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mTOR regulates phagosome and entotic vacuole fission.

Krajcovic M, Krishna S, Akkari L, Joyce JA, Overholtzer M - Mol. Biol. Cell (2013)

Bottom Line: Here we find that phagosomes and entotic vacuoles undergo a late maturation step characterized by fission, which redistributes vacuolar contents into lysosomal networks.Vacuole fission is regulated by the serine/threonine protein kinase mammalian target of rapamycin complex 1 (mTORC1), which localizes to vacuole membranes surrounding engulfed cells.These data identify a late stage of phagocytosis and entosis that involves processing of large vacuoles by mTOR-regulated membrane fission.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065 Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, NY 10065 Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065 BCMB Allied Program, Weill Cornell Medical College, New York, NY 10065.

ABSTRACT
Macroendocytic vacuoles formed by phagocytosis, or the live-cell engulfment program entosis, undergo sequential steps of maturation, leading to the fusion of lysosomes that digest internalized cargo. After cargo digestion, nutrients must be exported to the cytosol, and vacuole membranes must be processed by mechanisms that remain poorly defined. Here we find that phagosomes and entotic vacuoles undergo a late maturation step characterized by fission, which redistributes vacuolar contents into lysosomal networks. Vacuole fission is regulated by the serine/threonine protein kinase mammalian target of rapamycin complex 1 (mTORC1), which localizes to vacuole membranes surrounding engulfed cells. Degrading engulfed cells supply engulfing cells with amino acids that are used in translation, and rescue cell survival and mTORC1 activity in starved macrophages and tumor cells. These data identify a late stage of phagocytosis and entosis that involves processing of large vacuoles by mTOR-regulated membrane fission.

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mTOR is recruited to lysosomal vacuoles harboring engulfed cells but not latex beads. (A) Top, mTOR (left) is recruited to corpse-containing entotic vacuole (arrow) (MCF10A), where it colocalizes with Lamp1 (right); inset, colocalization of immunostained mTOR (red) and Lamp1 (green). DAPI-stained nucleus is shown in blue. Bottom, mTOR does not recruit to bead-containing vacuole (left, arrow), which is marked by Lamp1 (right) (MCF10A); inset, merged image with DAPI-stained nucleus in blue. All are confocal microscopic images. Bars, 10 μm. (B) mTOR localizes to corpse-containing entotic vacuole (labeled corpse with arrow; 70% were positive for mTOR, n = 37) but not to bead-containing lysosomal vacuole (bead, arrow; 2.3% positive for mTOR, n = 84) in the same MCF10A cell. Top and middle, confocal images of immunofluorescence for mTOR and Lamp1; bottom, merge with DAPI-stained nucleus (blue); inset, DIC. (C) mTOR localizes to apoptotic cell phagosomes (52% positive for mTOR, n = 54) but not latex bead phagosomes (1.9% positive for mTOR, n = 154) in J774.1 macrophages. Confocal microscopic images show macrophage with an engulfed apoptotic corpse and two beads, as indicated, stained for mTOR and Lamp1 by immunofluorescence. Right, merged image with DAPI-stained nucleus (blue) and DIC.
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Figure 2: mTOR is recruited to lysosomal vacuoles harboring engulfed cells but not latex beads. (A) Top, mTOR (left) is recruited to corpse-containing entotic vacuole (arrow) (MCF10A), where it colocalizes with Lamp1 (right); inset, colocalization of immunostained mTOR (red) and Lamp1 (green). DAPI-stained nucleus is shown in blue. Bottom, mTOR does not recruit to bead-containing vacuole (left, arrow), which is marked by Lamp1 (right) (MCF10A); inset, merged image with DAPI-stained nucleus in blue. All are confocal microscopic images. Bars, 10 μm. (B) mTOR localizes to corpse-containing entotic vacuole (labeled corpse with arrow; 70% were positive for mTOR, n = 37) but not to bead-containing lysosomal vacuole (bead, arrow; 2.3% positive for mTOR, n = 84) in the same MCF10A cell. Top and middle, confocal images of immunofluorescence for mTOR and Lamp1; bottom, merge with DAPI-stained nucleus (blue); inset, DIC. (C) mTOR localizes to apoptotic cell phagosomes (52% positive for mTOR, n = 54) but not latex bead phagosomes (1.9% positive for mTOR, n = 154) in J774.1 macrophages. Confocal microscopic images show macrophage with an engulfed apoptotic corpse and two beads, as indicated, stained for mTOR and Lamp1 by immunofluorescence. Right, merged image with DAPI-stained nucleus (blue) and DIC.

Mentions: Amino acids are known to signal to mTORC1 through Rag GTPases that recruit mTORC1 to lysosomal membranes where the upstream activator Rheb resides (Sancak et al., 2008). Given that activated mTORC1 localizes to lysosomes, we examined whether mTOR would localize to phagosome and entotic vacuole membranes when reactivated by corpse degradation in amino acid–free media. Indeed, endogenous mTOR localized prominently to the lysosomal vacuoles harboring degrading entotic corpses but not to lysosomal vacuoles harboring latex beads, which did not reactivate mTORC1 (Figure 2A). Of interest, when cells were allowed to engulf both neighboring cells and latex beads, mTOR localized to the lysosomal vacuoles harboring degrading cells and not to those with engulfed beads (Figure 2B), even though RagC GTPase localized to both compartments (Supplemental Figure S4). Similarly, mTOR localized to phagosomes harboring degrading apoptotic cells in macrophages but not to those with uncoated or PS-coated latex beads, even within the same cell (Figure 2C and Supplemental Figure S1E), demonstrating compartment-specific reactivation of mTORC1, which is consistent with a recently proposed inside-out model of signaling to mTORC1 by amino acids from within lysosomes (Zoncu et al., 2011).


mTOR regulates phagosome and entotic vacuole fission.

Krajcovic M, Krishna S, Akkari L, Joyce JA, Overholtzer M - Mol. Biol. Cell (2013)

mTOR is recruited to lysosomal vacuoles harboring engulfed cells but not latex beads. (A) Top, mTOR (left) is recruited to corpse-containing entotic vacuole (arrow) (MCF10A), where it colocalizes with Lamp1 (right); inset, colocalization of immunostained mTOR (red) and Lamp1 (green). DAPI-stained nucleus is shown in blue. Bottom, mTOR does not recruit to bead-containing vacuole (left, arrow), which is marked by Lamp1 (right) (MCF10A); inset, merged image with DAPI-stained nucleus in blue. All are confocal microscopic images. Bars, 10 μm. (B) mTOR localizes to corpse-containing entotic vacuole (labeled corpse with arrow; 70% were positive for mTOR, n = 37) but not to bead-containing lysosomal vacuole (bead, arrow; 2.3% positive for mTOR, n = 84) in the same MCF10A cell. Top and middle, confocal images of immunofluorescence for mTOR and Lamp1; bottom, merge with DAPI-stained nucleus (blue); inset, DIC. (C) mTOR localizes to apoptotic cell phagosomes (52% positive for mTOR, n = 54) but not latex bead phagosomes (1.9% positive for mTOR, n = 154) in J774.1 macrophages. Confocal microscopic images show macrophage with an engulfed apoptotic corpse and two beads, as indicated, stained for mTOR and Lamp1 by immunofluorescence. Right, merged image with DAPI-stained nucleus (blue) and DIC.
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Figure 2: mTOR is recruited to lysosomal vacuoles harboring engulfed cells but not latex beads. (A) Top, mTOR (left) is recruited to corpse-containing entotic vacuole (arrow) (MCF10A), where it colocalizes with Lamp1 (right); inset, colocalization of immunostained mTOR (red) and Lamp1 (green). DAPI-stained nucleus is shown in blue. Bottom, mTOR does not recruit to bead-containing vacuole (left, arrow), which is marked by Lamp1 (right) (MCF10A); inset, merged image with DAPI-stained nucleus in blue. All are confocal microscopic images. Bars, 10 μm. (B) mTOR localizes to corpse-containing entotic vacuole (labeled corpse with arrow; 70% were positive for mTOR, n = 37) but not to bead-containing lysosomal vacuole (bead, arrow; 2.3% positive for mTOR, n = 84) in the same MCF10A cell. Top and middle, confocal images of immunofluorescence for mTOR and Lamp1; bottom, merge with DAPI-stained nucleus (blue); inset, DIC. (C) mTOR localizes to apoptotic cell phagosomes (52% positive for mTOR, n = 54) but not latex bead phagosomes (1.9% positive for mTOR, n = 154) in J774.1 macrophages. Confocal microscopic images show macrophage with an engulfed apoptotic corpse and two beads, as indicated, stained for mTOR and Lamp1 by immunofluorescence. Right, merged image with DAPI-stained nucleus (blue) and DIC.
Mentions: Amino acids are known to signal to mTORC1 through Rag GTPases that recruit mTORC1 to lysosomal membranes where the upstream activator Rheb resides (Sancak et al., 2008). Given that activated mTORC1 localizes to lysosomes, we examined whether mTOR would localize to phagosome and entotic vacuole membranes when reactivated by corpse degradation in amino acid–free media. Indeed, endogenous mTOR localized prominently to the lysosomal vacuoles harboring degrading entotic corpses but not to lysosomal vacuoles harboring latex beads, which did not reactivate mTORC1 (Figure 2A). Of interest, when cells were allowed to engulf both neighboring cells and latex beads, mTOR localized to the lysosomal vacuoles harboring degrading cells and not to those with engulfed beads (Figure 2B), even though RagC GTPase localized to both compartments (Supplemental Figure S4). Similarly, mTOR localized to phagosomes harboring degrading apoptotic cells in macrophages but not to those with uncoated or PS-coated latex beads, even within the same cell (Figure 2C and Supplemental Figure S1E), demonstrating compartment-specific reactivation of mTORC1, which is consistent with a recently proposed inside-out model of signaling to mTORC1 by amino acids from within lysosomes (Zoncu et al., 2011).

Bottom Line: Here we find that phagosomes and entotic vacuoles undergo a late maturation step characterized by fission, which redistributes vacuolar contents into lysosomal networks.Vacuole fission is regulated by the serine/threonine protein kinase mammalian target of rapamycin complex 1 (mTORC1), which localizes to vacuole membranes surrounding engulfed cells.These data identify a late stage of phagocytosis and entosis that involves processing of large vacuoles by mTOR-regulated membrane fission.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065 Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, NY 10065 Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065 BCMB Allied Program, Weill Cornell Medical College, New York, NY 10065.

ABSTRACT
Macroendocytic vacuoles formed by phagocytosis, or the live-cell engulfment program entosis, undergo sequential steps of maturation, leading to the fusion of lysosomes that digest internalized cargo. After cargo digestion, nutrients must be exported to the cytosol, and vacuole membranes must be processed by mechanisms that remain poorly defined. Here we find that phagosomes and entotic vacuoles undergo a late maturation step characterized by fission, which redistributes vacuolar contents into lysosomal networks. Vacuole fission is regulated by the serine/threonine protein kinase mammalian target of rapamycin complex 1 (mTORC1), which localizes to vacuole membranes surrounding engulfed cells. Degrading engulfed cells supply engulfing cells with amino acids that are used in translation, and rescue cell survival and mTORC1 activity in starved macrophages and tumor cells. These data identify a late stage of phagocytosis and entosis that involves processing of large vacuoles by mTOR-regulated membrane fission.

Show MeSH
Related in: MedlinePlus