Katanins and Spastin in Trypanosome Cytokinesis Figure 4. SPA is essential for abscission in bloodstream stage T. brucei. A. Cumulative growth curves of three independent bloodstream SPA RNAi clones (C1: clone 1; C2: clone 2; C3: clone 3) cultured in the presence or absence of tetracycline (tet). Inset: Western blotting showing downregulation of SPA expression in clone 3 following RNAi induction. One allele of SPA in the RNAi cell line was replaced with an epitope tagged copy of the gene (3xha:GFP:SPA). Whole cell lysates (12 hours post-induction with tetracycline (tet)) were analysed by Western blotting with anti-HA antibody (top panel). Lysates were also blotted with anti-oligopeptidase B (OPB) antibody as a loading control (lower panel). B-G show data for clone 3. B. DAPI staining. The nucleus (N) and kinetoplast (K) configurations of .200 cells per time point following RNAi induction are shown. C. DNA content analysis. The fluorescence of 10,000 propidium iodide-stained cells was analysed by flow cytometry at the time points (in hours) indicated. The ploidies of peaks are given. D. Cytokinesis stage analysis. 2N2K cells (n.100 per time point) were analysed for their cytokinesis stage. Error bars represent standard deviations of 3 biological replicates. E. Example images of 2N2K and 3N3K cells in abscission. Left panels: DIC image; middle panels: DAPI image; right panels: merge. F. SEM analysis of induced RNAi cells (t = 12.5 hours) in abscission. Scale bars: 2 mm. G. TEM analysis of SPA RNAi cells in abscission following induction with tetracycline (t = 12.5 hours), enlarged as indicated to visualise subpellicular microtubules (Mt) present at the contact region between the two daughter cell bodies. Scale bars: 2 mm (upper panels); 500 nm (lower panels). doi:10.1371/journal.pone.0030367.g004 protein remaining after RNAi induction may be sufficient for cytokinesis until cells reach furrow ingression/abscission, or alternatively, there could be redundancy of function between the microtubule severing enzymes during some stages of cytokinesis but not others. However, since the phenotypes between indepen- dent RNAi cell lines for any given protein were highly consistent and very distinctive phenotypes were observed for the different severing proteins, we favour a model where these severing proteins play distinct roles at different stages of cytokinesis. It is possible that each protein has different microtubule cleavage specificities, for example due to differences in microtu- bule post-translational modifications and/or differential interac- tions with microtubule-binding proteins, which vary through cytokinesis. Indeed, it has been proposed that Tetrahymena Kat1 preferentially severs older post-translationally modified microtu- bules, which would account for its specificity for only a subset of microtubules [52] and acetylation and polyglutamylation of tubulin have been shown to enhance katanin and spastin- mediated microtubule severing, respectively in mammalian cells [29,30]. Further, microtubule-associated proteins XMAP230 in Xenopus, Tau and MAP2 in mammalian cells and MAP65-1c in tobacco have been shown to reduce katanin-mediated severing of microtubules [26–28], most likely by blocking katanin binding sites on the microtubules. T. brucei microtubules are known to be modified by acetylation, tyrosination and polyglutamylation [53]. Acetylation of lysine 40 of a-tubulin is a stable modification found on all microtubules - the stable subpellicular and axonemal microtubules as well as the transient spindle microtubules - that is incorporated during microtubule polymerisation [54]. Recently, depletion of the GTPase ARL2 was shown to lead to reduced levels of acetylated a-tubulin and defects in cytokinesis [16]; whether the two observations are directly linked, perhaps by PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e30367