Wilhelm bernhard workshop on the cell nucleus

FLUORESCENCE ENERGY TRANSFER FOR STUDYING CHROMATIN ARRANGEMENT IN G₀/G₁ TRANSITION AND APOPTOSIS

Croce AC.¹, Lussignoli S. ¹, Bottone M.G. ², Pellicciari C. ^{1, 2}, Barni S. ^{1, 2}, Bottiroli G. <sup>1

1</sup>Istituto di Genetica Molecolare del CNR, Sezione Istochimica e Citometria, and ²Dipartimento di Biologia Animale, University of Pavia, Italy
Fluorescence Resonance Energy Transfer (FRET) is a photo-physical process by which a fluorophore (the donor, D) in an excited state transfers its energy to a neighbouring fluorophore (the acceptor, A) through a non-radiative dipole-dipole interaction. FRET occurs in a spatial range of about 120 Å, and its efficiency depends on both distance and reciprocal orientation of the dye molecules. Provided that the dyes specifically bind the target biomolecules, FRET analysis can give information on the supramolecular arrangements of the biological structures. FRET process was exploited to investigate conformational changes of chromatin in cells under different functional activity: cultured human fibroblasts in G₀ (quiescent) or G₁ (activation of genes which are specific for cell cycle progression) phases; cultured rat thymocytes, under normal conditions or undergoing spontaneous or drug induced apoptosis (chromatin condensation, DNA cleavage). Hoechst 33258 (a non-intercalating, AT specific dye) and Propidium Iodide (an intercalating, base unspecific dye) were selected as a D/A couple due to their very suitable excitation/emission spectral properties. FRET efficiency (which was defined through the enhancement of the acceptor sensitised emission) was measured in single cells by microspectrofluorometry, and proved to be higher in G₁ than in G₀ fibroblasts. The increase of FRET efficiency was already appreciable at short time after cell cycle stimulation, when changes in the proliferation marker expression statin and Ki67 are still absent. FRET imaging on single nuclei showed a different spatial distribution of FRET efficiency between G₀ and G₁ fibroblasts. In thymocytes the induction of apoptosis resulted in an increase in FRET efficiency, that preceded the occurrence of cell morphology alterations. Due to its high sensitivity, FRET analysis provides a suitable tool for detecting very early structural changes of chromatin in different physiological expression of the cell.