Yeast cell responses and survival during periodic osmotic stress are controlled by glucose availability.

Natural environments of living organisms are often dynamic and multifactorial, with multiple parameters fluctuating over time. To better understand how cells respond to dynamically interacting factors, we quantified the effects of dual fluctuations of osmotic stress and glucose deprivation on yeast cells using microfluidics and time-lapse microscopy. Strikingly, we observed that cell proliferation, survival, and signaling depend on the phasing of the two periodic stresses. Cells divided faster, survived longer, and showed decreased transcriptional response when fluctuations of hyperosmotic stress and glucose deprivation occurred in phase than when the two stresses occurred alternatively. Therefore, glucose availability regulates yeast responses to dynamic osmotic stress, showcasing the key role of metabolic fluctuations in cellular responses to dynamic stress. We also found that mutants with impaired osmotic stress response were better adapted to alternating stresses than wild-type cells, showing that genetic mechanisms of adaptation to a persistent stress factor can be detrimental under dynamically interacting conditions.

A new look at an old classic: implementation of a SERS-based water hardness titration.

The routine use of SERS as an analytical technique has been hindered by practical considerations among which the irreproducibility of its signals and the lack of robustness of its calibration. In the present work, we examine a strategy to perform quantitative SERS without the need for calibration. The method reinvests a colorimetric volumetric titration procedure to determine water hardness but involves monitoring the progression of the titration through the SERS signal of a complexometric indicator. Upon reaching the equivalence between the chelating titrant and the metal analytes, the SERS signal abruptly jumps, which conveniently serves as an end-point marker. Three mineral waters spanning divalent metal concentrations varying by a factor of 25 were successfully titrated in this way, with satisfactory accuracy. Remarkably, the developed procedure can be run in less than an hour, without laboratory-grade carrying capacity and would be relevant for field measurements.

CyberSco.Py an open-source software for event-based, conditional microscopy.

Timelapse fluorescence microscopy imaging is routinely used in quantitative cell biology. However, microscopes could become much more powerful investigation systems if they were endowed with simple unsupervised decision-making algorithms to transform them into fully responsive and automated measurement devices. Here, we report CyberSco.Py, Python software for advanced automated timelapse experiments. We provide proof-of-principle of a user-friendly framework that increases the tunability and flexibility when setting up and running fluorescence timelapse microscopy experiments. Importantly, CyberSco.Py combines real-time image analysis with automation capability, which allows users to create conditional, event-based experiments in which the imaging acquisition parameters and the status of various devices can be changed automatically based on the image analysis. We exemplify the relevance of CyberSco.Py to cell biology using several use case experiments with budding yeast. We anticipate that CyberSco.Py could be used to address the growing need for smart microscopy systems to implement more informative quantitative cell biology experiments.

Observing Nutrient Gradients, Gene Expression and Growth Variation Using the "Yeast Machine" Microfluidic Device.

The natural environment of microbial cells like bacteria and yeast is often a complex community in which growth and internal organization reflect morphogenetic processes and interactions that are dependent on spatial position and time. While most of research is performed in simple homogeneous environments (e.g., bulk liquid cultures), which cannot capture full spatiotemporal community dynamics, studying biofilms or colonies is complex and usually does not give access to the spatiotemporal dynamics at single cell level. Here, we detail a protocol for generation of a microfluidic device, the "yeast machine", with arrays of long monolayers of yeast colonies to advance the global understanding of how intercellular metabolic interactions affect the internal structure of colonies within defined and customizable spatial dimensions. With Saccharomyces cerevisiae as a model yeast system we used the "yeast machine" to demonstrate the emergence of glucose gradients by following expression of fluorescently labelled hexose transporters. We further quantified the expression spatial patterns with intra-colony growth rates and expression of other genes regulated by glucose availability. In addition to this, we showed that gradients of amino acids also form within a colony, potentially opening similar approaches to study spatiotemporal formation of gradients of many other nutrients and metabolic waste products. This approach could be used in the future to decipher the interplay between long-range metabolic interactions, cellular development, and morphogenesis in other same species or more complex multi-species systems at single-cell resolution and timescales relevant to ecology and evolution.

A microfluidic device for inferring metabolic landscapes in yeast monolayer colonies.

Microbial colonies are fascinating structures in which growth and internal organization reflect complex morphogenetic processes. Here, we generated a microfluidics device with arrays of long monolayer yeast colonies to further global understanding of how intercellular metabolic interactions affect the internal structure of colonies within defined boundary conditions. We observed the emergence of stable glucose gradients using fluorescently labeled hexose transporters and quantified the spatial correlations with intra-colony growth rates and expression of other genes regulated by glucose availability. These landscapes depended on the external glucose concentration as well as secondary gradients, for example amino acid availability. This work demonstrates the regulatory genetic networks governing cellular physiological adaptation are the key to internal structuration of cellular assemblies. This approach could be used in the future to decipher the interplay between long-range metabolic interactions, cellular development and morphogenesis in more complex systems.

An integrative modeling approach to the age-performance relationship in mammals at the cellular scale.

Physical and cognitive performances change across lifespan. Studying cohorts of individuals in specific age ranges and athletic abilities remains essential in assessing the underlying physiological mechanisms that result in such a drop in performance. This decline is now viewed as a unique phenotypic biomarker and a hallmark of the aging process. The rates of decline are well documented for sets of traits such as running or swimming but only a limited number of studies have examined the developmental and senescent phases together. Moreover, the few attempts to do so are merely descriptive and do not include any meaningful biological features. Here we propose an averaged and deterministic model, based on cell population dynamics, replicative senescence and functionality loss. It describes the age-related change of performance in 17 time-series phenotypic traits, including human physical and cognitive skills, mouse lemur strength, greyhound and thoroughbred speed, and mouse activity. We demonstrate that the estimated age of peak performance occurs in the early part of life (20.5% ± 6.6% of the estimated lifespan) thus emphasizing the asymmetrical nature of the relationship. This model is an initial attempt to relate performance dynamics to cellular dynamics and will lead to more sophisticated models in the future.

Age-Related Upper Limits in Physical Performances.

Maximal physical performances are powerful and accurate biomarkers in the understanding of age-related changes during the aging process. Previous studies have characterized age-related changes from Caenorhabditis elegans to Homo sapiens. We characterized changes in this pattern for H. sapiens, decade by decade, from 1970 to 2017. Using 286,916 performances related to age from the world's best performances in each age group, we measured the relative change of 10 different running and jumping events for both women and men. We compared the change in sexual dimorphism with age and showed that the gender gap in maximal performance regarding age increases gradually, especially after the age of 50. Between 1970 and 2017, the performances for all age groups in all events have slightly progressed. However, during the last decades, the relative progression of the best performances for all age groups has decreased in both range and frequency, suggesting that age-related maximal physical performances for H. sapiens are reaching their physiological limits.

Uncooked spaghetti in a colander: Injection of semiflexible polymers in a nanopore.

We study the flow injection of semiflexible polymers in a nanopore with a diameter smaller than the persistence length of the macromolecules. The suction model from de Gennes and Brochard is modified to take into account the effect of the rigidity of the polymer in the Odijk regime. We show that in this case of extreme confinement the flow threshold vanishes slowly and that in the limit of infinitely small nanopore the free energy barrier eventually disappears.

Age-Related Changes in Locomotor Performance Reveal a Similar Pattern for Caenorhabditis elegans, Mus domesticus, Canis familiaris, Equus caballus, and Homo sapiens.

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.

Structural and multi-scale rheophysical investigation of diphasic magneto-sensitive materials based on biopolymers.

We present a structural and a multi-scale rheophysical investigation of magneto-sensitive materials based on biopolymers, namely aqueous solutions of sodium alginate incorporating magnetic maghemite nanoparticles, functionalized with adsorbed negative citrate ions. The large alginate ionic strength impacts the structure and the rheology of these nanocomposites in zero magnetic field. In given physico-chemical conditions, the system is fluid and homogeneous on macroscopic scales while it is diphasic on microscopic ones, containing micro-droplets coming from the demixion of the system. These micro-droplets are liquid and deformable under magnetic field. Their under-field elongation and their zero-field relaxation are directly observed by optical microscopy to determine their interfacial tension, their magnetic susceptibility and their internal viscosity. A structural analysis of the solutions of alginate chains and of the phase-separated mixtures of alginate and nanoparticles by Small Angle Scattering completes the local description of the system.

Zero-mode waveguide detection of flow-driven DNA translocation through nanopores.

We directly measure the flow-driven injection of DNA through nanopores at the level of single molecule and single pore using a modified zero-mode waveguide method. We observe a flow threshold independent of the pore radius, the DNA concentration, and length. We demonstrate that the flow injection of DNA in nanopores is controlled by an energy barrier as proposed in the de Gennes-Brochard suction model. Finally, we show that the height of the energy barrier is modulated by functionalizing the nanopores.

Growing yeast into cylindrical colonies.

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.

Micropatterned porous membranes for combinatorial cell-based assays.

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.

Twisting and buckling: a new undulation mechanism for artificial swimmers.

Among the various locomotion strategies of the animal kingdom, the undulation locomotion is of particular interest for biomimetic applications. In this paper, we present an artificial swimmer set into motion by a new and non-trivial undulation mechanism, based on the twisting and buckling of its body. The swimmer consists of a long cylinder of ferrogel which is polarized transversely and in opposite directions at each extremity. When it is placed on a water film and submitted to a transverse oscillating magnetic field, the worm-like swimmer undulates and swims. Whereas symmetry breaking is due to the field gradient, the undulations of the worm result from a torsional buckling instability as the polarized ends tend to align with the applied magnetic field. The critical magnetic field above which buckling and subsequent swimming is observed may be predicted using elasticity equations including the effect of the magnetic torque. As the length of the worm is varied, several undulation modes are observed which are in good agreement with the bending modes of an elastic rod with free ends.

Locomotion control of Caenorhabditis elegans through confinement.

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.

Micro-patterned porous substrates for cell-based assays.

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 (µPM). 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. µPMs 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.

Running worms: C. elegans self-sorting by electrotaxis.

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.

Mechanics of cell spreading within 3D-micropatterned environments.

Most 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 extracellular 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 microposts, 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 pillar sides, spanning between the elastic micropillars and applying large forces on the substrate. Interestingly, the architecture of the actin cytoskeleton and 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 cell 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.

Diffusion of latex and DNA chains in 2D confined media.

We report a study on the dynamics of latex polystyrene beads and of DNA molecules confined in two dimensions, using fluorescence video-microscopy. We particularly focus on the character of the confined objects (hard or soft) and on the nature of the confinement: liquid (in a soap film) or solid (between two glass plates). For weak confinements, whatever the nature of confinement, we observe that DNA molecules and latex beads behave very similarly: the tighter the confinement, the slower the diffusion with a good agreement with theory. For strong confinements between solid walls (thickness of confinement smaller than the bulk radius of gyration), DNA coils are not immobilized and still diffuse. We show in this case that the conformation of DNA chains is in good agreement with the predictions of De Gennes and Brochard (radius approximately e (-1/4), with e the confinement gap); on the other hand, we cannot really check the theoretical predictions for the diffusion coefficient. Interestingly, strong confinement of latex beads in a soap film leads to a anomalous slow diffusion, certainly associated with an additional viscous drag generated by the interfaces.