Fig. 6. Diffusion-mobility of ts045-G-FP in pre-budding complexes and GPI-FP in the ER membrane. (A) Cells expressing ts045-G and Sar1p^(H79G) were imaged before (pre-bleach) and after photobleaching of the central boxed area. Cells were imaged for five frames before bleaching a central region of the cell and monitoring recovery by measuring fluorescence intensity (arbitrary units) within the bleached area (the first post-bleach frame is defined as time zero on the x-axis). Frames were 0.677 seconds apart; bleaching was performed using four scans of a central region of interest with maximum laser power. The first post-bleach image was acquired approximately 4 seconds after the end of the bleaching step. (B) Cells expressing ts045-G-GFP and Sar1p^(H79G) at 32°C (), GPI-GFP and Sar1p^(H79G) at 32°C (), ts045-G-GFP at 39.5°C (), or ts045-G-GFP at 32°C () were analysed using fluorescence recovery after photobleaching. Data shown is the average from six cells of three independent experiments. Error bars showthe standard error of the mean (s.e.m.). (C) Cells expressing ts045-G-FP were temperature-shifted from 39.5°C to 32°C for 5 minutes and imaged by using FRAP. The cell was bleached within the boxed region and 18 seconds later imaged every 1.3 seconds. The top right hand corner of each panel shows a 2x zoom of the bleached region. Arrowheads with asterisks highlight structures that do not show fluorescence recovery, arrowhead shows a structure that shows limited recover. Arrows highlight structures that move into the bleached region during the post-bleach period. (D) Cells expressing lumFP and Sar1p^(H79G) were sequentially imaged at low laser intensity and high laser intensity (FLIP) (within the boxed region only). Fluorescence loss occurs outside the bleached region. In control cells, in which a bleach region was selected away from any cells, no significant loss of fluorescence occurs.