Katanins and Spastin in Trypanosome Cytokinesis Figure 1. KAT80 is essential for cytokinesis in bloodstream stage T. brucei. A. Cumulative growth curves of two independent bloodstream KAT80 RNAi clones (C1: clone 1; C2: clone 2) cultured in the presence or absence of tetracycline (tet). B–G show data for clone 1; data for clone 2 is comparable (not shown). B. Real time PCR analysis showing downregulation of KAT80 transcript 14 hours post-induction with tetracycline (tet). Error bars represent standard deviations. C. DAPI staining. The nucleus (N) and kinetoplast (K) configurations of .200 cells per time point following RNAi induction are shown. ‘Other’ comprises mainly multinucleate and/or multi-kinetoplast cells. D. 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. E. Cytokinesis stage analysis. 2N2K cells (n.100 cells per time point) were analysed for their cytokinesis stage. Error bars represent standard deviations of 3 biological replicates. Example image of 2N2K furrowing cell shown on right. Top panel: DIC image; middle panel: DAPI image; bottom panel: merge. Arrow indicates the furrow. F. SEM analysis of induced KAT80 RNAi cells (t = 12.5 hours). i-ii: furrowing cells; iii: furrowing cell with rounded posterior end; iv: cell with posterior end folded back towards the anterior. Arrowheads point to furrows while the asterisk indicates the posterior end in (iv). Scale bars: 2 mm. G. TEM analysis of a furrowing cell following induction (t = 12.5 hours) of KAT80 RNAi, enlarged as indicated to visualise subpellicular microtubules (Mt). N: nucleus. Scale bars: 1 mm. doi:10.1371/journal.pone.0030367.g001 spastin was essentially undetectable by 12 hours post-induction (Fig. 4A). Similar to the downregulation of KAT80 and KAT60, depletion of SPA resulted in a steady increase in 2N2K cells over time as observed by DAPI staining, reaching 32% at 18 hours post-induction (Fig. 4B) although, compared to depletion of the KAT60a and KAT60c, relatively few multinucleate cells were observed. Flow cytometry data was consistent with the DAPI data (Fig. 4C). Examination of the 2N2K cells revealed a highly significant increase in the proportion of 2N2K cells undergoing abscission from 13% to 41% at 12 hours post-induction ((one way ANOVA and Dunnett’s post-hoc test; F = 106.80, df = 8, p = 0.00); Fig. 4D, 4E). Amongst the multinucleate cells, cells that had not yet completed abscission were observed re-replicating their DNA, reinitiating cytokinesis and again being blocked at abscission (see 3N3K cell in Fig. 4E). SEM analysis supported the DAPI data, showing cells with 2 full length flagella undergoing abscission (Fig. 4F, image i) as well as cells with multiple flagella apparently blocked in abscission (Fig. 4F, image ii-iv). SEM also revealed a few cells undergoing abscission to reveal two different posterior ends, a flat end and a pointed end (Fig. 4, image iii). TEM analysis of SPA RNAi cells undergoing abscission demonstrated that the two cell bodies remained joined by the plasma membrane and subpellicular microtubules, indicating that final microtubule severing and membrane remodelling had yet to occur (Fig. 4G). PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30367