PCV - Programme interdiciplinaire en physique et chimie du vivant

Photocontrôle de protéines à l'échelle de la cellule unique dans un organisme vivant – Applications au développement et à la régénération – PROTEOPHANE

Submission summary

Scientific background and objectives Controlling gene expression in space and time is a much sought after goal in biology. Inducible gene expression systems have been used but they remain limited by the technology associated to the inducer delivery. Ideally, the latter should be noninvasive, fast, local, and tunable. We want to implement a technology fulfilling all those criteria. It makes use of two-photon excitation in the infra red range to release a biologically active inducer upon photoactivation of a nonactive precursor distributed in the organism after appropriate conditioning. In this project, we will use this technology to study a number of processes active in development and tissue regeneration. These processes will be investigated at the single cell level in a live zebrafish. Description of the project, methodology This project closely integrates biological, chemical and physical contributions. The biologists will build transgenic fish strains to study cell lineage during regeneration and to the investigation of the extent and dynamics of action of a number of transcription factors (engrailed, pax6) and kinases (p38alpha). These proteins (fused to a steroid receptor) will be activated locally (at the single cell level) by uncaging an appropriate inducer belonging to the nonendogeneous tamoxifen series. These transgenic strains will express a fluorescent marker under control by the induced proteins (Cre-ERT for cell lineage, engrailed-ERT,pax6-ERT, etc.). Some of this strains exist already (e.g. ngn1 :GFP for pax6), others will have to be engineered (e.g. Fgf8 :GFP for engrailed). Moreover cell lines will also be made in order to characterize the action of the induced proteins and their control by the inducing molecules (synthesized by the chemists) in order to select the most efficient inducer. The chemists will design, synthesize and study appropriate photolabile protecting groups, inducers of the tamoxifen series, and inhibitors of the kinase p38alpha, caged or not. They will develop a series of caging groups that fulfill a collection of criteria making them suitable for the present project. A thorough in vitro photophysical/photochemical investigation on model systems will precede the in vivo experiments. We shall be looking for inducers designed to easily penetrate into the whole body in their caged state, but that will be retained within the targeted cell after uncaging. To test for these demanding requirements, we will use the previously mentioned cell lines developed by the biologists. The physicists will build an upright versatile microscope. It will allow for i) direct transmission or epifluorescence observations with application-dependent light sources; ii) local two-photon excitation in the targeted cell. With regards to the latter point, it will be necessary to design and build an appropriate excitation setup making uncaging efficient without being detrimental because of the heating from possible weak residual one-photon absorption of the cell contents at the wavelength used for two-photon excitation. Expected results In the final biological constructs, photoactivation will not only yield control over gene expression in the targeted cell, but also make the latter and its progeny irreversibly fluorescent. Thus, it will be easy to follow in vivo the proliferation and/or the migration of the activated labeled cells. In-situ hybridization studies will allow us to monitor the spatio-temporal activation of various targets of the studied transcription factors (and even their putative activation cascade in neighboring cells). Tissue disruption and selective sorting of the fluorescent cells will allow us to further analyze their transcriptome/proteome. Histological studies will thus be completed by investigations of expression profiles. In the study of development and regeneration in zebrafish, the present technology will be unique to analyze the events occurring between the beginning of morphogene expression or activation and the following phases of cellular differentiation both at the cellular and at the molecular level. The relevance of a zebrafish platform in which it is possible to control the level of gene expression and the degree of activity of certain kinases and transcription factors goes beyond the biological issues considered here. Zebrafish is a model organism in which to study vertebrate development (due to the transparency of its embryo) and tissue regeneration (due to its ability to regenerate many tissues, such as the tail or the eye). The techniques developed here will open a new vista on the investigation of these important issues.

Project coordination

Ludovic JULLIEN (Autre établissement d’enseignement supérieur)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

INSTITUT CURIE - SECTION DE RECHERCHE

Help of the ANR 750,000 euros
Beginning and duration of the scientific project: - 48 Months

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