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Imagerie des déclins de fluorescence pour l'étude de la dynamique et des interactions de macromolécules en cellules vivantes

Abstract : The aim of our work was to develop fluorescence decay imaging and to demonstrate its utility in the study of macromolecular dynamics and the interactions between macromolecules in live cells. Our approach consisted of simultaneous time-correlated single photon counting (TCSPC) and characterisation of spatial localisation of emission area along a line. Monodirectional fluorescence decay imaging was obtained that was characteristic of fluorescence kinetics in different subcellular regions. Fluorescence anisotropy decays from a small subcellular volume (1 µm3) were also measured under a microscope in confocal mode. The results show that this technological approach is useful for investigating the biology of living cells. Endogenous fluorescent labelling of proteins was carried out by GFP or spectral mutants fusion. Protein-protein interactions were studied by measuring donor fluorescence lifetime diminution (heteroFRET) or fluorescence depolarisation kinetics (homoFRET). We demonstrated the formation of (i) heterodimers of the p45 protein of NF-E2 transcription factor with two partners in different subcellular compartments by heteroFRET, and (ii) herpes simplex type 1 thymidine kinase dimer. In the latter case, homoFRET was more efficient at demonstrating dimerisation than heteroFRET. Moreover, measurement of ethidium fluorescence anisotropy decay demonstrated that DNA torsional dynamics, are restricted inside non-perturbed chromatin. Further developments in bidimensional and tri-dimensional imaging are promising.
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https://tel.archives-ouvertes.fr/tel-00003477
Contributor : Marc Tramier <>
Submitted on : Friday, October 3, 2003 - 9:10:08 PM
Last modification on : Wednesday, December 9, 2020 - 3:05:25 PM
Long-term archiving on: : Friday, April 2, 2010 - 7:07:35 PM

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  • HAL Id : tel-00003477, version 1

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Marc Tramier. Imagerie des déclins de fluorescence pour l'étude de la dynamique et des interactions de macromolécules en cellules vivantes. Biologie cellulaire. Université Pierre et Marie Curie - Paris VI, 2001. Français. ⟨tel-00003477⟩

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