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. 2008 Oct;19(10):4492–4505. doi: 10.1091/mbc.E08-04-0363

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© 2008 by The American Society for Cell Biology

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Figure 3. — Detection of intravacuolar free-floating PMN vesicles by fluorescence microscopy. To trace the nuclear content of PMN vesicles a NLS-mcherry fusion protein consisting of the nuclear localization sequence of Nab2 and a tandem repeat of the fluorescent mcherry protein was used. (A) The lipase-like Atg15 protein is required for intravacuolar lysis of autophagic bodies. atg15Δ cells expressing NLS-mcherry were starved for 4 h in SD(-N) and examined with a Zeiss Axioscope2 microscope (see also Supplemental Video 1). The red fluorescent vesicles were absent in atg1Δ atg15Δ cells (A and Supplemental Video 2); bar, 5 μm. atg15Δ cells expressing NLS-mcherry and the autophagosomal marker GFP-Atg8 (B) or the cytosolic marker 3-phosphoglycerate kinase (PGK; C) showed after 4-h starvation in SD-(N) medium significantly more green than red fluorescent intravacuolar vesicles. This suggests that the red fluorescent PMN vesicles, which contain nuclear material are distinct from autophagic bodies. The images shown in B and C were taken simultaneously with a Leica TCS SP2 AOBS confocal laser scan microscope. (D) The indicated mutant cells coexpressing mcherry-NLS and the autophagic marker GFP-Atg8 were starved for 4 h in SD(-N) medium in the presence of 1 mM of the proteinase B inhibitor PMSF. The accumulation of red fluorescent PMN vesicles is in accordance with the Western blot analysis of PMN. The absence of intravacuolar PMN vesicles in vac8Δ cells, with the synchronous appearance of GFP-Atg8 in their vacuoles further confirms that PMN vesicles are distinct from autophagic bodies. Bar, 10 μm.

Figure 3. — Detection of intravacuolar free-floating PMN vesicles by fluorescence microscopy. To trace the nuclear content of PMN vesicles a NLS-mcherry fusion protein consisting of the nuclear localization sequence of Nab2 and a tandem repeat of the fluorescent mcherry protein was used. (A) The lipase-like Atg15 protein is required for intravacuolar lysis of autophagic bodies. atg15Δ cells expressing NLS-mcherry were starved for 4 h in SD(-N) and examined with a Zeiss Axioscope2 microscope (see also Supplemental Video 1). The red fluorescent vesicles were absent in atg1Δ atg15Δ cells (A and Supplemental Video 2); bar, 5 μm. atg15Δ cells expressing NLS-mcherry and the autophagosomal marker GFP-Atg8 (B) or the cytosolic marker 3-phosphoglycerate kinase (PGK; C) showed after 4-h starvation in SD-(N) medium significantly more green than red fluorescent intravacuolar vesicles. This suggests that the red fluorescent PMN vesicles, which contain nuclear material are distinct from autophagic bodies. The images shown in B and C were taken simultaneously with a Leica TCS SP2 AOBS confocal laser scan microscope. (D) The indicated mutant cells coexpressing mcherry-NLS and the autophagic marker GFP-Atg8 were starved for 4 h in SD(-N) medium in the presence of 1 mM of the proteinase B inhibitor PMSF. The accumulation of red fluorescent PMN vesicles is in accordance with the Western blot analysis of PMN. The absence of intravacuolar PMN vesicles in vac8Δ cells, with the synchronous appearance of GFP-Atg8 in their vacuoles further confirms that PMN vesicles are distinct from autophagic bodies. Bar, 10 μm.