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. 2024 Mar 19;13:e92900. doi: 10.7554/eLife.92900

Figure 8. Four-dimensional (4D) dynamics and 3D distribution of Imh1, Gga1, and Apl6.

(A–H) Dual-color 4D super-resolution confocal live imaging microscopy (SCLIM) imaging of yeast cells expressing EGFP-Imh1 and Sys1-iRFP (A–D), and Gga1-GFP and Sec7-tagRFP (E–H). (A and E) Low-magnification images of the cells. The white broken lines indicate the edge of the cells. (B and F) Time-lapse images of the single cisternae (white arrows in A and D, respectively) in the cells. (C and G) Time course changes in relative fluorescence intensities (F/Fpeak) of green and red channels in B and E, respectively. (D and H) Averaged time course changes in F/Fpeak of green and red channels (mean ± SEM). Time 0 was set as the midpoint between the green and red fluorescence peaks of each cisterna (n=16 and 13 cisternae for D and H, respectively). (I and J) 3D co-localization analyses of Apl6 versus Golgi/trans-Golgi network (TGN) marker proteins. (I) GFP-Apl6 was co-expressed with Mnn9-mCherry (cis-Golgi), Sec21-2xmCherry (cis/medial-Golgi), Sys1-iRFP (trans-Golgi), and Sec7-tagRFP (TGN) and imaged by dual-color 3D SCLIM. (J) Pearson’s correlation coefficient (r) for co-localization of the indicated proteins versus Apl6. Numbers in parentheses indicate the number of cells examined. Each value represents mean ± SD. (K–O) Dual-color time-lapse SCLIM imaging of yeast cells expressing Apl6-GFP and Sys1-iRFP. (K) Low-magnification image of the cells. The white broken lines indicate the edge of the cells. (L) Time-lapse images of the single cisterna (white arrows in K) in the cell. (M) Time course changes in relative fluorescence intensities (F/Fpeak) of green and red channels in L. (N) Averaged time course changes in F/Fpeak of green and red channels (mean ± SEM). Time 0 was set as the midpoint between the green and red fluorescence peaks of each cisterna (n=13 cisternae). (O) Magnified image and line scan analyses of Apl6-GFP and Sys1-iRFP signals in the single maturing cisterna (blue rectangle in L). The F/Fpeak values (green and red channels) along the white broken lines in the upper panels are profiled in the graphs below. Scale bars: 2 μm (A, E, I, and L) and 1 μm (B, F, and L).

Figure 8—source data 1. Data used for graphs presented in Figure 8D, H, J, and N.

Figure 8.

Figure 8—figure supplement 1. Individual data of fluorescence time courses and peak-to-peak times shown in Figure 8.

Figure 8—figure supplement 1.

(A–C) Time course changes in relative fluorescence intensities (F/Fpeak) of green and red channels in six representative cisternae (cisterna 1–6) expressing Sys1-iRFP and Imh1-EGFP (A), Sec7-tagRFP and Gga1-GFP (B), and Sys1-iRFP and Apl6-GFP (C). (D) Scatter plots of the peak-to-peak times (s). Red lines with error bars indicate mean ± SEM.
Figure 8—figure supplement 2. Spatial distribution of Apl6 and Sys1 within maturing cisterna.

Figure 8—figure supplement 2.

Left panels show time-lapse super-resolution confocal live imaging microscopy (SCLIM) images of six representative cisternae (cisterna 1–6) expressing Apl6-GFP and Sys1-iRFP. Scale bars: 1 μm. Magnified images (blue rectangles) and their line scan analyses are shown in the right panels. The F/Fpeak values (green and red channels) along the white broken lines are profiled.