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      Kinesin 9 family members perform separate functions in the trypanosome flagellum

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          Abstract

          KIF9B localizes to the axoneme and basal body and is needed for flagella assembly, whereas KIF9A localizes only to the axoneme and controls flagella motility without affecting their structure.

          Abstract

          Numerous eukaryote genome projects have uncovered a variety of kinesins of unknown function. The kinesin 9 family is limited to flagellated species. Our phylogenetic experiments revealed two subfamilies: KIF9A (including Chlamydomonas reinhardtii KLP1) and KIF9B (including human KIF6). The function of KIF9A and KIF9B was investigated in the protist Trypanosoma brucei that possesses a single motile flagellum. KIF9A and KIF9B are strongly associated with the cytoskeleton and are required for motility. KIF9A is localized exclusively in the axoneme, and its depletion leads to altered motility without visible structural modifications. KIF9B is found in both the axoneme and the basal body, and is essential for the assembly of the paraflagellar rod (PFR), a large extra-axonemal structure. In the absence of KIF9B, cells grow abnormal flagella with excessively large blocks of PFR-like material that alternate with regions where only the axoneme is present. The functional diversity of the kinesin 9 family illustrates the capacity for adaptation of organisms to suit specific cytoskeletal requirements.

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          Most cited references 33

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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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            TREEFINDER: a powerful graphical analysis environment for molecular phylogenetics

            Background Most analysis programs for inferring molecular phylogenies are difficult to use, in particular for researchers with little programming experience. Results TREEFINDER is an easy-to-use integrative platform-independent analysis environment for molecular phylogenetics. In this paper the main features of TREEFINDER (version of April 2004) are described. TREEFINDER is written in ANSI C and Java and implements powerful statistical approaches for inferring gene tree and related analyzes. In addition, it provides a user-friendly graphical interface and a phylogenetic programming language. Conclusions TREEFINDER is a versatile framework for analyzing phylogenetic data across different platforms that is suited both for exploratory as well as advanced studies.
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              Flagellar motility is required for the viability of the bloodstream trypanosome.

              The 9 + 2 microtubule axoneme of flagella and cilia represents one of the most iconic structures built by eukaryotic cells and organisms. Both unity and diversity are present among cilia and flagella on the evolutionary as well as the developmental scale. Some cilia are motile, whereas others function as sensory organelles and can variously possess 9 + 2 and 9 + 0 axonemes and other associated structures. How such unity and diversity are reflected in molecular repertoires is unclear. The flagellated protozoan parasite Trypanosoma brucei is endemic in sub-Saharan Africa, causing devastating disease in humans and other animals. There is little hope of a vaccine for African sleeping sickness and a desperate need for modern drug therapies. Here we present a detailed proteomic analysis of the trypanosome flagellum. RNA interference (RNAi)-based interrogation of this proteome provides functional insights into human ciliary diseases and establishes that flagellar function is essential to the bloodstream-form trypanosome. We show that RNAi-mediated ablation of various proteins identified in the trypanosome flagellar proteome leads to a rapid and marked failure of cytokinesis in bloodstream-form (but not procyclic insect-form) trypanosomes, suggesting that impairment of flagellar function may provide a method of disease control. A postgenomic meta-analysis, comparing the evolutionarily ancient trypanosome with other eukaryotes including humans, identifies numerous trypanosome-specific flagellar proteins, suggesting new avenues for selective intervention.
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                Author and article information

                Journal
                J Cell Biol
                J. Cell Biol
                jcb
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                30 November 2009
                : 187
                : 5
                : 615-622
                Affiliations
                [1 ]Adaptation Processes of Protozoa to their Environment, Centre National de la Recherche Scientifique FRE3206 and [2 ]Nucleic acids: Dynamics, Targeting and Biological Functions, UMR5153, Muséum National d'Histoire Naturelle, 75231 Paris, France
                [3 ]Trypanosome Cell Biology, Centre National de la Recherche Scientifique URA 2581 and [4 ]Plate-forme de Microscopie Ultrastructurale, Institut Pasteur, 75015 Paris, France
                Author notes
                Correspondence to Linda Kohl: lkohl@ 123456mnhn.fr
                Article
                200903139
                10.1083/jcb.200903139
                2806587
                19948486
                © 2009 Demonchy et al.

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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