6th ESACP Congress, Heidelberg, April 7-11, 1999

A007
FLUORESCENCE ANISOTROPY DECAY AS PROBE OF DYNAMICS OF MACROMOLECULES IN LIVING CELLS
Tramier M, Moisan I, Pansu R, Kemnitz K, Coppey, Durieux Ch, Alainquant B, Coppey M

Lab.Biochimie Acides Nucléiques, Institut Curie, Paris, ENS de Cachan, Cachan, France Europhoton, Berlin, Germany, ENS Paris, France

The measurement of fluorescence dynamics after a very short excitation pulse allows to i) increase the sensitivity of detection, ii) indentify several fluorescent species from their life-time, iii) study internal molecular dynamics of each species by anisotropy decays. This time-correlated fluorescence detection is extremely well suited for living cell studies in confocal and standard microscopy.
The rotational mobilities of green fluorescent protein alone or fused with thymidine kinase from Herpes virus were determined in different subcellular compartments of mammalian living cells. Cytoplasmic and nuclear microviscosities and the apparent volume, occupied by the chimeric protein inside cells, could be estimated. Moreover, dimers of TK-GFP in subcellular aggregates were unveiled.
Taking advantage of the differing fluorescence kinetics of ethidium molecules free or intercalated in double-stranded nucleic acids, as few as one ethidium molecule per 10 000 base pairs could be detected intercalated in nuclear DNA and/or double-stranded regions of RNA, inside living mammalian cell. Measurements of fluorescence anisotropy decay of intercalated ethidium were carried out on sub-nuclear regions in proliferative and resting cells. Using the above ultra-sensitive technique of time-correlated single photon counting, various informations of biological relevance were evidenced : i) free ethidium concentrations equilibrate between extracellular medium and nucleus, ii) a restriction of intercalation in nuclear DNA takes place in unperturbed cell, in contrast to naked DNA or permeabilized cell, iii) torsional dynamics of nuclear DNA is strongly and globaly restrained in both types of cells, likely due to protein interactions, iv) by increasing ethidium concentration inside the nucleus, the torsional dynamics representing naked DNA is recovered in neuronal cells, presumably due to enlargement of intercalation sites by protein dissociation.This is not the case in proliferative cells, where the torsional dynamics of nuclear DNA remains restrained, independent of ethidium concentration : this means that the strength of protein-DNA interactions, restraining torsional dynamics of DNA, is higher for proliferative cells than for neuronal cells.
In a near future, analogs experiments will be carried out in imaging mode, by using novel time- and space-correlated single photon-counting detectors.