Cybergenetics is a novel field of research aiming at remotely pilot cellular processes in realtime with to leverage the biotechnological potential of synthetic biology. Yet, the control of only a small number of genetic circuits has been tested so far. Here we investigate the control of multistable gene regulatory networks, which are ubiquitously found in nature and play critical roles in cell differentiation and decision-making. Using an in silico feedback control loop, we demonstrate that a bistable genetic toggle switch can be dynamically maintained near its unstable equilibrium position for extended periods of time. Importantly, we show that a direct method based on dual periodic forcing is sufficient to simultaneously maintain many cells in this undecided state. These findings pave the way for the control of more complex cell decision-making systems at both the single cell and the population levels, with vast fundamental and biotechnological applications.

Although myosin II filaments are known to exist in non-muscle cells their dynamics and organization are incompletely understood. Here, we combined structured illumination microscopy with pharmacological and genetic perturbations, to study the process of actomyosin cytoskeleton self-organization into arcs and stress fibres. A striking feature of the myosin II filament organization was their ‘registered’ alignment into stacks, spanning up to several micrometres in the direction orthogonal to the parallel actin bundles. While turnover of individual myosin II filaments was fast (characteristic half-life time 60 s) and independent of actin filament turnover, the process of stack formation lasted a longer time (in the range of several minutes) and required myosin II contractility, as well as actin filament assembly/disassembly and crosslinking (dependent on formin Fmnl3, cofilin1 and a-actinin-4). Furthermore, myosin filament stack formation involved long-range movements of individual myosin filaments towards each other suggesting the existence of attractive forces between myosin II filaments. These forces, possibly transmitted via mechanical deformations of the intervening actin filament network, may in turn remodel the actomyosin cytoskeleton and drive its self-organization.

Extracellular vesicles (EVs) released by cells and circulating in body fluids are recognized as potent vectors of intercellular self-communication. Due to their cellular origin, EVs hold promise as naturally targeted “personalized” drug delivery system insofar as they can be engineered with drugs or theranostic nanoparticles. However, technical hurdles related to their production, drug loading, purification, and characterization restrain the translation of selfderived EVs into a clinical drug delivery system. Herein, different methods are compared to generate and to purify EVs encapsulating iron oxide nanoparticles and a clinical photosensitizer drug (Foscan) as biocamouflaged agents for photodynamic therapy, magnetic resonance imaging, magnetic manipulation, and hyperthermia. Theranostic EVs are produced from drugand nanoparticle-loaded endothelial cells either by spontaneous release in complete medium, by starvation in serum-free medium or by mechanical stress in a microfluidic chip mimicking vessel shear stress, and purified by ultracentrifugation or magnetic sorting. The impact of the production and purification protocols is investigated on EV yield and size, nanoparticle and drug cargo, and finally on their therapeutic efficacy. EV production by starvation combined with purification by ultracentrifugation may be considered a reasonable trade-off between loading, yield, and purity for biogeneration of theranostic EVs.

Extracellular vesicles (EVs) released by cells and circulating in body fluids are recognized as potent vectors of intercellular self-communication. Due to their cellular origin, EVs hold promise as naturally targeted “personalized” drug delivery system insofar as they can be engineered with drugs or theranostic nanoparticles. However, technical hurdles related to their production, drug loading, purification, and characterization restrain the translation of selfderived EVs into a clinical drug delivery system. Herein, different methods are compared to generate and to purify EVs encapsulating iron oxide nanoparticles and a clinical photosensitizer drug (Foscan) as biocamouflaged agents for photodynamic therapy, magnetic resonance imaging, magnetic manipulation, and hyperthermia. Theranostic EVs are produced from drugand nanoparticle-loaded endothelial cells either by spontaneous release in complete medium, by starvation in serum-free medium or by mechanical stress in a microfluidic chip mimicking vessel shear stress, and purified by ultracentrifugation or magnetic sorting. The impact of the production and purification protocols is investigated on EV yield and size, nanoparticle and drug cargo, and finally on their therapeutic efficacy. EV production by starvation combined with purification by ultracentrifugation may be considered a reasonable trade-off between loading, yield, and purity for biogeneration of theranostic EVs.

The exploration of the phase diagram of a minimal model for barchan fields leads to the description of three distinct phases for the system: stationary, percolable, and unstable. In the stationary phase the system always reaches an out-of-equilibrium, fluctuating, stationary state, independent of its initial conditions. This state has a large and continuous range of dynamics, from dilute—where dunes do not interact—to dense, where the system exhibits both spatial structuring and collective behavior leading to the selection of a particular size for the dunes. In the percolable phase, the system presents a percolation threshold when the initial density increases. This percolation is unusual, as it happens on a continuous space for moving, interacting, finite lifetime dunes. For extreme parameters, the system exhibits a subcritical instability, where some of the dunes in the field grow without bound. We discuss the nature of the asymptotic states and their relations to well-known models of statistical physics.

Locomotion is one of the major physiological functions for most animals. Previous studies have described aging mechanisms linked to locomotor performance among different species. However, the precise dynamics of these age-related changes, and their interactions with development and senescence, are largely unknown. Here, we use the same conceptual framework to describe locomotor performances in Caenorhabditis elegans, Mus domesticus, Canis familiaris, Equus caballus, and Homo sapiens We show that locomotion is a consistent biomarker of age-related changes, with an asymmetrical pattern throughout life, regardless of the type of effort or its duration. However, there is variation (i) among species for the same mode of locomotion, (ii) within species for different modes of locomotion, and (iii) among individuals of the same species for the same mode of locomotion. Age-related patterns are modulated by genetic (such as selective breeding) as well as environmental conditions (such as temperature). However, in all cases, the intersection of the rising developmental phase and the declining senescent phase reveals neither a sharp transition nor a plateau, but a smooth transition, emphasizing a crucial moment: the age at peak performance. This transition may define a specific target for future investigations on the dynamics of such biological interactions.

not available yet.

Significant cell-to-cell heterogeneity is ubiquitously observed in isogenic cell populations. Consequently, parameters of models of intracellular processes, usually fitted to populationaveraged data, should rather be fitted to individual cells to obtain a population of models of similar but non-identical individuals. Here, we propose a quantitative modeling framework that attributes specific parameter values to single cells for a standard model of gene expression. We combine high quality single-cell measurements of the response of yeast cells to repeated hyperosmotic shocks and state-of-the-art statistical inference approaches for mixed-effects models to infer multidimensional parameter distributions describing the population, and then derive specific parameters for individual cells. The analysis of single-cell parameters shows that single-cell identity (e.g. gene expression dynamics, cell size, growth rate, mother-daughter relationships) is, at least partially, captured by the parameter values of gene expression models (e.g. rates of transcription, translation and degradation). Our approach shows how to use the rich information contained into longitudinal single-cell data to infer parameters that can faithfully represent single-cell identity.

Although many factors required for the formation of export-competent mRNPs have been described, an integrative view of the spatiotemporal coordinated cascade leading mRNPs from their site of transcription to their site of nuclear exit, at a single cell level, is still partially missing due to technological limitations. Here we report that the RNA Spinach aptamer is a powerful tool for mRNA imaging in live S. cerevisiae with high spatial-temporal resolution and no perturbation of the mRNA biogenesis properties. Dedicated image processing workflows are developed to allow detection of very low abundance of transcripts, accurate quantitative dynamic studies, as well as to provide a localization precision close to 100 nm at consistent time scales. Combining these approaches has provided a state-of-the-art analysis of the osmotic shock response in live yeast by localizing induced transcription factors, target gene loci and corresponding transcripts.

The ability to mechanically interact with the extracellular matrix is a fundamental feature of adherent eukaryotic cells. Cell–matrix adhesion in many cell types is mediated by protein complexes called focal adhesions (FAs). Recent progress in super resolution microscopy revealed FAs possess an internal organization, yet such methods do not enable observation of the formation and dynamics of their internal structure in living cells. Here, we combine structured illumination microscopy (SIM) with total internal reflection fluorescence microscopy (TIRF) to show that the proteins inside FA patches are distributed along elongated subunits, typically 300 +/- 100 nm wide, separated by 400 +/- 100 nm, and individually connected to actin cables. We further show that the formation and dynamics of these linear subunits are intimately linked to radial actin fiber formation and actomyosin contractility. We found FA growth to be the result of nucleation of new linear subunits and their coordinated elongation. Taken together, this study reveals that the basic units of mature focal adhesion are 300-nm-wide elongated, dynamic structures. We anticipate this ultrastructure to be relevant to investigation of the function of FAs and their behavior in response to mechanical stress.

Here, we describe a protocol for producing micropatterned porous membranes which can be used for combinatorial cell-based assays. We use contact printing to pattern the surface of a porous filter membrane with a thin layer of polydimethylsiloxane (PDMS). This allows the porosity of the filter membrane to be altered at selected locations. Cells can be grown on one side of the filter membrane, while drugs and reagents can be deposited on the porous areas of the other side of the membrane. The reagents can diffuse through the pores of the membrane to the cells. The first part of the protocol describes how to design a stamp and use it to contact print PDMS. The second part describes how to create microprinted membranes for cell-based assays. The method is simple, highly customizable, can be performed at the bench, and can be used to perform combinatorial or time-dependent cell-based assays.

Microfluidic platforms are ideal for generating dynamic temporal and spatial perturbations in extracellular environments. Single cells and organisms can be trapped and maintained in microfluidic platforms for long periods of time while their responses to stimuli are measured using appropriate fluorescence reporters and time-lapse microscopy. Such platforms have been used to study problems as diverse as C. elegans olfaction (1), cancer cell migration (2), and E. coli chemotaxis (3). In this paper we describe how to construct and use a microfluidic chip to study the response of single yeast cells to dynamic perturbations of their fluid environment. The method involves creation of a photoresist master mold followed by subsequent creation of a polydimethylsiloxane (PDMS) microfluidic chip for maintaining live yeast cells in a channel with two inputs for stimulating the cells. We emphasize simplicity and the methods discussed here are accessible to the average biological laboratory. We cover the basic toolbox for making microfluidic lab-on-a-chip devices, and the techniques discussed serve as a starting point for creating sophisticated microfluidic devices capable of implementing more complicated experimental protocols.

By implementing an external feedback loop one can tightly control the expression of a gene over many cell generations with quantitative accuracy. Controlling precisely the level of a protein of interest will be useful to probe quantitatively the dynamical properties of cellular processes and to drive complex, synthetically-engineered networks. In this chapter we describe a platform for real-time closed-loop control of gene expression in yeast that integrates microscopy for monitoring gene expression at the cell level, microfluidics to manipulate the cells environment and original software for automated imaging, quantification and model predictive control. By using an endogenous osmo-stress responsive promoter and playing with the osmolarity of the cells environment, we demonstrate that long-term control can indeed be achieved for both time-constant and time-varying target profiles, at the population level, and even at the single-cell level.

Recent works have demonstrated the experimental feasibility of real-time gene expression control based on deterministic controller designs. By taking control of the level of intracellular proteins, one can probe the cell dynamics with unprecedented flexibility. However, single-cell dynamics are stochastic in nature, and a control framework explicitly accounting for this variability is lacking. In this work we devise a stochastic Model Predictive Control (MPC) framework that fills this gap. Based on stochastic modelling of gene response dynamics, our approach combines a full state-feedback receding-horizon controller with a real-time estimation method that compensates for unobserved state variables. Using previously developed models of osmostress inducible gene expression in yeast, we show in silico that our stochastic control approach may outperform deterministic control design in the control of single cells. Application of the proposed framework to real experiments on yeast is envisioned.

Individual lifespans of isogenic organisms, such as Caenorhabditis elegans nematodes, fruit flies, and mice, vary greatly even under identical environmental conditions. To study the molecular mechanisms responsible for such variability, we used an assay based on the measurement of post-reproductive nematode movements stimulated by a moderate electric field. This assay allows for the separation of individual nematodes based on their speed. We show that this phenotype could be used as a biomarker for aging because it is a better predictor of lifespan than chronological age. Fast nematodes have longer lifespans, fewer protein carbonyls, higher heatshock resistance, and higher transcript levels of the daf-16 and hsf-1 genes, which code for the stress response transcription factors, than slow nematodes. High transcript levels of the genes coding for heat-shock proteins observed in slow nematodes correlate with lower heat-shock resistance, more protein carbonyls, and shorter lifespan. Taken together, our data suggests that shorter lifespan results from early-life damage accumulation that causes subsequent faster age-related deterioration.

Microorganisms often form complex multicellular assemblies such as biofilms and colonies. Understanding the interplay between assembly expansion, metabolic yield, and nutrient diffusion within a freely growing colony remains a challenge. Most available data on microorganisms are from planktonic cultures, due to the lack of experimental tools to control the growth of multicellular assemblies. Here, we propose a method to constrain the growth of yeast colonies into simple geometric shapes such as cylinders. To this end, we designed a simple, versatile culture system to control the location of nutrient delivery below a growing colony. Under such culture conditions, yeast colonies grow vertically and only at the locations where nutrients are delivered. Colonies increase in height at a steady growth rate that is inversely proportional to the cylinder radius. We show that the vertical growth rate of cylindrical colonies is not defined by the single-cell division rate, but rather by the colony metabolic yield. This contrasts with cells in liquid culture, in which the single-cell division rate is the only parameter that defines the population growth rate. This method also provides a direct, simple method to estimate the metabolic yield of a colony. Our study further demonstrates the importance of the shape of colonies on setting their expansion. We anticipate that our approach will be a starting point for elaborate studies of the population dynamics, evolution, and ecology of microbial colonies in complex landscapes.

In order to test parameters of the peculiar dynamics occurring in barchan fields, and compute statistical analysis over large numbers of dunes, we build and study an agent-based model, which includes the well-known physics of an isolated barchan, and observations of interactions between dunes. We showed in a previous study that such a model, where barchans interact through short-range sand recapture and collisions, reproduces the peculiar behaviours of real fields, namely its spatial structuring along the wind direction, and the size selection by the local density. In this paper we focus on the mechanisms that drives these features. In particular, we show that eolian remote sand transfer between dunes ensures that a dense field structures itself into a very heterogeneous pattern, which alternates dense and diluted stripes in the wind direction. In these very dense clusters of dunes, the accumulation of collisions leads to the local emergence of a new size for the dunes.

Crescent-shaped barchan dunes are highly mobile dunes which are ubiquitous on Earth and other bodies deserts. Although they have theoretically been shown to be unstable without equilibrium size when considered separately, they form large assemblies in deserts and may spatially organize in narrow corridors that extend in the wind direction. Collisions of barchans have been proposed as a mechanism to redistribute sand between dunes and prevent the formation of very large dunes. Here, we use an agent-based model with elementary rules of sand redistribution during collisions to access the full dynamics of very large barchan fields. We tune the dune field density by changing the sand load/lost ratio and follow the transition between dilute fields, where barchans barely interact, and dense fields, where dune collisions control and stabilize the dune field. In this dense regime, flocks of barchans with a small, well selected size form: the dune field self-organizes in narrow corridors of dunes, as it is observed in real dense barchan deserts.

Regulation of the cellular volume is fundamental for cell survival and function. Deviations from equilibrium trigger dedicated signaling and transcriptional responses that mediate water homeostasis and volume recovery. Cells are densely packed with proteins, and molecular crowding may play an important role in cellular processes. Indeed, increasing molecular crowding has been shown to modify the kinetics of biochemical reactions in vitro; however, the effects of molecular crowding in living cells are mostly unexplored. Here, we report that, in yeast, a sudden reduction in cellular volume, induced by severe osmotic stress, slows down the dynamics of several signaling cascades, including the stress-response pathways required for osmotic adaptation. We show that increasing osmotic compression decreases protein mobility and can eventually lead to a dramatic stalling of several unrelated signaling and cellular processes. The rate of these cellular processes decreased exponentially with protein density when approaching stalling osmotic compression. This suggests that, under compression, the cytoplasm behaves as a soft colloid undergoing a glass transition. Our results shed light on the physical mechanisms that force cells to cope with volume fluctuations to maintain an optimal protein density compatible with cellular functions.

Gene expression plays a central role in the orchestration of cellular processes. The use of inducible promoters to change the expression level of a gene from its physiological level has significantly contributed to the understanding of the functioning of regulatory networks. However, from a quantitative point of view, their use is limited to short-term, population-scale studies to average out cell-to-cell variability and gene expression noise and limit the nonpredictable effects of internal feedback loops that may antagonize the inducer action. Here, we show that, by implementing an external feedback loop, one can tightly control the expression of a gene over many cell generations with quantitative accuracy. To reach this goal, we developed a platform for real-time, closed-loop control of gene expression in yeast that integrates microscopy for monitoring gene expression at the cell level, microfluidics to manipulate the cells’ environment, and original software for automated imaging, quantification, and model predictive control. By using an endogenous osmostress responsive promoter and playing with the osmolarity of the cells environment, we show that long-term control can, indeed, be achieved for both time-constant and time-varying target profiles at the population and even the single-cell levels. Importantly, we provide evidence that real-time control can dynamically limit the effects of gene expression stochasticity. We anticipate that our method will be useful to quantitatively probe the dynamic properties of cellular processes and drive complex, synthetically engineered networks.

The model organism Caenorhabditis elegans shows two distinct locomotion patterns in laboratory situations: it swims in low viscosity liquids and it crawls on the surface of an agar gel. This provides a unique opportunity to discern the respective roles of mechanosensation (perception and proprioception) and mechanics in the regulation of locomotion and in the gait selection. Using an original device, we present what to our knowledge are new experiments where the confinement of a worm between a glass plate and a soft agar gel is controlled while recording the worm’s motion. We observed that the worm continuously varied its locomotion characteristics from free swimming to slow crawling with increasing confinement so that it was not possible to discriminate between two distinct intrinsic gaits. This unicity of the gait is also proved by the fact that wild-type worms immediately adapted their motion when the imposed confinement was changed with time. We then studied locomotory deficient mutants that also exhibited one single gait and showed that the light touch response was needed for the undulation propagation and that the ciliated sensory neurons participated in the joint selection of motion period and undulation-wave velocity. Our results reveal that the control of maximum curvature, at a sensory or mechanical level, is a key ingredient of the locomotion regulation.

The role of geometrical confinement on collective cell migration has been recognized but has not been elucidated yet. Here, we show that the geometrical properties of the environment regulate the formation of collective cell migration patterns through cell–cell interactions. Using microfabrication techniques to allow epithelial cell sheets to migrate into strips whose width was varied from one up to several cell diameters, we identified the modes of collective migration in response to geometrical constraints. We observed that a decrease in the width of the strips is accompanied by an overall increase in the speed of the migrating cell sheet. Moreover, large-scale vortices over tens of cell lengths appeared in the wide strips whereas a contraction-elongation type of motion is observed in the narrow strips. Velocity fields and traction force signatures within the cellular population revealed migration modes with alternative pulling and/or pushing mechanisms that depend on extrinsic constraints. Force transmission through intercellular contacts plays a key role in this process because the disruption of cell–cell junctions abolishes directed collective migration and passive cell–cell adhesions tend to move the cells uniformly together independent of the geometry. Altogether, these findings not only demonstrate the existence of patterns of collective cell migration depending on external constraints but also provide a mechanical explanation for how large-scale interactions through cell–cell junctions can feed back to regulate the organization of migrating tissues.

Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that lowcurvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion.

Cell migration plays a major role in many fundamental biological processes, such as morphogenesis, tumor metastasis, and wound healing. As they anchor and pull on their surroundings, adhering cells actively probe the stiffness of their environment. Current understanding is that traction forces exerted by cells arise mainly at mechanotransduction sites, called focal adhesions, whose size seems to be correlated to the force exerted by cells on their underlying substrate, at least during their initial stages. In fact, our data show by direct measurements that the buildup of traction forces is faster for larger substrate stiffness, and that the stress measured at adhesion sites depends on substrate rigidity. Our results, backed by a phenomenological model based on active gel theory, suggest that rigidity-sensing is mediated by a large-scale mechanism originating in the cytoskeleton instead of a local one. We show that large-scale mechanosensing leads to an adaptative response of cell migration to stiffness gradients. In response to a step boundary in rigidity, we observe not only that cells migrate preferentially toward stiffer substrates, but also that this response is optimal in a narrow range of rigidities. Taken together, these findings lead to unique insights into the regulation of cell response to external mechanical cues and provide evidence for a cytoskeleton-based rigidity-sensing mechanism.

In the search for new therapeutic chemicals, lab-on-a-chip systems have recently emerged as innovative and efficient tools for cell-based assays and high throughput screening. Here, we describe a novel, versatile and simple device for cell-based assays at the bench-top. We created spatial variations of porosity on the surface of a membrane filter by microcontact printing with a biocompatible polymer (PDMS). We called such systems Micro-Printed Membranes (mPM). Active compounds dispensed on the porous areas, where the membrane pores are not clogged by the polymer, can cross the membrane and reach cells growing on the opposite side. Only cells immediately below those porous areas could be stimulated by chemicals. We performed proof-of-principle experiments using Hoechst nuclear staining, calcein-AM cell viability assay and destabilization of the cytoskeleton organisation by cytochalasin B. Resulting fluorescent staining properly matched the drops positioning and no cross-contaminations were observed between adjacent tests. This well-less cell-based screening system is highly flexible by design and it enables multiple compounds to be tested on the same cell tissue. Only low sample volumes in the microlitre range are required. Moreover, chemicals can be delivered sequentially and removed at any time while cells can be monitored in real time. This allows the design of complex, sequential and combinatorial drug assays. mPMs appear as ideal systems for cell-based assays. We anticipate that this lab-on-chip device will be adapted for both manual and automated high content screening experiments.

To decipher the dynamical functioning of cellular processes, the method of choice is to observe the time response of cells subjected to well controlled perturbations in time and amplitude. Efficient methods, based on molecular biology, are available to monitor quantitatively and dynamically many cellular processes. In contrast, it is still a challenge to perturb cellular processes - such as gene expression - in a precise and controlled manner. Here, we propose a first step towards in vivo control of gene expression: in real-time, we dynamically control the activity of a yeast signaling cascade thanks to an experimental platform combining a micro-fluidic device, an epi-fluorescence microscope and software implementing control approaches.We experimentally demonstrate the feasibility of this approach, and we investigate computationally some possible improvements of our control strategy using a model of the yeast osmo-adaptation response fitted to our data.

The High Osmolarity Glycerol (HOG) MAP kinase pathway in the budding yeast Saccharomyces cerevisiae is one of the best characterized model signaling pathways. The pathway processes external signals of increased osmolarity into appropriate physiological responses within the yeast cell. Recent advances in microfluidic technology coupled with quantitative modeling and techniques from reverse systems engineering have allowed yet further insight into this already well understood pathway. These new techniques are essential for understanding the dynamical processes at play when cells process external stimulus into biological responses. They are widely applicable to other signaling pathways of interest. Here we review the recent advances brought by these approaches in the context of understanding the dynamics of the HOG pathway signaling.

Determining the in vivo kinetics of a signaling pathway is a challenging task.We can measure a property we termed pathway bandwidth to put in vivo bounds on the kinetics of the mitogen-activated protein kinase(MAPk) signaling cascade in Saccharomyces cerevisiae that responds to hyperosmotic stress [the High Osmolarity Glycerol (HOG) pathway]. Our method requires stimulating cells with square waves of oscillatory hyperosmotic input (1 M sorbitol) over a range of frequencies and measuring the activity of the HOG pathway in response to this oscillatory input. The input frequency at which the pathway’s steady-state activity drops precipitously because the stimulus is changing too rapidly for the pathway to respond faithfully is defined as the pathway bandwidth. In this chapter, we provide details of the techniques required to measure pathway bandwidth in the HOG pathway. These methods are generally useful and can be applied to signaling pathways in S. cerevisiae and other organisms whenever a rapid reporter of pathway activity is available.

The nematode C. elegans displays complex dynamical behaviors that are commonly used to identify relevant phenotypes. Although its maintenance is straightforward, sorting large populations of worms when looking for a behavioral phenotype is difficult, time consuming and hardly quantitative when done manually. Interestingly, when submitted to a moderate electric field, worms move steadily along straight trajectories. Here, we report an inexpensive method to measure worms crawling velocities and sort them within a few minutes by taking advantage of their electrotactic skills. This method allows to quantitatively measure the effect of mutations and aging on worm’s crawling velocity. We also show that worms with different locomotory phenotypes can be spatially sorted, fast worms traveling away from slow ones. Group of nematodes with comparable locomotory fitness could then be isolated for further analysis. C. elegans is a growing model for neurodegenerative diseases and using electrotaxis for self-sorting can improve the high-throughput search of therapeutic bio-molecules.

Most of tissue cells evolve in vivo in a three-dimensional (3D) microenvironment including complex topographical patterns. Cells exert contractile forces to adhere and migrate through the extra-cellular matrix (ECM). Although cell mechanics has been extensively studied on 2D-surfaces, there are too few approaches that give access to the traction forces that are exerted in 3D environments. Here, we describe an approach to measure dynamically the contractile forces exerted by fibroblasts while they spread within arrays of large flexible micropillars coated with ECM proteins. Contrary to very dense arrays of micropost, the density of the micropillars has been chosen to promote cell adhesion in between the pillars. Cells progressively impale onto the micropatterned substrate. They first adhere on the top of the pillars without applying any detectable forces. Then, they spread along the pillars sides, spanning between the elastic micropillars and applying large forces on the substrate. Interestingly, the architecture of the actin cytoskeleton and of the adhesion complexes vary over time as cells pull on the pillars. In particular, we observed less stress fibers than for cells spread on flat surfaces. However, prominent actin stress fibers are observed at cell edges surrounding the micropillars. They generate increasing contractile forces during cells spreading. Cells treated with blebbistatin, a myosin II inhibitor, relax their internal tension, as observed by the release of pillar deformations. Moreover cell spreading on pillars coated with ECM proteins only on their tops are not able to generate significant traction forces. Taken together, these findings highlight the dynamic relationship between cellular forces and acto-myosin contractility in 3D-environments, the influence of cytoskeletal network mechanics on cell shape as well as the importance of cell-ECM contact area in the generation of traction forces.

During migration, cells interact with their environment by exerting mechanical forces on it. A combination of two techniques shows that they do so in all three dimensions by a push–pull mechanism.

We present a study of polygons forming on the free surface of a water flow confined to a stationary cylinder and driven by a rotating bottom plate as described by Jansson et al. (2006). In particular, we study the case of a triangular structure, either completely wet or with a dry centre. For the dry structures, we present measurements of the surface shapes and the process of formation. We show experimental evidence that the formation can take place as a two-stage process: first the system approaches an almost stable rotationally symmetric state and from there the symmetry-breaking proceeds like a low-dimensional linear instability. We show that the circular state and the unstable manifold connecting it with the polygon solution are universal in the sense that very different initial conditions lead to the same circular state and unstable manifold. For a wet triangle we measure the surface flows by PIV and show that there are three vortices present, but that the strength of these vortices are far too weak to account for the rotation velocity of the polygon. We show that partial blocking of the surface flow destroys the polygons and reestablishes the rotational symmetry. For the rotationally symmetric state our theoretical analysis of the surface flow shows that it consists of two distinct regions: an inner, rigidly rotating centre and an outer annulus, where the surface flow is that of a point vortex with a weak secondary flow. This prediction is consistent with the experimentally determined surface flow.

Abstract: One major goal of systems biology is to understand the dynamical functioning of biological systems at the cellular level. A common approach to investigate the dynamics of a system is to observe its response to perturbations. To improve our capacity to perturb cellular processes via the expression of a given protein with a chosen temporal expression profile, we develop an experimental platform for the real time control of gene expression. In short, this platform allows for applying short osmotic stresses to yeast cells, that trigger the expression of a target gene via the activation of the HOG signal transduction pathway, and for observing in real time the cellular response. In Uhlendorf et al. (PSB’11), we describe preliminary experimental results on the control of the signal transduction pathway obtained using a simple proportional-integral controller. However, the control of the full system, including the much slower transcription and translation processes, necessitates more elaborate control methods. In this paper, we propose a model based control strategy tailored to the specificities of the biological system, notably to its perfect adaptation to osmotic stress. The practical feasibility and the robustness of the proposed approach with respect to gene expression noise is tested in silico using a simple, switched linear model of the osmostress response in yeast.

Magnetic actuated microdevices can be used to achieve several complex functions in microfluidics and microfabricated devices. For example, magnetic mixers and magnetic actuators have been proposed to help handling fluids at a small scale. Here, we present a strategy to create magnetically actuated micropillar arrays. We combined microfabrication techniques and the dispersion of magnetic aggregates embedded inside polymeric matrices to design micrometre scale magnetic features. By creating a magnetic field gradient in the vicinity of the substrate, well-defined forces were applied on these magnetic aggregates which in turn induced a deflection of the micropillars. By dispersing either spherical aggregates or magnetic nanowires into the gels, we can induce synchronized motions of a group of pillars or the movement of isolated pillars under a magnetic field gradient. When combined with microfabrication processes, this versatile tool leads to local as well as global substrate actuations within a range of dimensions that are relevant for microfluidics and biological applications.

Whereas the adhesion and migration of individual cells have been well described in terms of physical forces, the mechanics of multicellular assemblies is still poorly understood. Here, we study the behavior of epithelial cells cultured on microfabricated substrates designed to measure cell-to-substrate interactions. These substrates are covered by a dense array of flexible micropillars whose deflection enables us to measure traction forces. They are obtained by lithography and soft replica molding. The pillar deflection is measured by video microscopy and images are analyzed with home-made multiple particle tracking software. First, we have characterized the temporal and spatial distributions of traction forces of cellular assemblies of various sizes. The mechanical force balance within epithelial cell sheets shows that the forces exerted by neighboring cells strongly depend on their relative position in the monolayer: the largest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. The average traction stress rapidly decreases from its maximum value at the edge but remains much larger than the inherent noise due to the force resolution of our pillar tracking software, indicating an important mechanical activity inside epithelial cell islands. Moreover, these traction forces vary linearly with the rigidity of the substrate over about two decades, suggesting that cells exert a given amount of deformation rather than a force. Finally, we engineer micropatterned substrates supporting pillars with anisotropic stiffness. On such substrates cellular growth is aligned with respect to the stiffest direction in correlation with the magnitude of the applied traction forces.

Traction forces between adhesive cells play an important role in a number of collective cell processes. Intercellular contacts, in particular cadherin-based intercellular junctions, are the major means for force transmission within tissues. We investigated the effect of cellular tension on the formation of cadherin-cadherin contacts by spreading cells on substrates with tunable stiffness coated with N-cadherin homophilic ligands. On the most rigid substrates, cells appear well spread and present similar cadherin adhesions and cytoskeleton organization than the ones classically observed on cadherin-coated glass substrates. However, when cells are cultured on softer substrates, a change in morphology is observed: cells are less spread with a more disorganized actin network. By a quantitative analysis of the cells adhering on the cadherin coated surfaces, we show that the areas of high forces are correlated with the cadherin adhesions. The stiffer the substrates, the larger the average traction forces and the more developed the cadherin adhesions are. When cells are treated with blebbistatin to inhibit myosin II, the forces decrease and the cadherin adhesions disappear. Together, these findings are consistent with a mechano-sensitive regulation of cadherins-mediated intercellular junctions through the cellular contractile machinery.

The shape of dunes depends on the history of wind regimes and sand availability. In deserts exposed to winds from two different directions but with comparable magnitude, dunes are found to be linear ridges, which are either perpendicular or parallel to the mean wind direction, depending on the angle between the two wind directions. These dunes, respectively observed for small and large angles between winds, are called transverse and longitudinal dunes. In both cases, their large width (hundreds of meters) and evolution time scale (years) strongly limit the investigation of their dynamics and thus our understanding of such structures. Here we show that, under water, similar structures can be obtained but at much smaller space and time scales. Performing controlled experiments together with numerical simulations, we highlight the physical mechanisms at play in the formation and long-term evolution of these structures. We show in particular that, while longitudinal dunes are stable and extend in time, transverse dunes are unstable. They evolve into wavy ridges and eventually break into barchans if the sand supply is too low. This fundamental difference is understood through the study of single sand piles and bars exposed to two winds. In the case of a large angle between winds, a sand pile grows a fi nger pointing in the average wind direction and transforms into a longitudinal dune. Such an elongation does not occur for a small angle where a sand pile evolves into a barchan. These results explain the morphological differences between straight and long longitudinal dunes and sinuous transverse dunes, while giving keys to infer the wind history or pattern state of development from the observation of dune shapes in the field.

In a three-dimensional environment, cells migrate through complex topographical features. Using microstructured substrates, we investigate the role of substrate topography in cell adhesion and migration. To do so, fibroblasts are platted on chemically identical substrates composed of microfabricated pillars. When the dimensions of the pillars (i.e., the diameter, length, and spacing) are varied, migrating cells encounter alternating flat and rough surfaces that depend on the spacing between the pillars. Consequently, we show that substrate topography affects cell shape and migration by modifying cell-to-substrate interactions. Cells on micropillar substrates exhibit more elongated and branched shapes with fewer actin stress fibers compared with cells on flat surfaces. By analyzing the migration paths in various environments, we observe different mechanisms of cell migration, including a persistent type of migration, that depend on the organization of the topographical features. These responses can be attributed to a spatial reorganization of the actin cytoskeleton due to physical constraints and a preferential formation of focal adhesions on the micropillars, with an increased lifetime compared to that observed on flat surfaces. By changing myosin II activity, we show that actomyosin contractility is essential in the cellular response to micron-scale topographic signals. Finally,the analysis of cell movements at the frontier between flat and micropillar substrates shows that cell transmigration through the micropillar substrates depends on the spacing between the pillars.

Genetic and biochemical studies yield information about the component proteins and interactions involved in a cellular signaling pathway. However this parts inventory often does not immediately reveal the in vivo signal processing capabilities and function of the pathway. Signaling pathways are complex systems with dynamic behavior and a systems level approach is needed to understand the physiological roles they play within the cell. We recently used such an approach to measure the signal processing behavior of the budding yeast HOG MAP kinase pathway in response to precisely varied temporal stimuli controlled with a microfluidic device. Despite being a well-studied pathway with well-known components the signaling dynamics and biochemical parameters of this pathway were not known. Our approach allowed us to characterize the pathway’s in vivo signal processing and put bounds on all of the in vivo reaction rates. The experimental and theoretical techniques used in our study are general and can be applied to understanding other signaling pathways in a range of biological systems.

Les cascades de signalisation permettent aux organismes vivants de transformer une information extracellulaire en une suite de réactions biochimiques, conduisant in fine à une réponse biologique adaptée. Les voies de signalisations de type MAPK (Mitogen activated protein kinases) [1, 2] sont impliquées chez les eucaryotes dans de nombreux processus cellulaires, allant de la réponse aux stress (JNK, p38/HOG) au contrôle des processus de prolifération et de différenciation cellulaires (ERK/MAPK). Ces chemins de signalisation sont composés autour d'™un bloc de trois kinases successives, formant une cascade véhiculant l'information extracellulaire depuis les récepteurs transmembranaires jusqu'€™au noyau, coeur de la machinerie cellulaire [...]

Signaling pathways relay information about changes in the external environment so that cells can respond appropriately. How much information a pathway can carry depends on its bandwidth. We designed a microfluidic device to reliably change the environment of single cells over a range of frequencies. Using this device, we measured the bandwidth of the Saccharomyces cerevisiae signaling pathway that responds to high osmolarity. This prototypical pathway, the HOG pathway, is shown to act as a low-pass filter, integrating the signal when it changes rapidly and following it faithfully when it changes more slowly. We study the dependence of the pathway’s bandwidth on its architecture. We measure previously unknown bounds on all of the in vivo reaction rates acting in this pathway. We find that the two-component Ssk1 branch of this pathway is capable of fast signal integration, whereas the kinase Ste11 branch is not. Our experimental techniques can be applied to other signaling pathways, allowing the measurement of their in vivo kinetics and the quantification of their information capacity.

Competition between random genetic drift and natural selection play a central role in evolution: Whereas nonbeneficial mutations often prevail in small populations by chance, mutations that sweep through large populations typically confer a selective advantage. Here, however, we observe chance effects during range expansions that dramatically alter the gene pool even in large microbial populations. Initially well mixed populations of two fluorescently labeled strains of Escherichia coli develop well defined, sector-like regions with fractal boundaries in expanding colonies. The formation of these regions is driven by random fluctuations that originate in a thin band of pioneers at the expanding frontier. A comparison of bacterial and yeast colonies (Saccharomyces cerevisiae) suggests that this large-scale genetic sectoring is a generic phenomenon that may provide a detectable footprint of past range expansions.

According to the dictionary, a dune is “a mound or ridge of wind-blown sand”. But the shape of a dune is more than just a “mound”, even if it is often difficult to interpret the contours at first glance. Expanses of dunes are often compared to the sea. Firstly because of the multitude of crests, so similar to waves. But also because, just like waves, we sense intuitively that sand dunes are in movement, and it is their movement that determines their form. For if you look closer, the shape of dunes is not so random...But how can we describe this shape, understand it, determine its origin and dynamics? How can we bring to light the logic that links these forms to the countless grains of sand swept up by the wind, or understand what it is that gives this collective phenomenon its coherence?

We report a novel and spectacular instability of a fluid surface in a rotating system. In a flow driven by rotating the bottom plate of a partially filled, stationary cylindrical container, the shape of the free surface can spontaneously break the axial symmetry and assume the form of a polygon rotating rigidly with a speed different from that of the plate. With water, we have observed polygons with up to 6 corners. It has been known for many years that such flows are prone to symmetry breaking, but apparently the polygonal surface shapes have never been observed. The creation of rotating internal waves in a similar setup was observed for much lower rotation rates, where the free surface remains essentially flat [J. M. Lopez et al., J. Fluid Mech. 502, 99 (2004).]. We speculate that the instability is caused by the strong azimuthal shear due to the stationary walls and that it is triggered by minute wobbling of the rotating plate.

Since Marco Polo it has been known that some sand dunes have the peculiar ability to emit a loud sound with a well-defined frequency, sometimes for several minutes. The origin of this sustained sound has remained mysterious, partly because of its rarity in nature. It has been recognized that the sound is not due to the air flow around the dunes but to the motion of an avalanche, and not to an acoustic excitation of the grains but to their relative motion. By comparing singing dunes around the world and two controlled experiments, in the laboratory and the field, we prove that the frequency of the sound is the frequency of the relative motion of the sand grains. Sound is produced because moving grains synchronize their motions. The laboratory experiment shows that the dune is not needed for sound emission. A velocity threshold for sound emission is found in both experiments, and an interpretation is proposed.

Barchans are propagating crescentic dunes that form in arid regions where strong, unidirectional winds blow across a firm soil lightly covered with sand. Recently, solitary barchan dunes have been shown to be unstable towards sand flux variation either growing to form mega-dunes or shrinking and disappearing when perturbed from equilibrium. However, observations of large corridors of barchan dunes in the field suggest that these bedforms are stable over long periods of time. Since dunes’ migration rates are inversely proportional to their size, barchans of different size must collide and these collisions may be crucial in maintaining the stability of dunes in nature. Here, we first explain the unstable behavior of solitary barchans and then illustrate, using down-scaled physical experiments, the qualitative dynamics of binary collisions. A stability analysis, inspired by these experiments, suggests that collisions may indeed regulate the size of barchans migrating in a corridor by redistributing sand from large dunes to smaller ones.

Barchans are aeolian sand dunes that form where unidirectional winds blow on firm ground with limited sand supply. Although they have been analyzed for decades, the difficulty of conducting experiments in the field, due to the large lengthscale and timescale involved, is a considerable obstacle to understand their dynamics. However, crescentic patterns also appear underwater on a much smaller scale, allowing easier study. It is shown here that down-scaled solitary barchans can be created in the laboratory using the periodic and asymmetric motion of a plate underwater to simulate the effect of a unidirectional flow. Those subaqueous barchans compare very well to their aeolian relatives. Their speeds scale as the cube of the speed of the flow times the inverse of their typical dimension. Finally, the exploration of new topics, such as the influence of the directionality of the wind or that of binary collisions, illustrate that barchans can eventually adapt to any external perturbations by emitting one or several small barchans.

Aeolian sand dunes originate from wind flow and sand bed interactions. According to wind properties and sand availability, they can adopt different shapes, ranging from huge motion-less star dunes to small and mobile barchan dunes. The latter are crescentic and emerge under a unidirectional wind, with a low sand supply. Here, a 3d model for barchan based on existing 2d model is proposed. After describing the intrinsic issues of 3d modeling, we show that the deflection of particules in reptation due to the shape of the dune leads to a lateral sand flux deflection, which takes the mathematical form of a non-linear diffusive process. This simple and physically meaningful coupling method is used to understand the shape of barchan dunes.

Barchans are crescentic dunes propagating on a solid ground. They form dune fields in the shape of elongated corridors in which the size and spacing between dunes are rather well selected. We show that even very realistic models for solitary dunes do not reproduce these corridors. Instead, two instabilities take place. First, barchans receive a sand flux at their back proportional to their width while the sand escapes only from their horns. Large dunes proportionally capture more sand than they lose, while the situation is reversed for small ones: therefore, solitary dunes cannot remain in a steady state. Second, the propagation speed of dunes decreases with the size of the dune: this leads, through the collision process, to a coarsening of barchan fields. We show that these phenomena are not specific to the model, but result from general and robust mechanisms. The length scales needed for these instabilities to develop are derived and discussed. They turn out to be much smaller than the dune field length. As a conclusion, there should exist further, yet unknown, mechanisms regulating and selecting the size of dunes.

A new experiment can create small scale barchan dunes under water: some sand is put on a tray moving periodically and asymmetrically in a water tank, and barchans rapidly form.We measure basic morphological and dynamical properties of these dunes and compare them to field data. These favorable results demonstrate experimentally the relevance of the so-called ‘‘saturation length’’ for the control of the dunes physics.