Vers une étude de la division asymétrique des cellules à l'échelle de la molécule unique

The goal of this project is to develop novel tools to explore dynamic organizational processes in living cells, with unprecedented detail and sensitivity. In particular, we are interested in the process of asymmetric cell division (ACD), during which a mother cell divides asymmetrically into two different daughter cells. This process is a key mechanism in the generation of cellular diversity in organisms of all levels. During the course of ACD, prior to the separation of the mother cell into daughter cells, it needs to generate an asymmetric spatial distribution for some of its content. This symmetry-breaking process is called polarization i.e. the generation of a polarized structure. One of the ways to study polarization is to characterize the intra-cellular transport mechanisms that generate it. As a means to observe intra-cellular transport in detail, we employ single-molecule methods, which allow us to separately follow (or track) the movement of single molecules. By observing one event at a time we can obtain much more information about the process, compared to simultaneous measurements of the entire population. We are using novel and powerful fluorophores, quantum dots (QDs), which allow for single molecule detection and tracking. On the technical level, we developed methods to cope with certain technical difficulties associated with intra-cellular, single-molecule, tracking experiments. These include the delivery of such fluorophores into living cells, the targeting of specific molecules of interest, as well as control over the stoichiometry of QD-protein complexes. The project combines different disciplines, such as single molecule imaging, molecular biology, and primary cell culture, thereby covering almost all aspects of an in-vivo single-molecule experiment.Le but de ce projet est de développer de nouveaux outils pour explorer des processus d'organisation intracellulaire dynamique dans les cellules vivantes, avec une sensibilité sans précédent. Ce travail se concentre sur deux aspects principaux : le développement d'outils pour l'étude en molécule unique de la division cellulaire asymétrique, et la mise au point de sondes monovalentes qui permettent le suivi d'une protéine individuelle utilisant un nanocristal semiconducteur (ou quantum dot, QD). La division cellulaire asymétrique (DCA) est définie comme une division cellulaire dans laquelle une cellule mère donne naissance à deux cellules filles avec des destins différents (ce qui se manifeste à travers par exemple la taille, le contenu ou le profil d'expression). Notre étude se concentre sur la division cellulaire asymétrique dans les cellules souches neurales de Drosophila melanogaster, appelés neuroblastes. Au cours de la division asymétrique des neuroblasts, avant la séparation de la cellule-mère en deux cellules-filles, certaines molécules dans le cytoplasme se redistribuent de façon asymétrique (polarisée). Ce travail a montré la faisabilité de l'étude de la division cellulaire asymétrique à l'échelle de la molécule unique. Les méthodes ont été conçues et développées pour la conjugaison et la caractérisation des complexes QD-protéines. Nous avons réussi à cibler les protéines localisées de manière asymétrique dans des neuroblastes en division. Cela ouvre la voie à des études intracellulaires de ce phénomène, en utilisant des QDs individuels Ce travail a également mis en évidence la limite principale de ce système expérimental : la nature tridimensionnelle des mouvements. En raison de l'épaisseur de la neuroblate, les QDs sortent du plan focal très souvent. En conséquence, l'obtention des trajectoires suffisamment longues pour le calcul des paramètres de transport, devient très difficile. Toutefois, certaines informations peuvent encore être extraites des données que nous avons obtenues, en analysant la répartition spatiale de "courts-déplacements" dans les films obtenus. Les déplacements des QDs entre deux images consécutives sont regroupés et analysés en fonction de leur emplacement par rapport à une carte polaire normalisée d'un neuroblaste polarisé. Une telle analyse n'a pas besoin des trajectoires longues mais peut, quand même, révèler des differences dans la mobilité des protéines entre les differents domaines de la cellule. Cette analyse est actuellement en cours. Nous avons aussi réussi à produire des sondes monovalentes pour le suivi des proteines membranaires extracellulaires. Ces sondes sont basées sur un fragment de chaîne variable d'anticorp (ScFv). Ces sondes doivent avoir des nombreuses applications dans le suivi des diverses protéines membranaires, mais doivent être améliorées afin de répondre aux exigences rigoureuses du suivi intracellulaire

Toward a single molecule study of asymmetric experiment

Le but de ce projet est de développer de nouveaux outils pour explorer des processus d'organisation intracellulaire dynamique dans les cellules vivantes, avec une sensibilité sans précédent. Ce travail se concentre sur deux aspects principaux : le développement d'outils pour l'étude en molécule unique de la division cellulaire asymétrique, et la mise au point de sondes monovalentes qui permettent le suivi d'une protéine individuelle utilisant un nanocristal semiconducteur (ou quantum dot, QD). La division cellulaire asymétrique (DCA) est définie comme une division cellulaire dans laquelle une cellule mère donne naissance à deux cellules filles avec des destins différents (ce qui se manifeste à travers par exemple la taille, le contenu ou le profil d'expression). Notre étude se concentre sur la division cellulaire asymétrique dans les cellules souches neurales de Drosophila melanogaster, appelés neuroblastes. Au cours de la division asymétrique des neuroblasts, avant la séparation de la cellule-mère en deux cellules-filles, certaines molécules dans le cytoplasme se redistribuent de façon asymétrique (polarisée). Ce travail a montré la faisabilité de l'étude de la division cellulaire asymétrique à l'échelle de la molécule unique. Les méthodes ont été conçues et développées pour la conjugaison et la caractérisation des complexes QD-protéines. Nous avons réussi à cibler les protéines localisées de manière asymétrique dans des neuroblastes en division. Cela ouvre la voie à des études intracellulaires de ce phénomène, en utilisant des QDs individuels Ce travail a également mis en évidence la limite principale de ce système expérimental : la nature tridimensionnelle des mouvements. En raison de l'épaisseur de la neuroblate, les QDs sortent du plan focal très souvent. En conséquence, l'obtention des trajectoires suffisamment longues pour le calcul des paramètres de transport, devient très difficile. Toutefois, certaines informations peuvent encore être extraites des données que nous avons obtenues, en analysant la répartition spatiale de "courts-déplacements" dans les films obtenus. Les déplacements des QDs entre deux images consécutives sont regroupés et analysés en fonction de leur emplacement par rapport à une carte polaire normalisée d'un neuroblaste polarisé. Une telle analyse n'a pas besoin des trajectoires longues mais peut, quand même, révèler des differences dans la mobilité des protéines entre les differents domaines de la cellule. Cette analyse est actuellement en cours. Nous avons aussi réussi à produire des sondes monovalentes pour le suivi des proteines membranaires extracellulaires. Ces sondes sont basées sur un fragment de chaîne variable d'anticorp (ScFv). Ces sondes doivent avoir des nombreuses applications dans le suivi des diverses protéines membranaires, mais doivent être améliorées afin de répondre aux exigences rigoureuses du suivi intracellulaire.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Toxin-independent virulence of Bacillus anthracis in rabbits.

The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play a major role in pathogenicity. In the guinea pig (GP) model we have previously shown that deletion of all three toxin components results in a relatively moderate attenuation in virulence, indicating that B. anthracis possesses an additional toxin-independent virulence mechanism. To characterize this toxin-independent mechanism in anthrax disease, we developed a new rabbit model by intravenous injection (IV) of B. anthracis encapsulated vegetative cells, artificially creating bacteremia. Using this model we were able to demonstrate that also in rabbits, B. anthracis mutants lacking the toxins are capable of killing the host within 24 hours. This virulent trait depends on the activity of AtxA in the presence of pXO2, as, in the absence of the toxin genes, deletion of either component abolishes virulence. Furthermore, this IV virulence depends mainly on AtxA rather than the whole pXO1. A similar pattern was shown in the GP model using subcutaneous (SC) administration of spores of the mutant strains, demonstrating the generality of the phenomenon. The virulent strains showed higher bacteremia levels and more efficient tissue dissemination; however our interpretation is that tissue dissemination per se is not the main determinant of virulence whose exact nature requires further elucidation

Improved production of monoclonal antibodies against the LcrV antigen of Yersinia pestis using FACS-aided hybridoma selection

 For about four decades, hybridoma technologies have been the “work horse” of monoclonal antibody production. These techniques proved to be robust and reliable, albeit laborious. Over the years, several major improvements have been introduced into the field, but yet, antibody production still requires many hours of labor and considerable resources. In this work, we present a leap forward in the advancement of hybridoma-based monoclonal antibody production, which saves labor and time and increases yield, by combining hybridoma technology, fluorescent particles and fluorescence-activated cell sorting (FACS). By taking advantage of the hybridomas’ cell-surface associated antibodies, we can differentiate between antigen-specific and non-specific cells, based on their ability to bind the particles. The speed and efficiency of antibody discovery, and subsequent cell cloning, are of high importance in the field of infectious diseases. Therefore, as a model system, we chose the protein LcrV, a major virulence factor of the plague pathogen Yersinia pestis, an important re-emerging pathogen and a possible bioterror agent

Using old antibiotics to treat ancient bacterium-β-lactams for Bacillus anthracis meningitis.

As Bacillus anthracis spores pose a proven bio-terror risk, the treatment focus has shifted from exposed populations to anthrax patients and the need for effective antibiotic treatment protocols increases. The CDC recommends carbapenems and Linezolid (oxazolidinone), for the treatment of anthrax, particularly for the late, meningeal stages of the disease. Previously we demonstrated that treatment with Meropenem or Linezolid, either as a single treatment or in combination with Ciprofloxacin, fails to protect rabbits from anthrax-meningitis. In addition, we showed that the failure of Meropenem was due to slow BBB penetration rather than low antibacterial activity. Herein, we tested the effect of increasing the dose of the antibiotic on treatment efficacy. We found that for full protection (88% cure rate) the dose should be increased four-fold from 40 mg/kg to 150 mg/kg. In addition, B. anthracis is a genetically stable bacterium and naturally occurring multidrug resistant B. anthracis strains have not been reported. In this manuscript, we report the efficacy of classical β-lactams as a single treatment or in combination with β-lactamase inhibitors in treating anthrax meningitis. We demonstrate that Ampicillin based treatment of anthrax meningitis is largely efficient (66%). The high efficacy (88-100%) of Augmentin (Amoxicillin and Clavulonic acid) and Unasyn (Ampicillin and Sulbactam) makes them a favorable choice due to reports of β-lactam resistant B. anthracis strains. Tazocin (Piperacillin and Tazobactam) proved inefficient compared to the highly efficient Augmentin and Unasyn

Non-specific interactions govern cytosolic diffusion of nanosized objects in mammalian cells

International audienceThe diffusivity of macromolecules in the cytoplasm of eukaryotic cells varies over orders of magnitude and dictates the kinetics of cellular processes. However, a general description that associates the Brownian or anomalous nature of intracellular diffusion to the architectural and biochemical properties of the cytoplasm has not been achieved. Here we measure the mobility of individual fluorescent nanoparticles in living mammalian cells to obtain a comprehensive analysis of cytoplasmic diffusion. We identify a correlation between tracer size, its biochemical nature and its mobility. Inert particles with size equal or below 50 nm behave as Brownian particles diffusing in a medium of low viscosity with negligible effects of molecular crowding. Increasing the strength of non-specific interactions of the nanoparticles within the cytoplasm gradually reduces their mobility and leads to subdiffusive behaviour. These experimental observations and the transition from Brownian to subdiffusive motion can be captured in a minimal phenomenological model

Infection with a Nonencapsulated <i>Bacillus anthracis</i> Strain in Rabbits—The Role of Bacterial Adhesion and the Potential for a Safe Live Attenuated Vaccine

Nonencapsulated (∆pXO2) Bacillus anthracis strains are commonly used as vaccines and for anthrax research, mainly in the mouse model. Previously, we demonstrated that the infection of rabbits, intranasally or subcutaneously, with the spores of a fully virulent strain results in the systemic dissemination of the bacteria, meningitis, and death, whereas ∆pXO2 strains are fully attenuated in this animal model. We used the intravenous inoculation of rabbits to study the pathogenicity of the ∆pXO2 strain infection. Bacteremia, brain bacterial burden, and pathology were used as criteria to compare the Vollum∆pXO2 disease to the wild type Vollum infection. To test the role of adhesion in the virulence of Vollum∆pXO2, we deleted the major adhesion protein BslA and tested the virulence and immunogenicity of this mutant. We found that 50% of the rabbits succumb to Vollum∆pXO2 strain following i.v. infection, a death that was accompanied with significant neurological symptoms. Pathology revealed severe brain infection coupled with an atypical massive bacterial growth into the parenchyma. Contrary to the Vollum strain, deletion of the bslA gene fully attenuated the ∆pXO2 strain. Though the Vollum∆pXO2 cannot serve as a model for B. anthracis pathogenicity in rabbits, deletion of the bslA gene prevents central nervous system (CNS) infections, possibly leading to the generation of a safer vaccine

Pathology of wild-type and toxin-independent Bacillus anthracis meningitis in rabbits.

Hemorrhagic meningitis is considered a complication of anthrax and was reported in about 50% of deadly cases in humans and non-human primates (NHP). Recently we demonstrated in Guinea pigs and rabbits that 100% of the B. anthracis-infected animals presented histopathology of meningitis at the time of death, some without any sign of hemorrhage. A similar pathology was observed in animals that succumbed following infection with the toxin deficient mutant, thus indicating that anthrax meningitis is a toxin-independent phenomenon. In this manuscript we describe a histopathological study of the B. anthracis infection of the central nervous system (CNS). Though we could find sporadic growth of the bacteria around blood vessels in the cortex, we report that the main infiltration route is the choroid plexus. We found massive destruction of entire sections of the choroid plexus coupled with massive aggregation of bacilli in the ventricles, in close proximity to the parenchyma. The choroid plexus also contained significant amounts of intravascular bacterial aggregates, often enclosed in what appear to be fibrin-like clots. The high concentration of these aggregates in areas of significant tissue destruction combined with the fact that capsular B. anthracis bacteria have a low tendency to adhere to endothelial cells, might suggest that these clots are used as an adherence mechanism by the bacteria. The major histopathological finding is meningitis. We find massive bacterial growth in the meninges without evidence of encephalitis, even when the bacteria emerge from a parenchymal blood vessel. Erythrocytes were present within the meningeal space but no clear vasculitis could be detected. Histology of the brain stem indicates meningitis, edema and hemorrhages that might explain death from suffocation due to direct damage to the respiratory center. All of these processes are toxin-independent, since they were observed following infection with either the wild type strain or the toxin-deficient mutant. Herein, we propose that the first step of anthrax-meningitis is bacterial adhesion to the blood vessels by manipulating coagulation, mainly in the choroid plexus. The trapped bacteria then destroy sections of the choroid plexus, resulting in penetration into the CSF, leading to meningitis and hemorrhage. Death could be the result of increased intracranial pressure and/or damage to the brain stem

Publisher Correction: Non-specific interactions govern cytosolic diffusion of nanosized objects in mammalian cells

International audienceIn the version of this Article originally published, Supplementary Videos 3-5 were incorrectly labelled; 3 should have been 5, 4 should have been 3 and 5 should have been 4. This has now been corrected

Alternate atxA and acpA dependent response of Bacillus anthracis to serum, HCO3- and CO2.

Bacillus anthracis overcomes host immune responses by producing capsule and secreting toxins. Production of these virulence factors in response to entering the host environment was shown to be regulated by atxA, the major virulence regulator, known to be activated by HCO3- and CO2. While toxin production is regulated directly by atxA, capsule production is independently mediated by two regulators; acpA and acpB. In addition, it was demonstrated that acpA has at least two promotors, one of them shared with atxA. We used a genetic approach to study capsule and toxin production under different conditions. Unlike previous works utilizing NBY, CA or R-HCO3- medium under CO2 enriched conditions, we used a sDMEM-based medium. Thus, toxin and capsule production can be induced in ambient or CO2 enriched atmosphere. Using this system, we could differentiate between induction by 10% NRS, 10% CO2 or 0.75% HCO3-. In response to high CO2, capsule production is induced by acpA based response in an atxA-independent manner, with little to no toxin (protective antigen PA) production. atxA based response is activated in response to serum independently of CO2, inducing toxin and capsule production in an acpA or acpB dependent manner. HCO3- was also found to activate atxA based response, but in non-physiological concentrations. Our findings may help explain the first stages of inhalational infection, in which spores germinating in dendritic cells require protection (by encapsulation) without affecting cell migration to the draining lymph-node by toxin secretion