Université PSL



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Contact dependence and velocity crossover in friction between microscopic solid/solid contacts
McGraw, A. Niguès, A. Chennevière, A. Siria
Nano Lett. - 17 (10) 6335–6339 - DOI: 10.1021/acs.nanolett.7b03076 - 2017
Friction at the nanoscale differs markedly from that between surfaces of macroscopic extent. Characteristically, the velocity dependence of friction between apparent solid/solid contacts can strongly deviate from the classically assumed velocity independence. Here, we show that a nondestructive friction between solid tips with radius on the scale of hundreds of nanometers and solid hydrophobic self-assembled monolayers has a strong velocity dependence. Specifically, using laterally oscillating quartz tuning forks, we observe a linear scaling in the velocity at the lowest accessed velocities, typically hundreds of micrometers per second, crossing over into a logarithmic velocity dependence. This crossover is consistent with a general multicontact friction model that includes thermally activated breaking of the contacts at subnanometric elongation. We find as well a strong dependence of the friction on the dimensions of the frictional probe.
Nano-on-Micro Fibrous Extracellular Matrices for Scalable Expansion of Human Es/Ips Cells
L. Liu, K.-i. Kamei, M. Yoshioka, M. Nakajima, J. Li, N. Fujimoto, S. Terada, Y. Tokunaga, Y. Koyama, H. Sato, K. Hasegawa, N. Nakatsuji and Y. Chen
Biomaterials - 124 47-54 - DOI: 10.1016/j.biomaterials.2017.01.039 - 2017
Human pluripotent stem cells (hPSCs) hold great potential for industrial and clinical applications. Clinical-grade scaffolds and high-quality hPSCs are required for cell expansion as well as easy handling and manipulation of the products. Current hPSC culture methods do not fulfill these requirements because of a lack of proper extracellular matrices (ECMs) and cell culture wares. We developed a layered nano-on-micro fibrous cellular matrix mimicking ECM, named "fiber-on-fiber (FF)" matrix, which enables easy handling and manipulation of cultured cells. While non-woven sheets of cellulose and polyglycolic acid were used as a microfiber layer facilitating mechanical stability, electrospun gelatin nanofibers were crosslinked on the microfiber layer, generating a mesh structure with connected nanofibers facilitating cell adhesion and growth. Our results showed that the FF matrix supports effective hPSC culture with maintenance of their pluripotency and normal chromosomes over two months, as well as effective scaled-up expansion, with fold increases of 54.1 ± 15.6 and 40.4 ± 8.4 in cell number per week for H1 human embryonic stem cells and 253G1 human induced pluripotent stem cells, respectively. This simple approach to mimick the ECM may have important implications after further optimization to generate lineage-specific products.
Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes
Mohammad Soltani, Cesar A. Vargas-Garcia, Duarte Antunes, and Abhyudai Singh
PLoS Comp. Biol. - 12(8) e1004972 - doi: 10.1371/journal.pcbi.1004972 - 2016
Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells.
Transient compartmentalization of RNA replicators prevents extinction due to parasites
Matsumura S, Kun Á, Ryckelynck M, Coldren F, Szilágyi A, Jossinet F, Rick C, Nghe P, Szathmáry E, Griffiths A.
Science - 354(6317): 1293-1296 - DOI: 10.1126/science.aag1582 - 2016
The appearance of molecular replicators (molecules that can be copied) was probably a critical step in the origin of life. However, parasitic replicators would take over and would have prevented life from taking off unless the replicators were compartmentalized in reproducing protocells. Paradoxically, control of protocell reproduction would seem to require evolved replicators. We show here that a simpler population structure, based on cycles of transient compartmentalization (TC) and mixing of RNA replicators, is sufficient to prevent takeover by parasitic mutants. TC tends to select for ensembles of replicators that replicate at a similar rate, including a diversity of parasites that could serve as a source of opportunistic functionality. Thus, TC in natural, abiological compartments could have allowed life to take hold.
Efficient laboratory evolution of computationally designed enzymes with low starting activities using fluorescence-activated droplet sorting
Obexer R, Pott M, Zeymer C, Griffiths A, Hilvert D.
Protein Eng Des Sel - 29(9) 355-66 - doi: 10.1093/protein/gzw032 - 2016
De novo biocatalysts with non-natural functionality are accessible by computational enzyme design. The catalytic activities obtained for the initial designs are usually low, but can be optimized significantly by directed evolution. Nevertheless, rate accelerations approaching the level of natural enzymes can only be achieved over many rounds of tedious and time-consuming laboratory evolution. In this work, we show that microfluidic-based screening using fluorescence-activated droplet sorting (FADS) is ideally suited for efficient optimization of designed enzymes with low starting activity, essentially straight out of the computer. We chose the designed retro-aldolase RA95.0, which had been previously evolved by conventional microtiter plate screening, as an example and reoptimized it using the microfluidic-based assay. Our results show that FADS is sufficiently sensitive to detect enzyme activities as low as kcat/Km = 0.5 M(-1)s(-1) The ultra-high throughput of this system makes screening of large mutant libraries possible in which clusters of up to five residues are randomized simultaneously. Thus, combinations of beneficial mutations can be identified directly, leading to large jumps in catalytic activity of up to 80-fold within a single round of evolution. By exploring several evolutionary trajectories in parallel, we identify alternative active site arrangements that exhibit comparably enhanced efficiency but opposite enantioselectivity
Hierarchy and extremes in selections from pools of randomized proteins
Boyer S, Biswas D, Kumar Soshee A, Scaramozzino N, Nizak C2, Rivoire O.
Proc. Nat. Acad. Sci. USA - 113(13) 3482-7 - doi: 10.1073/pnas. - 2016
Variation and selection are the core principles of Darwinian evolution, but quantitatively relating the diversity of a population to its capacity to respond to selection is challenging. Here, we examine this problem at a molecular level in the context of populations of partially randomized proteins selected for binding to well-defined targets. We built several minimal protein libraries, screened them in vitro by phage display, and analyzed their response to selection by high-throughput sequencing. A statistical analysis of the results reveals two main findings. First, libraries with the same sequence diversity but built around different "frameworks" typically have vastly different responses; second, the distribution of responses of the best binders in a library follows a simple scaling law. We show how an elementary probabilistic model based on extreme value theory rationalizes the latter finding. Our results have implications for designing synthetic protein libraries, estimating the density of functional biomolecules in sequence space, characterizing diversity in natural populations, and experimentally investigating evolvability (i.e., the potential for future evolution).
Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution
Denis Cottinet , Florence Condamine, Nicolas Bremond, Andrew D. Griffiths, Paul B. Rainey, J. Arjan G. M. de Visser, Jean Baudry, Jérôme Bibette
Nature Biotechnology - 11(4) e0152395 - doi.org/10.1371/journal.pone.0152395 - 2016
Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes–via growth–over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.
Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells.
Vargas P, Maiuri P, Bretou M, Sáez PJ, Pierobon P, Maurin M, Chabaud M, Lankar D, Obino D, Terriac E, Raab M, Thiam H-R, Brocker T, Kitchen-Goosen SM, Alberts AS, Sunareni P, Xia S, Li R, Voituriez R, Piel M, Lennon-Duménil A-M
Nat. Cell Biol. - 18(1): 43-53 - DOI: 10.1016/j.jim.2015.12.005 - 2016
Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined. Here, we show that the migration of immature DCs depends on two main actin pools: a RhoA-mDia1-dependent actin pool located at their rear, which facilitates forward locomotion; and a Cdc42-Arp2/3-dependent actin pool present at their front, which limits migration but promotes antigen capture. Following TLR4-MyD88-induced maturation, Arp2/3-dependent actin enrichment at the cell front is markedly reduced. Consequently, mature DCs switch to a faster and more persistent mDia1-dependent locomotion mode that facilitates chemotactic migration to LVs and lymph nodes. Thus, the differential use of actin-nucleating machineries optimizes the migration of immature and mature DCs according to their specific function.
Deterministic patterns in cell motility
Ido Lavi, Matthieu Piel, Ana-Maria Lennon-Duménil , Raphaël Voituriez and Nir S. Gov
Nature Physics - 12 1146–1152 - DOI: : 10.1038/NPHYS3836 - 2016
Cell migration paths are generally described as random walks, associated with both intrinsic and extrinsic noise. However, complex cell locomotion is not merely related to such fluctuations, but is often determined by the underlying machinery. Cell motility is driven mechanically by actin and myosin, two molecular components that generate contractile forces. Other cell functions make use of the same components and, therefore, will compete with the migratory apparatus. Here, we propose a physical model of such a competitive system, namely dendritic cells whose antigen capture function and migratory ability are coupled by myosin II. The model predicts that this coupling gives rise to a dynamic instability, whereby cells switch from persistent migration to unidirectional self-oscillation, through a Hopf bifurcation. Cells can then switch to periodic polarity reversals through a homoclinic bifurcation. These predicted dynamic regimes are characterized by robust features that we identify through in vitro trajectories of dendritic cells over long timescales and distances. We expect that competition for limited resources in other migrating cell types can lead to similar deterministic migration modes.
A new microfluidic approach for the one-step capture, amplification and label-free quantification of bacteria from raw samples
Iago Pereiro, Amel Bendali, Sanae Tabnaoui, Lucile Alexandre, Jana Srbova, Zuzana Bilkova, Shane Deegan, Lokesh Joshi, Jean-Louis Viovy, Laurent Malaquin, Bruno Dupuy and Stéphanie Descroix
Chem. Sci. - 8(2) 1329-1336 - DOI: 10.1039/C6SC03880H - 2016
A microfluidic method to specifically capture and detect infectious bacteria based on immunorecognition and proliferative power is presented. It involves a microscale fluidized bed in which magnetic and drag forces are balanced to retain antibody-functionalized superparamagnetic beads in a chamber during sample perfusion. Captured cells are then cultivated in situ by infusing nutritionally-rich medium. The system was validated by the direct one-step detection of Salmonella Typhimurium in undiluted unskimmed milk, without pre-treatment. The growth of bacteria induces an expansion of the fluidized bed, mainly due to the volume occupied by the newly formed bacteria. This expansion can be observed with the naked eye, providing simple low-cost detection of only a few bacteria and in a few hours. The time to expansion can also be measured with a low-cost camera, allowing quantitative detection down to 4 cfu (colony forming unit), with a dynamic range of 100 to 107 cfu ml−1 in 2 to 8 hours, depending on the initial concentration. This mode of operation is an equivalent of quantitative PCR, with which it shares a high dynamic range and outstanding sensitivity and specificity, operating at the live cell rather than DNA level. Specificity was demonstrated by controls performed in the presence of a 500× excess of non-pathogenic Lactococcus lactis. The system's versatility was demonstrated by its successful application to the detection and quantitation of Escherichia coli O157:H15 and Enterobacter cloacae. This new technology allows fast, low-cost, portable and automated bacteria detection for various applications in food, environment, security and clinics.

278 publications.