Figure 3

Coronin dynamics in particle uptake and release. Cells expressing GFP-coronin (green) and mRFP-LimEΔ (red) are exposed to budded yeast. (a) Complete uptake of budded yeast. In the frames at 0 and 85 seconds, brightfield images are superimposed on the fluorescence images to show particle shape. At 0 to 53 seconds, the extension of a cup occurs around the yeast mother cell, with a clear layering of coronin at the cytoplasmic face of the actin layer. During cup protrusion, actin precedes coronin at the edges of the cup (16 and 49 seconds, arrowheads). During expansion of the cup over the bud of the particle, the coronin label is again layered on the cytoplasmic face of the actin label (85 and 89 seconds). Subsequently, actin is disassembled (142 and 173 seconds), and finally coronin is dispersed upon closure of the cup (197 seconds). At stages of local retraction, coronin transiently replaces the actin at the rim (24 and 28 seconds, arrowheads). The complete series is shown in Additional file 4. (b) Partial uptake followed by release of budded yeast. The release starts with the green coronin label becoming prominent at the edge of the cup (0 seconds, arrowhead), followed by stratified accumulation of actin and coronin at the neck (85 and 121 seconds). Actin disappears gradually before coronin (178 seconds) before retraction of the cup is finished and the particle released (295 seconds). (c) Positions of the line scan displayed in (d) and (e) that measured the dynamics of coronin relative to actin disassembly. The scan has a width of 3 pixels. (d, e) Fluorescence intensities of (D) GFP-coronin and (E) mRFP-LimEΔ at the neck of a budded particle before its release, measured at intervals of 20 seconds. The fluorescence intensities are normalized to the highest value in the first frame of each of the two channels, which is set to 100. The scan direction from the phagosome to the cytoplasm is plotted from left to right. Because the phagosome was moving during the run, the scan positions had to be slightly readjusted to the actual border of the cup. Bars = 10 μm.