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A Vps21 endocytic module regulates autophagy.

Chen Y, Zhou F, Zou S, Yu S, Li S, Li D, Song J, Li H, He Z, Hu B, Björn LO, Lipatova Z, Liang Y, Xie Z, Segev N - Mol. Biol. Cell (2014)

Bottom Line: These GTPases function in modules that include guanine-nucleotide exchange factor (GEF) activators and downstream effectors.We propose that the endocytic Vps21 module also regulates autophagy.These findings support the idea that the two pathways leading to the lysosome--endocytosis and autophagy--converge through the Vps21 and Ypt7 GTPase modules.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Key Laboratory of Agricultural Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China.

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Atg1-dependent accumulation of GFP-Atg8 crescent-like structures in vps21∆ mutant cells. (A) Vps21 suppresses accumulation of GFP-Atg8 crescents in vps21∆ mutant cells under nitrogen starvation. Wild-type and vps21∆ mutant cells expressing GFP-Atg8 were transformed with a plasmid for expression of Vps21 (as in Figure 1B). The cells were shifted from rich (YPD, top) to starvation medium (SD-N, bottom), their vacuoles were stained with FM4-64, and they were analyzed by live-cell fluorescence microscopy. In wild-type and vps21∆ mutant cells grown in rich medium (YPD), GFP-Atg8 is diffuse and occasionally localizes to a single dot next to the vacuole. In wild-type cells starved for nitrogen (SD-N), GFP-Atg8 localizes inside the vacuole membrane, which is marked by FM4-64, and to a single dot of the AP per cell. However, whereas GFP-Atg8 can be seen in the vacuole of some vps21∆ mutant cells under nitrogen starvation, in ∼45% of these cells, GFP-Atg8 accumulates in crescent-like structures, which overlaps with the FM4-64 signal. Expression of Vps21 in mutant cells suppresses this accumulation (number of cells visualized for each strain: >300 in YPD; >800 in SD-N). Left to right, PhC, GFP-Atg8, FM4-64, merge, and percentage of cells with a GFP-Atg8 crescent. Arrows point to GFP-Atg8 crescents in vps21∆ mutant cells. (B) Accumulation of GFP-Atg8 crescents in vps21∆ mutant cells depends on Atg1. ATG1 was deleted in wild-type and vps21∆ mutant cells. GFP-Atg8 accumulation and vacuolar morphology were determined as described in A. In atg1∆ mutant cells, GFP-Atg8 accumulates in a single dot outside the vacuole, and crescent-like structures of GFP-Atg8 were not observed in the atg1∆ vps21∆ double-mutant cells (number of cells visualized for each strain, >500). Bar, 2 μm. Results represent two independent experiments.
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Figure 2: Atg1-dependent accumulation of GFP-Atg8 crescent-like structures in vps21∆ mutant cells. (A) Vps21 suppresses accumulation of GFP-Atg8 crescents in vps21∆ mutant cells under nitrogen starvation. Wild-type and vps21∆ mutant cells expressing GFP-Atg8 were transformed with a plasmid for expression of Vps21 (as in Figure 1B). The cells were shifted from rich (YPD, top) to starvation medium (SD-N, bottom), their vacuoles were stained with FM4-64, and they were analyzed by live-cell fluorescence microscopy. In wild-type and vps21∆ mutant cells grown in rich medium (YPD), GFP-Atg8 is diffuse and occasionally localizes to a single dot next to the vacuole. In wild-type cells starved for nitrogen (SD-N), GFP-Atg8 localizes inside the vacuole membrane, which is marked by FM4-64, and to a single dot of the AP per cell. However, whereas GFP-Atg8 can be seen in the vacuole of some vps21∆ mutant cells under nitrogen starvation, in ∼45% of these cells, GFP-Atg8 accumulates in crescent-like structures, which overlaps with the FM4-64 signal. Expression of Vps21 in mutant cells suppresses this accumulation (number of cells visualized for each strain: >300 in YPD; >800 in SD-N). Left to right, PhC, GFP-Atg8, FM4-64, merge, and percentage of cells with a GFP-Atg8 crescent. Arrows point to GFP-Atg8 crescents in vps21∆ mutant cells. (B) Accumulation of GFP-Atg8 crescents in vps21∆ mutant cells depends on Atg1. ATG1 was deleted in wild-type and vps21∆ mutant cells. GFP-Atg8 accumulation and vacuolar morphology were determined as described in A. In atg1∆ mutant cells, GFP-Atg8 accumulates in a single dot outside the vacuole, and crescent-like structures of GFP-Atg8 were not observed in the atg1∆ vps21∆ double-mutant cells (number of cells visualized for each strain, >500). Bar, 2 μm. Results represent two independent experiments.

Mentions: To gain insight into the autophagic step blocked in vps21∆ mutant cells under starvation, we determined the vacuolar morphology and localization of GFP-Atg8 by live-cell fluorescence microscopy. GFP-Atg8 that resides on the inner membrane of APs is delivered to the vacuole after the AP and vacuole fuse, where it is degraded (Kirisako et al., 1999; Huang et al., 2000). When grown in rich medium (YPD), GFP-Atg8 is diffuse, with an occasional dot near the vacuole of both wild-type and vps21∆ mutant cells. Under starvation (synthetic defined medium that lacks nitrogen and amino acid [SD-N]), wild-type cells generally contain one or two dots per cell of GFP-Atg8 near the vacuole representing the AP, and a substantial amount is delivered to the vacuole for degradation (as indicated by GFP fluorescence observed inside the vacuoles). In vps21∆ mutant cells starved for nitrogen, some cells contain green fluorescence in their vacuoles (Figure 2A), which is in agreement with the ∼50% processed GFP found in these cells (Figure 1B). Of importance, after 4 h of nitrogen starvation, ∼45% of the vps21∆ mutant cells accumulate GFP-Atg8 in crescent-like structures near the vacuole (stained by FM4-64). This GFP-Atg8 accumulation phenotype of vps21∆ mutant cells occurs only under starvation and can be complemented by expression of Vps21 from a plasmid (Figure 2A). A similar phenotype was observed in vps21ts mutant cells at 37°C (Supplemental Figure S1B). Analysis of the two other Rab5-related genes, YPT52 and YPT53, revealed that in cells deleted for these genes, the GFP-Atg8 pattern is similar to that seen in wild- type cells (Supplemental Figure S2D).


A Vps21 endocytic module regulates autophagy.

Chen Y, Zhou F, Zou S, Yu S, Li S, Li D, Song J, Li H, He Z, Hu B, Björn LO, Lipatova Z, Liang Y, Xie Z, Segev N - Mol. Biol. Cell (2014)

Atg1-dependent accumulation of GFP-Atg8 crescent-like structures in vps21∆ mutant cells. (A) Vps21 suppresses accumulation of GFP-Atg8 crescents in vps21∆ mutant cells under nitrogen starvation. Wild-type and vps21∆ mutant cells expressing GFP-Atg8 were transformed with a plasmid for expression of Vps21 (as in Figure 1B). The cells were shifted from rich (YPD, top) to starvation medium (SD-N, bottom), their vacuoles were stained with FM4-64, and they were analyzed by live-cell fluorescence microscopy. In wild-type and vps21∆ mutant cells grown in rich medium (YPD), GFP-Atg8 is diffuse and occasionally localizes to a single dot next to the vacuole. In wild-type cells starved for nitrogen (SD-N), GFP-Atg8 localizes inside the vacuole membrane, which is marked by FM4-64, and to a single dot of the AP per cell. However, whereas GFP-Atg8 can be seen in the vacuole of some vps21∆ mutant cells under nitrogen starvation, in ∼45% of these cells, GFP-Atg8 accumulates in crescent-like structures, which overlaps with the FM4-64 signal. Expression of Vps21 in mutant cells suppresses this accumulation (number of cells visualized for each strain: >300 in YPD; >800 in SD-N). Left to right, PhC, GFP-Atg8, FM4-64, merge, and percentage of cells with a GFP-Atg8 crescent. Arrows point to GFP-Atg8 crescents in vps21∆ mutant cells. (B) Accumulation of GFP-Atg8 crescents in vps21∆ mutant cells depends on Atg1. ATG1 was deleted in wild-type and vps21∆ mutant cells. GFP-Atg8 accumulation and vacuolar morphology were determined as described in A. In atg1∆ mutant cells, GFP-Atg8 accumulates in a single dot outside the vacuole, and crescent-like structures of GFP-Atg8 were not observed in the atg1∆ vps21∆ double-mutant cells (number of cells visualized for each strain, >500). Bar, 2 μm. Results represent two independent experiments.
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Figure 2: Atg1-dependent accumulation of GFP-Atg8 crescent-like structures in vps21∆ mutant cells. (A) Vps21 suppresses accumulation of GFP-Atg8 crescents in vps21∆ mutant cells under nitrogen starvation. Wild-type and vps21∆ mutant cells expressing GFP-Atg8 were transformed with a plasmid for expression of Vps21 (as in Figure 1B). The cells were shifted from rich (YPD, top) to starvation medium (SD-N, bottom), their vacuoles were stained with FM4-64, and they were analyzed by live-cell fluorescence microscopy. In wild-type and vps21∆ mutant cells grown in rich medium (YPD), GFP-Atg8 is diffuse and occasionally localizes to a single dot next to the vacuole. In wild-type cells starved for nitrogen (SD-N), GFP-Atg8 localizes inside the vacuole membrane, which is marked by FM4-64, and to a single dot of the AP per cell. However, whereas GFP-Atg8 can be seen in the vacuole of some vps21∆ mutant cells under nitrogen starvation, in ∼45% of these cells, GFP-Atg8 accumulates in crescent-like structures, which overlaps with the FM4-64 signal. Expression of Vps21 in mutant cells suppresses this accumulation (number of cells visualized for each strain: >300 in YPD; >800 in SD-N). Left to right, PhC, GFP-Atg8, FM4-64, merge, and percentage of cells with a GFP-Atg8 crescent. Arrows point to GFP-Atg8 crescents in vps21∆ mutant cells. (B) Accumulation of GFP-Atg8 crescents in vps21∆ mutant cells depends on Atg1. ATG1 was deleted in wild-type and vps21∆ mutant cells. GFP-Atg8 accumulation and vacuolar morphology were determined as described in A. In atg1∆ mutant cells, GFP-Atg8 accumulates in a single dot outside the vacuole, and crescent-like structures of GFP-Atg8 were not observed in the atg1∆ vps21∆ double-mutant cells (number of cells visualized for each strain, >500). Bar, 2 μm. Results represent two independent experiments.
Mentions: To gain insight into the autophagic step blocked in vps21∆ mutant cells under starvation, we determined the vacuolar morphology and localization of GFP-Atg8 by live-cell fluorescence microscopy. GFP-Atg8 that resides on the inner membrane of APs is delivered to the vacuole after the AP and vacuole fuse, where it is degraded (Kirisako et al., 1999; Huang et al., 2000). When grown in rich medium (YPD), GFP-Atg8 is diffuse, with an occasional dot near the vacuole of both wild-type and vps21∆ mutant cells. Under starvation (synthetic defined medium that lacks nitrogen and amino acid [SD-N]), wild-type cells generally contain one or two dots per cell of GFP-Atg8 near the vacuole representing the AP, and a substantial amount is delivered to the vacuole for degradation (as indicated by GFP fluorescence observed inside the vacuoles). In vps21∆ mutant cells starved for nitrogen, some cells contain green fluorescence in their vacuoles (Figure 2A), which is in agreement with the ∼50% processed GFP found in these cells (Figure 1B). Of importance, after 4 h of nitrogen starvation, ∼45% of the vps21∆ mutant cells accumulate GFP-Atg8 in crescent-like structures near the vacuole (stained by FM4-64). This GFP-Atg8 accumulation phenotype of vps21∆ mutant cells occurs only under starvation and can be complemented by expression of Vps21 from a plasmid (Figure 2A). A similar phenotype was observed in vps21ts mutant cells at 37°C (Supplemental Figure S1B). Analysis of the two other Rab5-related genes, YPT52 and YPT53, revealed that in cells deleted for these genes, the GFP-Atg8 pattern is similar to that seen in wild- type cells (Supplemental Figure S2D).

Bottom Line: These GTPases function in modules that include guanine-nucleotide exchange factor (GEF) activators and downstream effectors.We propose that the endocytic Vps21 module also regulates autophagy.These findings support the idea that the two pathways leading to the lysosome--endocytosis and autophagy--converge through the Vps21 and Ypt7 GTPase modules.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Key Laboratory of Agricultural Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China.

Show MeSH
Related in: MedlinePlus