and p60 katanin can be recruited to mammalian centrosomes by other means [25]. Katanin activity is also regulated by microtubule binding proteins [26–28], which can inhibit its activity, and the activities of both katanin and spastin are regulated by post-translational modification of microtubules [27,29,30]. Spastin and fidgetin have high homology to katanin p60, but have not been reported to interact with a regulatory subunit. p60 katanin and spastin are known to oligomerise into hexameric rings in the presence of ATP, stimulating their ATPase activity and creating a central pore into which it is thought the C- terminal tail of tubulin is pulled, generating a mechanical force which breaks the microtubule [19,31]. Katanin, spastin and fidgetin control microtubule flux during mitosis in Drosophila melanogaster [20], and recently, Drosophila katanin has also been shown to depolymerise microtubules and to regulate the interactions of microtubule plus ends with the cell cortex and to negatively regulate cell migration [32,33]. Mammalian katanin is crucial for the redistribution of c-tubulin at mitosis [34] while in Chlamydomonas, katanin is required for severing the flagellum from the basal bodies before mitosis [35] and in Arabidopsis thaliana, katanin was recently shown to be important for cell division [36]. Further, katanin protein, but not its microtubule severing activity is required for meiotic spindle formation in Caenorhabditis elegans [37,38]. Spastin localises to centrosomes and spindle poles in dividing cells [39], and to the distal ends of axons, where it regulates axon branching [40]; mutations in the spastin gene, SPG4, account for 40% of cases of autosomal dominant Hereditary Spastic Paraplegia (HSP), which is characterised by the retrograde degeneration of corticospinal tracts (the longest axons), leading to progressive weakness and spastic paralysis of the lower limbs [41]. Additionally, spastin functions in the endosomal system in human cells, where two spastin isoforms are expressed. The 68 kDa isoform regulates endoplasmic reticulum (ER) to Golgi trafficking [42], and may be involved in ER shaping via interactions with ER membrane protein, receptor expression enhancing protein 1 (REEP1), and atlastin [43]. The shorter 60 kDa isoform localises to endosomes and is recruited to the midbody via an interaction with the endosomal sorting complex required for transport (ESCRT)-III machinery, where it seems to be required for severing microtu- bules to accomplish abscission [42,44,45]. Spastin knockdown has also been reported to result in the disorganization of central spindle microtubules and defective delivery of FIP3-containing endosomes to the intracellular bridge [46]. Since the trypanosome cytoskeleton needs to be bisected and remodelled during cell division, we reasoned that microtubule severing might play a major role in this process. Previously, the existence of homologues of the katanin p80 regulatory subunit (KAT80) and p60 catalytic subunit (KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in T. brucei was reported [47]. RNA interference (RNAi) studies of these proteins in procyclic trypanosomes, however, only yielded a discernible phenotype for KAT80; depletion of KAT80 slowed proliferation and was accompanied by the appearance of multi-nucleate cells and zoids (anucleate cells with a single kinetoplast), suggesting a possible role for KAT80 in cytokinesis [47]. The same study also investigated the subcellular localisation of these proteins in procyclic T. brucei using ectopically expressed tagged proteins. KAT60c could not be localised, but KAT80:GFP and KA- T60a:GFP displayed a diffuse cytoplasmic localisation, and ectopic expression of KAT80:GFP disrupted cell division. Tagged KAT60b proteins localised to the flagellum and their expression reduced flagellar length by 20%. FID:GFP, GFP:SPA and SPA:GFP localised to the nucleus; FID:GFP had a punctate Katanins and Spastin in Trypanosome Cytokinesis distribution during much of the cell cycle but localised to the spindle during mitosis while the tagged spastin proteins formed puncta around the trypanosome nucleolus throughout the cell cycle and their expression led to abnormalities in nucleus morphology [47]. Given the functional variability of several cell cycle and cytokinesis regulators between trypanosome life cycle stages [5,14], and differences in the molecular composition of the cytoskeleton [48–50], we sought to investigate the function of the katanins, spastin and fidgetin in bloodstream stage T. brucei. While RNAi of fidgetin did not yield any discernible phenotype, depletion of the katanin and spastin proteins revealed that they play essential, non-redundant and discrete functions in cytokinesis in bloodstream trypanosomes. Results To investigate the roles of katanins, spastin and fidgetin in bloodstream T. brucei, two or three independent RNAi cell lines were generated for each microtubule severing protein. Equivalent procyclic RNAi cell lines were also generated for comparative purposes. Following the induction of RNAi by the addition of tetracycline to the growth medium, the efficiency of the RNAi was assessed either by performing quantitative PCR to determine mRNA knockdown, or by replacing one allele of the gene in question with a tagged version, allowing determination of protein knockdown via Western blotting. The effects of microtubule severing protein depletion on proliferation and cell cycle progres- sion were then monitored. Trypanosomes are easily classified into different cell cycle stages by staining the nucleus and mitochondrial genome (the kinetoplast). In early G1 phase, cells have one nucleus and one kinetoplast (1N1K). Kinetoplast S phase starts before nuclear S phase and is also substantially shorter, resulting in cells with a 1N2K configuration in early G2 phase. During mitosis, the nucleus divides to generate a 2N2K cell, which then undergoes cytokinesis to give rise to two daughter 1N1K cells. RNAi analysis revealed no discernible phenotype for fidgetin in either life cycle stage (data not shown), and hence this protein will not be considered further here. RNAi of the KAT60 proteins or spastin in procyclic T. brucei also did not yield any discernible phenotype (data not shown), in line with previous observations [47]. KAT80 is required for normal proliferation and cell division in procyclic T. brucei Depletion of KAT80 in procyclic T. brucei indicated that it is essential for normal in vitro growth (Fig. S1A and S1B), confirming previous observations [47]. Following KAT80 depletion, DAPI staining showed that 2N1K and 0N1K (zoid) cells slowly accumulated in equal numbers over the first 5 days of induction (Fig. S1C), suggesting that these abnormal cells were the progeny of a 2N2K cell, which had divided aberrantly. The zoid population continued to increase, comprising about 20% of the total population by 7 days post-induction (Fig. S1C) and flow cytometry profiles of propidium iodide-stained cells showed a peak of ,1C DNA content appearing from 3 days post-induction, consistent with the zoid population observed by DAPI staining (Fig. S1D). Taken together, these data are consistent with KAT80 being important for accurate positioning of the cleavage furrow during cytokinesis in procyclic T. brucei. KAT80 is essential for proliferation and cleavage furrow ingression in bloodstream T. brucei RNAi of KAT80 in bloodstream trypanosomes resulted in a more dramatic phenotype than in procyclic parasites. A rapid PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e30367