Cytokinesis in Bloodstream Stage Trypanosoma brucei Requires a Family of Katanins and Spastin Corinna Benz, Caroline Clucas, Jeremy C. Mottram, Tansy C. Hammarton* Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom 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. Citation: Benz C, Clucas C, Mottram JC, Hammarton TC (2012) Cytokinesis in Bloodstream Stage Trypanosoma brucei Requires a Family of Katanins and Spastin. PLoS ONE 7(1): e30367. doi:10.1371/journal.pone.0030367 Editor: Dan Zilberstein, Technion-Israel Institute of Technology, Israel Received August 10, 2011; Accepted December 19, 2011; Published January 18, 2012 Copyright: 2012 Benz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Medical Research Council (via a Career Development Fellowship (ref. G120/1001) and New Investigator Research Grant (ref. GO900239) to TCH), Research Councils UK (Academic Fellowship to TCH) and The Wellcome Trust (Project grant ref. 070231 to JCM and TCH, and Value In People funding ref. 084546/Z/07/Z to TCH for CB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Tansy.Hammarton@glasgow.ac.uk Introduction The protozoan parasite, Trypanosoma brucei, is the causative agent of the human and animal African trypanosomiases, which are frequently fatal if untreated. It has a digenetic life cycle, replicating in the alimentary canal of its vector, the tsetse fly, and the bloodstream of the mammal. During its life cycle, the trypanosome undergoes a series of differentiation steps yielding forms that differ morphologically and biochemically, as well as with respect to their replicative status [1]. The long slender bloodstream stage and the procyclic and epimastigote forms in the tsetse fly are proliferative; other life cycle stages are either cell cycle arrested (the short stumpy bloodstream stage and the metacyclic form in the tsetse salivary glands), or, in the case of the long proventricular form, undergo a single asymmetric cell division [2,3]. Considerable differences in cell cycle regulation are known to exist between the long slender bloodstream stage and the procyclic form, and the subcellular organisation of different life cycle stages also varies significantly [3–6]. The vermiform morphology of T. brucei is conferred by a subpellicular microtubule cytoskeleton [7]. Microtubules within this cytoskeletal corset are arranged in parallel with their plus ends orientated to the posterior of the cell [8]. Microtubule-associated proteins (MAPs) crosslink the microtubules to each other and to the plasma membrane, stabilising the cytoskeleton [7,9,10]. During cell division, the cytoskeleton is inherited semi-conservatively; the cytoskeleton as a whole does not break down at any point, and newly forming microtubules interdigitate between existing ones [11]. This therefore imposes a barrier to cell division, since the microtubules and their linking MAPs must be broken and remodelled at the site of cleavage. Cytokinesis in T. brucei involves the ingression of a cleavage furrow from the anterior end of the cell to the posterior, following the longitudinal axis of the cell. This is a very divergent mode of division compared to the contractile actomyosin ring used by mammals and yeasts to pinch the cell in two. Indeed, no role for actin or myosin in trypanosome cytokinesis has been uncovered, and instead these proteins are required for endocytosis in the bloodstream stage [12,13]. Multiple regulators of trypanosome cytokinesis have been identified [14–18], but their interplay and targets remain largely unexplored. Moreover, information about the mechanics and effectors of cell division is lacking [6,14]. Katanin, spastin and fidgetin are members of a family of AAA ATPases (ATPases Associated with various cellular Activities) that influence microtu- bule dynamics in a variety of organisms [19–21]. These enzymes sever microtubules along their length, thus shortening them and increasing the overall number of microtubules, as well as increasing the pool of free tubulin molecules, which can nucleate new microtubules. Katanin is a heterodimer, consisting of a regulatory p80 and a catalytic p60 subunit [22,23]. The regulatory subunit is important for substrate recognition and targeting of the catalytic subunit, although it is not essential for its activity [24], PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e30367