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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A007
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.
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 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.