportements collectifs cellulaires n’est pas bien comprise. Nous l’abordons dans deux systèmes biologiques. A l'échelle du micromètre lors de la constriction de l’anneau cytokinétique, nous montrons que des complexes d’acto-myosine s’auto-organisent sous forme d’agrégats dans la levure à fission et dans la cellule de mammifères. Ces auto-organisations découlent de règles d'interactions communes mais pour des fonctions distinctes, le transport et la génération de stress respectivement. A l'échelle de 100 micromètres, nous observons des pulsations corrélées de cellules épithéliales. Nous montrons les rôles du frottement avec la surface, et le couplage entre l’aire cellulaire, sa hauteur et sa contractilité. Nous présentons aussi deux études, des polyamines synthétiques pour étudier la polymérisation d'actine in vivo, puis l’inversion de sens dans la migration - la ratchetaxie. Cette thèse illustre l'importance des phénomènes physiques dans la dynamique cellulaire

Digital in-line holographic imaging of intracellular nanoparticles tagged with TAT peptide

As the field of nanoparticles for biological and medical applications moves on apace, their interaction with cellular membranes remains an enigma. Internalization of nanoparticles in the cell are being studied extensively to acquire knowledge on various physiological properties (i.e. size, charge, permeability and receptor density) governing their interactions with the cell. Analysis and comparisons of various kinds of endocytotic studies are being carried out using several optical techniques. The need to track the temporal efficiency of these molecular movements combined with their positional information at different time frames demands a three-dimensional imaging approach. The resolution of the three-dimensional imaging system should be capable of recording nanoparticle movements whose step size would be in the range of nanometres. In this context, this project is aimed at exploring the capability of Digital In-line holography for imaging the intracellular nanoparticles in fixed cells with a view towards developing this technology for real time temporal investigations in the near future. The experiments involved fluorescence and holographic imaging of two different cell lines with internalized functionalized nanoparticles. A customized digital in-line holography set up was assembled to acquire the holograms of nanoparticle distributions inside the cell. The holograms and fluorescence images were compared to confirm the absence of artifacts due to imaging systems. Then the holograms were reconstructed using an algorithm implemented through MATLAB. The analysis of the images clearly revealed the utility Of in-line holography for acquiring holograms of intracellular nanoparticles and in reconstructing them at different axial distances thereby apparently predicting their axial and spatial distributions. Hence, it is perceived that this project can be further improvised to extract the valuable temporal details at molecular level by applying it in real time imaging. These investigations would lead to a better understanding of transport phenomena across membranes and can be employed in the process of designing and delivering drugs to the targeted domain at the desired instant of time.Master of Science (Biomedical Engineering

Interplay between cell height variations and planar pulsations in epithelial monolayers

International audienceBiological tissues change their shapes through collective interactions of cells. This coordination sets length and time scales for dynamics where precision is essential, in particular during morphogenetic events. However, how these scales emerge remains unclear. Here, we address this question using the pulsatile domains observed in confluent epithelial MDCK monolayers where cells exhibit synchronous contraction and extension cycles of [Formula: see text] h duration and [Formula: see text] length scale. We report that the monolayer thickness changes gradually in space and time by more than twofold in order to counterbalance the contraction and extension of the incompressible cytoplasm. We recapitulate these pulsatile dynamics using a continuum model and show that incorporation of cell stiffness dependent height variations is critical both for generating temporal pulsations and establishing the domain size. We propose that this feedback between height and mechanics could be important in coordinating the length scales of tissue dynamics

A New Test/Diagnosis/Rework Model for Use in Technical Cost Modeling of Electronic Systems Assembly

This paper presents a test/diagnosis/rework analysis model for use in technical cost modeling of electronic assemblies. The approach includes a model of test operations characterized by fault coverage, false positives, and defects introduced in test, in addition to rework and diagnosis operations that have variable success rates and their own defect introduction mechanisms. The model can accommodate an arbitrary number of rework attempts on any given assembly and can be used to optimize the fault coverage and rework investment during system tradeoff analyses. The model’s implementation allows all inputs to the model to be represented as probability distributions thereby accommodating inevitable uncertainties in input data present during tradeoff activities and uses Monte Carlo methods to determine model outputs

Synthetic polyamines: new compounds specific to actin dynamics for mammalian cell and fission yeast.

International audienceActin is a major actor in the determination of cell shape. On the one hand, site-directed assembly/disassembly cycles of actin filaments drive protrusive force leading to lamellipodia and filopodia dynamics. Force produced by actin similarly contributes in membrane scission in endocytosis or Golgi remodeling. On the other hand, cellular processes like adhesion, immune synapse, cortex dynamics or cytokinesis are achieved by combining acto-myosin contractility and actin assembly in a complex and not fully understood manner. New chemical compounds are therefore needed to disentangle acto-myosin and actin dynamics. We have found that synthetic, cell permeant, short polyamines are promising new actin regulators in this context. They generate growth and stabilization of lamellipodia within minutes by slowing down the actin assembly/disassembly cycle and facilitating nucleation. We now report that these polyamines also slow down cytokinetic ring closure in fission yeast. This shows that these synthetic compounds are active also in yeasts, and these experiments specifically highlight that actin depolymerization is involved in the ring closure. Thus, synthetic polyamines appear to be potentially powerful agents in a quantitative approach to the role of actin in complex processes in cell biology, developmental biology and potentially cancer research

Pulsations and flows in tissues as two collective dynamics with simple cellular rules

International audienceCollective motions of epithelial cells in vivo are essential for morphogenesis in developmental biology. Tissues elongate, contract, flow, and oscillate, thus sculpting embryos. These tissue level dynamics are known, but the physical mechanisms at the cellular level are unclear, with various behaviors depending on the tissues and species. Moreover, investigations on in vitro tissue behavior usually focus on only one type of cell dynamics and use diverse theoretical approaches, making systematic comparisons between studies challenging. Here, we show that a single epithelial monolayer of Madin Darby Canine Kidney (MDCK) cells can exhibit two types of local tissue kinematics, pulsations and long range coherent flows. We analyzed these distinct motions by using quantitative live imaging. We also report that these motions can be controlled with internal and external cues such as specific inhibitors, and friction modulation of the substrate by microcontact printing method. We further demonstrate with a unified vertex model that both behaviors depend on the competition between velocity alignment and random diffusion of cell polarization. When alignment and diffusion are comparable, a pulsatile flow emerges, whereas the tissue undergoes long-range flows when velocity alignment dominates. We propose that environmental friction, acto-myosin distributions, and cell polarization kinetics are important in regulating the dynamics of tissue morphogenesis

Mechanics of cell integration in vivo

During embryonic development, regeneration and homeostasis, cells have to physically integrate into their target tissues, where they ultimately execute their function. Despite a significant body of research on how mechanical forces instruct cellular behaviors within the plane of an epithelium, very little is known about the mechanical interplay at the interface between migrating cells and their surrounding tissue, which has its own dynamics, architecture and identity. Here, using quantitative in vivo imaging and molecular perturbations, together with a theoretical model, we reveal that multiciliated cell (MCC) precursors in the Xenopus embryo form dynamic filopodia that pull at the vertices of the overlying epithelial sheet to probe their stiffness and identify the preferred positions for their integration into the tissue. Moreover, we report a novel function for a structural component of vertices, the lipolysis-stimulated lipoprotein receptor (LSR), in filopodia dynamics and show its critical role in cell intercalation. Remarkably, we find that pulling forces equip the MCCs to remodel the epithelial junctions of the neighboring tissue, enabling them to generate a permissive environment for their integration. Our findings reveal the intricate physical crosstalk at the cell-tissue interface and uncover previously unknown functions for mechanical forces in orchestrating cell integration

Cell motion as a stochastic process controlled by focal contacts dynamics

Paru sous le titre publié :Collective Dynamics of Focal Adhesions Regulate Direction of Cell MotionInternational audienceDirected cell motion is essential in physiological and pathological processes such as morphogenesis, wound healing, and cancer spreading. Chemotaxis has often been proposed as the driving mechanism, even though evidence of long-range gradients is often lacking in vivo. By patterning adhesive regions in space, we control cell shape and the potential to move along one direction in another migration mode coined ratchetaxis. We report that focal contact distributions collectively dictate cell directionality, and bias is non-linearly increased by gap distance between adhesive regions. Focal contact dynamics on micro-patterns allow to integrate these phenomena in a model where each focal contact is translated into a force with known amplitude and direction, leading to quantitative predictions for cell motion in new conditions with their successful experimental tests. Altogether, our study shows how local and minute timescale dynamics of focal adhesions and their distribution lead to long-term cellular motion with simple geometric rules

Spatial Fluctuations at Vertices of Epithelial Layers: Quantification of Regulation by Rho Pathway

In living matter, shape fluctuations induced by acto-myosin are usually studied in vitro via reconstituted gels, whose properties are controlled by changing the concentrations of actin, myosin, and cross-linkers. Such an approach deliberately avoids consideration of the complexity of biochemical signaling inherent to living systems. Acto-myosin activity inside living cells is mainly regulated by the Rho signaling pathway which is composed of multiple layers of coupled activators and inhibitors. Here, we investigate how such a pathway controls the dynamics of confluent epithelial tissues by tracking the displacements of the junction points between cells. Using a phenomenological model to analyze the vertex fluctuations, we rationalize the effects of different Rho signaling targets on the emergent tissue activity by quantifying the effective diffusion coefficient, and the persistence time and length of the fluctuations. Our results reveal an unanticipated correlation between layers of activation/inhibition and spatial fluctuations within tissues. Overall, this work connects regulation via biochemical signaling with mesoscopic spatial fluctuations, with potential application to the study of structural rearrangements in epithelial tissues