In vivo Study of the Histone Chaperone Activity of Nucleolin by FRAP

Nucleolin is a major nucleolar protein involved in various aspects of ribosome biogenesis such as regulation of polymerase I transcription, pre-RNA maturation, and ribosome assembly. Nucleolin is also present in the nucleoplasm suggesting that its functions are not restricted to nucleoli. Nucleolin possesses, in vitro, chromatin co-remodeler and histone chaperone activities which could explain numerous functions of nucleolin related to the regulation of gene expression. The goal of this report was to investigate the consequences of nucleolin depletion on the dynamics of histones in live cells. Changes in histone dynamics occurring in nucleolin silenced cells were measured by FRAP experiments on eGFP-tagged histones (H2B, H4, and macroH2A). We found that nuclear histone dynamics was impacted in nucleolin silenced cells; in particular we measured higher fluorescence recovery kinetics for macroH2A and H2B but not for H4. Interestingly, we showed that nucleolin depletion also impacted the dissociation constant rate of H2B and H4. Thus, in live cells, nucleolin could play a role in chromatin accessibility by its histone chaperone and co-remodeling activities

Localisation and functions of nucleolin at the centrosome

La nucléoline est une des protéines les plus abondantes des nucléoles. Ses fonctions ne sont cependant pas restreintes à la biogénèse des ribosomes. En absence de nucléoline, un phénotype d’amplification du nombre de centrosomes en mitose, associé à des fuseaux multipolaires a été récemment rapporté. Notre étude vise à comprendre l’implication de la nucléoline dans l’apparition de ce phénotype et notamment ses conséquences sur l’organisation des microtubules.Par immunofluorescence, nous mettons en évidence que la fraction centrosomale de la nucléoline est spécifiquement associée au centriole mature en interphase, alors qu’en mitose seule une forme phosphorylée y est détectée.En interphase, les cellules déplétées en nucleoline présentent une amplification de leurs centrioles immatures, entourés par un réseau anormal de péricentrine, dénotant une perte de structuration de la matrice péricentriolaire. De plus, une désorientation du réseau microtubulaire causée par une capacité de nucléation ralentie et une perte d’ancrage des microtubules au centrosome mature est observée. Par des expériences de co-immunoprécipitation avec la tubuline γ, un lien avec le complexe d’initiation de la nucléation des microtubules est dévoilé.En conclusion, les résultats de ma thèse révèlent que structurellement la nucléoline est associée au centriole mature des cellules en interphase et que fonctionnellement elle stimule la nucléation des microtubules et participe à leur ancrage au centrosome mature pour orienter le réseau microtubulaire en interphase. La nucléoline pourrait ainsi être un des acteurs de la synchronicité entre centrosomes et nucléoles pour la régulation du cycle cellulaire.Nucleolin is an abundant non-ribosomal protein of the nucleolus. Nevertheless its functions are not restricted to ribosome biogenesis. Without nucleolin, a phenotype of abnormally high centrosome numbers was recently reported in mitosis, associated with multipolar spindle formation. The purpose of our study is to understand nucleolin’s involvement in the appearance of this phenotype and specifically consequences on microtubule network organisation. By immunofluorescence, visual evidences of a centrosomal fraction of nucleolin are provided, specifically associated with the mature centriole of interphase cells. In mitosis, only a phosphorylated form of nucleolin is detected at the spindle poles.In interphase, nucleolin depleted cells exhibit immature centriole amplification surrounded by an abnormal mesh of pericentrine, showing a loss of pericentriolar matrix structuration. Furthermore, in most nucleolin depleted cells, a complete disorganisation of microtubule network is observed, caused by a slower microtubule nucleation capacity and a loss of microtubule anchoring at the mature centriole. Using co-immunoprecipitation with γ-tubulin, a major centrosomal protein, a link with the microtubule nucleation complex was highlighted.Taken together my thesis results reveal that in interphase cells, nucleolin is structurally associated with the mature centriole, and functionally stimulates microtubule nucleation and participates in their anchoring at the mature centrosome to orient microtubule network. Thus, nucleolin could be a major actor in the synchronicity between centrosome and nucleoli for cell cycle regulation

Expression of Nucleolin Affects Microtubule Dynamics

International audienc

Centrosomal nucleolin is required for microtubule network organization

Nucleolin is a pleiotropic protein involved in a variety of cellular processes. Although multipolar spindle formation has been observed after nucleolin depletion, the roles of nucleolin in centrosome regulation and functions have not been addressed. Here we report using immunofluorescence and biochemically purified centrosomes that nucleolin co-localized only with one of the centrioles during interphase which was further identified as the mature centriole. Upon nucleolin depletion, cells exhibited an amplification of immature centriole markers surrounded by irregular pericentrin staining; these structures were exempt from maturation markers and unable to nucleate microtubules. Furthermore, the microtubule network was disorganized in these cells, exhibiting frequent non-centrosomal microtubules. At the mature centriole a reduced kinetics in the centrosomal microtubule nucleation phase was observed in live silenced cells, as well as a perturbation of microtubule anchoring. Immunoprecipitation experiments showed that nucleolin belongs to protein complexes containing 2 key centrosomal proteins, γ-tubulin and ninein, involved in microtubule nucleation and anchoring steps. Altogether, our study uncovered a new role for nucleolin in restricting microtubule nucleation and anchoring at centrosomes in interphase cells

VIPV: Process development of integrated photovoltaic cells in a double-curved composite structure for automotive application

International audienceIn response to the marked increase in research activity and publications about Vehicle Integrated PhotoVoltaics (VIPV), this article is an attempt to identify the main constraints relative to the manufacturing of curved PV modules for automotive application and the interests of composite modules in this context. Two processes have been selected as better adapted to the automotive industrialization and in-mold integration of PV cells: Resin Transfer Molding (RTM) and thermocompression. A finite element analysis (FEA) to evaluate the mechanical strength of the resulting module is proposed. The aim of this numerical approach is to optimize the mechanical performance of the structure. First results were validated by a comparison of simulation outputs with 3-point bending test measurements

Microtubule growth parameters.

<p>Microtubule growth parameters.</p

Process development of integrated photovoltaic cells in a double-curved composite structure for Vehicle-Integrated PhotoVoltaics (VIPV) application

National audienceTopics and investigations The automotive industry is changing to lightweight materials and more energy-efficient vehicles. PV modules have a potential market in this transition. Rigid PV panels integrating silicon cells built directly into the car body outperform thin flexible solar films in terms of efficiency and reliability. To increase the integration of PV modules into the car, the use of composite materials is an interesting option, as they offer an efficient combination of mechanical performances and lightweight. However, the high heterogeneity of the components, the impermeability of the cells and the necessary achievement of certain optical characteristics make it necessary to adapt the usual manufacturing processes of composite panels. Thus, this study focuses on the adaptation of manufacturing processes for the production of composite double-curved PV modules. Moreover, it treats with a coupled numerical and experimental approach the aspect of thermomechanical stresses resulting from these processes. Methods Two processes are being developed, that use composite materials and not only polymers or glass, like most VIPV actors do. These processes-thermocompression and Resin Transfer Molding (RTM)are more adapted to the automotive industry constraints than lamination. As a first step towards the development of the RTM process, the early prototypes were produced using vacuum bagging process, which facilitates the selection of materials and stacks for injection processes. A new numerical model is developed with a Finite Element Analysis to optimize the process parameters and the layup for a one-cell composite PV module. Results A double curved composite PV module was manufactured using vacuum bagging process. This step enabled to select the most appropriate materials and process parameters for the RTM process, in terms of transparency, adhesion between the encapsulant and plies, diffusion of the resin, ageing behavior … A first numerical model (Figure 2) was developed. This model is validated by 3-point bending tests. The correlation between tests and numerical simulation is very good, with a relative error of around 5%. The thermocompression process is under development. It still represents a challenge in terms of residual stresses resulting from thermomechanical loading. The ongoing developments on the numerical model aim at optimizing the stack and thermocompression parameters to minimize these stresses. Conclusions and perspectives Two processes are developed. This work enabled to size a double-curved PV module with composite front and back sheets. It is applicable to either the automotive industry or any application needing lightweight and curved PV modules. The experimental tests validate the mechanical model and provide other properties like the photoelectric performance and the ageing behaviour of these curved panels with respect to IEC61215 standard. Further researches include the optimization of the composite layout and processes parameters to minimize weight while keeping high mechanical strength, as well as the optimization of the reliability and performance of the module

Nucleolin can affect microtubules stability through multiple pathways.

<p>Based on previous studies and our unpublished observations, four hypotheses can be drawn to explain nucleolin’s role on microtubule dynamics, simplified in the current scheme by polymerization and depolymerization arrows (respectively in purple and yellow) at the Minus and plus Ends. The first hypothesis derives from nucleolin’s role on centrosome function (A-centrosome associated function, in blue). Indeed, nucleolin (in red) localizes at the mature centriole, interacting with ninein and γTuRC, where it is involved in microtubule nucleation and anchoring [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157534#pone.0157534.ref015" target="_blank">15</a>]. The second hypothesis is based on the control of RNAP II gene transcription (B- transcriptome associated function, in orange), which is regulated by nucleolin binding to a specific gene regulating element (our unpublished results). The third hypothesis is through binding to microtubule associated proteins or with actin cytoskeleton, known to interact with microtubule (C-Actin cytoskeleton interaction, in green). The fourth hypothesis is that nucleolin could directly interact with microtubules (D- Direct interaction, in red), thereby resulting in slower and longer-lasting polymerizing microtubules.</p