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Biogenesis of the trypanosome endo-exocytotic organelle is cytoskeleton mediated.

Bonhivers M, Nowacki S, Landrein N, Robinson DR - PLoS Biol. (2008)

Bottom Line: Remarkably, RNA interference (RNAi)-mediated ablation of BILBO1 in insect procyclic-form parasites prevents FP biogenesis and induces vesicle accumulation, Golgi swelling, the aberrant repositioning of the new flagellum, and cell death.Cultured bloodstream-form parasites are also nonviable when subjected to BILBO1 RNAi.These results provide the first molecular evidence for cytoskeletally mediated FP biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Microbiologie Cellulaire et Moléculaire et Pathogénicité, UMR-CNRS 5234, University Bordeaux 2, Bordeaux, Cedex France.

ABSTRACT
Trypanosoma brucei is a protozoan parasite that is used as a model organism to study such biological phenomena as gene expression, protein trafficking, and cytoskeletal biogenesis. In T. brucei, endocytosis and exocytosis occur exclusively through a sequestered organelle called the flagellar pocket (FP), an invagination of the pellicular membrane. The pocket is the sole site for specific receptors thus maintaining them inaccessible to components of the innate immune system of the mammalian host. The FP is also responsible for the sorting of protective parasite glycoproteins targeted to, or recycling from, the pellicular membrane, and for the removal of host antibodies from the cell surface. Here, we describe the first characterisation of a flagellar pocket cytoskeletal protein, BILBO1. BILBO1 functions to form a cytoskeleton framework upon which the FP is made and which is also required and essential for FP biogenesis and cell survival. Remarkably, RNA interference (RNAi)-mediated ablation of BILBO1 in insect procyclic-form parasites prevents FP biogenesis and induces vesicle accumulation, Golgi swelling, the aberrant repositioning of the new flagellum, and cell death. Cultured bloodstream-form parasites are also nonviable when subjected to BILBO1 RNAi. These results provide the first molecular evidence for cytoskeletally mediated FP biogenesis.

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BILBO1 Localisation and FPC Biogenesis(A) A thin-section electron micrograph of a T. brucei PF cell. The image shows the structural characteristics of a 1K1N cell. Note; only trans-Golgi vesicles are observed, as the Golgi itself is not in the plane of the section. Asterisk (*) denotes the flagellum transition zone. Scale bar indicates 1 μm. A. axoneme; BB, basal body; G, Golgi; K, kinetoplast; M, mitochondria; N, nucleus.(B) Phase/fluorescence-micrograph of a procyclic cytoskeleton expressing BILBO1-eGFP. BILBO1-eGFP protein localises to the FPC. Scale bar indicates 2.5 μm.(C) Electron micrograph of a thin-sectioned WT cytoskeleton probed with anti-BILBO1 antiserum, followed by immunogold labelling. This illustrates the precise location of BILBO1 on the FPC. Asterisk (*) denotes the flagellum transition zone. Arrow denotes an unlabelled portion of the FPC. Scale bar indicates 100 nm.(D–G) Immunofluorescence of cytoskeletons probed with anti-BILBO1 antibody and counterstained with DAPI showing the FPC label and the duplication-segregation of the FPC during the cell cycle. Arrowhead in (F) denotes the kinetoplast, which has not completed S phase, and illustrates that the FPC is duplicated prior to kinetoplast S phase completion. Scale bar in (D–G) indicates 5 μm.
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pbio-0060105-g001: BILBO1 Localisation and FPC Biogenesis(A) A thin-section electron micrograph of a T. brucei PF cell. The image shows the structural characteristics of a 1K1N cell. Note; only trans-Golgi vesicles are observed, as the Golgi itself is not in the plane of the section. Asterisk (*) denotes the flagellum transition zone. Scale bar indicates 1 μm. A. axoneme; BB, basal body; G, Golgi; K, kinetoplast; M, mitochondria; N, nucleus.(B) Phase/fluorescence-micrograph of a procyclic cytoskeleton expressing BILBO1-eGFP. BILBO1-eGFP protein localises to the FPC. Scale bar indicates 2.5 μm.(C) Electron micrograph of a thin-sectioned WT cytoskeleton probed with anti-BILBO1 antiserum, followed by immunogold labelling. This illustrates the precise location of BILBO1 on the FPC. Asterisk (*) denotes the flagellum transition zone. Arrow denotes an unlabelled portion of the FPC. Scale bar indicates 100 nm.(D–G) Immunofluorescence of cytoskeletons probed with anti-BILBO1 antibody and counterstained with DAPI showing the FPC label and the duplication-segregation of the FPC during the cell cycle. Arrowhead in (F) denotes the kinetoplast, which has not completed S phase, and illustrates that the FPC is duplicated prior to kinetoplast S phase completion. Scale bar in (D–G) indicates 5 μm.

Mentions: T. brucei has proven to be an excellent model for the study of cytoskeletal biogenesis [10,27]. T. brucei has several single-copy organelles, such as the mitochondrion, the kinetoplast (mitochondrial genome), a single Golgi apparatus, and a single FP. The endocytotic and exocytotic activity is limited to the posterior region of the cell via the FP. Figure 1A illustrates the morphology of a PF cell and shows the overall position of the FP within the cell. In PF cells, the FP is always closely associated with the flagellum, and both structures are always located in the posterior of the cell.


Biogenesis of the trypanosome endo-exocytotic organelle is cytoskeleton mediated.

Bonhivers M, Nowacki S, Landrein N, Robinson DR - PLoS Biol. (2008)

BILBO1 Localisation and FPC Biogenesis(A) A thin-section electron micrograph of a T. brucei PF cell. The image shows the structural characteristics of a 1K1N cell. Note; only trans-Golgi vesicles are observed, as the Golgi itself is not in the plane of the section. Asterisk (*) denotes the flagellum transition zone. Scale bar indicates 1 μm. A. axoneme; BB, basal body; G, Golgi; K, kinetoplast; M, mitochondria; N, nucleus.(B) Phase/fluorescence-micrograph of a procyclic cytoskeleton expressing BILBO1-eGFP. BILBO1-eGFP protein localises to the FPC. Scale bar indicates 2.5 μm.(C) Electron micrograph of a thin-sectioned WT cytoskeleton probed with anti-BILBO1 antiserum, followed by immunogold labelling. This illustrates the precise location of BILBO1 on the FPC. Asterisk (*) denotes the flagellum transition zone. Arrow denotes an unlabelled portion of the FPC. Scale bar indicates 100 nm.(D–G) Immunofluorescence of cytoskeletons probed with anti-BILBO1 antibody and counterstained with DAPI showing the FPC label and the duplication-segregation of the FPC during the cell cycle. Arrowhead in (F) denotes the kinetoplast, which has not completed S phase, and illustrates that the FPC is duplicated prior to kinetoplast S phase completion. Scale bar in (D–G) indicates 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2365980&req=5

pbio-0060105-g001: BILBO1 Localisation and FPC Biogenesis(A) A thin-section electron micrograph of a T. brucei PF cell. The image shows the structural characteristics of a 1K1N cell. Note; only trans-Golgi vesicles are observed, as the Golgi itself is not in the plane of the section. Asterisk (*) denotes the flagellum transition zone. Scale bar indicates 1 μm. A. axoneme; BB, basal body; G, Golgi; K, kinetoplast; M, mitochondria; N, nucleus.(B) Phase/fluorescence-micrograph of a procyclic cytoskeleton expressing BILBO1-eGFP. BILBO1-eGFP protein localises to the FPC. Scale bar indicates 2.5 μm.(C) Electron micrograph of a thin-sectioned WT cytoskeleton probed with anti-BILBO1 antiserum, followed by immunogold labelling. This illustrates the precise location of BILBO1 on the FPC. Asterisk (*) denotes the flagellum transition zone. Arrow denotes an unlabelled portion of the FPC. Scale bar indicates 100 nm.(D–G) Immunofluorescence of cytoskeletons probed with anti-BILBO1 antibody and counterstained with DAPI showing the FPC label and the duplication-segregation of the FPC during the cell cycle. Arrowhead in (F) denotes the kinetoplast, which has not completed S phase, and illustrates that the FPC is duplicated prior to kinetoplast S phase completion. Scale bar in (D–G) indicates 5 μm.
Mentions: T. brucei has proven to be an excellent model for the study of cytoskeletal biogenesis [10,27]. T. brucei has several single-copy organelles, such as the mitochondrion, the kinetoplast (mitochondrial genome), a single Golgi apparatus, and a single FP. The endocytotic and exocytotic activity is limited to the posterior region of the cell via the FP. Figure 1A illustrates the morphology of a PF cell and shows the overall position of the FP within the cell. In PF cells, the FP is always closely associated with the flagellum, and both structures are always located in the posterior of the cell.

Bottom Line: Remarkably, RNA interference (RNAi)-mediated ablation of BILBO1 in insect procyclic-form parasites prevents FP biogenesis and induces vesicle accumulation, Golgi swelling, the aberrant repositioning of the new flagellum, and cell death.Cultured bloodstream-form parasites are also nonviable when subjected to BILBO1 RNAi.These results provide the first molecular evidence for cytoskeletally mediated FP biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Microbiologie Cellulaire et Moléculaire et Pathogénicité, UMR-CNRS 5234, University Bordeaux 2, Bordeaux, Cedex France.

ABSTRACT
Trypanosoma brucei is a protozoan parasite that is used as a model organism to study such biological phenomena as gene expression, protein trafficking, and cytoskeletal biogenesis. In T. brucei, endocytosis and exocytosis occur exclusively through a sequestered organelle called the flagellar pocket (FP), an invagination of the pellicular membrane. The pocket is the sole site for specific receptors thus maintaining them inaccessible to components of the innate immune system of the mammalian host. The FP is also responsible for the sorting of protective parasite glycoproteins targeted to, or recycling from, the pellicular membrane, and for the removal of host antibodies from the cell surface. Here, we describe the first characterisation of a flagellar pocket cytoskeletal protein, BILBO1. BILBO1 functions to form a cytoskeleton framework upon which the FP is made and which is also required and essential for FP biogenesis and cell survival. Remarkably, RNA interference (RNAi)-mediated ablation of BILBO1 in insect procyclic-form parasites prevents FP biogenesis and induces vesicle accumulation, Golgi swelling, the aberrant repositioning of the new flagellum, and cell death. Cultured bloodstream-form parasites are also nonviable when subjected to BILBO1 RNAi. These results provide the first molecular evidence for cytoskeletally mediated FP biogenesis.

Show MeSH
Related in: MedlinePlus