Localization of I-BARa-GFP during yeast uptake. (a) Uptake of an unbudded yeast. No accumulation of fluorescent IBARa at the cup that was formed around the spherical particle was detectable against the small, highly mobile clusters in the cytoplasm. (b-d) Partial uptake of budded yeast. Clusters of IBARa-GFP are indicated by arrowheads, positions of yeast mother cell and bud by circles. The IBARa protein disappeared from the neck region of the particle when uptake turned into release: 20 seconds in (b), and 10 seconds in (c). See Additional file 7 for the sequence shown in (d). In the 0 second images of (b, d) and the 74 seconds image of (c), the brightfield illumination is superimposed on the fluorescence image to show the budded yeast. (e) Quantification of fluorescence intensities of IBARa-GFP at the neck region of particles using a look-up table (color bar). Left panel, the cup in (b); middle panel, the cup in (c); right panel, the cup in (d). These images represent average projections over 10 frames (left), six frames (middle) or 14 frames (right) recorded at intervals of 0.5 seconds. For averaging, periods were selected in which the cup did not significantly move. Magnified regions of interest are shown in the inserts, demonstrating that the high fluorescence intensities are concentrated on about 2 pixels2. The pixel size is 168 × 168 nm. Time is indicated in seconds. Bar for (a-d) = 10 μm; Bar in (e) = 2 μm.