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Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein.

López-Reyes I, García-Rivera G, Bañuelos C, Herranz S, Vincent O, López-Camarillo C, Marchat LA, Orozco E - J. Biomed. Biotechnol. (2010)

Bottom Line: The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites.In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties.The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México, CP 07360, Mexico.

ABSTRACT
Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT). Here, we showed the presence and expression of homologous ESCRT genes in Entamoeba histolytica. We cloned and expressed the Ehvps4 gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence.

No MeSH data available.


Related in: MedlinePlus

Cellular localization of exogenous EhVps4 in transfected trophozoites. Trophozoites transfected with pNEO ((a) and (b)), pEhVps4 ((c) and (d)) and pEhVps4-E211Q ((e) and (f)) were incubated with mouse anti-FLAG antibodies, treated with FITC-labeled secondary antibodies, counterstained with DAPI and analyzed through confocal laser microscopy. pEhVps4-E211Q transfected trophozoites incubated with FITC-labeled secondary antibodies were used as control ((g) and (h)). (a), (c), (e) and (g) Cells observed in phase contrast; (b), (d), (f) and (h) Merge, trophozoites observed in the green (FITC) and blue (DAPI) channels. Arrowheads, EhVps4 signal in plasma membrane and cytoplasmic dots.
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fig6: Cellular localization of exogenous EhVps4 in transfected trophozoites. Trophozoites transfected with pNEO ((a) and (b)), pEhVps4 ((c) and (d)) and pEhVps4-E211Q ((e) and (f)) were incubated with mouse anti-FLAG antibodies, treated with FITC-labeled secondary antibodies, counterstained with DAPI and analyzed through confocal laser microscopy. pEhVps4-E211Q transfected trophozoites incubated with FITC-labeled secondary antibodies were used as control ((g) and (h)). (a), (c), (e) and (g) Cells observed in phase contrast; (b), (d), (f) and (h) Merge, trophozoites observed in the green (FITC) and blue (DAPI) channels. Arrowheads, EhVps4 signal in plasma membrane and cytoplasmic dots.

Mentions: To investigate the cellular localization of exogenous wild type EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins in transfected trophozoites, we performed immunofluorescence assays using anti-FLAG antibodies (see Figure 6). In pEhVps4 transfected trophozoites, anti-FLAG antibodies detected the overexpressed exogenous protein diffuse in the cytoplasm and at the plasma membrane (see Figure 6(d)). In contrast, in mutant pEhVps4-E211Q transfected cells, signal was not observed at the plasma membrane, but it was present as cytoplasmic structures of varying sizes (see Figure 6(f)). As expected, no signal was detected by anti-FLAG antibodies in pNEO transfected cells (see Figure 6(b)). Control assays without anti-FLAG primary antibodies did not show any signal in transfected trophozoites (see Figure 6(h)).


Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein.

López-Reyes I, García-Rivera G, Bañuelos C, Herranz S, Vincent O, López-Camarillo C, Marchat LA, Orozco E - J. Biomed. Biotechnol. (2010)

Cellular localization of exogenous EhVps4 in transfected trophozoites. Trophozoites transfected with pNEO ((a) and (b)), pEhVps4 ((c) and (d)) and pEhVps4-E211Q ((e) and (f)) were incubated with mouse anti-FLAG antibodies, treated with FITC-labeled secondary antibodies, counterstained with DAPI and analyzed through confocal laser microscopy. pEhVps4-E211Q transfected trophozoites incubated with FITC-labeled secondary antibodies were used as control ((g) and (h)). (a), (c), (e) and (g) Cells observed in phase contrast; (b), (d), (f) and (h) Merge, trophozoites observed in the green (FITC) and blue (DAPI) channels. Arrowheads, EhVps4 signal in plasma membrane and cytoplasmic dots.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2875786&req=5

fig6: Cellular localization of exogenous EhVps4 in transfected trophozoites. Trophozoites transfected with pNEO ((a) and (b)), pEhVps4 ((c) and (d)) and pEhVps4-E211Q ((e) and (f)) were incubated with mouse anti-FLAG antibodies, treated with FITC-labeled secondary antibodies, counterstained with DAPI and analyzed through confocal laser microscopy. pEhVps4-E211Q transfected trophozoites incubated with FITC-labeled secondary antibodies were used as control ((g) and (h)). (a), (c), (e) and (g) Cells observed in phase contrast; (b), (d), (f) and (h) Merge, trophozoites observed in the green (FITC) and blue (DAPI) channels. Arrowheads, EhVps4 signal in plasma membrane and cytoplasmic dots.
Mentions: To investigate the cellular localization of exogenous wild type EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins in transfected trophozoites, we performed immunofluorescence assays using anti-FLAG antibodies (see Figure 6). In pEhVps4 transfected trophozoites, anti-FLAG antibodies detected the overexpressed exogenous protein diffuse in the cytoplasm and at the plasma membrane (see Figure 6(d)). In contrast, in mutant pEhVps4-E211Q transfected cells, signal was not observed at the plasma membrane, but it was present as cytoplasmic structures of varying sizes (see Figure 6(f)). As expected, no signal was detected by anti-FLAG antibodies in pNEO transfected cells (see Figure 6(b)). Control assays without anti-FLAG primary antibodies did not show any signal in transfected trophozoites (see Figure 6(h)).

Bottom Line: The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites.In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties.The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México, CP 07360, Mexico.

ABSTRACT
Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT). Here, we showed the presence and expression of homologous ESCRT genes in Entamoeba histolytica. We cloned and expressed the Ehvps4 gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence.

No MeSH data available.


Related in: MedlinePlus