Figure 8.
Figure 8. Another transmembrane cargo Mid2-GFP is also transported from cis-Golgi to the TGN while maintained in a maturing cisterna. uso1-1 cells expressing heat-shock–inducible Mid2-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. Mid2-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 Mid2-GFP (cargo, green), mRFP-Sed5 (cis, red), and Sec7-iRFP (TGN, blue) are shown. Scale bar, 250 nm. (B) Representative 4D observation. 3D images were reconstructed from 26 optical slices 200 nm apart taken at 9.2-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 on the right. (C) Quantification of Golgi/TGN markers and cargo Mid2 during the cisternal maturation from cis-Golgi to the TGN. Fluorescence intensities of cargo, cis-Golgi, and TGN markers of selected cisternae from seven 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 seven maturing cisternae, the maximum fluorescence intensities of mRFP-Sed5 or Sec7-iRFP over time was set to 1 (left). For the trace of cargo, Mid2-GFP fluorescence intensity at time 0 in each of the seven 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 five cisternae at −92.0 s, six cisternae at −82.8 to −46.0 s, seven cisternae from −36.8 to 0 s, five cisternae from 9.2 to 27.6 s, and four cisternae from 36.8 to 46.0 s. Error bars represent standard errors of means. (D) Top panels show merged images of cargo (green), cis-Golgi (red), and TGN (blue) markers during the transitional period of cisternal maturation (−46.0 to 0 s) in B. Graphs show relative fluorescence profiles of cargo (green), cis-Golgi (red), and TGN (blue) markers along the white lines in the cisterna shown above. Cargo moved from the cis-Golgi zone to the TGN zone completely (−46.0 to −27.6 s, see arrowheads). Note that a small peak of cargo reappeared in the cis-Golgi zone (arrow at −9.2 s) but disappeared and merged again with TGN (0 s). An enlarged image of the red dotted region at −9.2 s is shown on the right. Arrowheads indicate the peak positions of Axl2-GFP fluorescence intensities.

Another transmembrane cargo Mid2-GFP is also transported from cis-Golgi to the TGN while maintained in a maturing cisterna. uso1-1 cells expressing heat-shock–inducible Mid2-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. Mid2-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 Mid2-GFP (cargo, green), mRFP-Sed5 (cis, red), and Sec7-iRFP (TGN, blue) are shown. Scale bar, 250 nm. (B) Representative 4D observation. 3D images were reconstructed from 26 optical slices 200 nm apart taken at 9.2-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 on the right. (C) Quantification of Golgi/TGN markers and cargo Mid2 during the cisternal maturation from cis-Golgi to the TGN. Fluorescence intensities of cargo, cis-Golgi, and TGN markers of selected cisternae from seven 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 seven maturing cisternae, the maximum fluorescence intensities of mRFP-Sed5 or Sec7-iRFP over time was set to 1 (left). For the trace of cargo, Mid2-GFP fluorescence intensity at time 0 in each of the seven 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 five cisternae at −92.0 s, six cisternae at −82.8 to −46.0 s, seven cisternae from −36.8 to 0 s, five cisternae from 9.2 to 27.6 s, and four cisternae from 36.8 to 46.0 s. Error bars represent standard errors of means. (D) Top panels show merged images of cargo (green), cis-Golgi (red), and TGN (blue) markers during the transitional period of cisternal maturation (−46.0 to 0 s) in B. Graphs show relative fluorescence profiles of cargo (green), cis-Golgi (red), and TGN (blue) markers along the white lines in the cisterna shown above. Cargo moved from the cis-Golgi zone to the TGN zone completely (−46.0 to −27.6 s, see arrowheads). Note that a small peak of cargo reappeared in the cis-Golgi zone (arrow at −9.2 s) but disappeared and merged again with TGN (0 s). An enlarged image of the red dotted region at −9.2 s is shown on the right. Arrowheads indicate the peak positions of Axl2-GFP fluorescence intensities.

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