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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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The New Flagellum of BILBO1 RNAi-Induced Cells Loses Physical Contact with the Old Flagellum Early in the Cell Cycle(A–F) Uninduced BILBO1 RNAi cytoskeletons probed with DAPI and double-labelled with anti-Flagellum Connector AB1 (green) and anti-PFR2 L8C4 (red) antibodies showing the attachment of the new flagellum to the maternal old flagellum and movement of the FC with the growth of the new flagellum.(A and D) Merged images of a 1K1N cytoskeleton in which the FC is located in the FP of the old flagellum.(B and E) Merged images of a 2K1N2F (two kinetoplasts, one nucleus, and two flagella) cytoskeleton in which the FC is present at the distal end of the new flagellum.(C and F) Merged images of a 2K2N2F postmitotic cytoskeleton in which the FC is present at the distal end of the new flagellum. (D, E, and F) are phase contrast merged images of (A, B, and C), respectively.(G–L) 2K2N2F-induced BILBO1 RNAi cytoskeletons showing the extended posterior end of the cell and the new flagellum-to-cell body attachment is disrupted. The FC is present only in the FP of the old flagellum. (J, K, and L) are phase contrast merged images of (G, H, and I), respectively.(M–R) 2K1N2F BILBO1 RNAi-induced cytoskeletons showing early stages in the phenotype in which the new flagellum is clearly not attached to the old flagellum. The FC is present only in the FP of the old flagellum. In (M and P), no PFR2 signal is detectable on the new flagellum. This new flagellum is at a very early stage of growth; it is not attached to the old flagellum and has a detached flagellum-to-cell body phenotype. The FC remains in the FP. (P, Q, and R) are phase contrast merges of (M, N, and O) respectively. Arrowheads denote the FC signal, and arrows denote the new flagellum.Scale bar indicates 5 μm.
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pbio-0060105-g005: The New Flagellum of BILBO1 RNAi-Induced Cells Loses Physical Contact with the Old Flagellum Early in the Cell Cycle(A–F) Uninduced BILBO1 RNAi cytoskeletons probed with DAPI and double-labelled with anti-Flagellum Connector AB1 (green) and anti-PFR2 L8C4 (red) antibodies showing the attachment of the new flagellum to the maternal old flagellum and movement of the FC with the growth of the new flagellum.(A and D) Merged images of a 1K1N cytoskeleton in which the FC is located in the FP of the old flagellum.(B and E) Merged images of a 2K1N2F (two kinetoplasts, one nucleus, and two flagella) cytoskeleton in which the FC is present at the distal end of the new flagellum.(C and F) Merged images of a 2K2N2F postmitotic cytoskeleton in which the FC is present at the distal end of the new flagellum. (D, E, and F) are phase contrast merged images of (A, B, and C), respectively.(G–L) 2K2N2F-induced BILBO1 RNAi cytoskeletons showing the extended posterior end of the cell and the new flagellum-to-cell body attachment is disrupted. The FC is present only in the FP of the old flagellum. (J, K, and L) are phase contrast merged images of (G, H, and I), respectively.(M–R) 2K1N2F BILBO1 RNAi-induced cytoskeletons showing early stages in the phenotype in which the new flagellum is clearly not attached to the old flagellum. The FC is present only in the FP of the old flagellum. In (M and P), no PFR2 signal is detectable on the new flagellum. This new flagellum is at a very early stage of growth; it is not attached to the old flagellum and has a detached flagellum-to-cell body phenotype. The FC remains in the FP. (P, Q, and R) are phase contrast merges of (M, N, and O) respectively. Arrowheads denote the FC signal, and arrows denote the new flagellum.Scale bar indicates 5 μm.

Mentions: AB1 labelling of noninduced cells showed the normal attachment of the new flagellum to the old flagellum within the FP, a similar observation to those published previously [40]. The immunofluorescence data presented in Figure 5A show a noninduced PF cell that has been probed with AB1 and a monoclonal antibody (L8C4) that targets the PFR. The merged immunofluorescence and phase contrast images of this cell indicate that a short new flagellum has formed, and the AB1 anti-FC staining shows the presence of the FC new-to-old flagellar attachment site. However, no L8C4 signal is observed on this new flagellum, indicating that it is only a few microns long and is located within the FP. As cells progress through the cell cycle (Figure 5B, 5C, 5E, and 5F), the new flagellum emerges from the FP. The distal tip of the new flagellum remains attached to, and moves along, the old flagellum (Figure 5A–5C).


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

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

The New Flagellum of BILBO1 RNAi-Induced Cells Loses Physical Contact with the Old Flagellum Early in the Cell Cycle(A–F) Uninduced BILBO1 RNAi cytoskeletons probed with DAPI and double-labelled with anti-Flagellum Connector AB1 (green) and anti-PFR2 L8C4 (red) antibodies showing the attachment of the new flagellum to the maternal old flagellum and movement of the FC with the growth of the new flagellum.(A and D) Merged images of a 1K1N cytoskeleton in which the FC is located in the FP of the old flagellum.(B and E) Merged images of a 2K1N2F (two kinetoplasts, one nucleus, and two flagella) cytoskeleton in which the FC is present at the distal end of the new flagellum.(C and F) Merged images of a 2K2N2F postmitotic cytoskeleton in which the FC is present at the distal end of the new flagellum. (D, E, and F) are phase contrast merged images of (A, B, and C), respectively.(G–L) 2K2N2F-induced BILBO1 RNAi cytoskeletons showing the extended posterior end of the cell and the new flagellum-to-cell body attachment is disrupted. The FC is present only in the FP of the old flagellum. (J, K, and L) are phase contrast merged images of (G, H, and I), respectively.(M–R) 2K1N2F BILBO1 RNAi-induced cytoskeletons showing early stages in the phenotype in which the new flagellum is clearly not attached to the old flagellum. The FC is present only in the FP of the old flagellum. In (M and P), no PFR2 signal is detectable on the new flagellum. This new flagellum is at a very early stage of growth; it is not attached to the old flagellum and has a detached flagellum-to-cell body phenotype. The FC remains in the FP. (P, Q, and R) are phase contrast merges of (M, N, and O) respectively. Arrowheads denote the FC signal, and arrows denote the new flagellum.Scale bar indicates 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2365980&req=5

pbio-0060105-g005: The New Flagellum of BILBO1 RNAi-Induced Cells Loses Physical Contact with the Old Flagellum Early in the Cell Cycle(A–F) Uninduced BILBO1 RNAi cytoskeletons probed with DAPI and double-labelled with anti-Flagellum Connector AB1 (green) and anti-PFR2 L8C4 (red) antibodies showing the attachment of the new flagellum to the maternal old flagellum and movement of the FC with the growth of the new flagellum.(A and D) Merged images of a 1K1N cytoskeleton in which the FC is located in the FP of the old flagellum.(B and E) Merged images of a 2K1N2F (two kinetoplasts, one nucleus, and two flagella) cytoskeleton in which the FC is present at the distal end of the new flagellum.(C and F) Merged images of a 2K2N2F postmitotic cytoskeleton in which the FC is present at the distal end of the new flagellum. (D, E, and F) are phase contrast merged images of (A, B, and C), respectively.(G–L) 2K2N2F-induced BILBO1 RNAi cytoskeletons showing the extended posterior end of the cell and the new flagellum-to-cell body attachment is disrupted. The FC is present only in the FP of the old flagellum. (J, K, and L) are phase contrast merged images of (G, H, and I), respectively.(M–R) 2K1N2F BILBO1 RNAi-induced cytoskeletons showing early stages in the phenotype in which the new flagellum is clearly not attached to the old flagellum. The FC is present only in the FP of the old flagellum. In (M and P), no PFR2 signal is detectable on the new flagellum. This new flagellum is at a very early stage of growth; it is not attached to the old flagellum and has a detached flagellum-to-cell body phenotype. The FC remains in the FP. (P, Q, and R) are phase contrast merges of (M, N, and O) respectively. Arrowheads denote the FC signal, and arrows denote the new flagellum.Scale bar indicates 5 μm.
Mentions: AB1 labelling of noninduced cells showed the normal attachment of the new flagellum to the old flagellum within the FP, a similar observation to those published previously [40]. The immunofluorescence data presented in Figure 5A show a noninduced PF cell that has been probed with AB1 and a monoclonal antibody (L8C4) that targets the PFR. The merged immunofluorescence and phase contrast images of this cell indicate that a short new flagellum has formed, and the AB1 anti-FC staining shows the presence of the FC new-to-old flagellar attachment site. However, no L8C4 signal is observed on this new flagellum, indicating that it is only a few microns long and is located within the FP. As cells progress through the cell cycle (Figure 5B, 5C, 5E, and 5F), the new flagellum emerges from the FP. The distal tip of the new flagellum remains attached to, and moves along, the old flagellum (Figure 5A–5C).

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