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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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RNAi Knockdown of BILBO1 Induces Loss of Basal Body–Mediated Golgi Segregation and Causes Defects of Important Cytoskeletal Structures(A–C) A nontransformed 2K2N cell probed with anti-GRASP (green) and DAPI (blue), illustrating two major GRASP signals (arrowheads) located between the segregated kinetoplasts and nuclei.(D–F) A BILBO1 RNAi-induced (36 h) 2K2N cell probed with anti-GRASP. The two GRASP signals are observed near the nuclei. Despite a limited degree of Golgi segregation, no GRASP signal is observed near the new kinetoplast. The kinetoplast and the new flagellum (asterisk) are located in the extreme posterior end of the cell.(G–I) BILBO1 and the FPC are important for cytoskeleton organisation. Immunofluorescence micrograph of a PF cytoskeleton probed with L3B2 (anti-FAZ) antibody after BILBO1 RNAi knockdown (36 h). The flagellum-to-cell body attachment is lost, and the new flagellum is located at the posterior region of the cell. No new FAZ is formed, whereas the old FAZ remains associated with the old flagellum. The kinetoplast (asterisk) is located in the extreme posterior of the cell.Scale bar indicates 5 μm.
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pbio-0060105-g004: RNAi Knockdown of BILBO1 Induces Loss of Basal Body–Mediated Golgi Segregation and Causes Defects of Important Cytoskeletal Structures(A–C) A nontransformed 2K2N cell probed with anti-GRASP (green) and DAPI (blue), illustrating two major GRASP signals (arrowheads) located between the segregated kinetoplasts and nuclei.(D–F) A BILBO1 RNAi-induced (36 h) 2K2N cell probed with anti-GRASP. The two GRASP signals are observed near the nuclei. Despite a limited degree of Golgi segregation, no GRASP signal is observed near the new kinetoplast. The kinetoplast and the new flagellum (asterisk) are located in the extreme posterior end of the cell.(G–I) BILBO1 and the FPC are important for cytoskeleton organisation. Immunofluorescence micrograph of a PF cytoskeleton probed with L3B2 (anti-FAZ) antibody after BILBO1 RNAi knockdown (36 h). The flagellum-to-cell body attachment is lost, and the new flagellum is located at the posterior region of the cell. No new FAZ is formed, whereas the old FAZ remains associated with the old flagellum. The kinetoplast (asterisk) is located in the extreme posterior of the cell.Scale bar indicates 5 μm.

Mentions: Electron microscopy reveals that induced PF cells possess what appear to be stacks of membranes that resemble a Golgi apparatus. These cells also amass large numbers of vesicles (Figure S1D and S1F). Since Golgi duplication in procyclic T. brucei cells involves Centrin-2 [33], and Golgi separation in T. brucei is basal body mediated [8], we wanted to test whether the observed Golgi swelling influenced Golgi duplication or segregation in induced BILBO1 RNAi-elongated cells. BILBO1-induced cells (36-h induction) were therefore probed with anti-GRASP antibody (Golgi marker) [33] and viewed by immunofluorescence to observe Golgi duplication and segregation. Similar to previous studies on WT cells, we observed two or more major separate Golgi-positive signals in all induced 2K2N phenotypes (Figure 4A–4F) [8,33]. The extended posterior portion of induced 2K2N cells varied in length; therefore, we scored Golgi signals of induced cells that were present in the extreme posterior distal half of the extension as “basal body segregation positive” and Golgi signals in the proximal anterior half as “basal body segregation negative.” In 2K2N WT cells, 98.56% (SE ± 0.26%, n = 764) were segregation positive, whereas 19.98% (SE ± 3.31%, n = 456) of induced cells (36-h induction) were segregation positive. These data indicate that in BILBO1 RNAi cells, Golgi duplication is not inhibited, but the basal body-dependent Golgi segregation machinery is disrupted. This latter observation is due, most likely, to malformations observed in the duplicated Golgi that may block the formation of essential components of the segregation machinery, but also probably related to the loss of cytoskeleton organisation and function in the absence of a FPC at the relocated posterior flagellum.


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

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

RNAi Knockdown of BILBO1 Induces Loss of Basal Body–Mediated Golgi Segregation and Causes Defects of Important Cytoskeletal Structures(A–C) A nontransformed 2K2N cell probed with anti-GRASP (green) and DAPI (blue), illustrating two major GRASP signals (arrowheads) located between the segregated kinetoplasts and nuclei.(D–F) A BILBO1 RNAi-induced (36 h) 2K2N cell probed with anti-GRASP. The two GRASP signals are observed near the nuclei. Despite a limited degree of Golgi segregation, no GRASP signal is observed near the new kinetoplast. The kinetoplast and the new flagellum (asterisk) are located in the extreme posterior end of the cell.(G–I) BILBO1 and the FPC are important for cytoskeleton organisation. Immunofluorescence micrograph of a PF cytoskeleton probed with L3B2 (anti-FAZ) antibody after BILBO1 RNAi knockdown (36 h). The flagellum-to-cell body attachment is lost, and the new flagellum is located at the posterior region of the cell. No new FAZ is formed, whereas the old FAZ remains associated with the old flagellum. The kinetoplast (asterisk) is located in the extreme posterior of the cell.Scale bar indicates 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0060105-g004: RNAi Knockdown of BILBO1 Induces Loss of Basal Body–Mediated Golgi Segregation and Causes Defects of Important Cytoskeletal Structures(A–C) A nontransformed 2K2N cell probed with anti-GRASP (green) and DAPI (blue), illustrating two major GRASP signals (arrowheads) located between the segregated kinetoplasts and nuclei.(D–F) A BILBO1 RNAi-induced (36 h) 2K2N cell probed with anti-GRASP. The two GRASP signals are observed near the nuclei. Despite a limited degree of Golgi segregation, no GRASP signal is observed near the new kinetoplast. The kinetoplast and the new flagellum (asterisk) are located in the extreme posterior end of the cell.(G–I) BILBO1 and the FPC are important for cytoskeleton organisation. Immunofluorescence micrograph of a PF cytoskeleton probed with L3B2 (anti-FAZ) antibody after BILBO1 RNAi knockdown (36 h). The flagellum-to-cell body attachment is lost, and the new flagellum is located at the posterior region of the cell. No new FAZ is formed, whereas the old FAZ remains associated with the old flagellum. The kinetoplast (asterisk) is located in the extreme posterior of the cell.Scale bar indicates 5 μm.
Mentions: Electron microscopy reveals that induced PF cells possess what appear to be stacks of membranes that resemble a Golgi apparatus. These cells also amass large numbers of vesicles (Figure S1D and S1F). Since Golgi duplication in procyclic T. brucei cells involves Centrin-2 [33], and Golgi separation in T. brucei is basal body mediated [8], we wanted to test whether the observed Golgi swelling influenced Golgi duplication or segregation in induced BILBO1 RNAi-elongated cells. BILBO1-induced cells (36-h induction) were therefore probed with anti-GRASP antibody (Golgi marker) [33] and viewed by immunofluorescence to observe Golgi duplication and segregation. Similar to previous studies on WT cells, we observed two or more major separate Golgi-positive signals in all induced 2K2N phenotypes (Figure 4A–4F) [8,33]. The extended posterior portion of induced 2K2N cells varied in length; therefore, we scored Golgi signals of induced cells that were present in the extreme posterior distal half of the extension as “basal body segregation positive” and Golgi signals in the proximal anterior half as “basal body segregation negative.” In 2K2N WT cells, 98.56% (SE ± 0.26%, n = 764) were segregation positive, whereas 19.98% (SE ± 3.31%, n = 456) of induced cells (36-h induction) were segregation positive. These data indicate that in BILBO1 RNAi cells, Golgi duplication is not inhibited, but the basal body-dependent Golgi segregation machinery is disrupted. This latter observation is due, most likely, to malformations observed in the duplicated Golgi that may block the formation of essential components of the segregation machinery, but also probably related to the loss of cytoskeleton organisation and function in the absence of a FPC at the relocated posterior 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