1852 Research Article Introduction The never in mitosis gene A (NIMA)-related kinases (NEKs) belong to a family of ubiquitous serine/threonine kinases that promote cell cycle events and have been implicated in driving G2-M progression (Fry, 2002; Fry et al., 1998a; Fry et al., 1998b; Rhee and Wolgemuth, 1997). Members of this family are also implicated in polycystic kidney diseases (PKD) (Liu et al., 2002; Mahjoub et al., 2002; Mahjoub et al., 2004). Although NEKs are the most closely related kinases to NIMA they serve different functions. Mammalian NEK1 is highly expressed in meiotic germ cells and is considered to have a role in controlling meiotic events (Letwin et al., 1992); it is also one of the key proteins involved in PKD (Upadhya et al., 2000) and recovery from DNA damage (Polci et al., 2004). Mammalian NEK2 is one of the most characterised of the NEKs and has, across the catalytic kinase domain, 44% amino acid identity with NIMA. The splice variant NEK2A is enriched at the centrosome and functional studies in homologues of NEK2 in mammalian and Xenopus cells suggest a role in centrosome maintenance, structure and centriole splitting (Fry et al., 2000; Fry et al., 1998a; Fry et al., 1998b; Hames et al., 2001; Twomey et al., 2004). The first non-vertebrate orthologue of human NEK2 (HsNEK2) was identified in Dictyostelium discoidium (DdNek2). DdNek2 shares 43% overall amino acid identity with HsNEK2 and 54% identity within the catalytic domain. Overexpression of DdNek2 stimulates the formation of supernumerary asters and microtubule organising centres (MTOCs) (Graf, 2002). Trypanosoma brucei is protozoan parasite responsible for sleeping sickness in humans and Nagana in animals (Chappuis et al., 2005; Kioy et al., 2004; Vickerman, 1985). The T. brucei procyclic insect form coordinates the replication and segregation of its single-copy organelles: the nucleus, mitochondria (including its genome, the kinetoplast) and flagellum. Wild-type G1 cells have a mature basal body that forms a flagellum and an immature basal body, which has not yet formed its own flagellum. As the cell passes through the cell cycle, the immature basal body matures and initiates the growth of a new flagellum. This is then accompanied by the formation of two immature basal bodies. Consequently, immature basal bodies are always very closely physically associated with a mature basal body and hence a flagellum (Sherwin and Gull, 1989). Basal bodies are also physically linked to each other in G1 of the cell cycle and are always physically linked to the kinetoplast by proteins that have yet to be identified (Ogbadoyi et al., 2003; Robinson and Gull, 1991). To date the only cytoskeletal cell cycle marker related to the control of cytokinesis in trypanosomes is the separation of its basal bodies. If basal body separation is blocked, the cell does not undergo cytokinesis (Ploubidou et al., 1999). Although the roles of NEK proteins have been studied in mammals, Xenopus and Drosophila, little is known about the mechanistic basis for these phenomena in protists. Previous searches for T. brucei NIMA-related kinases (NRKs) resulted in the identification two kinase genes, namely NRKA and B. They have highest expression levels and activity in bloodstream form, G0-arrested, stumpy cells and are considered to be involved in parasite differentiation (Gale et al., 1994; Gale and Parsons, 1993). The location of these proteins within the cell is unknown. In this study we have identified the first T. brucei basal body NRK protein, which we have named TbNRKC. NRKA and NRKB have high amino acid identity with the N-terminus of TbNRKC (~37% identity). The NIMA-related kinase 2 (NEK 2) has important cell cycle functions related to centriole integrity and splitting. Trypanosoma brucei does not possess centrioles, however, cytokinesis is coupled to basal body separation events. Here we report the first functional characterisation of a T. brucei basal body-cytoskeletal NIMA-related kinase (NRK) protein, TbNRKC. The TbNRKC kinase domain has high amino acid identity with the human NEK1 kinase domain (50%) but also shares 42% identity with human NEK2. TbNRKC is expressed in bloodstream and procyclic cells and functions as a bona fide kinase in vitro. Remarkably, RNAi knockdown of TbNRKC and overexpression of kinase-dead TbNRKC in procyclic forms induces the accumulation of cells with four basal bodies, whereas overexpression of active protein produces supernumary basal bodies and blocks cytokinesis. TbNRKC is located on mature and immature basal bodies and is the first T. brucei NRK to be found associated with the basal body cytokinesis pathway. Key words: NEK, Basal body, Cytokinesis, T. brucei, Cell cycle Summary NIMA-related kinase TbNRKC is involved in basal body separation in Trypanosoma brucei Lydie C. Pradel, Mélanie Bonhivers, Nicolas Landrein and Derrick R. Robinson* Laboratoire de Génomique Fonctionnelle des Trypanosomatides, CNRS UMR 5162, Université Bordeaux 2, 146 rue Léo Saignat, Bât. 3A, 33076 Bordeaux CEDEX, France *Author for correspondence (e-mail: Derrick.Robinson@parasitmol.u-bordeaux2.fr) Accepted 20 January 2006 Journal of Cell Science 119, 1852-1863 Published by The Company of Biologists 2006 doi:10.1242/jcs.02900 Journal of Cell Science