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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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Related in: MedlinePlus

No Endocytotic Activity Is Associated with the New FlagellumA 2K2N WT cell (A and B) and a BILBO1 RNAi-induced cell for 36 h (C and D) were labelled with DAPI (blue) and the red fluorescent lipophilic dye FM4-64X (red). Arrowheads in (A) denote areas of endocytotic activity associated with both FPs of this cell. In (C), a large area of endocytotic activity, in the region of the old FP, is labelled, but no activity is associated with the new flagellum at the posterior end of the cell. (B and D) are DAPI-phase-fluorescence merged images of (A and C). Scale bar indicates 5 μm.
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pbio-0060105-g006: No Endocytotic Activity Is Associated with the New FlagellumA 2K2N WT cell (A and B) and a BILBO1 RNAi-induced cell for 36 h (C and D) were labelled with DAPI (blue) and the red fluorescent lipophilic dye FM4-64X (red). Arrowheads in (A) denote areas of endocytotic activity associated with both FPs of this cell. In (C), a large area of endocytotic activity, in the region of the old FP, is labelled, but no activity is associated with the new flagellum at the posterior end of the cell. (B and D) are DAPI-phase-fluorescence merged images of (A and C). Scale bar indicates 5 μm.

Mentions: To test whether induced cells were capable of orthodox endocytotic activity, we carried out live PF cell endocytosis analysis using the fixable fluorescent lipophylic dye FM4-64X. WT 2K2N cells showed strong endocytotic activity at the base of both flagella, suggesting endocytosis activity via the old and the new FP (Figure 6A and 6B). In induced cells, we observed no endocytotic activity at the site of the new flagellum but considerable activity at the old FP (Figure 6C and 6D). Additionally, induced cells at the site of the new flagellum were negative for markers of early endocytosis such as clathrin or Rab5A (Figure S2), illustrating that in the absence of the FPC, and despite the fact that the new flagellum is still formed, no endocytotic activity is associated with this new flagellum.


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

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

No Endocytotic Activity Is Associated with the New FlagellumA 2K2N WT cell (A and B) and a BILBO1 RNAi-induced cell for 36 h (C and D) were labelled with DAPI (blue) and the red fluorescent lipophilic dye FM4-64X (red). Arrowheads in (A) denote areas of endocytotic activity associated with both FPs of this cell. In (C), a large area of endocytotic activity, in the region of the old FP, is labelled, but no activity is associated with the new flagellum at the posterior end of the cell. (B and D) are DAPI-phase-fluorescence merged images of (A and C). Scale bar indicates 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0060105-g006: No Endocytotic Activity Is Associated with the New FlagellumA 2K2N WT cell (A and B) and a BILBO1 RNAi-induced cell for 36 h (C and D) were labelled with DAPI (blue) and the red fluorescent lipophilic dye FM4-64X (red). Arrowheads in (A) denote areas of endocytotic activity associated with both FPs of this cell. In (C), a large area of endocytotic activity, in the region of the old FP, is labelled, but no activity is associated with the new flagellum at the posterior end of the cell. (B and D) are DAPI-phase-fluorescence merged images of (A and C). Scale bar indicates 5 μm.
Mentions: To test whether induced cells were capable of orthodox endocytotic activity, we carried out live PF cell endocytosis analysis using the fixable fluorescent lipophylic dye FM4-64X. WT 2K2N cells showed strong endocytotic activity at the base of both flagella, suggesting endocytosis activity via the old and the new FP (Figure 6A and 6B). In induced cells, we observed no endocytotic activity at the site of the new flagellum but considerable activity at the old FP (Figure 6C and 6D). Additionally, induced cells at the site of the new flagellum were negative for markers of early endocytosis such as clathrin or Rab5A (Figure S2), illustrating that in the absence of the FPC, and despite the fact that the new flagellum is still formed, no endocytotic activity is associated with this new flagellum.

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