Figure 2.
Inhibition of endocytosis blocks Golgi maturation in cells lacking AP-1/Ent5.(A) Diagram of the experimental rationale. In wild-type cells, a set of transmembrane TGN proteins, indicated in blue, recycle with the aid of AP-1/Ent5 from terminally maturing TGN cisternae to nascent TGN cisternae. In apl4Δ ent5Δ cells, which lack AP-1/Ent5, those TGN proteins presumably reach the plasma membrane in secretory vesicles and then undergo endocytic recycling to nascent TGN cisternae. Inhibition of endocytosis in an apl4Δ ent5Δ strain should trap transmembrane TGN proteins in secretory vesicles or at the plasma membrane and should therefore perturb Golgi function. (B) Inhibition of endocytosis with CK-666. Cells expressing Abp1-GFP were mock treated or incubated with CK-666 for 15 min, then incubated for 5 min with FM 4-64FX, and then imaged by confocal microscopy. Shown are average projected Z-stacks. Individual fluorescence channels are shown in grayscale with merged images on the right. To preserve diffuse signals, the images were not deconvolved. Scale bar, 2 µm. (C) Quantification of the analysis in B. Projected images were manually scored for the presence of internalized dye. At least 40 cells were analyzed for each sample. (D) A typical Golgi maturation event in an untreated cell lacking AP-1 and Ent5. apl4Δ ent5Δ cells expressing the early Golgi marker GFP-Vrg4 and the TGN marker Sec7-mScarlet were grown to mid-log phase and imaged by 4D confocal microscopy. Shown are average projected Z-stacks at the indicated time points from part 1 of Video 2. The upper row shows the complete projections, the second row shows edited projections that include only the cisterna being tracked, and the subsequent rows show the individual fluorescence channels from the edited projections. Scale bar, 2 µm. (E) Quantification of the fluorescence signals from the cisterna analyzed in D. (F) Persistence of early or TGN markers in cisternae of apl4Δ ent5Δ cells after CK-666 treatment. Cells grown to mid-log phase were treated with CK-666 for 30 min before imaging as in D. Shown are average projected Z-stacks at the indicated time points for separate cisternae from Parts 2 and 3 of Video 2. For each analyzed cisterna, the upper row shows the complete projections, and the lower row shows edited projections that include only the cisterna being tracked. Scale bars, 2 µm. (G) Quantification of the fluorescence signals from the cisternae analyzed in F. (H) Quantification of Golgi maturation events in the absence or presence of CK-666. Cells expressing GFP-Vrg4 and Sec7-mScarlet in the indicated genetic backgrounds were either mock treated or treated with CK-666 for 30 min and were imaged as in D. Individual cisternae were scored according to whether or not they matured within 3 min. At least 36 cisternae from at least 29 cells were analyzed for each strain.

Inhibition of endocytosis blocks Golgi maturation in cells lacking AP-1/Ent5. (A) Diagram of the experimental rationale. In wild-type cells, a set of transmembrane TGN proteins, indicated in blue, recycle with the aid of AP-1/Ent5 from terminally maturing TGN cisternae to nascent TGN cisternae. In apl4Δ ent5Δ cells, which lack AP-1/Ent5, those TGN proteins presumably reach the plasma membrane in secretory vesicles and then undergo endocytic recycling to nascent TGN cisternae. Inhibition of endocytosis in an apl4Δ ent5Δ strain should trap transmembrane TGN proteins in secretory vesicles or at the plasma membrane and should therefore perturb Golgi function. (B) Inhibition of endocytosis with CK-666. Cells expressing Abp1-GFP were mock treated or incubated with CK-666 for 15 min, then incubated for 5 min with FM 4-64FX, and then imaged by confocal microscopy. Shown are average projected Z-stacks. Individual fluorescence channels are shown in grayscale with merged images on the right. To preserve diffuse signals, the images were not deconvolved. Scale bar, 2 µm. (C) Quantification of the analysis in B. Projected images were manually scored for the presence of internalized dye. At least 40 cells were analyzed for each sample. (D) A typical Golgi maturation event in an untreated cell lacking AP-1 and Ent5. apl4Δ ent5Δ cells expressing the early Golgi marker GFP-Vrg4 and the TGN marker Sec7-mScarlet were grown to mid-log phase and imaged by 4D confocal microscopy. Shown are average projected Z-stacks at the indicated time points from part 1 of Video 2. The upper row shows the complete projections, the second row shows edited projections that include only the cisterna being tracked, and the subsequent rows show the individual fluorescence channels from the edited projections. Scale bar, 2 µm. (E) Quantification of the fluorescence signals from the cisterna analyzed in D. (F) Persistence of early or TGN markers in cisternae of apl4Δ ent5Δ cells after CK-666 treatment. Cells grown to mid-log phase were treated with CK-666 for 30 min before imaging as in D. Shown are average projected Z-stacks at the indicated time points for separate cisternae from Parts 2 and 3 of Video 2. For each analyzed cisterna, the upper row shows the complete projections, and the lower row shows edited projections that include only the cisterna being tracked. Scale bars, 2 µm. (G) Quantification of the fluorescence signals from the cisternae analyzed in F. (H) Quantification of Golgi maturation events in the absence or presence of CK-666. Cells expressing GFP-Vrg4 and Sec7-mScarlet in the indicated genetic backgrounds were either mock treated or treated with CK-666 for 30 min and were imaged as in D. Individual cisternae were scored according to whether or not they matured within 3 min. At least 36 cisternae from at least 29 cells were analyzed for each strain.

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