Figure 4.
Figure 4. Secretory cargo Axl2-GFP is transported from cis- to trans-Golgi while being maintained within a maturing cisterna. uso1-1 cells expressing heat-shock–inducible Axl2-GFP (cargo, green) and constitutive Mnn9-mCherry (cis-Golgi, red) and Sys1-iRFP (trans-Golgi, blue) were incubated at 37°C for 30 min and then shifted down to 24°C and observed by SCLIM. (A) A representative 3D image of cargo and Golgi markers is shown on the left. Axl2-GFP (cargo, green) was detected in the ER and the Golgi cisternae in the right cell and on the plasma membrane in the left cell. Scale bar, 1 µm. Right panels show enlarged 3D images of the selected cisternae with cargo in the boxed areas of the left panel. Merged and individual images of Axl2-GFP (cargo, green), Mnn9-mCherry (cis, red), and Sys1-iRFP (trans, blue) are shown. Scale bar, 250 nm. (B) A representative 4D (3D time-lapse) observation of a maturing cisterna. 3D images were reconstructed from 16 optical slices 200 nm apart around the center of the cell taken at 4.8-s intervals. The frame where Mnn9-mCherry signals disappeared was set as time 0. Merged (cargo, cis, and trans) and individual images are shown. Cargo was maintained within the Golgi cisterna, which matured from cis- to trans-Golgi. Scale bar, 250 nm. Relative fluorescence intensities of cargo, cis-Golgi, and trans-Golgi markers of the selected cisterna are shown on the right. (C) Quantification of Golgi markers and cargo during the cisternal maturation from cis- to trans-Golgi. Fluorescence intensities of cargo, cis-Golgi, and trans-Golgi markers of selected cisternae from nine independent maturation events were quantified with Z-stacks collected every 5.6 s. The frames where Mnn9-mCherry signals disappeared were set as time 0. For each of the nine maturing cisternae, the maximum fluorescence intensities of Mnn9-mCherry or Sys1-iRFP over time were set to 1 (left). For the trace of cargo, Axl2-GFP fluorescence intensity at time 0 in each of the nine experiments was normalized to 1 (right). Mean values of all the experiments per each time point were subjected to statistical analysis. Because of setting time 0 as the frames where Mnn9-mCherry signals disappeared, the number of maturing cisternae at each time point were five cisternae from −50.4 s to −44.8 s, seven cisternae at −39.2 s, eight cisternae from 33.6 s to 22.4 s, nine cisternae from −22.4 s to 0 s, eight cisternae at 5.6 s, seven cisternae at 11.2 s, and six cisternae from 16.8 s to 22.4 s. Bars represent standard errors of means.

Secretory cargo Axl2-GFP is transported from cis- to trans-Golgi while being maintained within a maturing cisterna. uso1-1 cells expressing heat-shock–inducible Axl2-GFP (cargo, green) and constitutive Mnn9-mCherry (cis-Golgi, red) and Sys1-iRFP (trans-Golgi, blue) were incubated at 37°C for 30 min and then shifted down to 24°C and observed by SCLIM. (A) A representative 3D image of cargo and Golgi markers is shown on the left. Axl2-GFP (cargo, green) was detected in the ER and the Golgi cisternae in the right cell and on the plasma membrane in the left cell. Scale bar, 1 µm. Right panels show enlarged 3D images of the selected cisternae with cargo in the boxed areas of the left panel. Merged and individual images of Axl2-GFP (cargo, green), Mnn9-mCherry (cis, red), and Sys1-iRFP (trans, blue) are shown. Scale bar, 250 nm. (B) A representative 4D (3D time-lapse) observation of a maturing cisterna. 3D images were reconstructed from 16 optical slices 200 nm apart around the center of the cell taken at 4.8-s intervals. The frame where Mnn9-mCherry signals disappeared was set as time 0. Merged (cargo, cis, and trans) and individual images are shown. Cargo was maintained within the Golgi cisterna, which matured from cis- to trans-Golgi. Scale bar, 250 nm. Relative fluorescence intensities of cargo, cis-Golgi, and trans-Golgi markers of the selected cisterna are shown on the right. (C) Quantification of Golgi markers and cargo during the cisternal maturation from cis- to trans-Golgi. Fluorescence intensities of cargo, cis-Golgi, and trans-Golgi markers of selected cisternae from nine independent maturation events were quantified with Z-stacks collected every 5.6 s. The frames where Mnn9-mCherry signals disappeared were set as time 0. For each of the nine maturing cisternae, the maximum fluorescence intensities of Mnn9-mCherry or Sys1-iRFP over time were set to 1 (left). For the trace of cargo, Axl2-GFP fluorescence intensity at time 0 in each of the nine experiments was normalized to 1 (right). Mean values of all the experiments per each time point were subjected to statistical analysis. Because of setting time 0 as the frames where Mnn9-mCherry signals disappeared, the number of maturing cisternae at each time point were five cisternae from −50.4 s to −44.8 s, seven cisternae at −39.2 s, eight cisternae from 33.6 s to 22.4 s, nine cisternae from −22.4 s to 0 s, eight cisternae at 5.6 s, seven cisternae at 11.2 s, and six cisternae from 16.8 s to 22.4 s. Bars represent standard errors of means.

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