Molecular & Biochemical Parasitology 192 (2013) 49–54 Contents lists available at ScienceDirect Molecular & Biochemical Parasitology Short communication A Protein Phosphatase 1 involved in correct nucleus positioning in trypanosomes Cécile Gallet, Raphaël Demonchy, Carole Koppel, Philippe Grellier, Linda Kohl ∗ MNHN/CNRS UMR7245, RDDM, Muséum National d’Histoire Naturelle, 61, rue Buffon, 75231 Paris 05, France a r t i c l e i n f o Article history: Received 14 May 2013 Received in revised form 22 November 2013 Accepted 24 November 2013 Available online 1 December 2013 Keywords: Phosphatase PP1 Nucleus Position Trypanosome a b s t r a c t Reversible protein phosphorylation is a key regulator in intracellular functions. In the African try- panosome, Trypanosoma brucei, the serine–threonine phosphatase PP1-3, is localised in the cytoplasm. RNAi mediated knockdown of PP1-3 leads to a coordinated rearrangement of cellular organelles and com- partments in the procyclic trypanosome. These parasites display their nucleus at the very posterior end of the cell. The kinetoplast is very close to the nucleus, and often located in a more anterior position. The lysosomal compartment, which in a normal procyclic cell is situated between nucleus and kinetoplast, is now positioned towards the anterior end of the cell. The Flagellum Attachment Zone, essential for cytoki- nesis, is still constructed, allowing initiation of the cleavage furrow and cell division. These adaptations allow dividing cells to distribute their organelles among the daughter cells and to proliferate normally. PP1-3 is therefore essential in conserving the intracellular organisation of the procyclic trypanosome cell. © 2013 Elsevier B.V. All rights reserved. Protein phosphorylation is one of the major mechanisms for the control and regulation of cellular functions in eukaryotic cells. Phosphorylation occurs mainly on serine (Ser) or threonine (Thr) residues, making Ser/Thr protein phosphatases (PP) exceedingly important players in the eukaryotic cell. The Ser/Thr protein phos- phatases of type 1 (PP1) are the most frequent and among the best characterised PP [1]. The PP1 catalytic subunit can interact with a large number of regulatory proteins, resulting in a conformational change of the phosphatase and a specific dephosphorylation of the target. This association of various regulatory domains with the catalytic domain explains the involvement of PP1s in cellular regu- lations as diverse as glycogen metabolism, glycolysis, microtubular functions, cell cycle progression, mitosis, apoptosis, cell signalling or motility [2]. In protozoan parasites, the physiological activity of PP1 was mainly studied using potent inhibitors, such calyculin A or okadaic acid (OA). These inhibitors act on both PP1 and PP2A with differ- ent specificities and revealed an involvement of these enzymes in the attachment process of the parasites Trichomonas vaginalis [3], Toxoplasma gondii, [4] and Plasmodium falciparum [5] to their host cells and in the differentiation of the extracellular stage into the intracellular stage of Trypanosoma cruzi [6]. In contrast to these Abbreviations: PP, protein phophatase; PP1, protein phosphatase 1; PP2A, pro- tein phosphatase 2A; RNAi, RNA interference; K, kinetoplast; N, nucleus; OA, okadaic acid; FAZ, Flagellum Attachment Zone; Tet, tetracycline. ∗ Corresponding author. Tel.: +33 140493503; fax: +33 140793499. E-mail address: lkohl@mnhn.fr (L. Kohl). parasites, Trypanosoma brucei, the agent of human sleeping sickness, does not invade host cells. Treatment of procyclic T. brucei with OA resulted in multinucleated cells with a kinetoplast segrega- tion defect, indicating a role for PP1 and/or PP2a in cytokinesis and organelle positioning [7]. Calyculin A also inhibits T. brucei growth (C. Gallet, P. Grellier, unpublished). Using these inhibitors to deter- mine whether the responsible PP was a type 1 or a type 2A is not possible. Taking advantage of the inducible RNA interference sys- tem in T. brucei, Li and co-workers simultaneously knocked down the expression of 7 of the 8 PP1, as well as the expression of the PP2A catalytic subunit in procyclic trypanosomes [8]. The authors observed a slight reduction in growth of the cells, but no defect in cytokinesis or organelle positioning [8]. As the knockdown had been performed on several PP1 at the same time [8], it was not pos- sible to quantify the efficiency of the knockdown for a particular PP1 enzyme. Inefficient knockdown, different roles of PP1 enzymes at different life cycle stages or the presence of another OA-sensitive PP in T. brucei could explain the differences in phenotype observed between the inhibitor and the RNAi studies. In order to determine whether a specific PP1 was involved in organelle positioning and cytokinesis in procyclic T. brucei, we decided to specifically knock down the expression of the first PP1 identified in this organism (TriTrypDB: Tb927.4.5030 [9]). Since this protein was also studied in the simultaneous RNAi knockdown study [8], it will be referred to as ‘PP1-3’, according to the PP1 nomenclature introduced by Li and coworkers. We first determined the localisation of PP1-3 in procyclic T. brucei (Fig. 1A and B) using a specific anti-PP1-3 antiserum raised in mice against the complete recombinant T. cruzi PP1-3, which 0166-6851/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molbiopara.2013.11.001