APPLICATION OF FLUORESCENCE RESONANCE ENERGY-TRANSFER TECHNIQUES TO THE STUDY OF LECTIN-BINDING SITE DISTRIBUTION ON PARAMECIUM-PRIMAURELIA(PROTISTA, CILIOPHORA) CELL-SURFACE

Fluorescence resonance energy transfer (FRET) is a photophysical pheno menon occurring between the molecules of two fluorochromes with suitab le spectral characteristics (donor-acceptor dye pair), and consisting in an excitation energy migration through a non-radiative process. Sin ce the efficiency of the process is strictly dependent on the distance and reciprocal orientation of the donor and acceptor molecules, FRET- based techniques can be successfully applied to the study of biomolecu les and cell component organisation and distribution. These techniques have been employed in studying Paramecium primaurelia surface membran e for the reciprocal distribution of N-acetylneuraminic acid (NeuAc) a nd N-acetylglucosamine (GlcNAc) glycosidic residues, which were found to be involved in mating cell pairing. NeuAc and GlcNAc were detected by their specific binding lectins, Limulus polyphemus agglutinin (LPA) and wheat germ agglutinin (WGA), respectively. Microspectrofluorometr ic analysis afforded the choice of fluorescein isothiocyanate and Texa s red conjugated with LPA and WGA, respectively, as a suitable donor-a cceptor couple efficiently activating FRET processes. Studies pet-form ed both in solution and in cells allowed to define the experimental co nditions favourable for a FRET analysis. The comparative study carried out both on the conjugating-region and the non conjugating region of the surface membrane, indicates that FRET distribution appears quite h omogeneous in mating-competent mating type (mt) I, whereas, in mating- competent mt II cells, FRET distribution seems to be preferentially lo calised on the conjugating-region functionally involved in mating cell pairing. This difference in the distribution of lectin-binding sites is suggested to be related to mating-competence acquisition.