Université PSL

Publications

RECHERCHER

Laboratoire :
Auteur :
Revue :
Année :

A L'ATTENTION DES EQUIPES IPGG :

- Pour toute publication de résultats ayant reçu l’aide de l’IPGG (présence dans les locaux de l’IPGG, passage sur la plateforme technologique de l’IPGG, collaboration inter équipes IPGG, lié à une bourse doctorale ou postdoctorale IPGG, ou encore utilisation des espaces communs), il vous faut indiquer  cette phrase « Ce travail a été réalisé avec le soutien du laboratoire d’excellence Institut Pierre-Gilles de Gennes (programme Investissements d’avenir ANR-10-IDEX-0001-02 PSL et ANR-10-LABX-31). » / « This work has received the support of "Institut Pierre-Gilles de Gennes" (laboratoire d’excellence, “Investissements d’avenir” program ANR-10-IDEX-0001-02 PSL and ANR-10-LABX-31.). ».

- Pour toute publication de résultats obtenu via l'utilisation d’un équipement acheté par l’Equipex IPGG, il vous faut ajouter  la codification suivante : « ANR-10-EQPX-34 ».

Development of Immobilized Enzyme Reactors for the characterization of the glycosylation heterogeneity of a protein
Laboratoire Spectrométrie de masse biologique et protéomique - Stan Perchepied, Nicolas Eskenazi, Chiara Giangrande, Julien Camperi, Thierry Fournier, Joëlle Vinh, Nathalie Delaunay, Valérie Pichon
ELSEVIER - 209 120568 - doi.org/10.1016/j.talanta.2019.120171 - 2020
The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl
Characterization of home-made graphite/PDMS microband electrodes for amperometric detection in an original reusable glass-NOA®-PDMS electrophoretic microdevice
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - J.Gouyon F.d’Orlyé S.Griveau F.Bedioui A.Varenne
Electrochimica Acta - 329 135164 - doi.org/10.1016/j.electacta.2019.135164 - 2020
A new dismountable and reusable microchip for electrophoretic separation coupled to amperometric detection was developed. For this purpose, a new home made three-microbands electrode system was developed and microfabricated based on screen-printing for the inclusion of graphite/polydimethylsiloxane (C-PDMS) composite in microchannels down to 30 μm width. The composition of the composite as well as the fabrication methodology were optimized for an easy handling and an optimized electrochemical behavior. The electrochemical characterization of this composite material was first performed in bulk format (disc-shaped electrode, 6 mm diameter). It was then transposed to the micrometric scale for its integration in an original glass-NOA81®-PDMS microfluidic device allowing for reversible sealing. The microband electrodes were characterized by scanning electron microcopy and cyclic voltammetry, illustrating a good control of the microelectrode width. Then, the analytical performances of the C-PDMS composite microelectrodes were evaluated using Ru(NH3)63+ and FcMeOH as model electroactive molecules. The electrophoretic separation and quantitation of Ru(NH3)63+ were then performed in a background electrolyte made of hydrochloric acid and sodium chloride, leading to a LOD and a LOQ of 3.4 μmol L−1 and 11.3 μmol L−1, respectively. The re-openable NOA-based microdevice permits to regenerate the electrode surface by simply repositioning the microband on a new spot, allowing for robust analysis in a reusable system.
Hydrogel Matrix-Grafted Impedimetric Aptasensors for the Detection of Diclofenac
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - G. S. Kassahun, S. Griveau, S. Juillard, J. Champavert, A. Ringuedé, B. Bresson, Y. Tran*, F. Bedioui, and C. Slim
Langmuir - 36(4) 827–836 - doi.org/10.1021/acs.langmuir.9b02031 - 2020
Driven by the growing concern about the release of untreated emerging pollutants and the need for determining small amounts of these pollutants present in the environment, novel biosensors dedicated to molecular recognition are developed. We have designed biosensors using a novel class of grafted polymers, surface-attached hydrogel thin films, on conductive transducers as a biocompatible matrix for biomolecule immobilization. We showed that they can be dedicated to the molecular recognition of diclofenac (DCL). The immobilization of the aptamer onto surface-attached hydrogel thin films by covalent attachment provides a biodegradable shelter, providing the aptamer with excellent environments to preserve its active and functional structure while allowing the detection of DCL. The grafting of the aptamer is obtained using the formation of amide bonds via the activation of carboxylic acid groups of the poly(acrylic acid) hydrogel thin film. For improved sensitivity and higher stability of the sensor, a high density of the immobilized aptamer is enabled. The aptamer-modified electrode was then incubated with DCL solutions at different concentrations. The performances of the aptasensor were investigated by electrochemical impedance spectroscopy. The change in charge-transfer resistance was found to be linear with DCL concentration in the 30 pM to 1 μM range. The detection limit was calculated to be 0.02 nM. The improvement of the limit of detection can be mainly attributed to the three-dimensional environment of the hydrogel matrix which improves the grafting density of the aptamer and the affinity of the aptamer to DCL.
Speciation and quantitation of precious metals in model acidic leach liquors, theoretical and practical aspects of recycling
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Jérémie Gouyon, Fanny d’Orlyé, Julia Zimmerman, Sophie Griveau, Fethi Bedioui & Anne Varenne
Analytical and Bioanalytical Chemistry volume - 412 4595–4608 - doi.org/10.1007/s00216-020-02707-4 - 2020
Waste printed circuit boards are a major source of strategic materials such as platinum group metals since they are used for the fabrication of technological devices, such as hard drive discs, capacitors, and diodes. Because of the high cost of platinum, palladium, and gold (> 25 k€/kg), an economic and environmental challenge is their recycling from printed circuit boards that represent around 2% weight of electronic equipment. Hydrometallurgical treatments allow the recovery of these metals in solution, with a high recovery rate for a leaching liquor made of thiourea in hydrochloric acid. So as to develop an efficient recycling process from this leach liquor, one requires the speciation of these strategic metals, as well as their extraction and quantitation in the mixture. For this purpose, platinum, palladium, and gold were dissolved in model leach liquors made of hydrochloric acid and thiourea at low concentration. The identification of metal complexes was determined as a function of thiourea concentration (between 10 μmol/L and 10 mmol/L) by the combination of UV-visible spectrometry, cyclic voltammetry, and for the first time capillary electrophoresis. The electrokinetic method was then applied for the quantitation of trace metal analyses in leach samples from waste printed circuit boards reprocessing, demonstrating its applicability for industrializable recycling applications.

Reversible microfluidics device for precious metal electrodeposition and depletion yield studies
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Jérémie Gouyon Fanny d’Orlyé Craig Simon Sophie Griveau Catherine Sella Laurent Thouin Fethi Bediouia Anne Varenne
Electrochimica Acta - 352 136474 - doi.org/10.1016/j.electacta.2020.136474 - 2020
A new low-cost reversible Glass-NOA®-PDMS microfluidic device was designed for the study of recovery yield of precious metals present in acid media mimicking leach liquors for long-term recycling objectives. It offers the unique advantage of allowing easy washing of the microchannel and renewal of the electrode surface by simply repositioning the microband electrodes which allows this type of device to have a relatively much longer lifespan than irreversibly closed ones. It consists in a re-useable microchip with four graphite microbands electrodes, prepared by screen printing, to set-up an original amperometric device for both depletion and yield quantification. One upstream working electrode is devoted to the depletion of the metallic ions through their electrolysis by electrodeposition while the second downstream working microelectrode is used as real-time detection electrode to evaluate the depletion efficiency. The dimensions of the depletion electrode and of the channel were optimized thanks to numerical simulations for a given range of flow velocities. First, the performances of the device were assessed experimentally according to flow rate and applied potential under continuous flow, and then compared to theoretical predictions using an electrochemical probe, ferrocenemethanol. The proof of concept was then demonstrated for precious metal, by electroreduction of Pd(II) and Au(III) from acidic leach liquors under continuous flow, with a depletion yield of up to 89% and 71% respectively.
Multiple Zones Modification of Open Off-Stoichiometry Thiol-Ene Microchannel by Aptamers: A Methodological Study & A Proof of Concept
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Samantha Bourg, Fanny d’Orlyé, Sophie Griveau, Fethi Bedioui, Jose Alberto Fracassi da Silva, andAnne Varenne.
Electrochimica Acta - 8(2) 24 - doi.org/10.3390/chemosensors8020024 - 2020
Off-stoichiometry thiol-ene polymer (OSTE) is an emerging thermoset with interesting properties for the development of lab-on-a-chip (LOAC), such as easy microfabrication process, suitable surface chemistry for modification and UV-transparency. One of the challenges for LOAC development is the integration of all the analytical steps in one microchannel, and particularly, trace level analytes extraction/preconcentration steps. In this study, two strategies for the immobilization of efficient tools for this purpose, thiol-modified (C3-SH) aptamers, on OSTE polymer surfaces were developed and compared. The first approach relies on a direct UV-initiated click chemistry reaction to graft thiol-terminated aptamers on ene-terminated OSTE surfaces. The second strategy consists of the immobilization of thiol-terminated aptamers onto OSTE substrates covered by gold nanoparticles. The presence of an intermediate gold nanoparticle layer on OSTE has shown great interest in the efficient immobilization of aptamers, preserving their interaction with the target, and preventing non-specific adsorption. With this second innovative strategy, we proved, for the first time the concept of creating multiple functional zones for sample treatment in an open OSTE-microchannel thanks to the immobilization of aptamers in consecutive areas by the simple droplet deposition methodology. This methodological development allows further consideration of OSTE material for lab-on-a-chip designs, integrating multiple zones for sample pretreatment, based on molecular recognition by ligands, such as aptamers, in a specific zone of the microchannel and is adaptable to a large range of analytical applications for LOAC industrialization. View Full-Text
Label-free graphene oxide–based SPR genosensor for the quantification of microRNA21
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Michael López Mujica, Yuanyuan Zhang, Féthi Bédioui, Fabiana Gutiérrez & Gustavo Rivas
Analytical and Bioanalytical Chemistry - 412 3539–3546 - doi.org/10.1007/s00216-020-02593-w - 2020
This work is focused on the development of a genosensor for microRNA-21 quantification using surface plasmon resonance (SPR) to transduce the hybridization event. The biosensing platform was built by self-assembling two bilayers of poly(diallyldimethylammonium chloride) (PDDA) and graphene oxide (GO) at a gold surface modified with 3-mercaptopropane sulfonate (MPS), followed by the covalent attachment of the DNA probe. GO was used in two directions, to allow the anchoring of the probe DNA and to increase the sensitivity of the biosensing event due to its field enhancer effect. The new bioanalytical platform represents an interesting alternative for the label-free biosensing of microRNA-21, with a linear range between 1.0 fM and 10 nM, a sensitivity of 5.1 ± 0.1 moM−1 and a detection limit of 0.3fM. The proposed sensing strategy was successfully used for the quantification of microRNA-21 in enriched urine samples.

Anna Notaro, Angelo Frei, Riccardo Rubbiani, Marta Jakubaszek, Uttara Basu, Severin Koch, Cristina Mari, Mazzarine Dotou, Olivier Blacque, Jérémie Gouyon, Fethi Bedioui, Nils Rotthowe, Rainer F. Winter, Bruno Goud, Stefano Ferrari, Mickaël Tharaud, Marti
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Anna Notaro, Angelo Frei, Riccardo Rubbiani, Marta Jakubaszek, Uttara Basu, Severin Koch, Cristina Mari, Mazzarine Dotou, Olivier Blacque, Jérémie GouyonJérémie Gouyon Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sci
J. Med. Chem - 63(10) 5568–5584 - doi.org/10.1021/acs.jmedchem.0c00431 - 2020
Chemotherapy remains one of the dominant treatments to cure cancer. However, due to the many inherent drawbacks, there is a search for new chemotherapeutic drugs. Many classes of compounds have been investigated over the years to discover new targets and synergistic mechanisms of action including multicellular targets. In this work, we designed a new chemotherapeutic drug candidate against cancer, namely, [Ru(DIP)2(sq)](PF6) (Ru-sq) (DIP = 4,7-diphenyl-1,10-phenanthroline; sq = semiquinonate ligand). The aim was to combine the great potential expressed by Ru(II) polypyridyl complexes and the singular redox and biological properties associated with the catecholate moiety. Experimental evidence (e.g., X-ray crystallography, electron paramagnetic resonance, electrochemistry) demonstrates that the semiquinonate is the preferred oxidation state of the dioxo ligand in this complex. The biological activity of Ru-sq was then scrutinized in vitro and in vivo, and the results highlight the promising potential of this complex as a chemotherapeutic agent against cancer.


Ruthenium(II) Complex Containing a Redox-Active Semiquinonate Ligand as a Potential Chemotherapeutic Agent: From Synthesis to In Vivo Studies
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Anna Notaro, Angelo Frei, Riccardo Rubbiani, Marta Jakubaszek, Uttara Basu, Severin Koch, Cristina Mari, Mazzarine Dotou, Olivier Blacque, Jérémie GouyonJérémie Gouyon Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sci
J. Med. Chem - 63(10) 5568–5584 - doi.org/10.1021/acs.jmedchem.0c00431 - 2020
Chemotherapy remains one of the dominant treatments to cure cancer. However, due to the many inherent drawbacks, there is a search for new chemotherapeutic drugs. Many classes of compounds have been investigated over the years to discover new targets and synergistic mechanisms of action including multicellular targets. In this work, we designed a new chemotherapeutic drug candidate against cancer, namely, [Ru(DIP)2(sq)](PF6) (Ru-sq) (DIP = 4,7-diphenyl-1,10-phenanthroline; sq = semiquinonate ligand). The aim was to combine the great potential expressed by Ru(II) polypyridyl complexes and the singular redox and biological properties associated with the catecholate moiety. Experimental evidence (e.g., X-ray crystallography, electron paramagnetic resonance, electrochemistry) demonstrates that the semiquinonate is the preferred oxidation state of the dioxo ligand in this complex. The biological activity of Ru-sq was then scrutinized in vitro and in vivo, and the results highlight the promising potential of this complex as a chemotherapeutic agent against cancer.


Ruthenium(II) Complex Containing a Redox-Active Semiquinonate Ligand as a Potential Chemotherapeutic Agent: From Synthesis to In Vivo Studies
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Dr. Anna Notaro Marta Jakubaszek Severin Koch Dr. Riccardo Rubbiani Dr. Orsolya Dömötör Dr. Éva A. Enyedy Mazzarine Dotou Dr. Fethi Bedioui
J. Med. Chem - 26 22 - doi.org/10.26434/chemrxiv.10008917.v1. - 2020
Cancer is one of the main causes of death worldwide. Chemotherapy, despite its severe side effects, is to date one of the leading strategies against cancer. Metal‐based drugs present several potential advantages when compared to organic compounds and they have gained trust from the scientific community after the approval on the market of the drug cisplatin. Recently, we reported the ruthenium complex ([Ru(DIP)2(sq)](PF6) (where DIP is 4,7‐diphenyl‐1,10‐phenantroline and sq is semiquinonate) with a remarkable potential as chemotherapeutic agent against cancer, both in vitro and in vivo. In this work, we analyse a structurally similar compound, namely [Ru(DIP)2(mal)](PF6), carrying the flavour‐enhancing agent approved by the FDA, maltol (mal). To possess an FDA approved ligand is crucial for a complex, whose mechanism of action might include ligand exchange. Herein, we describe the synthesis and characterisation of [Ru(DIP)2(mal)](PF6), its stability in solutions and under conditions that resemble the physiological ones, and its in‐depth biological investigation. Cytotoxicity tests on different cell lines in 2D model and on HeLa MultiCellular Tumour Spheroids (MCTS) demonstrated that our compound has higher activity than cisplatin, inspiring further tests. [Ru(DIP)2(mal)](PF6) was efficiently internalised by HeLa cells through a passive transport mechanism and severely affected the mitochondrial metabolism.


Increasing the Cytotoxicity of Ru(II) Polypyridyl Complexes by Tuning the Electronic Structure of Dioxo Ligands
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Anna Notaro, Marta Jakubaszek, Nils Rotthowe, Federica Maschietto, Robin Vinck, Patrick S. Felder, Bruno Goud, Mickaël Tharaud, Ilaria Ciofini, Fethi Bedioui, Rainer F. Winter, and Gilles Gasser
J. Am. Chem. Soc. - 142(13) 6066–6084 - doi.org/10.1021/jacs.9b12464 - 2020
Due to the great potential expressed by an anticancer drug candidate previously reported by our group, namely, Ru-sq ([Ru(DIP)2(sq)](PF6) (DIP, 4,7-diphenyl-1,10-phenanthroline; sq, semiquinonate ligand), we describe in this work a structure–activity relationship (SAR) study that involves a broader range of derivatives resulting from the coordination of different catecholate-type dioxo ligands to the same Ru(DIP)2 core. In more detail, we chose catechols carrying either an electron-donating group (EDG) or an electron-withdrawing group (EWG) and investigated the physicochemical and biological properties of their complexes. Several pieces of experimental evidences demonstrated that the coordination of catechols bearing EDGs led to deep-red positively charged complexes 1–4 in which the preferred oxidation state of the dioxo ligand is the uninegatively charged semiquinonate. Complexes 5 and 6, on the other hand, are blue/violet neutral complexes, which carry an EWG-substituted dinegatively charged catecholate ligand. The biological investigation of complexes 1–6 led to the conclusion that the difference in their physicochemical properties has a strong impact on their biological activity. Thus, complexes 1–4 expressed much higher cytotoxicities than complexes 5 and 6. Complex 1 constitutes the most promising compound in the series and was selected for a more in depth biological investigation. Apart from its remarkably high cytotoxicity (IC50 = 0.07–0.7 μM in different cancerous cell lines), complex 1 was taken up by HeLa cells very efficiently by a passive transportation mechanism. Moreover, its moderate accumulation in several cellular compartments (i.e., nucleus, lysosomes, mitochondria, and cytoplasm) is extremely advantageous in the search for a potential drug with multiple modes of action. Further DNA metalation and metabolic studies pointed to the direct interaction of complex 1 with DNA and to the severe impairment of the mitochondrial function. Multiple targets, together with its outstanding cytotoxicity, make complex 1 a valuable candidate in the field of chemotherapy research. It is noteworthy that a preliminary biodistribution study on healthy mice demonstrated the suitability of complex 1 for further in vivo studies.



Mineral surfaces select for longer RNA molecules
Laboratoire Biochimie - Ryo Mizuuchi, Alex Blokhuis,b, Lena Vincent, Philippe Nghe, Niles Lehman, and David Baumd
Chem. Comm. - 55(14) 2090–2093. - 10.1039/c8cc10319d - 2019
We report empirically and theoretically that multiple prebiotic minerals can selectively accumulate longer RNAs, with selectivity enhanced at higher temperatures. We further demonstrate that surfaces can be combined with a catalytic RNA to form longer RNA polymers, supporting the potential of minerals to develop genetic information on the early Earth.
Large work extraction and the Landauer limit in a continuous Maxwell demon
Laboratoire Biochimie - Marco Ribezzi Crivellari
Nature Physics - 15(7) 93 - DOI: 10.1038/s41567-019-0481-0 - 2019
The relation between entropy and information dates back to the classical Maxwell demon paradox¹, a thought experiment proposed in 1867 by James Clerk Maxwell to violate the second law of thermodynamics. A variant of the classical Maxwell demon is the Szilard engine, proposed by Leo Szilard in 1929¹. In it, at a given time, the demon observes the compartment occupied by a single molecule in a vessel and extracts work by operating a pulley device. Here, we introduce the continuous Maxwell demon, a device capable of extracting arbitrarily large amounts of work per cycle by repeated measurements of the state of a system, and experimentally test it in single DNA hairpin pulling experiments. In the continuous Maxwell demon, the demon monitors the state of the DNA hairpin (folded or unfolded) by observing it at equally spaced time intervals, but it extracts work only when the molecule changes state. We demonstrate that the average maximum work per cycle that can be extracted by the continuous Maxwell demon is limited by the information content of the stored sequences, in agreement with the second law. Work extraction efficiency is found to be maximal in the large information-content limit where work extraction is fuelled by rare events.
High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer
Laboratoire Biochimie - Grosselin K1,2,3, Durand A4,5, Marsolier J, Poitou A, Marangoni E, Nemati F, Dahmani A, Lameiras S, Reyal F, Frenoy O, Pousse Y, Reichen M, Woolfe A, Brenan C, Griffiths AD, Vallot C, Gérard A.i
Nat Genet. - 51(6) 1060-1066 - doi: 10.1038/s41588-019-0424-9. - 2019
Modulation of chromatin structure via histone modification is a major epigenetic mechanism and regulator of gene expression. However, the contribution of chromatin features to tumor heterogeneity and evolution remains unknown. Here we describe a high-throughput droplet microfluidics platform to profile chromatin landscapes of thousands of cells at single-cell resolution. Using patient-derived xenograft models of acquired resistance to chemotherapy and targeted therapy in breast cancer, we found that a subset of cells within untreated drug-sensitive tumors share a common chromatin signature with resistant cells, undetectable using bulk approaches. These cells, and cells from the resistant tumors, have lost chromatin marks-H3K27me3, which is associated with stable transcriptional repression-for genes known to promote resistance to treatment. This single-cell chromatin immunoprecipitation followed by sequencing approach paves the way to study the role of chromatin heterogeneity, not just in cancer but in other diseases and healthy systems, notably during cellular differentiation and development.
Experimental evidence of symmetry breaking of transition-path times
Laboratoire Biochimie - J.Gladrow, M. Ribezzi-Crivellari, F. Ritort & U. F. Keyser
Nature Communications - 10 55 - doi.org/10.1038/s41467-018-07873-9 - 2019
While thermal rates of state transitions in classical systems have been studied for almost a century, associated transition-path times have only recently received attention. Uphill and downhill transition paths between states at different free energies should be statistically indistinguishable. Here, we systematically investigate transition-path-time symmetry and report evidence of its breakdown on the molecular- and meso-scale out of equilibrium. In automated Brownian dynamics experiments, we establish first-passage-time symmetries of colloids driven by femtoNewton forces in holographically-created optical landscapes confined within microchannels. Conversely, we show that transitions which couple in a path-dependent manner to fluctuating forces exhibit asymmetry. We reproduce this asymmetry in folding transitions of DNA-hairpins driven out of equilibrium and suggest a topological mechanism of symmetry breakdown. Our results are relevant to measurements that capture a single coordinate in a multidimensional free energy landscape, as encountered in electrophysiology and single-molecule fluorescence experiments.
Recent insights into the genotype–phenotype relationship from massively parallel genetic assays
Laboratoire Biochimie - Harry Kemble Philippe Nghe Olivier Tenaillon
Nature Physics - 9 12 - doi.org/10.1111/eva.12846 - 2019
With the molecular revolution in Biology, a mechanistic understanding of the genotype–phenotype relationship became possible. Recently, advances in DNA synthesis and sequencing have enabled the development of deep mutational scanning assays, capable of scoring comprehensive libraries of genotypes for fitness and a variety of phenotypes in massively parallel fashion. The resulting empirical genotype–fitness maps pave the way to predictive models, potentially accelerating our ability to anticipate the behaviour of pathogen and cancerous cell populations from sequencing data. Besides from cellular fitness, phenotypes of direct application in industry (e.g. enzyme activity) and medicine (e.g. antibody binding) can be quantified and even selected directly by these assays. This review discusses the technological basis of and recent developments in massively parallel genetics, along with the trends it is uncovering in the genotype–phenotype relationship (distribution of mutation effects, epistasis), their possible mechanistic bases and future directions for advancing towards the goal of predictive genetics.
Large scale control and programming of gene expression using CRISPR.
Laboratoire Biochimie - Deyell M, Ameta S, Nghe P
Semin Cell Dev Biol. - S1084-9521(18 30110-1 - doi: 10.1016/j.semcdb.2019.05.013 - 2019
The control of gene expression in cells and organisms allows to unveil gene to function relationships and to reprogram biological responses. Several systems, such as Zinc fingers, TALE (Transcription activator-like effectors), and siRNAs (small-interfering RNAs), have been exploited to achieve this. However, recent advances in Clustered Regularly Interspaced Short Palindromic Repeats and Cas9 (CRISPR-Cas9) have overshadowed them due to high specificity, compatibility with many different organisms, and design flexibility. In this review we summarize state-of-the art for CRISPR-Cas9 technology for large scale gene perturbation studies, including single gene and multiple genes knock-out, knock-down, knock-up libraries, and their associated screening assays. We feature in particular the combination of these methods with single-cell transcriptomics approaches. Finally, we highlight the application of CRISPR-Cas9 systems in building synthetic circuits that can be interfaced with gene networks to control cellular states.
Reconstitution of cell migration at a glance
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Juan Manuel Garcia-Arcos, Renaud Chabrier, Mathieu Deygas, Guilherme Nader, Lucie Barbier, Pablo José Sáez, Aastha Mathur, Pablo Vargas, Matthieu Piel
Frontiers in immunology - 188.e5 171 - DOI : jcs225565 - 2019
Single cells migrate in a myriad of physiological contexts, such as tissue patrolling by immune cells, and during neurogenesis and tissue remodeling, as well as in metastasis, the spread of cancer cells. To understand the basic principles of single-cell migration, a reductionist approach can be taken. This aims to control and deconstruct the complexity of different cellular microenvironments into simpler elementary constrains that can be recombined together. This approach is the cell microenvironment equivalent of reconstituted systems that combine elementary molecular players to understand cellular functions. In this Cell Science at a Glance article and accompanying poster, we present selected experimental setups that mimic different events that cells undergo during migration These include polydimethylsiloxane (PDMS) devices to deform whole cells or organelles, micro patterning, nano-fabricated structures like grooves, and compartmentalized collagen chambers with chemical gradients. We also outline the main contribution of each technique to the understanding of different aspects of single-cell migration.
Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells.
Laboratoire Biologie cellulaire systémique de la polarité et de la division - 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 - 2019
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.
Optical volume and mass measurements show that mammalian cells swell during mitosis.
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Zlotek-Zlotkiewicz E, Monnier S, Cappello G, Le Berre M, Piel M
J. Cell Biol. - 211( 4): 765-74 - DOI: 10.1016/j.jim.2015.12.005 - 2019
The extent, mechanism, and function of cell volume changes during specific cellular events, such as cell migration and cell division, have been poorly studied, mostly because of a lack of adequate techniques. Here we unambiguously report that a large range of mammalian cell types display a significant increase in volume during mitosis (up to 30%). We further show that this increase in volume is tightly linked to the mitotic state of the cell and not to its spread or rounded shape and is independent of the presence of an intact actomyosin cortex. Importantly, this volume increase is not accompanied by an increase in dry mass and thus corresponds to a decrease in cell density. This mitotic swelling might have important consequences for mitotic progression: it might contribute to produce strong pushing forces, allowing mitotic cells to round up; it might also, by lowering cytoplasmic density, contribute to the large change of physicochemical properties observed in mitotic cells.

638 publications.