Katanins and Spastin in Trypanosome Cytokinesis Figure 2. KAT60a, KAT60b and KAT60c are each essential for cytokinesis in bloodstream stage T. brucei. Panels A–D: KAT60a; panels E– H: KAT60b; panels I–L: KAT60c. A, E, I: cumulative growth curves of two independent bloodstream RNAi clones (C1: clone 1; C2: clone 2) cultured in the presence or absence of tetracycline (tet). B,F,J: real time PCR (KAT60a and KAT60c) or Western analysis (KAT60b), at 8 hours post-induction showing downregulation of the relevant transcript or protein (data presented for clone 1 in each case). Error bars represent standard deviations. For KAT60b RNAi cell lines (F), one allele of KAT60b was replaced with 3xha:GFP:KAT60b to allow visualisation of KAT60b downregulation by Western blotting with anti-HA antibody. The membrane was also probed with anti-elongation factor 1 alpha (EF1-a) antisera as a loading control. C, G, K: DAPI staining. The nucleus (N) and kinetoplast (K) configurations of .200 cells per time point following RNAi induction of clone 1 are shown. ‘Other’ comprises mainly multinucleate and/or multi-kinetoplast cells. D, H, L: DNA content analysis. The fluorescence of 10,000 propidium iodide-stained cells was analysed by flow cytometry in the FL2-A channel at the time points (in hours) indicated for KAT60a, KAT60b and KAT60c RNAi cell lines. The ploidies of peaks are given. Data for clone 2 are comparable (not shown). doi:10.1371/journal.pone.0030367.g002 No defects in microtubule spacing or membrane association were observed (Fig. S4). Taken together, these data provide strong evidence for SPA playing an essential role in abscission, and we propose that spastin is important for the final severing steps required to separate the daughter cell subpellicular microtubule cytoskeletons. Discussion One of the most remarkable, yet poorly understood, features of trypanosome cytokinesis is the cleavage and remodelling of the subpellicular microtubule cytoskeleton. During cytokinesis, the plasma membrane must be split to generate two daughter cells, and the underlying microtubule cytoskeleton divided. As the cleavage furrow ingresses along the longitudinal axis of the cell, the membrane invaginates and crosslinks between the microtubules situated either side of the division plane are presumably broken. The trypanosome has a vermiform shape, conferred by a corset of parallel microtubules running longitudinally. The wider the cell is at any point along its length, the greater the number of microtubules that are present at that point, and the longest microtubules are found in the middle of the cell. Thus, furrow ingression will require microtubules to be severed and shortened or re-formed on either side of the cleavage plane, to maintain the vermiform shape in the daughter cells. Indeed, an increase in new, short, tyrosinated (see below) microtubules is observed in the middle of post-mitotic procyclic cells [51], and microtubule severing may aid the synthesis of new microtubules by increasing the pool of free tubulin subunits. Crosslinks between the microtubules and the plasma membrane are also likely to be remodelled as the furrow ingresses. Our functional analysis of microtubule severing protein homologues reveals that KAT80, KAT60a, KAT60b, KAT60c and SPA are all essential (and therefore non-redundant) for cytokinesis in bloodstream trypanosomes and provides the first clues as to how division of the cytoskeleton is accomplished. Depletion of the katanin and spastin proteins each resulted in the accumulation of 2N2K cells but interfered with cytokinesis at PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e30367