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Macrophage Receptor with Collagenous Structure (MARCO) Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway.

Hirano S, Kanno S - PLoS ONE (2015)

Bottom Line: The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells.The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2-3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta.Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways.

View Article: PubMed Central - PubMed

Affiliation: NanoTox, RCER, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan.

ABSTRACT
The Macrophage Receptor with COllagenous structure (MARCO) protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome) generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2-3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways.

No MeSH data available.


Related in: MedlinePlus

Effects of ammonia and amine compounds on conversion of LC3-I to LC3-II (lipidated LC3-I) and autophagic puncta formation.(A) Western blot analysis for the detection of LC3-I and LC3-II. GFP-MARCO-CHO cells were cultured in F12 culture medium and exposed to (NH4)3CO3. (a) The cells were cultured in the presence of 4 mM (NH4)2CO3 for 0, 2, 6, and 20 hr. (b) The cells were cultured for 6 hr in the presence of 0, 0.1, 0.4, 1, and 4 mM (NH4)2CO3. α-Tubulin was adopted as loading control. The band for LC3-I was not clear in GFP-MARCO-CHO cells. (B) Jurkat (human T cell leukemia) and J774.1 (murine macrophages) cells were exposed for 6 hr to 4 mM (NH4)2CO3 or 50 μM chloroquine. (C) GFP-MARCO-CHO cells were grown for 8 hr in the absence (Control) or presence of 4 mM (NH4)2CO3, 8 mM trimethylamine hydrochloride (TMA-HCl), or 8 mM trimethylamine (TMA). M, Western marker lane. α-Tubulin was adopted as loading control and the amount of LC3-I, LC3-II and tubulin were measured by densitometry. LC3-I/tubulin, LC3-II/tubulin, and LC3-II/LC3-I ratios were presented as mean ± SEM (N = 3). *, Significantly diferent from the control value. (D) Formation of fluorescent autophagic puncta by amines. GFP-MARCO-CHO cells were cultured for 8 hr in complete F12 culture medium in the absence (Control) or presence of 8 mM trimethylamine hydrochloride (TMA-HCl) or of 8 mM trimethylamine (TMA).
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pone.0142062.g006: Effects of ammonia and amine compounds on conversion of LC3-I to LC3-II (lipidated LC3-I) and autophagic puncta formation.(A) Western blot analysis for the detection of LC3-I and LC3-II. GFP-MARCO-CHO cells were cultured in F12 culture medium and exposed to (NH4)3CO3. (a) The cells were cultured in the presence of 4 mM (NH4)2CO3 for 0, 2, 6, and 20 hr. (b) The cells were cultured for 6 hr in the presence of 0, 0.1, 0.4, 1, and 4 mM (NH4)2CO3. α-Tubulin was adopted as loading control. The band for LC3-I was not clear in GFP-MARCO-CHO cells. (B) Jurkat (human T cell leukemia) and J774.1 (murine macrophages) cells were exposed for 6 hr to 4 mM (NH4)2CO3 or 50 μM chloroquine. (C) GFP-MARCO-CHO cells were grown for 8 hr in the absence (Control) or presence of 4 mM (NH4)2CO3, 8 mM trimethylamine hydrochloride (TMA-HCl), or 8 mM trimethylamine (TMA). M, Western marker lane. α-Tubulin was adopted as loading control and the amount of LC3-I, LC3-II and tubulin were measured by densitometry. LC3-I/tubulin, LC3-II/tubulin, and LC3-II/LC3-I ratios were presented as mean ± SEM (N = 3). *, Significantly diferent from the control value. (D) Formation of fluorescent autophagic puncta by amines. GFP-MARCO-CHO cells were cultured for 8 hr in complete F12 culture medium in the absence (Control) or presence of 8 mM trimethylamine hydrochloride (TMA-HCl) or of 8 mM trimethylamine (TMA).

Mentions: The conversion of LC3-I to its lipidated form LC3-II is a benchmark for autophagosomal maturation. Fig 6A shows that the lipidation of LC3 occurred in GFP-MARCO-CHO cells grown in F12 supplemented with (NH4)3CO3 time- and dose-dependently. The LC3 lipidation in (NH4)3CO3- or chloroquine-supplemented medium was observed in other types of cells such as Jurkat and J774.1 cells (Fig 6B). It should be noted that the amount of LC3 varies among the cell lines and LC3-I was not remarkably changed as LC3-II was increased clearly by (NH4)3CO3 and chloroquine. LC3 was lipidated in GFP-MARCO-CHO cells grown in medium supplemented with other amines such as trimethylamine (8 mM) and trimethylamine hydrochloride (8 mM) (Fig 6C). Those amines also induced autophagic small puncta (Fig 6D). Rapamycin, an inhibitor of mTOR, neither induced autophagic small puncta nor attenuated a potency of (NH4)3CO3 to induce formation of the puncta (S2 Fig).


Macrophage Receptor with Collagenous Structure (MARCO) Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway.

Hirano S, Kanno S - PLoS ONE (2015)

Effects of ammonia and amine compounds on conversion of LC3-I to LC3-II (lipidated LC3-I) and autophagic puncta formation.(A) Western blot analysis for the detection of LC3-I and LC3-II. GFP-MARCO-CHO cells were cultured in F12 culture medium and exposed to (NH4)3CO3. (a) The cells were cultured in the presence of 4 mM (NH4)2CO3 for 0, 2, 6, and 20 hr. (b) The cells were cultured for 6 hr in the presence of 0, 0.1, 0.4, 1, and 4 mM (NH4)2CO3. α-Tubulin was adopted as loading control. The band for LC3-I was not clear in GFP-MARCO-CHO cells. (B) Jurkat (human T cell leukemia) and J774.1 (murine macrophages) cells were exposed for 6 hr to 4 mM (NH4)2CO3 or 50 μM chloroquine. (C) GFP-MARCO-CHO cells were grown for 8 hr in the absence (Control) or presence of 4 mM (NH4)2CO3, 8 mM trimethylamine hydrochloride (TMA-HCl), or 8 mM trimethylamine (TMA). M, Western marker lane. α-Tubulin was adopted as loading control and the amount of LC3-I, LC3-II and tubulin were measured by densitometry. LC3-I/tubulin, LC3-II/tubulin, and LC3-II/LC3-I ratios were presented as mean ± SEM (N = 3). *, Significantly diferent from the control value. (D) Formation of fluorescent autophagic puncta by amines. GFP-MARCO-CHO cells were cultured for 8 hr in complete F12 culture medium in the absence (Control) or presence of 8 mM trimethylamine hydrochloride (TMA-HCl) or of 8 mM trimethylamine (TMA).
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Related In: Results  -  Collection

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pone.0142062.g006: Effects of ammonia and amine compounds on conversion of LC3-I to LC3-II (lipidated LC3-I) and autophagic puncta formation.(A) Western blot analysis for the detection of LC3-I and LC3-II. GFP-MARCO-CHO cells were cultured in F12 culture medium and exposed to (NH4)3CO3. (a) The cells were cultured in the presence of 4 mM (NH4)2CO3 for 0, 2, 6, and 20 hr. (b) The cells were cultured for 6 hr in the presence of 0, 0.1, 0.4, 1, and 4 mM (NH4)2CO3. α-Tubulin was adopted as loading control. The band for LC3-I was not clear in GFP-MARCO-CHO cells. (B) Jurkat (human T cell leukemia) and J774.1 (murine macrophages) cells were exposed for 6 hr to 4 mM (NH4)2CO3 or 50 μM chloroquine. (C) GFP-MARCO-CHO cells were grown for 8 hr in the absence (Control) or presence of 4 mM (NH4)2CO3, 8 mM trimethylamine hydrochloride (TMA-HCl), or 8 mM trimethylamine (TMA). M, Western marker lane. α-Tubulin was adopted as loading control and the amount of LC3-I, LC3-II and tubulin were measured by densitometry. LC3-I/tubulin, LC3-II/tubulin, and LC3-II/LC3-I ratios were presented as mean ± SEM (N = 3). *, Significantly diferent from the control value. (D) Formation of fluorescent autophagic puncta by amines. GFP-MARCO-CHO cells were cultured for 8 hr in complete F12 culture medium in the absence (Control) or presence of 8 mM trimethylamine hydrochloride (TMA-HCl) or of 8 mM trimethylamine (TMA).
Mentions: The conversion of LC3-I to its lipidated form LC3-II is a benchmark for autophagosomal maturation. Fig 6A shows that the lipidation of LC3 occurred in GFP-MARCO-CHO cells grown in F12 supplemented with (NH4)3CO3 time- and dose-dependently. The LC3 lipidation in (NH4)3CO3- or chloroquine-supplemented medium was observed in other types of cells such as Jurkat and J774.1 cells (Fig 6B). It should be noted that the amount of LC3 varies among the cell lines and LC3-I was not remarkably changed as LC3-II was increased clearly by (NH4)3CO3 and chloroquine. LC3 was lipidated in GFP-MARCO-CHO cells grown in medium supplemented with other amines such as trimethylamine (8 mM) and trimethylamine hydrochloride (8 mM) (Fig 6C). Those amines also induced autophagic small puncta (Fig 6D). Rapamycin, an inhibitor of mTOR, neither induced autophagic small puncta nor attenuated a potency of (NH4)3CO3 to induce formation of the puncta (S2 Fig).

Bottom Line: The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells.The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2-3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta.Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways.

View Article: PubMed Central - PubMed

Affiliation: NanoTox, RCER, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan.

ABSTRACT
The Macrophage Receptor with COllagenous structure (MARCO) protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome) generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2-3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways.

No MeSH data available.


Related in: MedlinePlus