F 2500

For the investigations on the monomer exchange reaction for the dimer mutants, we used a fluorescence spectrometer (F-2500; Hitachi High Technologies, Tokyo, Japan). The excitation wavelength was 480 nm. The solution contained 20 mM Hepes, 50 mM KCl, 2 mM MgCl₂, 1 mM DTT, and 0.2 mg/mL BSA at pH 7.9. In order to prevent the adsorption of p53 to the cuvette, we coated it with the MPC polymer. After mixing 10 nM of the Alexa488-Alexa488 dimer and 80 nM of the Alexa594-Alexa594 dimer, both dissolved in the above solution, the time course of the fluorescence spectrum was measured at 25°C. We calculated FRET efficiency based on the spectrometric data, E_S, using the equation: $$E_S=\frac{I_a}{\frac{{\Phi }_a}{{\Phi }_d}{I}_d+I_a},$$ where I_a, I_d, Φ_a and Φ_d denote the fluorescence intensities and the quantum yields of the acceptor and the donor, respectively. The wavelength dependence of the spectrometer detection efficiency was corrected as previously described^{74 (link)}. The maximum intensity of the spectrum of a pure Alexa488 solution was adjusted to that of the donor fluorescence in the observed FRET spectrum. I_d was then calculated as the integrated intensity from 490 nm to 700 nm in the spectrum of the pure Alexa488 solution. I_a was calculated as the integrated intensity from 490 nm to 700 nm after subtraction of the pure Alexa488 spectrum from the observed FRET spectrum for the sample.

The uptake of cell membranes was measured by staining cells with 10 µM FM1-43 (Thermo Fisher Scientific, Tokyo, Japan), a fluorescent lipid analog. Since the nutrient medium hampered the staining, the cells were stained after the medium had been exchanged with 15 mM Na/K phosphate buffer (pH 6.4) containing 0.1 M sorbitol. Sorbitol was used to suppress the activity of contractile vacuoles^{44 (link)}. Ten minutes after staining, cells were washed twice with a Na/K phosphate buffer containing 15 mM sodium azide to suppress exocytosis. The fluorescence intensities (excitation at 470 nm and emission at 570 nm) were measured using a fluorescence spectrophotometer (F-2500, Hitachi High-Technologies, Corp., Tokyo, Japan).
To quantify the uptake of the cell membrane during phagocytosis, Dictyostelium cells (4 × 10⁶ cells/mL) were mixed with E. coli cells (1 × 10⁸ cells/mL) in Na/K phosphate buffer. Since the cell membrane of E. coli was also stained, the fluorescence intensity of E. coli cells was subtracted from that of Dictyostelium cells containing bacteria.

Ultraviolet fluorescence emitted from aromatic amino acids, especially tryptophan (Trp), was detected by monitoring the fluorescence spectrum of the protein. SA was prepared at the concentration of 2 μM in 67 mM PB. The fluorescence of each SA was measured at Ex/Em = 295/320–400 nm, and bandwidths were evaluated at the Ex/Em of 5 or 10 nm using a fluoro-spectrophotometer (F-2500, Hitachi High-Tech, Minato, Tokyo, Japan).