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Molecular & Biochemical Parasitology
Short communication
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Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei
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Karen G. Rothberg 1 , Neal Jetton, James G. Hubbard 2 , Daniel A. Powell 3 , Vidya Pandarinath, Larry Ruben ∗
Q2 Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275, United States
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Article history: Received 23 September 2013 Received in revised form 12 April 2014 Accepted 18 April 2014 Available online xxx
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Keywords: Phosphoprotein phosphatase PP2A Okadaic acid Cell cycle regulation Basal bodies Trypanosoma brucei
The cell cycle consists of an orderly sequence of events, whose purpose is to faithfully replicate and segregate cellular components. Many events in the cell cycle are triggered by protein kinases and counteracting phosphoprotein phosphatases (PPP). In Trypanosoma brucei, RNAi has been used to characterize numerous regulatory kinases, while the role of protein phosphatases has primarily been deduced with inhibitors such as okadaic acid and calyculin. In the present study, we identify for the first time a protein phosphatase 2A family member (TbPP2A-1) whose knockdown with RNAi phenocopies the effects of okadaic acid (OKA). In bloodstream forms (BF) and insect stage procyclic forms (PF) RNAi of TbPP2A-1 generates a cell population characterized by: an inhibition of cell growth, a block in cytokinesis; continued synthesis of nuclear DNA leading to aneuploidy; continued mitosis leading to cells with N > 2, and an unusual phenotype where number of kinetoplasts (and flagella) is less than the number of nuclei. An engineered cell line was constructed to further study TbPP2A-1 and to facilitate the discovery of other cell cycle regulatory genes. © 2014 Published by Elsevier B.V.
Trypanosoma brucei is a unicellular parasite that invades the fluid spaces of the mammalian host. Survival depends upon effective immune evasion coupled with continuous replenishment of cell populations by replication. The cell division cycle is only halted during transmission between hosts. All eukaryotic cells rely upon triggering processes to coordinate events during the cell cycle. Reversible cycles of phosphorylation and dephosphorylation are critical. In trypanosomes a role in cell division has been described for a range of kinases, including: cyclin/Crk complexes, polo-like kinase, Aurora kinase, tousled-like kinase, NDR kinases and NIMA family members [reviewed in 1,2]. In contrast little is known about the corresponding phosphatases. In general, the kinome of T. brucei is more extensive than its phosphatome [3,4]. With 156 eukaryotic kinases and only 78 phosphatase catalytic subunits, each phosphatase is generally used to
∗ Corresponding author. Tel.: +1 214 768 2321; fax: +1 214 768 2995. E-mail address: [email protected] (L. Ruben). 1 Current address: Live Cell Imaging Core, University of Texas Southwestern Medical School, 5323 Harry Hines Blvd, Dallas, TX 75390, United States. 2 Current address: Department of Biology, Texas Wesleyan University, Fort Worth, TX 76105, United States. 3 Current address: Department of Immunology, University of Arizona, Tucson, AZ 85724, United States.
reverse the effects of multiple kinases. In the phosphoprotein phosphatase family (PPP family) of Ser/Thr phosphatases, specificity is accomplished by binding the catalytic subunit to scaffolding subunits. The scaffolds regulate both the enzymatic activity and location of the phosphatase [reviewed in 5]. The trypanosome genome encodes multiple PPP catalytic subunits belonging to the PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7 families [4,6]. A role for PPP family members in T. brucei cell division was first demonstrated with okadaic acid (OKA); an inhibitor of both PP2A and PP1 family members. OKA has no effect on nuclear division, but prevents kinetoplast division from keeping pace with mitosis. Cytokinesis is inhibited, leading to cells with multiple nuclei, and diminished number of kinetoplasts [7]. In T. cruzi, similar observations have been made, except the PP1/PP2A inhibitor calyculin was used [8]. In an effort to identify cell cycle regulatory phosphatases in T. brucei, the RNAi-dependent knockdown of all 8 PP1 family members has been described, along with knockdown of a single PP2A family member [9]. The PP2A family member had previously been cloned [10] and was later reclassified as PP4 [4]. Neither the loss of PP1 nor of PP4 blocked cell cycle progression at a specific step, nor generated a phenotype that resembled the effects of OKA. However, a more recent study determined that PP1-3 plays a role in nuclear positioning [11]. The only other PPP member to be characterized by RNAi is TbPP5. This phosphatase appears to have stage-specific
http://dx.doi.org/10.1016/j.molbiopara.2014.04.006 0166-6851/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Rothberg KG, et al. Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei. Mol Biochem Parasitol (2014), http://dx.doi.org/10.1016/j.molbiopara.2014.04.006
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functions associated with growth-related sensitivity to serum factors and Hsp90 chaperone function [12,13]. In general, very little is known about the function of PPP family members and their scaffolding proteins in T. brucei. None of the proteins evaluated to date mimics the effects of OKA when knocked down by RNAi. It may be that: (a) RNAi generates an insufficient knockdown to exert an effect; (b) the OKA treatment may have inhibited multiple phosphatases whereas RNAi disrupts expression of only a single protein; or (c) an appropriate phosphatase has not yet been described. To test the latter hypothesis, we used RNAi to evaluate function of the two PP2A members that had not been studied previously. We refer to these proteins as TbPP2A-1 (Tb927.3.1240) and TbPP2A-2 (Tb11.01.3770). As outlined below, the depletion of TbPP2A-1 rapidly altered cell growth and produced phenotypic changes. In contrast, TbPP2A-2 did not generate an obvious phenotype and we did not characterize it further. For the knockdown experiment, nucleotides 138–770 of tbpp2a-1 were amplified by PCR. RNAit confirmed that this region did not generate any 20mers with overlap to other regions of the genome. The PCR fragment was cloned into pZJM and electroporated into monomorphic 90–13 BF and 29–13 PF as described previously [14]. Transformants were selected with phleomycin, and clonal cell lines were obtained by limiting dilution. To verify that our construct lowered transcript levels of tbpp2a-1, reverse transcriptase PCR (RT-PCR) was used. After growth in 1 g ml−1 tetracycline, the transcript level of tbpp2a-1 was lowered to approximately 30% of control values in BF and to 20% of control values in PF (Fig. 1A, insert). The level of tbrack1 was monitored as a loading control and (−)RT was used to assess contamination by DNA. TbPP2A-1 was essential for growth of BF and PF (Fig. 1A). In both BF and PF, growth arrest occurred within 24 h of tetracycline induction. Over a 4-day period, cell numbers declined gradually by three- to fourfold; indicating that loss of TbPP2A-1 was cytostatic and not lytic. When cells were examined microscopically, both PF and BF showed signs of failure to transition appropriately through the cell cycle. Complexity of the trypanosome cell cycle is affected by multiple single-copy structures (the basal body, flagellum, kinetoplast, and Golgi apparatus). The situation contrasts with that in mammalian cells, where multi-copy organelles can be segregated to the daughter cells by stochastic processes. In trypanosomes, a mechanism is needed to ensure that the single-copy structures replicate and then segregate to appropriate locations in the cell where the cleavage furrow can form between them. Basal bodies are critical regulators of the cell division cycle. They are responsible for events associated with kinetoplast replication and positioning, Golgi segregation, and assembly of the new flagellum [15–17]. Cleavage furrow ingression during cytokinesis occurs between the two flagella [reviewed in 18]. The replication cycles for the single-copy organelles are generally coordinated with events in the nucleus, and thus create a series of cytological markers for cell cycle progression [19]. G1, S, G2 and M are readily identified with fluorescent DNA-binding dyes. During the normal division cycle, replication of the kinetoplasts (K) and assembly of the flagellum (F) precedes nuclear division (N). Thus a typical cell has KF ≥ N. However, after knockdown of TbPP2A-1, an unusual cell population developed with KF ≤ N (Fig. 1B). The extent of this population shift was quantified (Fig. 1C). Cells in the G1 phase of the cell cycle (1N1K) declined rapidly. Within 24 h post-induction, 17% of the BF population and 27% of PF consisted of cells with more than 2N (>2N), while cells with greater than 2 K were not observed. This phenomenon was especially pronounced after 48 h post-induction, when cells with greater than 2N increased to 55% of the BF cell population and 63% of PF, while no BF cells contained greater than 2 K. In contrast, uninduced, Tet(−) cells exhibited normal morphology (Fig. 1C, and Fig. S1). To determine if the relatively low kinetoplast numbers resulted from a block in segregation of the kDNA disk, size of the disk was
evaluated in fixed, DAPI stained cells after 24 h induction (Fig. S2). No significant difference was observed between Tet(−) and Tet(+) cells with mean values of 0.82 m and 0.84 m, respectively, and median values of 0.74 m and 0.77 m, respectively. The relative position of the nuclei and basal bodies is essential for proper alignment of the cleavage furrow and completion of cytokinesis. Misalignment of the cleavage furrow can prevent ingression or result in progeny that contain 0N1K; referred to as a zooid. Although cytokinesis was generally inhibited, some aberrant cleavage furrows were generated, as evidenced by an increase in zooids to 10% of the BF population and 15% of the PF after 48 h. When DNA content was examined by flow cytometry, the aneuploid population increased to 57% of the PF population and 60% of the BF population. Thus nuclear S phase continued, despite disruption of the kinetoplast cycle, and inhibition of cytokinesis (Fig. 1D). Altogether these data demonstrate that TbPP2A-1 has the same function in both BF and PF, in contrast with several of the cell division kinases. The dephosphorylation event mediated by TbPP2A-1 is important for maintaining kinetoplast numbers and for assembly of new flagella. Because cell cycle disruption is often accompanied by abnormal accumulation of nuclei or flagella, we sought to create a fluorescent cell line where these changes could be observed directly. The goal was to (a) visualize cell cycle disruption in live trypanosomes; and (b) to facilitate the identification and validation of novel cell cycle regulatory genes. The cell line was initially used to study the knockdown of TbPP2A-1. Marker cells were constructed that constitutively express nucleus localized GFP (NUC-GFP), and flagellar localized DsRed (DsRed-PFR). NUC-GFP builds upon an aequorin vector we described previously to monitor nuclear calcium levels within live T. brucei [20]. Both constructs are outlined in Fig. 2A. Stable clonal cell lines were produced that express either NUC.GFP or DsRed.PFR, or both together. Additionally, each cell line was transformed with the TbPP2A-1 construct in pZJM. Growth of these cell lines was monitored in the absence of tetracycline. Cell growth was not affected by transformation of PF 29-13 with combinations of NUC.GFP, DsRed-PFR and the TbPP2A-1 RNAi constructs (−Tet) or the corresponding antibiotics that are required to maintain the integrated plasmids (Fig. 2B). To assess utility of the tagged cell lines, live trypanosomes were examined by fluorescence microscopy (Fig. 2C, upper panel). An aliquot of cell culture was placed on a microscope slide and immobilized with an equal volume of 2% low-melting point agarose previously cooled to 37 ◦ C. The cells were placed in the refrigerator for 5 min, and then viewed directly. The marker cells gave a strong, well-localized signal that was uniform in each organelle. NUC-GFP was soluble in the nucleoplasm and it diffused into a post-mitotic connector between nuclei (arrow, top panel). The fluorescence output of the live cell population was evaluated by flow cytometry (Fig. 2D, upper panel). The fluorescence signal from either NUCGFP or DsRed-PFR was significantly greater than autofluorescence from the parental 29-13 procyclic form (PF) cells (dotted lines). The marker cells were used to visualize morphologic changes that accompany a 24 h treatment with OKA (Fig. 2C, middle panel). The drug treated cells failed to initiate cytokinesis, failed to synthesize new flagella at a normal rate, but still underwent nuclear division. Consequently the cells had the unusual phenotype of N > F. The pZJM.TbPP2A-1 vector was introduced into the marker cells. Clonal cell lines were obtained. Following tetracycline induction, the phenotype was similar to the one observed with OKA, and was identical to the one observed in Fig. 1B. The fluorescent signals were robust and well localized. In preparation for a flow cytometry analysis of these marker cells, the number of flagella per cell was manually counted. The TbPP2A-1RNAi cells from Fig. 1 were induced with tetracycline for 48 h, fixed and labeled with anti-PFR. The cell population with F ≥ 2 increased from 28% of the population in control cells, to 70% of the population in the tetracycline induced
Please cite this article in press as: Rothberg KG, et al. Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei. Mol Biochem Parasitol (2014), http://dx.doi.org/10.1016/j.molbiopara.2014.04.006
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Fig. 1. Cell cycle disruption results from knockdown of TbPP2A-1. (A) Knockdown of TbPP2A-1 inhibits growth of BF and PF. A 632 bp fragment of TbPP2A-1 (Tb927.3.1240) was amplified by PCR and cloned into the BamH1/HindIII site of the dual promoter vector pZJM. The construct was electroporated into BF 90-13 and PF 39-13. Clonal cell lines were generated by limiting dilution. For growth studies, log phase cultures were diluted to 2 × 105 cells ml−1 (BF) or 1 × 106 cells ml−1 (PF). Tetracycline (1 g ml−1 ) was added to the culture and cell density was followed with a hemocytometer. Where needed, cultures were maintained in the log phase of growth by dilution. Each time point is the cell density multiplied by the dilution factor (average ± SD; n = 3 independent experiments). The inset is RT-PCR, using total RNA from 1 × 108 cells as a template. PCR was performed ±RT (reverse transcriptase) and ±Tet. The forward primer was residues 103–123 (5 -AAACCTCTCAGTGAGCCGCAA-3 ) and the reverse primer was residues 749–765 (5 -ATGCGCACGAGCAATTG-3 ). TbRACK1 was amplified as a loading control. Intensity of the bands was quantified with ImageJ v1.47. (B) Cell morphology of the RNAi cells. After 24 h growth with 1 g ml−1 tetracycline, cells were washed with phosphate buffered saline, fixed, permeabilized, incubated with antibodies and counterstained with 4,6-diamidino-2-phenylindole (DAPI) as described previously [14]. Microscopy was with a Nikon C1 Digital Eclipse E600 microscope and image capture was with the Metamorph software. The DIC/DAPI image of BF cells was modified in the following way: the background was smoothed in Photoshop. The bar is 10 m. (C) Changes in cell morphology after induction of RNAi. At the indicated days post-induction, cells were fixed and stained with DAPI. To quantify the configuration of nuclei (N) and kinetoplasts (K), between 100–300 cells were scored per time point. The histogram is the average value ± SE for 2 independent experiments. (D) DNA content of PF after induction of RNAi. Cells were harvested, fixed, treated with RNase A and stained with propidium iodide as described previously [14]. Fluorescence analysis was performed with the FACSCalibur flow cytometer (Becton Dickinson) and cell cycle analysis was with Modfit software.
Please cite this article in press as: Rothberg KG, et al. Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei. Mol Biochem Parasitol (2014), http://dx.doi.org/10.1016/j.molbiopara.2014.04.006
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Fig. 2. Cell cycle analysis with an engineered PF cell line expressing NUC-GFP and DsRed-PFR. (A) Schematic of the NUC-GFP and DsRed-PFR plasmids. NUC-GFP was constructed on pHD496. The NLS-luciferase fusion protein was described previously [19]. The GFP in pCDNA3.1.SuperGFP (Invitrogen) was used to complete the construct. The HYG resistance marker was replaced with PUR. Expression was from an rDNA promoter. The flagellar construct contained monomeric DsRed (Clontech) in frame with the paraflagellar rod protein PFR2. The DsRed-PFR construct was cloned into the constitutive expression vector pTSA.HYG. The HYG resistance marker was replaced with BSR. Expression was from promoter read-through in the tubulin locus. Each vector was introduced singly or combined into PF 29-13. Antibiotics, where applicable included: 15 g ml−1 G418, 50 g ml−1 hygromycin, 1 g ml−1 puromycin, 10 g ml−1 blasticidin and 2.5 g ml−1 phleomycin. (B) Growth of the clonal cell lines. Growth was monitored with a hemocytometer. Neither the maintenance antibiotics, nor the florescent tags, nor the pZJM-TbPP2A-1 construct affected culture growth in the absence of tetracycline. C. Morphology of live marker cells. Unfixed cells were viewed directly as described in the text. Panel A shows a log phase control population (Con). Arrows indicate mitotic cells where a post-mitotic connector is visible with the soluble NUC-GFP fusion protein. Panel B shows cells treated with 100 nM okadaic acid (OKA) and viewed after 24 h. The arrow indicates a zooid with 1F and 0N. Other cells exhibit the OKA phenotype. Panels C and D show RNAi cells, 24 h post-induction with 1 g ml−1 tetracycline. The arrow indicates a cell with two-pairs of synchronously dividing nuclei. (D) Flow cytometer analysis of the live marker cells. Cells were washed in PBS-G with Dulbecco’s salts (Invitrogen) and suspended to a density of 2.5 × 106 cells ml−1 . The live cells were analyzed with a FACSCalibur flow cytometer using D-PBSG as sheath fluid. The FL1-H channel (GFP) was set at 630 V and the FL2-H channel (red) at 582 V on a log setting, and Amp Gain of 1.0. Fluorescence output from 100,000 cells in each channel is shown. In the upper panels, autofluorescence of the parental 29-13 cells (dotted line) is lower than fluorescence of the marker cells (blue line). Transformation of these cells with the pZJM-TbPP2A-1 vector in the absence of tetracycline did not shift fluorescence. In the lower panel, TbPP2A-1 RNAi was induced with tetracycline. After 72 h a new cell population appeared whose fluorescence increased compared with the fluorescence of the uninduced cells (orange line). These results are consistent with a cell population Q4 containing more nuclei and more flagella than the control population. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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population (Fig. S3). When marker cells were analyzed by flow cytometry, the increased fluorescence per cell was readily apparent (Fig. 2D, lower panel). The marker cells thus appear suitable for live cell imaging or for screening of RNAi libraries by flow cytometry for novel cell cycle regulatory genes. Overall, this study identifies for the first time a protein phosphatase that is essential for cell cycle progression in T. brucei. Its presence was previously alluded to by treatment with OKA. In the absence of TbPP2A-1, kinetoplast triplication fails to keep pace with mitosis. Replication of nuclear DNA continues unabated, and mitotic divisions in the absence of cytokinesis generate cells
with a distinctive phenotype. The cells phenocopy treatment with OKA, suggesting that TbPP2A-1 is primarily responsible for the morphological changes that accompany exposure to this inhibitor. RNAi knockdown of PP2A-1 should cause hyperphosphorylation of its target substrates. A similar hyperphosphorylation would be expected from overexpression of the corresponding kinase; and the two events should phenocopy each other. The literature contains such a kinase in the form of NIMA-related kinase TbNRKC [21]. When the TY1-tagged TbNRKC is overexpressed, cells appear with multiple nuclei, and the N > K morphology. However, the relationship between TbNRKC and TbPP2A-1 was not explored in this
Please cite this article in press as: Rothberg KG, et al. Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei. Mol Biochem Parasitol (2014), http://dx.doi.org/10.1016/j.molbiopara.2014.04.006
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study. To assess the effects of RNAi knockdown in the context of live trypanosomes; a marker cell line was constructed. These cells have nuclei that fluoresce green and flagella that fluoresce red. When treated with OKA or transformed with the tbpp2a-1 RNAi construct, the cells exhibited the same morphological changes. Because these marker cells are suitable for live cell imaging or detection in a flow cytometer, the cells may have utility in screening RNAi libraries in the search for novel cell cycle regulatory genes. Acknowledgements
The authors wish to thank several colleagues for their generous gifts of essential reagents. Thomas Seebeck, University of Bern 227 provided us with the anti-PFR antibodies. David Campbell, UCLA 228 supplied the pHD494 expression plasmid. George Cross, The Rock229 efeller University provided the 29-13 and 90-13 cell lines. This work 230 231 Q3 was supported in part by NIH R15 AI082513 and by a Gerald J. Ford 232 Fellowship. 226
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.molbiopara.2014.04.006. References [1] Jones NG, Thomas EB, Brown E, Dickens NJ, Hammarton Tc, Mottram JC. Regulators of Trypanosoma brucei cell cycle progression and differentiation identified using a kinome-wide RNAi screen. PLoS Pathog 2014;10(1):e1003886. [2] Li Z. Regulation of the cell division cycle in Trypanosoma brucei. Eukaryot Cell 2012;11:1180–90. [3] Parsons M, Worthey EA, Ward PN, Mottram JC. Comparative analysis of the kinomes of three pathogenic trypanosomatids: Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. BMC Genomics 2005;6:127. [4] Brenchley R, Tariq H, McElhinney H, Szöör B, Huxley-Jones J, Stevens R, et al. The TriTryp phosphatome: analysis of the protein phosphatase catalytic domains. BMC Genomics 2007;8:434.
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[5] Barford D, Das AK, Egloff MP. The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu Rev Biophys Biomol Struct 1998;27:133–64. [6] Szöör B. Trypanosomatid protein phosphatases. Mol Biochem Parasitol 2010;173:53–63. [7] Das A, Gale Jr M, Carter V, Parsons M. The protein phosphatase inhibitor okadaic acid induces defects in cytokinesis and organellar genome segregation in Trypanosoma brucei. J Cell Sci 1994;107:3477–83. [8] Orr GA, Werner C, Xu J, Bennett M, Weiss LM, Takvorkan P, et al. Identification of novel serine/threonine protein phosphatases in Trypanosoma cruzi: a potential role in control of cytokinesis and morphology. Infect Immun 2000;68:1350– 8. [9] Li Z, Tu X, Wang CC. Okadaic acid overcomes the blocked cell cycle caused by depleting Cdc2-related kinases in Trypanosoma brucei. Exp Cell Res 2006;312:3504–16. [10] Erondu NE, Donelson JE. Characterization of trypanosome protein phosphatase 1 and 2A catalytic subunits. Mol Biochem Parasitol 1991;49:303–14. [11] Gallet C, Demonchy R, Koppel C, Grellier P, Kohl L. A protein phosphatase 1 involved in correct nucleus positioning in trypanosomes. Mol Biochem Parastiol 2013;192:49–54. [12] Anderson S, Jones C, Saha L, Chaudhuri M. Functional characterization of the serine/threonine protein phosphatase 5 from Trypanosoma brucei. J Parasitol 2006;92:1152–61. [13] Jones C, Anderson S, Singha UK, Chaudhuri M. Protein phosphatase 5 is required for Hsp90 function during proteotoxic stresses in Trypanosoma brucei. Parasitol Res 2008;102:835–44. [14] Rothberg KG, Burdette DL, Pfannstiel J, Jetton N, Singh R, Ruben L. The RACK1 homologue from Trypanosoma brucei is required for the onset and progression of cytokinesis. J Biol Chem 2006;281:9781–90. [15] Robinson DR, Gull K. Basal body movements as a mechanism for mitochondrial genome segregation in the trypanosome cell cycle. Nature 1991;35:731–3. [16] Gluenz E, Povelones ML, Englund PT, Gull K. The kinetoplast duplication cycle in Trypanosoma brucei is orchestrated by cytoskeleton-mediated cell morphogenesis. Mol Cell Biol 2011;31:1012–21. [17] He CY, Pypaert M, Warren G. Golgi duplication in Trypanosoma brucei requires Centrin2. Science 2005;310:1196–8. [18] Hammarton TC, Monnerat S, Mottram JC. Cytokinesis in trypanosomatids. Curr Opin Microbiol 2007;10:520–7. [19] Woodward R, Gull K. Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei. J Cell Sci 1990;95:49–57. [20] Xiong Z-H, Ruben L. Nuclear calcium flux in Trypanosoma brucei can be quantified with targeted aequorin. Mol Biochem Parasitol 1996;83:57–67. [21] Pradel LC, Bonhivers M, Landrein N, Robinson DR. NIMA-related kinase TbNRKC is involved in basal body separation in Trypanosoma brucei. J Cell Sci 2006;119:1852–63.
Please cite this article in press as: Rothberg KG, et al. Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei. Mol Biochem Parasitol (2014), http://dx.doi.org/10.1016/j.molbiopara.2014.04.006
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Contents lists available at ScienceDirect
Molecular & Biochemical Parasitology
Short communication
1
Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei
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3
4 5 6
Q1
Karen G. Rothberg 1 , Neal Jetton, James G. Hubbard 2 , Daniel A. Powell 3 , Vidya Pandarinath, Larry Ruben ∗
Q2 Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275, United States
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a b s t r a c t
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Article history: Received 23 September 2013 Received in revised form 12 April 2014 Accepted 18 April 2014 Available online xxx
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Keywords: Phosphoprotein phosphatase PP2A Okadaic acid Cell cycle regulation Basal bodies Trypanosoma brucei
The cell cycle consists of an orderly sequence of events, whose purpose is to faithfully replicate and segregate cellular components. Many events in the cell cycle are triggered by protein kinases and counteracting phosphoprotein phosphatases (PPP). In Trypanosoma brucei, RNAi has been used to characterize numerous regulatory kinases, while the role of protein phosphatases has primarily been deduced with inhibitors such as okadaic acid and calyculin. In the present study, we identify for the first time a protein phosphatase 2A family member (TbPP2A-1) whose knockdown with RNAi phenocopies the effects of okadaic acid (OKA). In bloodstream forms (BF) and insect stage procyclic forms (PF) RNAi of TbPP2A-1 generates a cell population characterized by: an inhibition of cell growth, a block in cytokinesis; continued synthesis of nuclear DNA leading to aneuploidy; continued mitosis leading to cells with N > 2, and an unusual phenotype where number of kinetoplasts (and flagella) is less than the number of nuclei. An engineered cell line was constructed to further study TbPP2A-1 and to facilitate the discovery of other cell cycle regulatory genes. © 2014 Published by Elsevier B.V.
Trypanosoma brucei is a unicellular parasite that invades the fluid spaces of the mammalian host. Survival depends upon effective immune evasion coupled with continuous replenishment of cell populations by replication. The cell division cycle is only halted during transmission between hosts. All eukaryotic cells rely upon triggering processes to coordinate events during the cell cycle. Reversible cycles of phosphorylation and dephosphorylation are critical. In trypanosomes a role in cell division has been described for a range of kinases, including: cyclin/Crk complexes, polo-like kinase, Aurora kinase, tousled-like kinase, NDR kinases and NIMA family members [reviewed in 1,2]. In contrast little is known about the corresponding phosphatases. In general, the kinome of T. brucei is more extensive than its phosphatome [3,4]. With 156 eukaryotic kinases and only 78 phosphatase catalytic subunits, each phosphatase is generally used to
∗ Corresponding author. Tel.: +1 214 768 2321; fax: +1 214 768 2995. E-mail address: [email protected] (L. Ruben). 1 Current address: Live Cell Imaging Core, University of Texas Southwestern Medical School, 5323 Harry Hines Blvd, Dallas, TX 75390, United States. 2 Current address: Department of Biology, Texas Wesleyan University, Fort Worth, TX 76105, United States. 3 Current address: Department of Immunology, University of Arizona, Tucson, AZ 85724, United States.
reverse the effects of multiple kinases. In the phosphoprotein phosphatase family (PPP family) of Ser/Thr phosphatases, specificity is accomplished by binding the catalytic subunit to scaffolding subunits. The scaffolds regulate both the enzymatic activity and location of the phosphatase [reviewed in 5]. The trypanosome genome encodes multiple PPP catalytic subunits belonging to the PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7 families [4,6]. A role for PPP family members in T. brucei cell division was first demonstrated with okadaic acid (OKA); an inhibitor of both PP2A and PP1 family members. OKA has no effect on nuclear division, but prevents kinetoplast division from keeping pace with mitosis. Cytokinesis is inhibited, leading to cells with multiple nuclei, and diminished number of kinetoplasts [7]. In T. cruzi, similar observations have been made, except the PP1/PP2A inhibitor calyculin was used [8]. In an effort to identify cell cycle regulatory phosphatases in T. brucei, the RNAi-dependent knockdown of all 8 PP1 family members has been described, along with knockdown of a single PP2A family member [9]. The PP2A family member had previously been cloned [10] and was later reclassified as PP4 [4]. Neither the loss of PP1 nor of PP4 blocked cell cycle progression at a specific step, nor generated a phenotype that resembled the effects of OKA. However, a more recent study determined that PP1-3 plays a role in nuclear positioning [11]. The only other PPP member to be characterized by RNAi is TbPP5. This phosphatase appears to have stage-specific
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Please cite this article in press as: Rothberg KG, et al. Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei. Mol Biochem Parasitol (2014), http://dx.doi.org/10.1016/j.molbiopara.2014.04.006
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functions associated with growth-related sensitivity to serum factors and Hsp90 chaperone function [12,13]. In general, very little is known about the function of PPP family members and their scaffolding proteins in T. brucei. None of the proteins evaluated to date mimics the effects of OKA when knocked down by RNAi. It may be that: (a) RNAi generates an insufficient knockdown to exert an effect; (b) the OKA treatment may have inhibited multiple phosphatases whereas RNAi disrupts expression of only a single protein; or (c) an appropriate phosphatase has not yet been described. To test the latter hypothesis, we used RNAi to evaluate function of the two PP2A members that had not been studied previously. We refer to these proteins as TbPP2A-1 (Tb927.3.1240) and TbPP2A-2 (Tb11.01.3770). As outlined below, the depletion of TbPP2A-1 rapidly altered cell growth and produced phenotypic changes. In contrast, TbPP2A-2 did not generate an obvious phenotype and we did not characterize it further. For the knockdown experiment, nucleotides 138–770 of tbpp2a-1 were amplified by PCR. RNAit confirmed that this region did not generate any 20mers with overlap to other regions of the genome. The PCR fragment was cloned into pZJM and electroporated into monomorphic 90–13 BF and 29–13 PF as described previously [14]. Transformants were selected with phleomycin, and clonal cell lines were obtained by limiting dilution. To verify that our construct lowered transcript levels of tbpp2a-1, reverse transcriptase PCR (RT-PCR) was used. After growth in 1 g ml−1 tetracycline, the transcript level of tbpp2a-1 was lowered to approximately 30% of control values in BF and to 20% of control values in PF (Fig. 1A, insert). The level of tbrack1 was monitored as a loading control and (−)RT was used to assess contamination by DNA. TbPP2A-1 was essential for growth of BF and PF (Fig. 1A). In both BF and PF, growth arrest occurred within 24 h of tetracycline induction. Over a 4-day period, cell numbers declined gradually by three- to fourfold; indicating that loss of TbPP2A-1 was cytostatic and not lytic. When cells were examined microscopically, both PF and BF showed signs of failure to transition appropriately through the cell cycle. Complexity of the trypanosome cell cycle is affected by multiple single-copy structures (the basal body, flagellum, kinetoplast, and Golgi apparatus). The situation contrasts with that in mammalian cells, where multi-copy organelles can be segregated to the daughter cells by stochastic processes. In trypanosomes, a mechanism is needed to ensure that the single-copy structures replicate and then segregate to appropriate locations in the cell where the cleavage furrow can form between them. Basal bodies are critical regulators of the cell division cycle. They are responsible for events associated with kinetoplast replication and positioning, Golgi segregation, and assembly of the new flagellum [15–17]. Cleavage furrow ingression during cytokinesis occurs between the two flagella [reviewed in 18]. The replication cycles for the single-copy organelles are generally coordinated with events in the nucleus, and thus create a series of cytological markers for cell cycle progression [19]. G1, S, G2 and M are readily identified with fluorescent DNA-binding dyes. During the normal division cycle, replication of the kinetoplasts (K) and assembly of the flagellum (F) precedes nuclear division (N). Thus a typical cell has KF ≥ N. However, after knockdown of TbPP2A-1, an unusual cell population developed with KF ≤ N (Fig. 1B). The extent of this population shift was quantified (Fig. 1C). Cells in the G1 phase of the cell cycle (1N1K) declined rapidly. Within 24 h post-induction, 17% of the BF population and 27% of PF consisted of cells with more than 2N (>2N), while cells with greater than 2 K were not observed. This phenomenon was especially pronounced after 48 h post-induction, when cells with greater than 2N increased to 55% of the BF cell population and 63% of PF, while no BF cells contained greater than 2 K. In contrast, uninduced, Tet(−) cells exhibited normal morphology (Fig. 1C, and Fig. S1). To determine if the relatively low kinetoplast numbers resulted from a block in segregation of the kDNA disk, size of the disk was
evaluated in fixed, DAPI stained cells after 24 h induction (Fig. S2). No significant difference was observed between Tet(−) and Tet(+) cells with mean values of 0.82 m and 0.84 m, respectively, and median values of 0.74 m and 0.77 m, respectively. The relative position of the nuclei and basal bodies is essential for proper alignment of the cleavage furrow and completion of cytokinesis. Misalignment of the cleavage furrow can prevent ingression or result in progeny that contain 0N1K; referred to as a zooid. Although cytokinesis was generally inhibited, some aberrant cleavage furrows were generated, as evidenced by an increase in zooids to 10% of the BF population and 15% of the PF after 48 h. When DNA content was examined by flow cytometry, the aneuploid population increased to 57% of the PF population and 60% of the BF population. Thus nuclear S phase continued, despite disruption of the kinetoplast cycle, and inhibition of cytokinesis (Fig. 1D). Altogether these data demonstrate that TbPP2A-1 has the same function in both BF and PF, in contrast with several of the cell division kinases. The dephosphorylation event mediated by TbPP2A-1 is important for maintaining kinetoplast numbers and for assembly of new flagella. Because cell cycle disruption is often accompanied by abnormal accumulation of nuclei or flagella, we sought to create a fluorescent cell line where these changes could be observed directly. The goal was to (a) visualize cell cycle disruption in live trypanosomes; and (b) to facilitate the identification and validation of novel cell cycle regulatory genes. The cell line was initially used to study the knockdown of TbPP2A-1. Marker cells were constructed that constitutively express nucleus localized GFP (NUC-GFP), and flagellar localized DsRed (DsRed-PFR). NUC-GFP builds upon an aequorin vector we described previously to monitor nuclear calcium levels within live T. brucei [20]. Both constructs are outlined in Fig. 2A. Stable clonal cell lines were produced that express either NUC.GFP or DsRed.PFR, or both together. Additionally, each cell line was transformed with the TbPP2A-1 construct in pZJM. Growth of these cell lines was monitored in the absence of tetracycline. Cell growth was not affected by transformation of PF 29-13 with combinations of NUC.GFP, DsRed-PFR and the TbPP2A-1 RNAi constructs (−Tet) or the corresponding antibiotics that are required to maintain the integrated plasmids (Fig. 2B). To assess utility of the tagged cell lines, live trypanosomes were examined by fluorescence microscopy (Fig. 2C, upper panel). An aliquot of cell culture was placed on a microscope slide and immobilized with an equal volume of 2% low-melting point agarose previously cooled to 37 ◦ C. The cells were placed in the refrigerator for 5 min, and then viewed directly. The marker cells gave a strong, well-localized signal that was uniform in each organelle. NUC-GFP was soluble in the nucleoplasm and it diffused into a post-mitotic connector between nuclei (arrow, top panel). The fluorescence output of the live cell population was evaluated by flow cytometry (Fig. 2D, upper panel). The fluorescence signal from either NUCGFP or DsRed-PFR was significantly greater than autofluorescence from the parental 29-13 procyclic form (PF) cells (dotted lines). The marker cells were used to visualize morphologic changes that accompany a 24 h treatment with OKA (Fig. 2C, middle panel). The drug treated cells failed to initiate cytokinesis, failed to synthesize new flagella at a normal rate, but still underwent nuclear division. Consequently the cells had the unusual phenotype of N > F. The pZJM.TbPP2A-1 vector was introduced into the marker cells. Clonal cell lines were obtained. Following tetracycline induction, the phenotype was similar to the one observed with OKA, and was identical to the one observed in Fig. 1B. The fluorescent signals were robust and well localized. In preparation for a flow cytometry analysis of these marker cells, the number of flagella per cell was manually counted. The TbPP2A-1RNAi cells from Fig. 1 were induced with tetracycline for 48 h, fixed and labeled with anti-PFR. The cell population with F ≥ 2 increased from 28% of the population in control cells, to 70% of the population in the tetracycline induced
Please cite this article in press as: Rothberg KG, et al. Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei. Mol Biochem Parasitol (2014), http://dx.doi.org/10.1016/j.molbiopara.2014.04.006
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Fig. 1. Cell cycle disruption results from knockdown of TbPP2A-1. (A) Knockdown of TbPP2A-1 inhibits growth of BF and PF. A 632 bp fragment of TbPP2A-1 (Tb927.3.1240) was amplified by PCR and cloned into the BamH1/HindIII site of the dual promoter vector pZJM. The construct was electroporated into BF 90-13 and PF 39-13. Clonal cell lines were generated by limiting dilution. For growth studies, log phase cultures were diluted to 2 × 105 cells ml−1 (BF) or 1 × 106 cells ml−1 (PF). Tetracycline (1 g ml−1 ) was added to the culture and cell density was followed with a hemocytometer. Where needed, cultures were maintained in the log phase of growth by dilution. Each time point is the cell density multiplied by the dilution factor (average ± SD; n = 3 independent experiments). The inset is RT-PCR, using total RNA from 1 × 108 cells as a template. PCR was performed ±RT (reverse transcriptase) and ±Tet. The forward primer was residues 103–123 (5 -AAACCTCTCAGTGAGCCGCAA-3 ) and the reverse primer was residues 749–765 (5 -ATGCGCACGAGCAATTG-3 ). TbRACK1 was amplified as a loading control. Intensity of the bands was quantified with ImageJ v1.47. (B) Cell morphology of the RNAi cells. After 24 h growth with 1 g ml−1 tetracycline, cells were washed with phosphate buffered saline, fixed, permeabilized, incubated with antibodies and counterstained with 4,6-diamidino-2-phenylindole (DAPI) as described previously [14]. Microscopy was with a Nikon C1 Digital Eclipse E600 microscope and image capture was with the Metamorph software. The DIC/DAPI image of BF cells was modified in the following way: the background was smoothed in Photoshop. The bar is 10 m. (C) Changes in cell morphology after induction of RNAi. At the indicated days post-induction, cells were fixed and stained with DAPI. To quantify the configuration of nuclei (N) and kinetoplasts (K), between 100–300 cells were scored per time point. The histogram is the average value ± SE for 2 independent experiments. (D) DNA content of PF after induction of RNAi. Cells were harvested, fixed, treated with RNase A and stained with propidium iodide as described previously [14]. Fluorescence analysis was performed with the FACSCalibur flow cytometer (Becton Dickinson) and cell cycle analysis was with Modfit software.
Please cite this article in press as: Rothberg KG, et al. Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei. Mol Biochem Parasitol (2014), http://dx.doi.org/10.1016/j.molbiopara.2014.04.006
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Fig. 2. Cell cycle analysis with an engineered PF cell line expressing NUC-GFP and DsRed-PFR. (A) Schematic of the NUC-GFP and DsRed-PFR plasmids. NUC-GFP was constructed on pHD496. The NLS-luciferase fusion protein was described previously [19]. The GFP in pCDNA3.1.SuperGFP (Invitrogen) was used to complete the construct. The HYG resistance marker was replaced with PUR. Expression was from an rDNA promoter. The flagellar construct contained monomeric DsRed (Clontech) in frame with the paraflagellar rod protein PFR2. The DsRed-PFR construct was cloned into the constitutive expression vector pTSA.HYG. The HYG resistance marker was replaced with BSR. Expression was from promoter read-through in the tubulin locus. Each vector was introduced singly or combined into PF 29-13. Antibiotics, where applicable included: 15 g ml−1 G418, 50 g ml−1 hygromycin, 1 g ml−1 puromycin, 10 g ml−1 blasticidin and 2.5 g ml−1 phleomycin. (B) Growth of the clonal cell lines. Growth was monitored with a hemocytometer. Neither the maintenance antibiotics, nor the florescent tags, nor the pZJM-TbPP2A-1 construct affected culture growth in the absence of tetracycline. C. Morphology of live marker cells. Unfixed cells were viewed directly as described in the text. Panel A shows a log phase control population (Con). Arrows indicate mitotic cells where a post-mitotic connector is visible with the soluble NUC-GFP fusion protein. Panel B shows cells treated with 100 nM okadaic acid (OKA) and viewed after 24 h. The arrow indicates a zooid with 1F and 0N. Other cells exhibit the OKA phenotype. Panels C and D show RNAi cells, 24 h post-induction with 1 g ml−1 tetracycline. The arrow indicates a cell with two-pairs of synchronously dividing nuclei. (D) Flow cytometer analysis of the live marker cells. Cells were washed in PBS-G with Dulbecco’s salts (Invitrogen) and suspended to a density of 2.5 × 106 cells ml−1 . The live cells were analyzed with a FACSCalibur flow cytometer using D-PBSG as sheath fluid. The FL1-H channel (GFP) was set at 630 V and the FL2-H channel (red) at 582 V on a log setting, and Amp Gain of 1.0. Fluorescence output from 100,000 cells in each channel is shown. In the upper panels, autofluorescence of the parental 29-13 cells (dotted line) is lower than fluorescence of the marker cells (blue line). Transformation of these cells with the pZJM-TbPP2A-1 vector in the absence of tetracycline did not shift fluorescence. In the lower panel, TbPP2A-1 RNAi was induced with tetracycline. After 72 h a new cell population appeared whose fluorescence increased compared with the fluorescence of the uninduced cells (orange line). These results are consistent with a cell population Q4 containing more nuclei and more flagella than the control population. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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population (Fig. S3). When marker cells were analyzed by flow cytometry, the increased fluorescence per cell was readily apparent (Fig. 2D, lower panel). The marker cells thus appear suitable for live cell imaging or for screening of RNAi libraries by flow cytometry for novel cell cycle regulatory genes. Overall, this study identifies for the first time a protein phosphatase that is essential for cell cycle progression in T. brucei. Its presence was previously alluded to by treatment with OKA. In the absence of TbPP2A-1, kinetoplast triplication fails to keep pace with mitosis. Replication of nuclear DNA continues unabated, and mitotic divisions in the absence of cytokinesis generate cells
with a distinctive phenotype. The cells phenocopy treatment with OKA, suggesting that TbPP2A-1 is primarily responsible for the morphological changes that accompany exposure to this inhibitor. RNAi knockdown of PP2A-1 should cause hyperphosphorylation of its target substrates. A similar hyperphosphorylation would be expected from overexpression of the corresponding kinase; and the two events should phenocopy each other. The literature contains such a kinase in the form of NIMA-related kinase TbNRKC [21]. When the TY1-tagged TbNRKC is overexpressed, cells appear with multiple nuclei, and the N > K morphology. However, the relationship between TbNRKC and TbPP2A-1 was not explored in this
Please cite this article in press as: Rothberg KG, et al. Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei. Mol Biochem Parasitol (2014), http://dx.doi.org/10.1016/j.molbiopara.2014.04.006
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study. To assess the effects of RNAi knockdown in the context of live trypanosomes; a marker cell line was constructed. These cells have nuclei that fluoresce green and flagella that fluoresce red. When treated with OKA or transformed with the tbpp2a-1 RNAi construct, the cells exhibited the same morphological changes. Because these marker cells are suitable for live cell imaging or detection in a flow cytometer, the cells may have utility in screening RNAi libraries in the search for novel cell cycle regulatory genes. Acknowledgements
The authors wish to thank several colleagues for their generous gifts of essential reagents. Thomas Seebeck, University of Bern 227 provided us with the anti-PFR antibodies. David Campbell, UCLA 228 supplied the pHD494 expression plasmid. George Cross, The Rock229 efeller University provided the 29-13 and 90-13 cell lines. This work 230 231 Q3 was supported in part by NIH R15 AI082513 and by a Gerald J. Ford 232 Fellowship. 226
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.molbiopara.2014.04.006. References [1] Jones NG, Thomas EB, Brown E, Dickens NJ, Hammarton Tc, Mottram JC. Regulators of Trypanosoma brucei cell cycle progression and differentiation identified using a kinome-wide RNAi screen. PLoS Pathog 2014;10(1):e1003886. [2] Li Z. Regulation of the cell division cycle in Trypanosoma brucei. Eukaryot Cell 2012;11:1180–90. [3] Parsons M, Worthey EA, Ward PN, Mottram JC. Comparative analysis of the kinomes of three pathogenic trypanosomatids: Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. BMC Genomics 2005;6:127. [4] Brenchley R, Tariq H, McElhinney H, Szöör B, Huxley-Jones J, Stevens R, et al. The TriTryp phosphatome: analysis of the protein phosphatase catalytic domains. BMC Genomics 2007;8:434.
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[5] Barford D, Das AK, Egloff MP. The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu Rev Biophys Biomol Struct 1998;27:133–64. [6] Szöör B. Trypanosomatid protein phosphatases. Mol Biochem Parasitol 2010;173:53–63. [7] Das A, Gale Jr M, Carter V, Parsons M. The protein phosphatase inhibitor okadaic acid induces defects in cytokinesis and organellar genome segregation in Trypanosoma brucei. J Cell Sci 1994;107:3477–83. [8] Orr GA, Werner C, Xu J, Bennett M, Weiss LM, Takvorkan P, et al. Identification of novel serine/threonine protein phosphatases in Trypanosoma cruzi: a potential role in control of cytokinesis and morphology. Infect Immun 2000;68:1350– 8. [9] Li Z, Tu X, Wang CC. Okadaic acid overcomes the blocked cell cycle caused by depleting Cdc2-related kinases in Trypanosoma brucei. Exp Cell Res 2006;312:3504–16. [10] Erondu NE, Donelson JE. Characterization of trypanosome protein phosphatase 1 and 2A catalytic subunits. Mol Biochem Parasitol 1991;49:303–14. [11] Gallet C, Demonchy R, Koppel C, Grellier P, Kohl L. A protein phosphatase 1 involved in correct nucleus positioning in trypanosomes. Mol Biochem Parastiol 2013;192:49–54. [12] Anderson S, Jones C, Saha L, Chaudhuri M. Functional characterization of the serine/threonine protein phosphatase 5 from Trypanosoma brucei. J Parasitol 2006;92:1152–61. [13] Jones C, Anderson S, Singha UK, Chaudhuri M. Protein phosphatase 5 is required for Hsp90 function during proteotoxic stresses in Trypanosoma brucei. Parasitol Res 2008;102:835–44. [14] Rothberg KG, Burdette DL, Pfannstiel J, Jetton N, Singh R, Ruben L. The RACK1 homologue from Trypanosoma brucei is required for the onset and progression of cytokinesis. J Biol Chem 2006;281:9781–90. [15] Robinson DR, Gull K. Basal body movements as a mechanism for mitochondrial genome segregation in the trypanosome cell cycle. Nature 1991;35:731–3. [16] Gluenz E, Povelones ML, Englund PT, Gull K. The kinetoplast duplication cycle in Trypanosoma brucei is orchestrated by cytoskeleton-mediated cell morphogenesis. Mol Cell Biol 2011;31:1012–21. [17] He CY, Pypaert M, Warren G. Golgi duplication in Trypanosoma brucei requires Centrin2. Science 2005;310:1196–8. [18] Hammarton TC, Monnerat S, Mottram JC. Cytokinesis in trypanosomatids. Curr Opin Microbiol 2007;10:520–7. [19] Woodward R, Gull K. Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei. J Cell Sci 1990;95:49–57. [20] Xiong Z-H, Ruben L. Nuclear calcium flux in Trypanosoma brucei can be quantified with targeted aequorin. Mol Biochem Parasitol 1996;83:57–67. [21] Pradel LC, Bonhivers M, Landrein N, Robinson DR. NIMA-related kinase TbNRKC is involved in basal body separation in Trypanosoma brucei. J Cell Sci 2006;119:1852–63.
Please cite this article in press as: Rothberg KG, et al. Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei. Mol Biochem Parasitol (2014), http://dx.doi.org/10.1016/j.molbiopara.2014.04.006
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