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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 RNAi Prevents FP Biogenesis(A). Western blot of BILBO1 RNAi-induced cells (+) probed with anti-BILBO1 antibody (upper panel) or control antibody L8C4 (anti-PFR2), 0–72 h of BILBO1 RNAi induction. This blot shows that BILBO1 protein levels diminish over time but are still present after 72 h of RNAi induction.(B) A phase-DAPI-immunofluorescence micrograph of a BILBO1 RNAi-induced 2K2N cytoskeleton probed with anti-PFR2 (L8C4) and anti-basal bodies (BBA4) (36-h induction). Basal body and kinetoplast are indicated by the arrowhead; the flagellum and site of PFR initiation are indicated by the arrow. Scale bar indicates 5 μm.(C) A micrograph of a thin section of the FP of a noninduced PF cell illustrating the transition zone (arrowhead) and the FPC (arrow).(D) A micrograph of BILBO1 RNAi-induced cell (48 h) at the new flagellum region. Note loss of flagellum-to-cell body attachment, PFR (asterisk), transition zone location (arrowhead), and absence of a FP. Scale bar indicates 500 nm.(E) Thin-section micrograph of BILBO1 RNAi-induced (48 h) cell illustrating the proximal end of the new flagellum and the absence of a FP. A portion of the basal body (BB) is located in the cell, whereas the transition zone (arrowhead) is external to the cell body. Note the presence of cytoplasmic microtubule(s) (absent in WT cells) at the proximal region of the basal body (asterisk). K, kinetoplast. Scale bar indicates 200 nm.
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pbio-0060105-g002: BILBO1 RNAi Prevents FP Biogenesis(A). Western blot of BILBO1 RNAi-induced cells (+) probed with anti-BILBO1 antibody (upper panel) or control antibody L8C4 (anti-PFR2), 0–72 h of BILBO1 RNAi induction. This blot shows that BILBO1 protein levels diminish over time but are still present after 72 h of RNAi induction.(B) A phase-DAPI-immunofluorescence micrograph of a BILBO1 RNAi-induced 2K2N cytoskeleton probed with anti-PFR2 (L8C4) and anti-basal bodies (BBA4) (36-h induction). Basal body and kinetoplast are indicated by the arrowhead; the flagellum and site of PFR initiation are indicated by the arrow. Scale bar indicates 5 μm.(C) A micrograph of a thin section of the FP of a noninduced PF cell illustrating the transition zone (arrowhead) and the FPC (arrow).(D) A micrograph of BILBO1 RNAi-induced cell (48 h) at the new flagellum region. Note loss of flagellum-to-cell body attachment, PFR (asterisk), transition zone location (arrowhead), and absence of a FP. Scale bar indicates 500 nm.(E) Thin-section micrograph of BILBO1 RNAi-induced (48 h) cell illustrating the proximal end of the new flagellum and the absence of a FP. A portion of the basal body (BB) is located in the cell, whereas the transition zone (arrowhead) is external to the cell body. Note the presence of cytoplasmic microtubule(s) (absent in WT cells) at the proximal region of the basal body (asterisk). K, kinetoplast. Scale bar indicates 200 nm.

Mentions: We used the tetracycline-inducible RNAi system to assess the function of BILBO1 in PF cells and BSF cells [29,30]. Cell growth was arrested in the PF cells after 24 h of induction, followed by cell death (as judged by a reduction in cell numbers over time) after 48–72 h of induction. In induced BSF cells, cell death, (as judged by a reduction in cell numbers over time), began after approximately 24 h of induction (Figure S1G and S1H). Note that western blot studies show that BILBO1 protein was not completely depleted in PF cells at 72 h after induction (Figure 2A). Densitometry data of PF cells indicate that at 24 h of induction, BILBO1 protein levels had dropped to 44.2% of parental levels and to 27.5% and 19.6% at 48 h and 72 h, respectively. When we observed PF cells by immunofluorescence after 36 h of BILBO1 RNAi induction, the BILBO1 signal was weak and only detectable on the mother FPC (unpublished data).


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

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

BILBO1 RNAi Prevents FP Biogenesis(A). Western blot of BILBO1 RNAi-induced cells (+) probed with anti-BILBO1 antibody (upper panel) or control antibody L8C4 (anti-PFR2), 0–72 h of BILBO1 RNAi induction. This blot shows that BILBO1 protein levels diminish over time but are still present after 72 h of RNAi induction.(B) A phase-DAPI-immunofluorescence micrograph of a BILBO1 RNAi-induced 2K2N cytoskeleton probed with anti-PFR2 (L8C4) and anti-basal bodies (BBA4) (36-h induction). Basal body and kinetoplast are indicated by the arrowhead; the flagellum and site of PFR initiation are indicated by the arrow. Scale bar indicates 5 μm.(C) A micrograph of a thin section of the FP of a noninduced PF cell illustrating the transition zone (arrowhead) and the FPC (arrow).(D) A micrograph of BILBO1 RNAi-induced cell (48 h) at the new flagellum region. Note loss of flagellum-to-cell body attachment, PFR (asterisk), transition zone location (arrowhead), and absence of a FP. Scale bar indicates 500 nm.(E) Thin-section micrograph of BILBO1 RNAi-induced (48 h) cell illustrating the proximal end of the new flagellum and the absence of a FP. A portion of the basal body (BB) is located in the cell, whereas the transition zone (arrowhead) is external to the cell body. Note the presence of cytoplasmic microtubule(s) (absent in WT cells) at the proximal region of the basal body (asterisk). K, kinetoplast. Scale bar indicates 200 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2365980&req=5

pbio-0060105-g002: BILBO1 RNAi Prevents FP Biogenesis(A). Western blot of BILBO1 RNAi-induced cells (+) probed with anti-BILBO1 antibody (upper panel) or control antibody L8C4 (anti-PFR2), 0–72 h of BILBO1 RNAi induction. This blot shows that BILBO1 protein levels diminish over time but are still present after 72 h of RNAi induction.(B) A phase-DAPI-immunofluorescence micrograph of a BILBO1 RNAi-induced 2K2N cytoskeleton probed with anti-PFR2 (L8C4) and anti-basal bodies (BBA4) (36-h induction). Basal body and kinetoplast are indicated by the arrowhead; the flagellum and site of PFR initiation are indicated by the arrow. Scale bar indicates 5 μm.(C) A micrograph of a thin section of the FP of a noninduced PF cell illustrating the transition zone (arrowhead) and the FPC (arrow).(D) A micrograph of BILBO1 RNAi-induced cell (48 h) at the new flagellum region. Note loss of flagellum-to-cell body attachment, PFR (asterisk), transition zone location (arrowhead), and absence of a FP. Scale bar indicates 500 nm.(E) Thin-section micrograph of BILBO1 RNAi-induced (48 h) cell illustrating the proximal end of the new flagellum and the absence of a FP. A portion of the basal body (BB) is located in the cell, whereas the transition zone (arrowhead) is external to the cell body. Note the presence of cytoplasmic microtubule(s) (absent in WT cells) at the proximal region of the basal body (asterisk). K, kinetoplast. Scale bar indicates 200 nm.
Mentions: We used the tetracycline-inducible RNAi system to assess the function of BILBO1 in PF cells and BSF cells [29,30]. Cell growth was arrested in the PF cells after 24 h of induction, followed by cell death (as judged by a reduction in cell numbers over time) after 48–72 h of induction. In induced BSF cells, cell death, (as judged by a reduction in cell numbers over time), began after approximately 24 h of induction (Figure S1G and S1H). Note that western blot studies show that BILBO1 protein was not completely depleted in PF cells at 72 h after induction (Figure 2A). Densitometry data of PF cells indicate that at 24 h of induction, BILBO1 protein levels had dropped to 44.2% of parental levels and to 27.5% and 19.6% at 48 h and 72 h, respectively. When we observed PF cells by immunofluorescence after 36 h of BILBO1 RNAi induction, the BILBO1 signal was weak and only detectable on the mother FPC (unpublished data).

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