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An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis.

Mari M, Griffith J, Rieter E, Krishnappa L, Klionsky DJ, Reggiori F - J. Cell Biol. (2010)

Bottom Line: Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations.The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes.We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Institute of Biomembranes, University Medical Centre Utrecht, Utrecht University, 3584 CX Utrecht, The Netherlands.

ABSTRACT
Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations. The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes. We found that yeast Atg9 concentrates in a novel compartment comprising clusters of vesicles and tubules, which are derived from the secretory pathway and are often adjacent to mitochondria. We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore. In addition, genetic analyses indicate that Atg1, Atg13, and phosphatidylinositol-3-phosphate are involved in the further rearrangement of these initial membranes. Thus, our data reveal that the Atg9-positive compartments are important for the de novo formation of the PAS and the sequestering vesicle that are the hallmarks of autophagy.

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Live cell imaging of an Atg9 reservoir becoming the PAS. Atg9-mChe GFP-Atg8 (MMY120) cells were grown to log phase and transferred to SD-N medium for 20 min before being imaged as described in Materials and methods. Sequential images acquired with a time lapse of 5 s are shown. The arrow highlights the Atg9 reservoirs that ultimately colocalize with Atg8 in the process of becoming the PAS. The complete movie reconstruction is presented in Video 1. An identical result was obtained with cells expressing endogenous Atg9-GFP (FRY172) and carrying the pCumCheV5ATG8415 plasmid, which expresses mChe-Atg8 (Video 2). DIC, differential interference contrast. Bar, 2 µm.
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fig9: Live cell imaging of an Atg9 reservoir becoming the PAS. Atg9-mChe GFP-Atg8 (MMY120) cells were grown to log phase and transferred to SD-N medium for 20 min before being imaged as described in Materials and methods. Sequential images acquired with a time lapse of 5 s are shown. The arrow highlights the Atg9 reservoirs that ultimately colocalize with Atg8 in the process of becoming the PAS. The complete movie reconstruction is presented in Video 1. An identical result was obtained with cells expressing endogenous Atg9-GFP (FRY172) and carrying the pCumCheV5ATG8415 plasmid, which expresses mChe-Atg8 (Video 2). DIC, differential interference contrast. Bar, 2 µm.

Mentions: To further demonstrate that the Atg9 reservoirs are the PAS precursor, we analyzed the evolution of this compartment by time-lapse fluorescence microscopy. At present, the primary distinguishing feature of the PAS is that most of the Atg proteins associate at least transiently with this site. Thus, the idea behind this experiment is that an Atg9 reservoir should acquire the rest of the conserved Atg proteins as it becomes a PAS. To test this hypothesis, we generated a strain expressing higher levels of Atg9-monomeric cherry (mChe) to avoid rapid bleaching of the red fluorescent protein, allowing longer recording intervals. In addition, ATG8 was genomically tagged with GFP in the same cells. We selected Atg8 as a PAS protein marker because this factor is the last Atg protein to be recruited at this site (Suzuki et al., 2007), and consequently, its presence at the PAS reflects the complete assembly of the Atg machinery. The engineered cells were then grown to logarithmic (log) phase before being transferred in SD-N medium for 20 min and imaged. Nitrogen starvation was used to induce autophagy because under these conditions, double-membrane vesicles form at a higher frequency, increasing the chance of capturing PAS biogenesis. As shown in Fig. 9 and Video 1, this approach allowed us to observe Atg9 reservoirs becoming the PAS; GFP-Atg8 was seen to move from a cytosolic location and ultimately colocalized with a reservoir. Importantly, the same result was obtained with cells expressing endogenous Atg9-GFP and carrying a plasmid expressing mChe-Atg8 (Video 2). These results demonstrate that the PAS originates from the Atg9 reservoirs, and consequently, this latter compartment supplies at least part of the phagophore membrane. They also further support the notion that the Atg9 reservoirs translocate en bloc to become the PAS.


An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis.

Mari M, Griffith J, Rieter E, Krishnappa L, Klionsky DJ, Reggiori F - J. Cell Biol. (2010)

Live cell imaging of an Atg9 reservoir becoming the PAS. Atg9-mChe GFP-Atg8 (MMY120) cells were grown to log phase and transferred to SD-N medium for 20 min before being imaged as described in Materials and methods. Sequential images acquired with a time lapse of 5 s are shown. The arrow highlights the Atg9 reservoirs that ultimately colocalize with Atg8 in the process of becoming the PAS. The complete movie reconstruction is presented in Video 1. An identical result was obtained with cells expressing endogenous Atg9-GFP (FRY172) and carrying the pCumCheV5ATG8415 plasmid, which expresses mChe-Atg8 (Video 2). DIC, differential interference contrast. Bar, 2 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3101592&req=5

fig9: Live cell imaging of an Atg9 reservoir becoming the PAS. Atg9-mChe GFP-Atg8 (MMY120) cells were grown to log phase and transferred to SD-N medium for 20 min before being imaged as described in Materials and methods. Sequential images acquired with a time lapse of 5 s are shown. The arrow highlights the Atg9 reservoirs that ultimately colocalize with Atg8 in the process of becoming the PAS. The complete movie reconstruction is presented in Video 1. An identical result was obtained with cells expressing endogenous Atg9-GFP (FRY172) and carrying the pCumCheV5ATG8415 plasmid, which expresses mChe-Atg8 (Video 2). DIC, differential interference contrast. Bar, 2 µm.
Mentions: To further demonstrate that the Atg9 reservoirs are the PAS precursor, we analyzed the evolution of this compartment by time-lapse fluorescence microscopy. At present, the primary distinguishing feature of the PAS is that most of the Atg proteins associate at least transiently with this site. Thus, the idea behind this experiment is that an Atg9 reservoir should acquire the rest of the conserved Atg proteins as it becomes a PAS. To test this hypothesis, we generated a strain expressing higher levels of Atg9-monomeric cherry (mChe) to avoid rapid bleaching of the red fluorescent protein, allowing longer recording intervals. In addition, ATG8 was genomically tagged with GFP in the same cells. We selected Atg8 as a PAS protein marker because this factor is the last Atg protein to be recruited at this site (Suzuki et al., 2007), and consequently, its presence at the PAS reflects the complete assembly of the Atg machinery. The engineered cells were then grown to logarithmic (log) phase before being transferred in SD-N medium for 20 min and imaged. Nitrogen starvation was used to induce autophagy because under these conditions, double-membrane vesicles form at a higher frequency, increasing the chance of capturing PAS biogenesis. As shown in Fig. 9 and Video 1, this approach allowed us to observe Atg9 reservoirs becoming the PAS; GFP-Atg8 was seen to move from a cytosolic location and ultimately colocalized with a reservoir. Importantly, the same result was obtained with cells expressing endogenous Atg9-GFP and carrying a plasmid expressing mChe-Atg8 (Video 2). These results demonstrate that the PAS originates from the Atg9 reservoirs, and consequently, this latter compartment supplies at least part of the phagophore membrane. They also further support the notion that the Atg9 reservoirs translocate en bloc to become the PAS.

Bottom Line: Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations.The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes.We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Institute of Biomembranes, University Medical Centre Utrecht, Utrecht University, 3584 CX Utrecht, The Netherlands.

ABSTRACT
Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations. The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes. We found that yeast Atg9 concentrates in a novel compartment comprising clusters of vesicles and tubules, which are derived from the secretory pathway and are often adjacent to mitochondria. We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore. In addition, genetic analyses indicate that Atg1, Atg13, and phosphatidylinositol-3-phosphate are involved in the further rearrangement of these initial membranes. Thus, our data reveal that the Atg9-positive compartments are important for the de novo formation of the PAS and the sequestering vesicle that are the hallmarks of autophagy.

Show MeSH
Related in: MedlinePlus