Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin

Abstract

Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell division.

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 µm. 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 µm.

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-α) 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).

Figure 3. Depletion of KAT60a, KAT60b or KAT60c delays cytokinesis at an early stage in bloodstream trypanosomes.

SEM images of RNAi cells (A: KAT60a; B: KAT60b; C: KAT60c) with 2 full length flagella. Cells were induced with tetracycline for 12.5 hours. Arrowheads indicate cleavage folds. Scale bars: 2 µm.

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 µm. 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 µm (upper panels); 500 nm (lower panels).