Fluorescence recovery after photobleaching (FRAP). Cells expressing GFP-actin were fed with budded yeast. For the analysis shown in (b) and (c) the cells expressed also mRFP-LimEΔ. The bleached areas are circumscribed by circles. (a) Fluorescence intensities of GFP-actin at the neck of a particle from the phagosome membrane (left) to the cytoplasmic space (right). Fluorescence recovery after bleaching was plotted at 2 second intervals and its temporal succession indicated by color-coding the plots from light green to blue. The pre-bleach scan (-1 second) is demarcated by a dotted line. (b) Fluorescence intensities integrated over the areas shown in the plot of (a). In this case, recovery even exceeded the pre-bleach values. Time of the first image after a bleaching pulse was set to zero. (c) Plot of integrated fluorescence intensities similar to (b), but recorded at a stage of net disassembly of actin. After the bleaching pulse, the LimEΔ marker bound to the remaining actin structures although the incorporation of fluorescent actin subunits had ceased. For (a to c) fluorescence intensities were determined along slices = 3 pixels in width through the actin-rich zone that surrounds the concave neck of a particle. In (b, c) fluorescence intensities were normalized by setting the pre-bleach values to100, and scan positions are shown in blue within the insets. Green curves represent fluorescence intensities of GFP-actin, red curves of mRFP-LimEΔ. (d, e) A cell that switches from (d) arrest of the cup at the neck of the particle to (e) cup progression around the entire particle. Mother cell (circle) and bud (dot) are demarcated in the panels at 0 and 58 seconds showing brightfield illumination superimposed on the fluorescence images. The entire sequence is shown in Additional file 5. Time is indicated in seconds after the first frame of (d). The first bleaching pulse was set between the frames taken at 11 and 12 seconds while the cup was arrested, and the second pulse between 70 and 71 seconds after cup extension had resumed. Bar = 10 μm. (f) Look-up table showing color coding of the eight-bit intensity scale used in (d) and (e).
Clarke et al. BMC Biology 2010 8:154 doi:10.1186/1741-7007-8-154