decrease in growth rate followed by a growth arrest was observed within 24 hours of RNAi induction (Fig. 1A), despite only a rather modest reduction (,40% at 14 hours post-induction) in KAT80 mRNA (Fig. 1B). DAPI staining revealed that the proportion of post-mitotic (2N2K) cells in the population increased substantially (from 13.2% to 26.4%) over the first 10 hours of induction, rising to 32.8% at 18 hours post-induction (Fig. 1C). Unlike KAT80 depletion in procyclic T. brucei, few 0N1K and 2N1K cells were observed, but other abnormal cell types (mainly multi-nucleate and/or multi-kinetoplast cells) started to appear in small numbers at 14 hours post-induction and constituted 10% of the population at 18 hours post-induction. Flow cytometry data were consistent with the DAPI data, showing a relative increase in cells with a 4C DNA content 10 hours post-induction and the appearance of an 8C peak from 14 hours post-induction (Fig. 1D). The increase in 2N2K cells and subsequent appearance of multinucleate cells indicated that cytokinesis was delayed, since cells that do not divide in a timely fashion are known to re-replicate their organelles [5]. To determine whether the 2N2K cells were arrested at a particular stage of cytokinesis, 2N2K cells were scored according to how far cytokinesis had progressed (Fig. 1E). At t = 0, the majority of 2N2K cells were in early cytokinesis, since only about 15% of cells displayed a visible cleavage furrow, or had reached abscission, the final stage of cytokinesis. At later time points, there was a significant increase (one way ANOVA and Dunnett’s post- hoc test; F = 55.30, df = 8, p = 0.00) in furrowing 2N2K cells (see example cell image in Fig. 1E) with a concomitant decrease in non-furrowing cells (Fig. 1E). Thus, depletion of KAT80 in bloodstream form cells appears to delay furrow ingression during cytokinesis. Scanning electron microscopy (SEM) was used to visualise KAT80 RNAi cells in more detail (Fig. 1F). In addition to cells undergoing furrow ingression (Fig. 1F, images i -iii) being clearly visible following RNAi induction, defects in posterior end morphology such as rounding or broadening were observed in some cells (Fig. 1F, image iii) and in a few cells, the posterior end was unusually folded back towards the anterior of the cell (Fig. 1F, image iv). Furrows were further examined by transmission electron microscopy (TEM) (Fig. 1G), which showed microtubules arranged in a herring bone pattern at the furrow either side of the undivided cell membrane. No defects in microtubule spacing or membrane association were observed at the furrows (Fig. 1G) or elsewhere in the cell (Fig. S2), arguing that, in addition to a possible effect on microtubules at the posterior end of the cell, the depletion of KAT80 specifically affects microtubule cleavage and membrane remodelling at the furrow, but does not cause global defects in microtubule spacing or membrane attachment. KAT60a, KAT60b and KAT60c are essential for proliferation and are required for early events during cytokinesis in bloodstream trypanosomes Depletion of any of the KAT60 proteins resulted in a growth arrest within 24 hours in bloodstream stage T. brucei (Fig. 2A, 2B, 2E, 2F, 2I, and 2J), indicating that these proteins are essential for proliferation and are non-redundant. As specific antibodies to the trypanosome katanins are not yet available, tagged copies of the katanin genes were introduced at the respective endogenous loci in the RNAi cell lines to allow monitoring of protein knockdown following RNAi induction. However, only tagged KAT60b protein could be reliably detected by Western blot, revealing a substantial depletion of KAT60b at 8 hours post-induction (Fig. 2F). Since tagged KAT60a and KAT60c could not be detected, presumably due to very low levels of expression, real time PCR was used to show that KAT60a and KAT60c transcripts were downregulated to ,60% at 8 hours post-induction (Fig. 2B, 2J). Katanins and Spastin in Trypanosome Cytokinesis In each case, depletion of KAT60a, KAT60b or KAT60c resulted in an increase in 2N2K cells, followed by the appearance at later time points of substantial numbers of multi-nucleate/multi- kinetoplast cells (Fig. 2C, 2G, and 2K), indicating defects in cytokinesis. Small numbers of 0N1K (zoid) and 2N1K cells were also present at later time points, and are also characteristic of defects in cytokinesis. Flow cytometry data were consistent with the DAPI data, showing an increase in the 4C peak relative to the 2C peak and the appearance of cells with .4C DNA content over time (Fig. 2D, 2H, and 2L). The kinetics of the cell cycle progression defects varied for the different KAT60 proteins. For example, depletion of KAT60b resulted in 2N2K cell numbers gradually increasing over the time course of the experiment with multinucleate cells only appearing later at 14–18 hours post- induction (Fig. 2G), while depletion of KAT60a or KAT60c saw 2N2K cell numbers peaking at ,14 hours post-induction, at which time there were also significant numbers of multinucleate cells present (Fig. 2C and 2K). The differences between the KAT60 proteins may reflect subtle differences in their function. Examination of the 2N2K cell populations that accumulated post-RNAi induction for all three KAT60 proteins revealed only minor changes in the proportion of cells at different stages of cytokinesis (no furrow, furrowing and abscission) over time (Fig. S3), with around 80% cells having no visible furrow. However, examination of KAT60a, KAT60b and KAT60c RNAi cells at higher magnification by SEM revealed qualitative differences between them. Examination of cells with two full length flagella (2F), likely to correspond to the 2N2K cells visualised by DAPI staining [10], at 12 hours post-induction, revealed that following KAT60a deple- tion, in many of these cells without a furrow, a cleavage fold had formed along the longitudinal axis of the cell (Fig. 3A, images i-iv), although the two daughter cell bodies had not been physically separated at any point along their length. This fold was not easily visible by light microscopy, and thus 2N2K cells classified as having ‘no furrow’ by DAPI staining are likely to have included cells with and without cleavage folds. Fold formation likely represents the early stages of furrow ingression, characterised by membrane ingression and (presumably) remodelling of the underlying micro- tubules. The apparent abundance of 2F cells with cleavage folds following KAT60a depletion may suggest a particular importance for KAT60a in the final cleavage of the cytoskeleton to separate the daughter cell bodies. In contrast, following KAT60b depletion, 2F ‘no furrow’ cells, in general, did not show such a pronounced cleavage fold (Fig. 3B; compare Fig. 3B, image i with Fig. 3A, images iii and iv), while defects in posterior end morphology eg rounding were evident in some cells (Fig. 3B, image ii). Additionally, despite the absence of cleavage furrow ingression from the anterior end of the cell, two daughter cell bodies were defined, but not separated at the posterior end of some cells (Fig. 3B, images iii–v). This may indicate that KAT60b is important for microtubule and membrane remodelling at the anterior of the cell, and following its depletion, remodelling occurs preferentially at the posterior end. KAT60c RNAi 2F ‘no furrow’ cells showed a variety of morphologies, including no membrane invagination or cleavage fold formation (Fig. 3C, image i), substantial cleavage fold formation (Fig. 3C, images ii–iv), and definition of the two daughter cell posterior ends in the absence of cleavage at the anterior end (Fig. 3C, images v–vi), which may suggest that KAT60c plays a role in cytoskeleton/membrane remodelling throughout cytokinesis. Spastin is required for abscission in bloodstream stage T. brucei Induction of SPA RNAi resulted in a rapid growth arrest, and depletion of SPA was confirmed by Western blotting, showing that PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e30367