For FRET experiments, fluorescence images were collected using a X20 objective from the animal pole of oocytes with a confocal microscope (Leica SP1) using laser excitation at 458 nm and 488 nm. Regions of interest were drawn by hand in MetaMorph (Molecular Devices), and background fluorescence was quantified from the blank area inside the cell and subtracted ( Supplementary Fig. S6 ). The pinhole was adjusted so that the brightest cells were within the linear range of the detector with a maximum pinhole setting of 1 Airy unit. Data from cells with very dim fluorescent signals were discarded. Emission spectra were constructed over a range of wavelengths with an emission window of 2 nm. Spectra closely resembled the published spectra 46, 47 , suggesting that the fluorescent properties of these fluorescent protein variants are retained despite their incorporation into fusion proteins. FRET calculations were made using the spectral FRET method, as previously described 7, 9, 23 .