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      Basal Body Positioning Is Controlled by Flagellum Formation in Trypanosoma brucei

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          Abstract

          To perform their multiple functions, cilia and flagella are precisely positioned at the cell surface by mechanisms that remain poorly understood. The protist Trypanosoma brucei possesses a single flagellum that adheres to the cell body where a specific cytoskeletal structure is localised, the flagellum attachment zone (FAZ). Trypanosomes build a new flagellum whose distal tip is connected to the side of the old flagellum by a discrete structure, the flagella connector. During this process, the basal body of the new flagellum migrates towards the posterior end of the cell. We show that separate inhibition of flagellum assembly, base-to-tip motility or flagella connection leads to reduced basal body migration, demonstrating that the flagellum contributes to its own positioning. We propose a model where pressure applied by movements of the growing new flagellum on the flagella connector leads to a reacting force that in turn contributes to migration of the basal body at the proximal end of the flagellum.

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          Most cited references39

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          A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei.

          First-generation inducible expression vectors for Trypanosoma brucei utilized a single tetracycline-responsive promoter to drive expression of an experimental gene, in tandem with a drug-resistance marker gene to select for integration (Wirtz E, Clayton CE. Science 1995; 268:1179-1183). Because drug resistance and experimental gene expression both depended upon the activity of the regulated promoter, this approach could not be used for inducible expression of toxic products. We have now developed a dual-promoter approach, for expressing highly toxic products and generating conditional gene knock-outs, using back-to-back constitutive T7 and tetracycline-responsive PARP promoters to drive expression of the selectable marker and test gene, respectively. Transformants are readily obtained with these vectors in the absence of tetracycline, in bloodstream or procyclic T. brucei cell lines co-expressing T7 RNA polymerase and Tet repressor, and consistently show tetracycline-responsive expression through a 10(3)-10(4)-fold range. Uninduced background expression of a luciferase reporter averages no more than one molecule per cell, enabling dominant-negative approaches relying upon inducible expression of toxic products. This tight regulation also permits the production of functional gene knock-outs through regulated expression of an experimental gene in a null-mutant background.
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            A motility in the eukaryotic flagellum unrelated to flagellar beating.

            We report a motility in the flagella of the green alga Chlamydomonas that is unrelated to dynein-based flagellar beating. This motility, referred to as intraflagellar transport, was observed as the rapid bidirectional movement of granule-like particles along the length of the flagella. Intraflagellar transport could be experimentally separated from other, previously reported, nonbeat flagellar motilities. EM of flagella showed groups of nonvesicular, lollipop-shaped structures positioned between the outer doublet microtubules and the flagellar membrane. Movement of these complexes along the length of the flagella may be responsible for intraflagellar transport.
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              Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies.

              The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS ONE
                plos
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2007
                9 May 2007
                : 2
                : 5
                : e437
                Affiliations
                [1 ]Dynamique et Régulation des Génomes, Muséum National d'Histoire Naturelle, INSERM and CNRS, Paris, France
                [2 ]Trypanosome Cell Biology Unit, Pasteur Institute and CNRS, Paris, France
                [3 ]Biologie Fonctionnelle des Protozoaires, Muséum National d'Histoire Naturelle, Paris, France
                [4 ]Biologie du Développement, CNRS, Station zoologique, Villefranche sur Mer, France
                [5 ]Génomique fonctionnelle des Trypanosomatides, Université Bordeaux 2 and CNRS, Bordeaux, France
                Dartmouth College, United States of America
                Author notes
                * To whom correspondence should be addressed. E-mail: pbastin@ 123456pasteur.fr

                Conceived and designed the experiments: PB SA LK CB. Performed the experiments: SA LK CB TB GT FR JC. Analyzed the data: PB SA LK CB TB FR DR. Contributed reagents/materials/analysis tools: MB DR. Wrote the paper: PB DR.

                Article
                06-PONE-RA-00508R1
                10.1371/journal.pone.0000437
                1857822
                17487282
                14c8f3e0-88ec-4bdc-b660-c728467b3896
                Absalon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 19 December 2006
                : 9 April 2007
                Page count
                Pages: 16
                Categories
                Research Article
                Cell Biology
                Cell Biology/Cell Growth and Division
                Cell Biology/Cell Signaling
                Cell Biology/Cytoskeleton
                Cell Biology/Morphogenesis and Cell Biology
                Microbiology/Parasitology
                Infectious Diseases/Protozoal Infections

                Uncategorized
                Uncategorized

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