32 Publications (Articles, Patents, Book chapters...)
[32] J. Uhlendorf, A. Miermont, T. Delaveau, G. Charvin, F. Fages, S. Bottani,
G. Batt*, P. Hersen* (* denotes corresponding authors)., Long-term model predictive control of gene expression at the population and single-cell levels; PNAS, , (2012).
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.
[31] F. Lebois, P. Sauvage, C. Py, O. Cardoso, B. Ladoux, P. Hersen, J-M. Di Meglio, The C. elegans gait is continuously variable and determined by ambient mechanical stress; Biophysical Journal, 102, 2791-98 (2012).
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.
[30] S.R.K. Vedula, M.C. Leong, T.L. Laic, P. Hersen, A.J. Kabla, C.T. Lim, B. Ladoux., Emerging modes of collective cell migration induced by geometrical constraints; PNAS, 109 (32), 12974-12979 (2012).
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.
[29] E. Anon, X. Serra-Picamal, P. Hersen, N.C. Gauthier, M.P. Sheetz, X. Trepat, B. Ladoux, Cell crawling mediates collective cell migration to close undamaged epithelial gaps; PNAS, 109 (27), 10891-10896 (2012).
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.
[28] L. Trichet, J. Le Digabel, R. Hawkins, RK Vedula, M Gupta, C. Ribrault, P. Hersen, R. Voituriez, B. Ladoux, Evidence of a large-scale mechanosensing mechanism for cellular adaptation to substrate stiffness; PNAS, 109(18), 6933-38 (2012).
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.
[27] F. Evenou, J-M. Di Meglio, B. Ladoux, P. Hersen*, Micropatterned porous substrate for combinatorial cell-based assays; Lab on Chip, 12, 1717 (2012).
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.
[26] Jannis Uhlendorf, Samuel Bottani, Francois Fages, Pascal Hersen and Gregory Batt
, towards real-time control of gene expression: controlling the hog signaling cascade ; Proceedings of the Pacific Symposium on Biocomputing, 16, 338-349 (2011).
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.
[25] Agnès Miermont, Jannis Uhlendorf, Megan Mc Clean and Pascal Hersen., The dynamical systems properties of the HOG signaling cascade; Journal of Signal Transduction, , (2011).
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.
[24] Megan N. McClean, Pascal Hersen, and Sharad Ramanathan, Measuring In Vivo Signaling Kinetics in a Mitogen-Activated Kinase Pathway Using Dynamic Input Stimulation; Yeast Genetic Networks: Methods and Protocols, Methods in Molecular Biology, , 734, 101-119 (2011).
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.
[23] Xavier Manière, Félix Lebois, Ivan Matic, Benoit Ladoux, Jean-Marc Di Meglio and Pascal Hersen., Running worms: C. elegans self-sorting by electrotaxis; PLoS One, 6(2), e16637 (2011).
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.
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