dynamics during mitosis [20], CG1193, which is involved in dendrite severing in sensory neurons during metamorphosis [76] and CG10793, which has not yet been characterised), the expression of three KAT60 proteins that play essential and non- redundant roles in cytokinesis likely reflects the challenges inherent in cleaving the stable microtubule cytoskeleton in T. brucei. It is interesting that KAT60c (and also spastin) are not present in L. major, and that in promastigote Leishmania, while the subcellular localisation of KAT60a suggests it may play a role in cytokinesis, KAT60b appeared to be involved in flagellar length regulation [47]. Presumably these differences reflect differences in the cell biology and structure of these two parasites. Depletion of the microtubule severing proteins in T. brucei did not appear to cause widespread disruption to the trypanosome cytoskeleton although more detailed electron microscopy investigations of the trypanosome cytoskeleton following depletion of katanins and spastin will be required to rule out subtle defects. Evidence for KAT80 regulating the KAT60 proteins, as has been found to be the case for all other p80 katanins studied to date, is still lacking in T. brucei, and the role of fidgetin remains enigmatic. To date, we have unfortunately been unable to detect any tagged katanin or spastin protein in bloodstream form trypanosomes via immunofluorescence, presumably due to them being present at very low levels in the cell, and thus have been unable to determine their subcellular localisation, which would shed further light on their functions. Investigating the interactions between the different katanins, and between the microtubule severing enzymes and the microtubule cytoskeleton will be challenging due to their very low abundance, but should be a priority in the future, as should purifying these proteins to allow their biochemical characterisation. Pairwise knockdown of different katanins may provide further insight into their overlapping roles in cytokinesis, and a fidgetin gene knockout should allow an assessment of its essentiality. Materials and Methods Cloning of RNAi, endogenous tagging and inducible expression constructs Gene products were amplified from T. brucei brucei strain EATRO 795 or Lister 427 genomic DNA using the polymerase chain reaction (PCR) with the oligonucleotides detailed in Table S1. PCR products were cloned into pSC-B (Stratagene), pGEMTeasy (Promega) or pCR2.1 TOPO (Invitrogen) and sequenced (by The Sequencing Service, University of Dundee) to check the fidelity of the PCR. Gene products were then subcloned into the appropriate destination vectors using the restriction sites incorporated into the primers (Table S1). For RNAi, unique fragments of KAT80, KAT60a, KAT60b, KAT60c, SPA and FID (selected by the programme RNAit [77], to minimise the possibility of off-target effects) were subcloned into p2T7ti:GFP [78] in place of the GFP sequence, generating pGL1103 (KAT80), pGL1049 (KAT60a), pHG96 (KAT60b), pGL1101 (KAT60c), pHG99 (SPA) and pHG94 (FID). Genes were also tagged at their 5` ends within their endogenous locus to retain the endogenous 3` UTR sequence and maintain native expression levels as closely as possible. The 5` end of the open reading frame (ORF) and a region encompassing the 5` untranslated region (UTR) just upstream of the start codon were amplified by PCR and subcloned via a three-way ligation into pEnT vectors [79] or their derivatives (pHG35 and pHG80) as described here. The endogenous tagging vectors generated were: pHG84 (3xha:eGFP:KAT80 in pHG80), pHG38 (ty:CFP:KAT60a in pEnT6-PURO-CFP-TY), pHG142 (3xha:eGFP:KAT60b in Katanins and Spastin in Trypanosome Cytokinesis pHG80), pHG47 (myc:mCherry:KAT60c in pHG35) and pHG111 (3xha:eGFP:SPA in pHG80). pHG35 was generated by amplifying c-myc:mCherry from p2686 [79] using PR7 and PR29 (Table S1) and cloning this sequence using Hind III/Xba I into pEnT6- BLAST-CFP-TY in place of ty:CFP. pHG80 was constructed by digesting pEnT6BLAST-eGFP-TY with Hind III and Spe I to remove the TY epitope coding sequence and replacing it with annealed oligonucleotides (PR9 and PR10) encoding a 3xHA epitope tag. Expression of tagged proteins following transfection of the constructs into T. brucei (see below) was verified by Western blotting with appropriate antibodies against the tags. Due to KAT60 tagged proteins being barely detectable or undetectable, presumably due to low expression levels, inducible expression constructs were generated for KAT60a (KAT60a:ty; pHG139) and KAT60c (KAT60c:myc; pGL1502) in a pHD675 background [80]; KAT60a still could not be detected by Western blotting with anti-TY antibody following induction. Culturing and transfection of T. brucei and analysis of cell lines Bloodstream and procyclic stage T. brucei brucei were cultured and transfected as described previously [4,81]. For RNAi, the procyclic 427 pLew13 pLew29 and the bloodstream form 427 pLew13 pLew90 cell lines [82] were transfected with the appropriate Not I-linearised, purified RNAi plasmids (described above) and cloned by limiting dilution. Induction of RNAi was achieved by adding 1 mgml-1 tetracycline to the culture medium. To assess the level of specific mRNA downregulation following induction of RNAi, real time PCR analysis was performed on cDNA prepared from RNAi cell lines at a suitable time point following induction, as described previously [83]. The gene specific primers used are given in Table S1; control primers were for GPI8 [83,84]. To enable downregulation of the targeted protein to be monitored by Western blotting, RNAi cell lines were transfected with the appropriate endogenous tagging constructs (described above), linearised with Xba I. Lysates were analysed by Western blotting with antibodies against the epitope tag (see below); loading controls were performed by staining the membrane with Ponceau S, or by blotting with anti-oligopepti- dase B (OPB) [85] or anti-elongation factor 1 alpha (EF1-a; clone CBP-KK1, Millipore) antisera. Following induction of RNAi cell lines, cell proliferation was monitored by counting cells at appropriate intervals using a Neubauer Improved haemocytom- eter and cell cycle progression was analysed via DAPI staining of methanol-fixed cells and flow cytometry of propidium iodide stained cells as described previously [4]. The cytokinesis stage of 2N2K cells was determined as previously described [86] and the results were analysed by one way ANOVA and a Dunnett’s post- hoc test. For analysis of subcellular localisation of katanins and spastin, and interactions between katanin proteins, Xho I-linearised endogenous tagging constructs were transfected into Lister 427 bloodstream stage cells. Where appropriate, T. brucei bloodstream stage cultures were synchronised using hydroxyurea as described previously [87] and the success of the synchronisation monitored by DAPI staining and flow cytometry (as above). Inducible expression constructs were Not I-linearised, transfected into the bloodstream stage cell line 427 pHD449 [80], and protein expression induced by adding 1 mgml21 tetracycline to the culture medium. Expression of tagged proteins was assessed using Western blotting of cell lysates with anti-TY [88], anti-GFP (Santa-Cruz), anti-HA (Roche) and anti-myc (GenScript) antibodies. To analyse interactions between katanin proteins, 1–56109 bloodstream form cells expressing 3xha:GFP:KAT80 and a tagged copy of KAT60b PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e30367