Limits...
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.

Show MeSH

Related in: MedlinePlus

RNAi of BILBO1 Disrupts Cytokinesis(A) WT and BILBO1 RNAi PF cells were scored for kinetoplast/nuclei by DAPI labelling at 0, 24 h, and 36 h postinduction (+ TET). After 36 h of induction, a large number of 2K2N cell types are produced, and the proportion of the 2K1N cell type diminishes significantly. Induced cells arrest in the 2K2N configuration. The “Round” category represents round PF cells in which neither the nucleus nor the kinetoplasts could be individually distinguished. The “Other” categories represent cells in which 2N or 2K could not be assessed.(B) The distribution of 2K2N cell types in WT and BILBO1 RNAi-induced cells (36 h). Cell morphology was scored by phase contrast microscopy and DAPI labelling as well as for the number of cells with flagella that had lost their flagellum-to-cell body attachment. The “Other” 2K2N category represents PF cells in which the position of the flagellum could not be assessed. Five distinctive 2K2N phenotypes were observed in induced PF cells: (1) 2K2N cells that appeared normal in kinetoplast and nuclear positioning (KNKN [8.96% SE ± 0.82%]); (2) KNKN cells with a loss of new flagellum-to-cell body attachment (20.56% SE ± 1.76%); (3) KKNN cells with a loss of new flagellum-to-cell body attachment (9.63% SE ± 0.63%); (4) elongated KNKN cells (18.33% SE ± 3.01%); and (5) elongated KKNN cells (41.73% SE ± 2.3%).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2365980&req=5

pbio-0060105-g003: RNAi of BILBO1 Disrupts Cytokinesis(A) WT and BILBO1 RNAi PF cells were scored for kinetoplast/nuclei by DAPI labelling at 0, 24 h, and 36 h postinduction (+ TET). After 36 h of induction, a large number of 2K2N cell types are produced, and the proportion of the 2K1N cell type diminishes significantly. Induced cells arrest in the 2K2N configuration. The “Round” category represents round PF cells in which neither the nucleus nor the kinetoplasts could be individually distinguished. The “Other” categories represent cells in which 2N or 2K could not be assessed.(B) The distribution of 2K2N cell types in WT and BILBO1 RNAi-induced cells (36 h). Cell morphology was scored by phase contrast microscopy and DAPI labelling as well as for the number of cells with flagella that had lost their flagellum-to-cell body attachment. The “Other” 2K2N category represents PF cells in which the position of the flagellum could not be assessed. Five distinctive 2K2N phenotypes were observed in induced PF cells: (1) 2K2N cells that appeared normal in kinetoplast and nuclear positioning (KNKN [8.96% SE ± 0.82%]); (2) KNKN cells with a loss of new flagellum-to-cell body attachment (20.56% SE ± 1.76%); (3) KKNN cells with a loss of new flagellum-to-cell body attachment (9.63% SE ± 0.63%); (4) elongated KNKN cells (18.33% SE ± 3.01%); and (5) elongated KKNN cells (41.73% SE ± 2.3%).

Mentions: Cell counts using DAPI-stained PF cells (Figure 3A) indicated that the ratio of cells with two kinetoplasts and two nuclei (2K2N) increased from 11.73% (standard error [SE] ± 0.63%, n = 1,542) in the nontransformed parental cell line to 24.41% (SE ± 5.73%, n = 811) in induced cells after 36 h of induction. We also detected a decrease in the population of cells with one kinetoplast and one nucleus (1K1N) from 70.24% (SE ± 2.22%, n = 1,542) in the parental cell line to 42.62% (SE ± 4.33%, n = 811) in induced cells after 36-h induction. Interestingly, only 3.57 ± 1.43% of the population were multinucleated, as compared to 1.46% ± 0.7 in the noninduced cells, indicating that induced cells do not continue through mitosis but instead undergo a cell-cycle block at the 2K2N stage (Figure 3A). Within the induced 2K2N population (Figure 3B), 60.06% (SE ± 2.76%, n = 535) of cells possessed an elongated posterior end. Furthermore, 91.04% of induced 2K2N cells had the mispositioned flagellar phenotype, 8.96% of noninduced cells had mispositioned flagella, whereas 3.36% of WT cells had this phenotype. In all BILBO1 RNAi-induced cells, mispositioned new flagella always maintained a disrupted flagellum-to-cell body attachment.


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

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

RNAi of BILBO1 Disrupts Cytokinesis(A) WT and BILBO1 RNAi PF cells were scored for kinetoplast/nuclei by DAPI labelling at 0, 24 h, and 36 h postinduction (+ TET). After 36 h of induction, a large number of 2K2N cell types are produced, and the proportion of the 2K1N cell type diminishes significantly. Induced cells arrest in the 2K2N configuration. The “Round” category represents round PF cells in which neither the nucleus nor the kinetoplasts could be individually distinguished. The “Other” categories represent cells in which 2N or 2K could not be assessed.(B) The distribution of 2K2N cell types in WT and BILBO1 RNAi-induced cells (36 h). Cell morphology was scored by phase contrast microscopy and DAPI labelling as well as for the number of cells with flagella that had lost their flagellum-to-cell body attachment. The “Other” 2K2N category represents PF cells in which the position of the flagellum could not be assessed. Five distinctive 2K2N phenotypes were observed in induced PF cells: (1) 2K2N cells that appeared normal in kinetoplast and nuclear positioning (KNKN [8.96% SE ± 0.82%]); (2) KNKN cells with a loss of new flagellum-to-cell body attachment (20.56% SE ± 1.76%); (3) KKNN cells with a loss of new flagellum-to-cell body attachment (9.63% SE ± 0.63%); (4) elongated KNKN cells (18.33% SE ± 3.01%); and (5) elongated KKNN cells (41.73% SE ± 2.3%).
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0060105-g003: RNAi of BILBO1 Disrupts Cytokinesis(A) WT and BILBO1 RNAi PF cells were scored for kinetoplast/nuclei by DAPI labelling at 0, 24 h, and 36 h postinduction (+ TET). After 36 h of induction, a large number of 2K2N cell types are produced, and the proportion of the 2K1N cell type diminishes significantly. Induced cells arrest in the 2K2N configuration. The “Round” category represents round PF cells in which neither the nucleus nor the kinetoplasts could be individually distinguished. The “Other” categories represent cells in which 2N or 2K could not be assessed.(B) The distribution of 2K2N cell types in WT and BILBO1 RNAi-induced cells (36 h). Cell morphology was scored by phase contrast microscopy and DAPI labelling as well as for the number of cells with flagella that had lost their flagellum-to-cell body attachment. The “Other” 2K2N category represents PF cells in which the position of the flagellum could not be assessed. Five distinctive 2K2N phenotypes were observed in induced PF cells: (1) 2K2N cells that appeared normal in kinetoplast and nuclear positioning (KNKN [8.96% SE ± 0.82%]); (2) KNKN cells with a loss of new flagellum-to-cell body attachment (20.56% SE ± 1.76%); (3) KKNN cells with a loss of new flagellum-to-cell body attachment (9.63% SE ± 0.63%); (4) elongated KNKN cells (18.33% SE ± 3.01%); and (5) elongated KKNN cells (41.73% SE ± 2.3%).
Mentions: Cell counts using DAPI-stained PF cells (Figure 3A) indicated that the ratio of cells with two kinetoplasts and two nuclei (2K2N) increased from 11.73% (standard error [SE] ± 0.63%, n = 1,542) in the nontransformed parental cell line to 24.41% (SE ± 5.73%, n = 811) in induced cells after 36 h of induction. We also detected a decrease in the population of cells with one kinetoplast and one nucleus (1K1N) from 70.24% (SE ± 2.22%, n = 1,542) in the parental cell line to 42.62% (SE ± 4.33%, n = 811) in induced cells after 36-h induction. Interestingly, only 3.57 ± 1.43% of the population were multinucleated, as compared to 1.46% ± 0.7 in the noninduced cells, indicating that induced cells do not continue through mitosis but instead undergo a cell-cycle block at the 2K2N stage (Figure 3A). Within the induced 2K2N population (Figure 3B), 60.06% (SE ± 2.76%, n = 535) of cells possessed an elongated posterior end. Furthermore, 91.04% of induced 2K2N cells had the mispositioned flagellar phenotype, 8.96% of noninduced cells had mispositioned flagella, whereas 3.36% of WT cells had this phenotype. In all BILBO1 RNAi-induced cells, mispositioned new flagella always maintained a disrupted flagellum-to-cell body attachment.

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