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. 2012 May;56(5):2666–2682. doi: 10.1128/AAC.06450-11

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Fig 4 — Immunofluorescence staining experiments confirm observations of organelle disruption seen in electron microscopy studies, demonstrating differences between N-benzoyl-2-hydroxybenzamide-resistant and wild-type parasites, both with and without N-benzoyl-2-hyroxybenzamide pressure. The images show that the imides target the secretory pathway and that the acidocalcisome/plant-like vacuole is lost with this treatment and other secretory organelles develop unusual morphology and function. Organelles whose contents are secreted and thereby implicated in association with adaptin-3β include rhoptries, acidocalcisomes/plant-like vacuoles, micronemes, and dense granules. There is marked distortion of secretory organelles, including micronemes (A), rhoptries (B), functional dense granules (C), and fragmentation and then disappearance of the acidocalcisome/plant-like vacuole (D). These abnormalities were present at 4 h and more pronounced at 24 h. Mutant parasites exhibited a similar phenotype, with more pronounced abnormalities being seen in MP-IV-1-treated mutants, with the exception of dense granule proteins reaching the parasitophorous vacuole in the mutants. These results demonstrate an effect of MP-IV-1 on organelles that receive proteins via the secretory pathway, with the most profound effect being on the acidocalcisome/plant-like vacuole. Abbreviations for antibody labeling of parasite surface or organelle: SAG1, surface antigen; GRA1, dense granules; ROP13, rhoptries; CPL, acidocalcisome/plant-like vacuole; and M2, micronemes. (E) Immunostaining for enoyl reductase, which demonstrates that the plastid may be modestly elongated in the MP-IV-1-treated parasites, especially at 24 h. Since targeting to the plastid differs from targeting to secretory organelles, this may be a secondary effect. This finding is subtle and not marked as for the micronemes, rhoptries, dense granules, and acidocalcisome/plant-like vacuole. (F) Higher magnification of merged images of acidocalcisomes/PLVs (immunostained with antibody to CPL), rhoptries (immunostained with antibody to ROP13), and micronemes (immunostained with antibody to MIC2) in wild-type (WT), N-hydroxy-2-benzamide-treated wild-type parasites (WT+MP-IV-1), insertional mutants (mut), and mutant parasites treated with N-hydroxy-2-benzimide (mut+MP-IV-1).

Fig 4 — Immunofluorescence staining experiments confirm observations of organelle disruption seen in electron microscopy studies, demonstrating differences between N-benzoyl-2-hydroxybenzamide-resistant and wild-type parasites, both with and without N-benzoyl-2-hyroxybenzamide pressure. The images show that the imides target the secretory pathway and that the acidocalcisome/plant-like vacuole is lost with this treatment and other secretory organelles develop unusual morphology and function. Organelles whose contents are secreted and thereby implicated in association with adaptin-3β include rhoptries, acidocalcisomes/plant-like vacuoles, micronemes, and dense granules. There is marked distortion of secretory organelles, including micronemes (A), rhoptries (B), functional dense granules (C), and fragmentation and then disappearance of the acidocalcisome/plant-like vacuole (D). These abnormalities were present at 4 h and more pronounced at 24 h. Mutant parasites exhibited a similar phenotype, with more pronounced abnormalities being seen in MP-IV-1-treated mutants, with the exception of dense granule proteins reaching the parasitophorous vacuole in the mutants. These results demonstrate an effect of MP-IV-1 on organelles that receive proteins via the secretory pathway, with the most profound effect being on the acidocalcisome/plant-like vacuole. Abbreviations for antibody labeling of parasite surface or organelle: SAG1, surface antigen; GRA1, dense granules; ROP13, rhoptries; CPL, acidocalcisome/plant-like vacuole; and M2, micronemes. (E) Immunostaining for enoyl reductase, which demonstrates that the plastid may be modestly elongated in the MP-IV-1-treated parasites, especially at 24 h. Since targeting to the plastid differs from targeting to secretory organelles, this may be a secondary effect. This finding is subtle and not marked as for the micronemes, rhoptries, dense granules, and acidocalcisome/plant-like vacuole. (F) Higher magnification of merged images of acidocalcisomes/PLVs (immunostained with antibody to CPL), rhoptries (immunostained with antibody to ROP13), and micronemes (immunostained with antibody to MIC2) in wild-type (WT), N-hydroxy-2-benzamide-treated wild-type parasites (WT+MP-IV-1), insertional mutants (mut), and mutant parasites treated with N-hydroxy-2-benzimide (mut+MP-IV-1).