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Actin, RhoA, and Rab11 participation during encystment in Entamoeba invadens.

Herrera-Martínez M, Hernández-Ramírez VI, Lagunes-Guillén AE, Chávez-Munguía B, Talamás-Rohana P - Biomed Res Int (2013)

Bottom Line: Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles.Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%.These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del I.P.N., Avenida Instituto Politécnico Nacional No. 2508, Colonia San Pedro Zacatenco, Delegación Gustavo A. Madero, 07360 México City, DF, Mexico.

ABSTRACT
In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

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Rab11 colocalizes with ENO during encystment. Trophozoites were induced to encyst by incubation in LG medium diluted to 47% containing 5% ABS. At 12, 24, 48, 72, and 96 h, aliquots were taken, fixed with 4% paraformaldehyde and blocked with 10% FBS, before staining to analyze the localization of enolase with anti-enolase mAb (1 : 50) (A-5, sc-271384 Santa Cruz Biotechnology), Rab 11 with anti-Rab-11 mAb (1 : 25) (C-19, sc-6565 Santa Cruz Biotechnology), and F-actin with Rhodamine phalloidin (1 : 50) (R415, Invitrogen). Cells were viewed with a Carl Zeiss LSM 700 microscope. Micrographs correspond to 3D optical images. Co-localization, of molecules of interest, is shown by arrows.
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fig8: Rab11 colocalizes with ENO during encystment. Trophozoites were induced to encyst by incubation in LG medium diluted to 47% containing 5% ABS. At 12, 24, 48, 72, and 96 h, aliquots were taken, fixed with 4% paraformaldehyde and blocked with 10% FBS, before staining to analyze the localization of enolase with anti-enolase mAb (1 : 50) (A-5, sc-271384 Santa Cruz Biotechnology), Rab 11 with anti-Rab-11 mAb (1 : 25) (C-19, sc-6565 Santa Cruz Biotechnology), and F-actin with Rhodamine phalloidin (1 : 50) (R415, Invitrogen). Cells were viewed with a Carl Zeiss LSM 700 microscope. Micrographs correspond to 3D optical images. Co-localization, of molecules of interest, is shown by arrows.

Mentions: Rab11 is a protein that participates in recycling during endosomes traffic; besides it participates in the transport of internalized receptors, from the Golgi to the plasma membrane. In Entamoeba a putative Golgi apparatus has been described, suggesting that it behaves similarly, with respect to vesicular transport, to Golgi apparatus present in higher eukaryotic cells [25–27]. Results obtained from the comparative genomic analysis of human Rab11 and EiRab11 (Figure 1) suggest a highly conserved vesicular trafficking machinery. During encystation of Giardia lamblia, Rab11 is involved in the transport of CWP1 to the periphery of the cyst through the actin cytoskeleton [13]. Therefore, it is possible to conclude that during encystment of E. invadens, Rab11 could also participate in the transport of proteins forming the cyst wall in this parasite. Consequently, the subcellular localizations of enolase (ENO), an enzyme found in encystment vesicles [12], F-actin, and Rab11 were determined during encystment (Figure 8) by confocal microscopy.


Actin, RhoA, and Rab11 participation during encystment in Entamoeba invadens.

Herrera-Martínez M, Hernández-Ramírez VI, Lagunes-Guillén AE, Chávez-Munguía B, Talamás-Rohana P - Biomed Res Int (2013)

Rab11 colocalizes with ENO during encystment. Trophozoites were induced to encyst by incubation in LG medium diluted to 47% containing 5% ABS. At 12, 24, 48, 72, and 96 h, aliquots were taken, fixed with 4% paraformaldehyde and blocked with 10% FBS, before staining to analyze the localization of enolase with anti-enolase mAb (1 : 50) (A-5, sc-271384 Santa Cruz Biotechnology), Rab 11 with anti-Rab-11 mAb (1 : 25) (C-19, sc-6565 Santa Cruz Biotechnology), and F-actin with Rhodamine phalloidin (1 : 50) (R415, Invitrogen). Cells were viewed with a Carl Zeiss LSM 700 microscope. Micrographs correspond to 3D optical images. Co-localization, of molecules of interest, is shown by arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3794519&req=5

fig8: Rab11 colocalizes with ENO during encystment. Trophozoites were induced to encyst by incubation in LG medium diluted to 47% containing 5% ABS. At 12, 24, 48, 72, and 96 h, aliquots were taken, fixed with 4% paraformaldehyde and blocked with 10% FBS, before staining to analyze the localization of enolase with anti-enolase mAb (1 : 50) (A-5, sc-271384 Santa Cruz Biotechnology), Rab 11 with anti-Rab-11 mAb (1 : 25) (C-19, sc-6565 Santa Cruz Biotechnology), and F-actin with Rhodamine phalloidin (1 : 50) (R415, Invitrogen). Cells were viewed with a Carl Zeiss LSM 700 microscope. Micrographs correspond to 3D optical images. Co-localization, of molecules of interest, is shown by arrows.
Mentions: Rab11 is a protein that participates in recycling during endosomes traffic; besides it participates in the transport of internalized receptors, from the Golgi to the plasma membrane. In Entamoeba a putative Golgi apparatus has been described, suggesting that it behaves similarly, with respect to vesicular transport, to Golgi apparatus present in higher eukaryotic cells [25–27]. Results obtained from the comparative genomic analysis of human Rab11 and EiRab11 (Figure 1) suggest a highly conserved vesicular trafficking machinery. During encystation of Giardia lamblia, Rab11 is involved in the transport of CWP1 to the periphery of the cyst through the actin cytoskeleton [13]. Therefore, it is possible to conclude that during encystment of E. invadens, Rab11 could also participate in the transport of proteins forming the cyst wall in this parasite. Consequently, the subcellular localizations of enolase (ENO), an enzyme found in encystment vesicles [12], F-actin, and Rab11 were determined during encystment (Figure 8) by confocal microscopy.

Bottom Line: Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles.Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%.These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del I.P.N., Avenida Instituto Politécnico Nacional No. 2508, Colonia San Pedro Zacatenco, Delegación Gustavo A. Madero, 07360 México City, DF, Mexico.

ABSTRACT
In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

Show MeSH
Related in: MedlinePlus