Figure 6.
Figure 6. Cargo is transported from cis-Golgi to the TGN while maintained in a maturing cisterna. uso1-1 cells expressing heat-shock–inducible Axl2-GFP (cargo, green) and constitutive mRFP-Sed5 (cis-Golgi, red) and Sec7-iRFP (TGN, blue) were incubated at 37°C for 15 min and then shifted down to 24°C and observed by SCLIM. (A) A representative 3D image is shown on the left. Axl2-GFP was detected on the Golgi cisternae and the plasma membrane. Scale bar, 1 µm. Right panels show enlarged 3D images of the selected cisternae shown in boxes in A. Merged and individual images of Axl2-GFP (cargo, green), mRFP-Sed5 (cis, red), and Sec7-iRFP (TGN, blue) are shown. Scale bar, 250 nm. (B) 4D observation of cisterna #1 of A. 3D images were reconstructed from 31 optical slices 200 nm apart taken at 9.5-s intervals. Merged (cargo, cis, and TGN) and individual images are shown. Scale bar, 250 nm. Relative fluorescence intensities of cargo, cis-Golgi, and TGN markers in the cisterna are shown in the lower graph. (C) Quantification of Golgi/TGN markers and cargo Axl2 during the cisternal maturation from cis-Golgi to the TGN. Fluorescence intensities of cargo, cis-Golgi, and TGN markers of selected cisternae from 17 independent maturation events were quantified with Z-stacks collected every 9.2 s. The frames where mRFP-Sed5 signals disappeared were set as time 0. For each of the 17 maturing cisternae, the maximum fluorescence intensities of mRFP-Sed5 or Sec7-iRFP over time was set to 1 (left). For the trace of cargo, Axl2-GFP fluorescence intensity at time 0 in each of the 17 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 mRFP-Sed5 signals disappeared, the number of maturing cisternae at each time point were 6 cisternae from −128.8 to −119.6 s, 8 cisternae at −110.4 s, 11 cisternae at 101.2 s, 12 cisternae at −92.0 s, 13 cisternae at −82.8 s, 15 cisternae from −73.6 to −55.2 s, 16 cisternae from −46.0 to −36.8 s, 17 cisternae from −27.6 to 0 s, 16 cisternae at 9.2 s, 15 cisternae at 18.4 s, 13 cisternae at 27.6 s, 12 cisternae at 36.8 s, and 11 cisternae at 46.0 s. Error bars represent standard errors of means.

Cargo is transported from cis-Golgi to the TGN while maintained in a maturing cisterna. uso1-1 cells expressing heat-shock–inducible Axl2-GFP (cargo, green) and constitutive mRFP-Sed5 (cis-Golgi, red) and Sec7-iRFP (TGN, blue) were incubated at 37°C for 15 min and then shifted down to 24°C and observed by SCLIM. (A) A representative 3D image is shown on the left. Axl2-GFP was detected on the Golgi cisternae and the plasma membrane. Scale bar, 1 µm. Right panels show enlarged 3D images of the selected cisternae shown in boxes in A. Merged and individual images of Axl2-GFP (cargo, green), mRFP-Sed5 (cis, red), and Sec7-iRFP (TGN, blue) are shown. Scale bar, 250 nm. (B) 4D observation of cisterna #1 of A. 3D images were reconstructed from 31 optical slices 200 nm apart taken at 9.5-s intervals. Merged (cargo, cis, and TGN) and individual images are shown. Scale bar, 250 nm. Relative fluorescence intensities of cargo, cis-Golgi, and TGN markers in the cisterna are shown in the lower graph. (C) Quantification of Golgi/TGN markers and cargo Axl2 during the cisternal maturation from cis-Golgi to the TGN. Fluorescence intensities of cargo, cis-Golgi, and TGN markers of selected cisternae from 17 independent maturation events were quantified with Z-stacks collected every 9.2 s. The frames where mRFP-Sed5 signals disappeared were set as time 0. For each of the 17 maturing cisternae, the maximum fluorescence intensities of mRFP-Sed5 or Sec7-iRFP over time was set to 1 (left). For the trace of cargo, Axl2-GFP fluorescence intensity at time 0 in each of the 17 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 mRFP-Sed5 signals disappeared, the number of maturing cisternae at each time point were 6 cisternae from −128.8 to −119.6 s, 8 cisternae at −110.4 s, 11 cisternae at 101.2 s, 12 cisternae at −92.0 s, 13 cisternae at −82.8 s, 15 cisternae from −73.6 to −55.2 s, 16 cisternae from −46.0 to −36.8 s, 17 cisternae from −27.6 to 0 s, 16 cisternae at 9.2 s, 15 cisternae at 18.4 s, 13 cisternae at 27.6 s, 12 cisternae at 36.8 s, and 11 cisternae at 46.0 s. Error bars represent standard errors of means.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal