PCV - Programme interdiciplinaire en physique et chimie du vivant

Etude, par microscopie à corrélation de fluorescence, de la réponse nucléaire au stress thermique ; aspects dynamiques. – Dynastress

Submission summary

An important current challenge in cellular biology is the understanding of the numerous networks of molecular interactions that regulate cellular processes. Traditionally, molecular interactions between cellular molecules have been measured by in vitro techniques. However, recent developments in confocal microscopy and about fluorescent probes (fusion proteins), make it possible to investigate, in living cells, various dynamical properties: mobility, interactions, compartmentation and aggregation, crowding, etc. From the instrumental point of view, the FCS (Fluorescence Correlation Spectroscopy) and FRAP (Fluorescence Recovery After Photobleaching) techniques are experiencing a strong interest. Note that, in comparison with single particle tracking, FCS and FRAP give access to faster molecular dynamics, since they do not imply to record wide field images and provide statistical information. In its usual implementation, FCS is limited to measurements done at a series of points, successively addressed during a long acquisition time. The present project is based on the development of a multiconfocal FCS (mFCS), the principle of which is to simultaneously collect the confocal signals coming from several points of a cellular environment and to analyze the fluorescence correlations to get molecular information at these points. In addition, statistical analysis methods inspired by FCS will make it possible to obtain, from wide field images, information about slower processes thanks to ICS (Image Correlation Spectroscopy), that intrinsically combine spatial and temporal correlation. The instrumental developments will be conducted around a Spatial Light Modulator (SLM). This is an optical device that makes it possible to create, from a single input laser beam, several illumination spots, thanks to the diffraction through a computer-addressed phase grating. It will also be possible to apply the same original technique of illumination control to FRAP measurements. Beyond two observation volumes we will use for detection, instead of Avalanche PhotoDiodes (APD), an Electron-Multiplied CCD camera (EM-CCD). With this camera it will also be possible to acquire, at high speed, wide field fluorescent images, to perform spatial and temporal ICS like analysis. These instrumental techniques will be applied to the study of the structural modifications of the nucleus in response to a thermal stress. We will be interested in the transcription factor HSF1 (Heat Shock Factor 1) that relocalizes, when heat shock, in a limited number of nuclear structures termed Nuclear Stress Bodies and also in the textural modifications of nuclear chromatin. The response to stress is a biological model especially interesting for a general study of gene expression and of modifications of chromatin structure as a response to external events.

Project coordination

Antoine DELON (Université)

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

Help of the ANR 250,000 euros
Beginning and duration of the scientific project: - 36 Months

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