Nanobiologie : la révolution diagnostique

National audienceDe la nanotechnologie ... Les nanotechnologies recouvrent l'ensemble des techniques, outils et procédés qui permettent de manipuler la matière à une échelle en dessous de la centaine de nanomètres (1 nanomètre = 1 milliardième de mètre), d'élaborer de nouveaux matériaux et composants toujours plus petits, de construire atome par atome de nouvelles molécules et d'exploiter leurs propriétés en vue de nouvelles applications. Les nanotechnologies devraient constituer dans les prochaines années un marché considérable. À l'horizon 2015, 15% de l'activité manufacturière mondiale serait concernée par des dispositifs ou des matériaux utilisant des avancées issues des nanotechnologies. En 2008, le montant du marché mondial est estimé à 500 milliards de dollars et pourrait doubler en 2015, selon la National science foundation. Toutefois, les nanotechnologies suscitent également des inquiétudes. Manipuler la matière à l'échelle moléculaire et interférer avec le monde du vivant soulève des questions éthiques et sanitaires qui doivent être prises en considération par les pouvoirs publics et les acteurs concernés. La nanotechnologie peut être considérée comme une évolution logique de la microélectronique dont l'objectif est la fabrication de circuits intégrés, familièrement appelés « puces électroniques » dont la référence en matériau de base est le silicium. A côté de la nanoélectronique industrielle a émergé la nanotechnologie moléculaire. Le but de cette dernière discipline est de pouvoir assurer un contrôle parfait sur la structure de la matière et de pouvoir construire des objets complexes avec une précision à l'échelle de l'atome ou de la molécule. ... aux nanoBIOtechnologies... Un important challenge dans les dispositifs de BIOcapteurs et de diagnostic est de savoir comment reconstituer/étudier des mécanismes biologiques pertinents à la surface d'une biopuce et quels outils analytiques sont convenables pour fournir rapidement des informations justes sur la structure des molécules attachées à la surface. Un meilleur contrôle dans la réalisation de biopuces peut être obtenu en combinant différentes approches interdisciplinaires, avec pour préoccupation de répondre à une question clé biologique. Les recherches menées au sein de la plateforme CLIPP (Clinical & Innovation Proteomic Platform), rassemblant des compétences multidisciplinaires, sont focalisées sur cet objectif. L'ensemble « conception, réalisation et caractérisation » de puces à protéines représente un des axes de recherche de la plateforme CLIPP. Nous développons différentes stratégies d'ingénierie/structuration de matériaux, de fonctionnalisation chimique et d'immobilisation de biomolécules qui conduit à l'optimisation d'évènements de reconnaissance et leur caractérisation à la surface de la puce. Dernièrement, nous avons développé une méthode simple pour la préparation de substrat d'or présentant des terrasses atomiquement planes, et permettant une caractérisation biomoléculaire à l'échelle nanométrique1. Le mode d'action du taxol sur les microtubules et les conséquences sur leur conformation a pu également être élucidé par observation par microscopie à force atomique (AFM) en conditions physiologiques2. Nous avons également développé des approches originales, consistant à combiner une analyse « globale » par Surface Plasmon Resonance (SPR) et un moyen de caractérisation à l'échelle nanométrique (AFM, Spectroscopie de Photo-électrons X, spectrométrie de masse (MS)). Nous avons en particulier démontré que l'orientation d'une protéine immobilisée en monocouche sur une surface, peut être déterminée par la détection de fragments secondaires spécifiques de la protéine par TOF-SIMS3. Ces développements ont contribué à l'établissement d'une nouvelle combinaison « Analyse des interactions Biomoléculaires / spectrométrie de masse (BIA-MS) basée sur une procédure totalement « on the chip »4. Nous proposons une approche bas coût combinant l'analyse sur un BIAcore 2000 sur puces synthétisées par nos soins (réalisation du substrat et greffage d'anticorps) avec l'identification par MS, directement sur la puce et sans étape d'élution. Utilisant cette technique, l'identification de complexes protéiques a pu être obtenue, et permet ainsi de compléter l'approche BIA-MS dans la découverte et l'analyse de complexes protéiques. Dernièrement, ces développements ont été transposés à de l'analyse multiplexée sur micro-arrays. Les premiers résultats de détection de biomarqueurs dans du plasma s'avèrent être très prometteurs pour une approche diagnostic moyen/haut débit

Preparation of flat gold terraces for protein chip developments

International audienceA simple method to prepare flat gold terraces on mica for atomic force microscopy biomolecular characterisation is described. The procedure includes preheating of the substrate, metal deposition and an annealing step. All of these steps are at elevated temperatures (300–420°C). This approach allows one to prepare large flat gold terraces (200– 500 nm), which constitute ideal substrates for visualisation and characterisation of a self-assembly monolayer of biomolecules at the nanoscale. The authors illustrated this potential of characterisation with the reconstitution of a protein monolayer

Straight GDP-Tubulin Protofilaments Form in the Presence of Taxol

International audienceMicrotubules exist in dynamic equilibrium, growing and shrinking by the addition or loss of tubulin dimers from the ends of protofilaments. The hydrolysis of GTP in b-tubulin destabilizes the microtubule lattice by increasing the curvature of protofilaments in the microtubule and putting strain on the lattice. The ob- servation that protofilament curvature depends on GTP hydrolysis suggests that microtubule destabil- izers and stabilizers work by modulating the curvature of the microtubule lattice itself. Indeed, the microtu- bule destabilizer MCAK has been shown to increase the curvature of protofilaments during depolymeriza- tion. Here, we show that the atomic force microscopy (AFM) of individual tubulin protofilaments provides sufficient resolution to allow the imaging of single pro- tofilaments in their native environment. By using this assay, we confirm previous results for the effects of GTP hydrolysis and MCAK on the conformation of pro- tofilaments. We go on to show that taxol stabilizes microtubules by straightening the GDP protofilament and slowing down the transition of protofilaments from straight to a curved configuration

Template-free 13-protofilament microtubule–MAP assembly visualized at 8 A resolution

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX’s unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin–tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX’s exquisite binding selectivity uncovers important insights into regulation of cellular MTs

Direct Observation of ATP-Induced Conformational Changes in Single P2X4 Receptors

The ATP-gated P2X4 receptor is a cation channel, which is important in various pathophysiological events. The architecture of the P2X4 receptor in the activated state and how to change its structure in response to ATP binding are not fully understood. Here, we analyze the architecture and ATP-induced structural changes in P2X4 receptors using fast-scanning atomic force microscopy (AFM). AFM images of the membrane-dissociated and membrane-inserted forms of P2X4 receptors and a functional analysis revealed that P2X4 receptors have an upward orientation on mica but lean to one side. Time-lapse imaging of the ATP-induced structural changes in P2X4 receptors revealed two different forms of activated structures under 0 Ca2+ conditions, namely a trimer structure and a pore dilation-like tripartite structure. A dye uptake measurement demonstrated that ATP-activated P2X4 receptors display pore dilation in the absence of Ca2+. With Ca2+, the P2X4 receptors exhibited only a disengaged trimer and no dye uptake was observed. Thus our data provide a new insight into ATP-induced structural changes in P2X4 receptors that correlate with pore dynamics

Gold/Silica biochips: applications to Surface Plasmon Resonance and fluorescence quenching

We report Gold/Silica biochips for low cost biosensor devices. Firstly, the study of biochemical interactions on silica by means of Surface Plasmon Resonance (SPR) is presented. Secondly, Gold/Silica biochips are employed to reduce the strong quenching that occurs when a fluorophore is close to the gold surface. Furthermore, the control of the Silica-like thickness allows optimizing the distance between the metallic surface and the fluorophore in order to enhance the fluorescent signal. These results represent the first steps towards highly sensitive, specific and low cost biosensors based, for example, on Surface Plasmon Coupled Emission (SPCE) techniques

Microtubules as a Critical Target for Arsenic Toxicity in Lung Cells in Vitro and in Vivo

To understand mechanisms for arsenic toxicity in the lung, we examined effects of sodium m-arsenite (As3+) on microtubule (MT) assembly in vitro (0–40 µM), in cultured rat lung fibroblasts (RFL6, 0–20 µM for 24 h) and in the rat animal model (intratracheal instillation of 2.02 mg As/kg body weight, once a week for 5 weeks). As3+ induced a dose-dependent disassembly of cellular MTs and enhancement of the free tubulin pool, initiating an autoregulation of tubulin synthesis manifest as inhibition of steady-state mRNA levels of βI-tubulin in dosed lung cells and tissues. Spindle MT injuries by As3+ were concomitant with chromosomal disorientations. As3+ reduced the binding to tubulin of [3H]N-ethylmaleimide (NEM), an -SH group reagent, resulting in inhibition of MT polymerization in vitro with bovine brain tubulins which was abolished by addition of dithiothreitol (DTT) suggesting As3+ action upon tubulin through -SH groups. In response to As3+, cells elevated cellular thiols such as metallothionein. Taxol, a tubulin polymerization agent, antagonized both As3+ and NEM induced MT depolymerization. MT–associated proteins (MAPs) essential for the MT stability were markedly suppressed in As3+-treated cells. Thus, tubulin sulfhydryls and MAPs are major molecular targets for As3+ damage to the lung triggering MT disassembly cascades

Tubulin-binding dibenz[c,e]oxepines: Part 2 Structural variation and biological evaluation as tumour vasculature disrupting agents

5,7-Dihydro-3,9,10,11-tetramethoxybenz[c,e]oxepin-4-ol 1, prepared from a dibenzyl ether precursor via Pd-catalysed intramolecular direct arylation, possesses broad-spectrum in vitro cytotoxicity towards various tumour cell lines, and induces vascular shutdown, necrosis and growth delay in tumour xenografts in mice at sub-toxic doses. The biological properties of 1 and related compounds can be attributed to their ability to inhibit microtubule assembly at the micromolar level, by binding reversibly to the same site of the tubulin αβ-heterodimer as colchicine 2 and the allocolchinol, N-acetylcolchinol 4

Mechanochemical modeling of dynamic microtubule growth involving sheet-to-tube transition

Microtubule dynamics is largely influenced by nucleotide hydrolysis and the resultant tubulin configuration changes. The GTP cap model has been proposed to interpret the stabilizing mechanism of microtubule growth from the view of hydrolysis effects. Besides, the microtubule growth involves the closure of a curved sheet at its growing end. The curvature conversion also helps to stabilize the successive growth, and the curved sheet is referred to as the conformational cap. However, there still lacks theoretical investigation on the mechanical-chemical coupling growth process of microtubules. In this paper, we study the growth mechanisms of microtubules by using a coarse-grained molecular method. Firstly, the closure process involving a sheet-to-tube transition is simulated. The results verify the stabilizing effect of the sheet structure, and the minimum conformational cap length that can stabilize the growth is demonstrated to be two dimers. Then, we show that the conformational cap can function independently of the GTP cap, signifying the pivotal role of mechanical factors. Furthermore, based on our theoretical results, we describe a Tetris-like growth style of microtubules: the stochastic tubulin assembly is regulated by energy and harmonized with the seam zipping such that the sheet keeps a practically constant length during growth.Comment: 23 pages, 7 figures. 2 supporting movies have not been uploaded due to the file type restriction

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points