The F-box protein CFB2 is required for cytokinesis of bloodstream-form Trypanosoma brucei

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Molecular & Biochemical Parasitology 156 (2007) 217–224

The F-box protein CFB2 is required for cytokinesis of bloodstream-form Trypanosoma brucei Corinna Benz, Christine E. Clayton ∗ Zentrum f¨ur Molekulare Biologie der Universit¨at Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120 Heidelberg, Germany Received 2 August 2007; received in revised form 14 August 2007; accepted 16 August 2007 Available online 21 August 2007

Abstract F-box proteins serve as mediators in targeting bound target proteins for ubiquitination and destruction. We here describe the roles of two Fbox proteins, CFB1 and CFB2, in the trypanosome cell cycle. Five almost identical copies of CFB1 are arranged in a direct tandem repeat on Trypanosoma brucei chromosome 1; immediately downstream is a single CFB2 gene. RNAi targeting CFB1 in bloodstream-form trypanosomes had a transient effect on growth and mitosis. Depletion of CFB2, in contrast, resulted in immediate growth arrest and rapid cell death. CFB2-depleted cells accumulated nuclei and kinetoplasts with the corresponding numbers of basal bodies and flagella. The CFB2 transcript was less abundant in procyclic-form trypanosomes, and RNAi against CFB2 in these forms had no effect on growth. These results suggest that CFB2 is required for bloodstream-form trypanosome cytokinesis. © 2007 Elsevier B.V. All rights reserved. Keywords: Trypanosoma; F-box; Cell cycle

1. Introduction The division cycle of eukaryotic cells is controlled by protein kinases which are activated by binding to cyclins. Cyclins are present only at particular times in the cell cycle; after they are no longer required they are destroyed by ubiquitination followed by digestion by the proteasome [1–3]. The ubiquitin chains are added by a cascade of enzymes called E1, E2 and E3 ubiquitin ligases, and specificity is determined by the E3 components. The E3 ligases that are important for cell cycle control are the anaphase promoting complex or cyclosome (APC/C) and the Skp1–Cdc53/cullin–F-box protein (SCF) complex [4]. In SCF complexes, proteins with an “F-box” domain (also called “cyclin-like F-box”) link targets to the degradation machinery. One domain of the protein – sometimes containing specific recognition motifs such as WD40 or leucine-rich repeats – binds to the doomed target. Meanwhile the F-box domain binds

Abbreviations: CFB1, cyclin F-box protein 1; CFB2, cyclin F-box protein 2; RNAi, RNA interference ∗ Corresponding author. E-mail address: [email protected] (C.E. Clayton). 0166-6851/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2007.08.005

to Skp1, which in turn links the target to the ubiquitination enzymes. Binding of kinase inhibitors and cyclins to F-box proteins usually depends on prior phosphorylation, although there are some exceptions [5]. The cell cycle of Trypanosoma brucei, like that of other eukaryotes, requires interactions between cyclins, cyclindependent kinases, and the ubiquitination machinery (reviewed in Refs. [6,7]). Trypanosomes in the G1 phase have one nucleus and one kinetoplast (1N1K). The first morphological event in the cell cycle is the maturation and duplication of the basal body, with nucleation of a new flagellum. The replication of the kinetoplast DNA starts just before nuclear S phase. Cells in early G2 have already duplicated and segregated their kinetoplast but still have a single nucleus, resulting in a 1N2K configuration. Following mitosis and just prior to cytokinesis, 2N2K cells are observed. Results of many experiments have shown that trypanosomes lack some checkpoints which in other organisms ensure accurate partition of the genetic material [6–8]. Thus, a variety of mutations, and treatment of the parasites with inhibitors, can result in cells whose complement of nuclei and kinetoplasts differs dramatically from the norm. Such deviant forms include “zoids” with a kinetoplast but no nucleus (0N1K), and cells with four or more nuclei and/or kinetoplasts

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[6,9,10]. Since the consequences of cell cycle dysregulation differ between bloodstream and procyclic forms, control of the cell cycle and the nature of checkpoints is probably to some extent developmentally regulated [8,9,11,12]. Regulated protein degradation via ubiquitin conjugation in T. brucei involves a classical 20S core proteasome and regulatory subunits [13,14]. As in other eukaryotes, cell cycle progression requires the proteasome. Depletion of any of the proteasome subunits by RNA interference inhibited cell growth and caused accumulation of ubiquitinated proteins [14–16]. Treatment of trypanosomes with lactacystin, a proteasome inhibitor, caused arrest at the G2/M boundary in procyclic-form cells, whereas bloodstream forms were arrested at both G1 and G2 stages of the cell cycle [17]. The involvement of the proteasome in cell cycle regulation was further underlined by the finding that two cyclin proteins have short half lives that can be prolonged by proteasome inhibitors [18]. RNAi against either of two of the seven T. brucei APC/C subunits, APC1 or CDC27, caused arrest in early mitosis in procyclic trypanosomes and at anaphase in bloodstream forms [19]. Roles for SCF complexes have, in contrast, not been demonstrated. In this paper, we investigate the functions of two F-box proteins encoded on trypanosome chromosome I. 2. Materials and methods 2.1. Trypanosome cell culture Bloodstream- and procyclic-form T. brucei were cultured and transfected as described previously [20]. Strain Lister 427 bloodstream- and procyclic-form cells constitutively expressing the tet repressor and T7 polymerase were used throughout this study [21]. G418 (2.5 ␮g/ml) and phleomycin (0.2 ␮g/ml) were added to the HMI-9 cell culture medium to maintain expression of the tet repressor and the T7 polymerase, respectively. The pleomorphic tet-repressor expressing cell line described in Ref. [20] was used for integrating a V5 epitope tag into the genome and cultured and differentiated as described previously. Recent microsatellite mapping results suggest that this line is probably T. b. brucei STIB247 (Turner, personal communication). 2.2. Inducible RNAi For RNAi-mediated knock down of the CFB proteins, p2T7TAblue -based vectors were used (pHD1722 for CFB1 and pHD1723 for CFB2) [21]. The CFB1 fragment corresponded to nt 1002–1500, for CFB2, nt 1051–1428 of the open reading frames. Trypanosomes were transfected with 10–15 ␮g of Not I-linearised plasmid DNA and clones were selected by the addition of 10 ␮g/ml hygromycin B. RNAi was induced with 0.5 ␮g of tetracycline and the efficiency of down-regulation of the target mRNAs was assessed by Northern blotting or (once the phenotype was established) by monitoring cell growth. 2.3. Northern blot and RNA analyses Total RNA was isolated from 5 × 107 log phase cells (density not exceeding 106 ml−1 for bloodstream-form cells and

5 × 106 ml−1 for procyclics) either using a RNeasy Mini kit (Qiagen, GmbH) or PeqGOLD TriFast (peqlab, GmbH). Ten micrograms of total RNA per lane were separated on a 1% agarose-formaldehyde gel and blotted onto a nylon membrane (Nytran-N, Schleicher & Schuell, GmbH) before hybridization with 32 P-labelled probes. Resulting bands were quantified using a phosphorimager and MacBas v2.0 software. 2.4. Flow cytometry 2 × 106 cells were suspended in 70% methanol, 30% PBS and left at 4 ◦ C for 0.5–7 days. Cells were washed and resuspended in PBS supplemented with 20 ␮g/ml RNase A and 30 ␮g/ml propidium iodide and incubated at 37 ◦ C for 30 min. A FACScan flow cytometer (Becton Dickinson) was used to analyze the DNA content of the cells and FL2-A fluorescence was recorded on 50,000 events gated according to forward and side scatter and analyzed using CellQuestPro software. 2.5. Immunofluorescence microscopy For immunofluorescent staining with KMX-1 (1:50 dilution), BBA4 (1:2 dilution) and L8C4 (1:2 dilution; all antibodies kind gifts from Keith Gull), cells were washed once with phosphatebuffered saline (PBS) and allowed to adhere to poly(l)lysine coated cover slips. The cells were then fixed in cold methanol at −20 ◦ C for 30 min before rehydration in PBS. Incubation with primary antibody was at room temperature for 1 h, followed by two washes with PBS and incubation with the anti mouse IgG AlexaFluor594-coupled secondary antibody (Molecular Probes, diluted 1:200) for another hour. After two more washes with PBS, cellular DNA was stained with 1 ␮g/ml of DAPI, and the slides were examined with a Leica DM RXA digital deconvolution microscope, and images were recorded using a digital charge-coupled-device camera (Hamamatsu). For the determination of cell cycle stages, cell samples were taken after 4, 8 and 12 h (CFB2), and after 1, 2, 3 and 4 days (CFB1), and after 8, 12, 24 and 30 h (CFB1 over-expression) before fixation in 4% paraformaldehyde. Cells were then permeabilised with 0.2% Triton X-100/PBS and stained with 1 ␮g/ml DAPI solution. The N–K configuration of at least 200 cells per time point was recorded. 3. Results 3.1. Expression of CFB1 and CFB2 mRNAs The CFB1 and CFB2 genes came to our attention because their expression is influenced by alterations in expression of two RNA-binding proteins, TbUBP1 and TbUBP2 [22]. On chromosome 1 (Fig. 1A), there are five copies of CFB1 which we have designated CFB1A-E. The predicted 58 kDa proteins are more than 99% identical. Downstream is a single gene encoding CFB2 (Tb927.1.4650), a 52 kDa protein. This differs from CFB1 at both the N- and C-termini (Fig. 1C and Supplementary section 5). The following 226 residues are well conserved and contain an F-box domain, which is recognised weakly by Pfam (E-value

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Fig. 1. Depletion of CFB1 or CFB2 mRNA inhibits trypanosome growth (A) Organization of the CFB1 and CFB2 genes on chromosome 1. Open reading frames are black bars, with the locus number beneath. The transcripts are shown beneath the genes; the 5 -ends are indicated as small black circles. (B) Northern blot showing developmental regulation of, and RNAi against, CFB2 mRNA. The identities of RNAs (and probes) are shown on the left. The blot was first probed for CFB2 (middle panel), then for tubulin (TUB, bottom), then stripped and reprobed for CFB1 (upper panel). (C) Alignment of CFB1 and CFB2 (details in Supplementary section 5). The cyclin F-box is cross-hatched. In the CFB2 diagram, the conserved central portion is black, regions of about 50% identity are grey, and the remainder is white. The percent identities are indicated below and an amino acid residue scale is above. (D) Effect of depleting CFB1 mRNA on cell growth. Cumulative growth curves are shown. Numbers for cells grown without tetracycline are shown with filled symbols and a solid line. Numbers for cells with tetracycline addition (at time = 0) are shown with open circles and a dashed line. Division times are also indicated; the value for the CFB1 RNAi cells is for days 1–3. A second cell line showed a similar effect; a third line had no RNAi. (E) Northern blot showing specific down-regulation of CFB1 mRNA. The probes are shown on the left. The control was SRP. (F) Effect of depleting CFB2 mRNA on cell growth; details as in (C).

0.22), and by InterPro (http://www.ebi.ac.uk/InterProScan/) both as a profile and as a member of a superfamily (SSF81383, IPR001810, P-value 0.0033); that of CFB2 is also recognised by Prosite (PS50181). The four T. cruzi homologues of CFB1/2 all have a predicted F-box domain but the single putative L. major homologue lacks it. In addition to the CFB array, the algorithms predict the presence of 7 other F-box domain protein genes in the trypanosome genome. The 3 kb CFB1 mRNAs are expressed at similar levels in both bloodstream and procyclic trypanosomes (Fig. 1B, lanes 1 and 2). The 3 -UTR of the 8 kb CFB2 transcript is unrelated to that of CFB1; CFB2 mRNA gave a weak Northern signal in bloodstream forms, and was even less abundant (5–10-fold) in procyclic forms (Figs. 1B and 4A, lanes 1 and 2). 3.2. Depletion of CFB1 or CFB2 inhibits cell growth The possession of a predicted F-box clearly does not guarantee that the CFB proteins are really involved in cell cycle regulation. To determine the function of the CFB proteins, we therefore reduced their expression by tetracyclineinducible RNA interference. Depletion of the CFB1 mRNA in bloodstream-form cells gave a moderate growth inhibition with

an escape from the RNAi effect beginning after 4 days of induction (Fig. 1D). (The tetracycline treatment alone does not affect the growth, transcriptome or proteome of trypanosomes [23].) The RNAi was poor since CFB1 transcript levels were reduced by only 50%; CFB2 mRNA was unaffected (Fig. 1E). During the TrypanoFan project, during which all predicted open reading frames on chromosome 1 were knocked down [24], depletion of CFB1 had no effect on growth, but a very slight effect on the cell cycle. The more pronounced effect in our experiments might have been due to more effective RNAi. We were unable to estimate the degree of CFB1 protein depletion. A polyclonal antibody against the C-terminal portion of CFB1 did not detect either CFB1 or CFB2 full-length protein in cell extracts either by Western blotting or by immunoprecipitation of metabolically labelled protein (see Supplementary section 6). Depletion of CFB2 RNA by RNAi in bloodstream-form trypanosomes caused immediate growth arrest followed by cell death (Fig. 1F). Specific reduction of the CFB2 transcript levels by 80–95% (three different lines) was confirmed by Northern blotting (Fig. 1B, lanes 3 and 4). We also made RNAi lines in procyclic forms. Addition of tetracycline to these cells had no effect on growth; induction of the dsRNA was clearly detectable on Northern blots but the CFB2 transcript was not detected at

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Fig. 2. Effect of CFB1 depletion on the cell cycle. (A) Analysis of nucleus and kinetoplast configurations of CFB1 RNAi cells over 4 days as revealed by DAPI staining; key on the graph. (B) Immunofluorescence staining for beta tubulin (TUB, antibody KMX-1), basal bodies (antibody BBA4) and the flagellum (paraflagellar rod, PFR) after 24 h RNAi. (C) Flow cytometry profiles of the same cells analyzed in the figure. Approximate positions of the different cell cycle phases are indicated. (D) Chart summarizing the percentage of cells in the respective cell cycle phases; key to symbols on the graph. The percentage of other combinations was too low to be visible at this scale.

all even without tetracycline (not shown), so we could not judge the effectiveness of the RNAi. 3.3. Effect of CFB1 and CFB2 depletion on the cell cycle CFB1 depletion had a minor effect on the bloodstream-form trypanosome cell cycle, with transient increases in 1N2K cells (Fig. 2A and B) and of S and G2/M phase cells (Fig. 2C and D). By tubulin staining, we saw no alteration in the proportion of spindle-containing cells (data not shown); this, combined with the increase in 1N2K cells, might be consistent with a delay in entry into mitosis. Division of basal bodies and flagella was however not inhibited (Fig. 2B). The effect of CFB2 depletion on bloodstream forms was dramatic, and visible after only 4 h (Fig. 3A). 2N4K accumulation started after 8 h, and by 12 h half of the cells had an NK configuration of 2N2K or more. After 24 h, most cells had also lost their normal morphology with an increasing proportion of rounded cells (Fig. 3B). Flow cytometry analysis confirmed these results, with two-thirds of cells in G2/M phase or beyond after 12 h of CFB2 depletion (Fig. 3C and D) and nearly 90%

at G2/M or beyond at 24 h (not shown). Microscopically, there was progressive accumulation of cells which appeared to be stuck together, then of “monsters” with several nuclei and kinetoplasts (Fig. 3B). Immunofluorescence analysis of basal bodies and flagella showed increases in the numbers of both structures in multinucleate and multi-kinetoplast cells, indicating that replication and separation of basal bodies and flagella could continue after CFB2 depletion (Fig. 3B). We did not see cells with half-completed cleavage furrows, either in phase-contrast or using tubulin staining, but to be really sure about this electron microscopy (or a specific stain for the furrow) would be needed. These results suggest that CFB2 is required, either directly or indirectly, for bloodstream-form trypanosome cytokinesis. 3.4. Over-expression of CFB1 inhibits bloodstream-form trypanosome growth We next made cells in which CFB1 could be over-expressed from a tetracycline-inducible RNA polymerase I promoter. Addition of tetracycline resulted in the appearance of two RNA bands of equivalent intensities from the inducible construct

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Fig. 3. Effect of CFB2 depletion on the cell cycle. (A) Analysis of nucleus and kinetoplast configurations of CFB2 RNAi cells over 12 h as revealed by DAPI staining. The “other” configurations were mostly 2N1K and 4N3K; 4N0K and 2N0K were also very rarely seen. (B) Immunofluorescence after 14–16 h depletion. Two panels for tubulin are shown. The upper one shows a “monster” cell with multiple flagella: cells of this type are alive since they still have flagellar motility. (Trypanosomes must lyse rapidly after death, since non-motile cells were not seen in culture.) The lower 2N2K cell appears to be stuck at cytokinesis without a visible cleavage furrow; by 12 h the proportion of such cells was approaching 40%. The lower two panels are stained for basal bodies and paraflagellar rod (PFR). (C) Flow cytometry. (D) Quantitation of (C). Details as for Fig. 2.

(Fig. 4A, lanes 4 and 6); this is normal for this construct and could be due to use of alternative polyadenylation sites. The abundance of the normal 3 kb CFB1 mRNA was increased three-fold after tetracycline addition (Fig. 4A, lanes 4 and 6). Either the CFB1 protein stabilises its own transcript, or the level of CFB1 mRNA increases as a consequence of growth arrest. CFB2 mRNA was in contrast unaffected (Fig. 4A). The extent of overproduction of CFB1 during these experiments could not be predicted from the mRNA abundance, since the 3 -UTR of the transgene mRNA is expected to influence translation efficiency [25]. We looked for the protein using our antibody but no band of the correct size was detected (see Supplementary section 6).

The induction of CFB1 transgene expression caused a severe and rapid effect on cell growth (Fig. 4B), with an increase in 2N2K cells (G2/M) at the expense of 1N1K and 1N2K cells (G1 and S) (Fig. 4C–E). The uninduced cells mysteriously have an excess of 1N2K cells (compare with Figs. 2 and 3) although their growth rate is completely normal. This, combined with the RNAi results, suggests that maintenance of the level of CFB1 protein within defined limits is important for normal growth. 4. Discussion In this paper we have shown that two proteins containing Fboxes, CFB1 and CFB2, are important in trypanosome growth.

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Fig. 4. CFB1 over-expression is lethal. (A) Northern blot from wild-type cells (lanes 1 and 2) and two cell lines (1 and 2) with tetracycline-inducible over-expression (upward arrow) of CFB1. The SRP RNA served as a loading control. (B) Over-expression of CFB1 is lethal. The cumulative growth curve is shown, solid symbols are cells without tetracycline, open symbols cells with tetracycline added at time = 0. The inset shows a Northern blot with the normal CFB1 transcript (top panel), two smaller inducible CFB1 transcripts of inhomogeneous length (middle panel, upward arrow) and an SRP control (bottom panel) (C) Analysis of nucleus and kinetoplast configurations. Cells in the “other” category included 2N4K, 4N4K, 3N2K, 2N3K, 5N2K, 3N3K, but not anucleate 0N1K cells (zoids). (D) Examples of flow cytometry profiles. (E) Quantitation of flow cytometry shown in (D).

Depletion of CFB1 mRNA resulted in a modest decrease in the growth rate, and a slight delay in nuclear division; and overexpression of CFB1 mRNA impaired growth more strongly. Although the observed increase in G2/M cells would be consistent with a requirement for precisely the right amount of CFB1 in mitosis or cytokinesis, many treatments of trypanosomes which affect growth result in similar phenotypes. It is therefore not possible to conclude that CFB1 is directly involved in cell cycle regulation. Depletion of CFB2 mRNA resulted in immediate cessation of cell division, manifest as an increase in binucleate and then multinucleate cells. The cells accumulated increasing numbers of kinetoplasts, flagella and basal bodies, so the impairment in cytokinesis was not caused by failure of basal body or flagel-

lum replication. The rapidity of the effect suggests (but certainly does not prove) that CFB2 is indeed directly involved in regulation of cytokinesis. Depletion of proteins that are certainly not directly involved in cell cycle regulation can also show quite specific effects at the morphological level. For example, inhibition of Variant Surface Glycoprotein synthesis prevents cytokinesis, with accumulation of internal flagellae [26]; and RNAi targeting a mitochondrial carrier protein homologue caused abnormalities in kinetoplast DNA division with consequent cell cycle irregularities [27]. Both CFB2 transcript (Fig. 1) and in situ-tagged CFB2 (Supplementary section 6) were less abundant in procyclic-form trypanosomes than in bloodstream forms, and CFB2 RNAi in that stage had no effect, so the function of CFB2 might be

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bloodstream-form specific. Since, however, we could not measure the protein level in any of the experiments, this conclusion is extremely tentative. F-box proteins often function in a complex with Skp1 [28]. There are two trypanosome genes encoding possible Skp1 homologues. We could not detect RNA from Tb10.26.0300, but RNAi targeting Tb11.02.3990 inhibited growth and caused an increase in CFB1 mRNA (Supplementary section 7). We were unable to express sufficient Tb11.02.3990 protein to identify binding partners (Supplementary section 6) so its status as an SCF component is unproven. Other possible homologues of SCF complex components are: Rbx1 (Tb10.70.6035), Sgt1 (Tb927.1.3200), Cdc53/Cul (five genes), and Cdc34 (at least seven genes). Apart from the F-box, neither CFB1 nor CFB2 contains recognisable protein–protein interaction domains. F-box proteins without such domains also exist in other organisms, but their functions are unclear [29]. To find out whether CFB1 and CFB2 are really involved in cell cycle regulation, it will be essential to characterise their interaction partners. To do this one must be able to detect the proteins. Our extensive attempts to do this included generation of a polyclonal antibody, and inducible or constitutive expression of tagged (V5, myc, TAP) and untagged CFB1 and CFB2 (see Supplementary section 6). We were never able to detect sufficient protein in order to find binding partners and the results suggested that CFB1 and CFB2 were extremely unstable. This is consistent with results from other organisms: Fbox proteins are often regulated by association with their respective complexes and by auto-ubiquitination [30]; they can also be unstable in the absence of F-box-dependent interactions [31]. In conclusion, our results indicate that both CFB1 and CFB2 are required for normal trypanosome growth, and that CFB2 may be directly required for bloodstream-form cytokinesis. Whether or not CFB1 and CFB2 are really involved in the formation of SCF complexes and cyclin degradation will only become clear when other components of the regulatory cascade have been identified. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SFB544). We thank Keith Gull (University of Oxford, UK) for the antibodies to tubulin, basal bodies and the paraflagellar rod and Prof. M. Inoue (Kurume University Medical School, Japan) for anti-14-3-3. The CFB1 in situ tag construct was made by Claudia Hartmann, whom we also thank, together with Ute Leibfried, for technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2007.08.005. References [1] Nandi D, Tahiliani P, Kumar A, Chandu D. The ubiquitin-proteasome system. J Biosci 2006;31:137–55.

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[2] Jentsch S. The ubiquitin-conjugation system. Annu Rev Genet 1992;26: 179–207. [3] Ardley HC, Robinson PA. E3 ubiquitin ligases. Essays Biochem 2005;41: 15–30. [4] Vodermaier HC. APC/C and SCF: controlling each other and the cell cycle. Curr Biol 2004;14:R787–96. [5] Willems AR, Schwab M, Tyers M. A hitchhiker’s guide to the cullin ubiquitin ligases: SCF and its kin. Biochim Biophys Acta 2004;1695:133–70. [6] McKean PG. Coordination of cell cycle and cytokinesis in Trypanosoma brucei. Curr Opin Microbiol 2003;6:600–7. [7] Hammarton TC. Cell cycle regulation in Trypanosoma brucei. Mol Biochem Parasitol 2007;153:1–8. [8] Ploubidou A, Robinson DR, Docherty RC, Ogbadoyi EO, Gull K. Evidence for novel cell cycle checkpoints in trypanosomes: kinetoplast segregation and cytokinesis in the absence of mitosis. J Cell Sci 1999;112(Pt 24):4641–50. [9] Tu X, Wang CC. The involvement of two cdc2-related kinases (CRKs) in Trypanosoma brucei cell cycle regulation and the distinctive stage-specific phenotypes caused by CRK3 depletion. J Biol Chem 2004;279:20519– 28. [10] Hammarton TC, Lillico SG, Welburn SC, Mottram JC. Trypanosoma brucei MOB1 is required for accurate and efficient cytokinesis but not for exit from mitosis. Mol Microbiol 2005;56:104–16. [11] Hammarton TC, Clark J, Douglas F, Boshart M, Mottram JC. Stagespecific differences in cell cycle control in Trypanosoma brucei revealed by RNA interference of a mitotic cyclin. J Biol Chem 2003;278:22877– 86. [12] Robinson DR, Sherwin T, Ploubidou A, Byard EH, Gull K. Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle. J Cell Biol 1995;128:1163–72. [13] Wang CC, Bozdech Z, Liu CL, et al. Biochemical analysis of the 20 S proteasome of Trypanosoma brucei. J Biol Chem 2003;278:15800–8. [14] Li Z, Zou CB, Yao Y, et al. An easily dissociated 26 S proteasome catalyzes an essential ubiquitin-mediated protein degradation pathway in Trypanosoma brucei. J Biol Chem 2002;277:15486–98. [15] Li Z, Wang C. Functional characterization of the 11 non-ATPase subunit proteins in the trypanosome 19 S proteasomal regulatory complex. J Biol Chem 2002;277:42686–93. [16] Li Y, Li Z, Wang CC. Differentiation of Trypanosoma brucei may be stage non-specific and does not require progression of cell cycle. Mol Microbiol 2003;49:251–65. [17] Mutomba MC, To WY, Hyun WC, Wang CC. Inhibition of proteasome activity blocks cell cycle progression at specific phase boundaries in African trypanosomes. Mol Biochem Parasitol 1997;90:491–504. [18] Van Hellemond JJ, Mottram JC. The CYC3 gene of Trypanosoma brucei encodes a cyclin with a short half-life. Mol Biochem Parasitol 2000;111:275–82. [19] Kumar P, Wang C. Depletion of anaphase-promoting complex or cyclosome (APC/C) subunit homolog APC1 or CDC27 of Trypanosoma brucei arrests the procyclic form in metaphase but the bloodstream form in anaphase. J Biol Chem 2005;280:31783–91. [20] van Deursen FJ, Shahi SH, Turner CMR, et al. Characterisation of the growth and differentiation in vivo and in vitro of bloodstreamform Trypanosoma brucei strain TREU 927. Mol Biochem Parasitol 2001;112:163–72. [21] Alibu VP, Storm L, Haile S, Clayton C, Horn D. A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei. Mol Biochem Parasitol 2004;139:75–82. [22] Hartmann C, Benz C, Brems S, et al. The small trypanosome RNA-binding proteins TbUBP1 and TbUBP2 influence expression of cyclin F box protein mRNAs in bloodstream trypanosomes, Eukaryot Cell, in press. [23] Luu VD, Brems S, Hoheisel J, Burchmore R, Guilbride D, Clayton C. Functional analysis of Trypanosoma brucei PUF1. Mol Biochem Parasitol 2006;150:340–9. [24] Subramaniam C, Veazey P, Redmond S, et al. Chromosome-wide analysis of gene function by RNA interference in the African trypanosome. Eukaryot Cell 2006;5:1539–49.

224

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[25] Clayton C, Shapira M. Post-transcriptional regulation of gene expression in trypanosomes and leishmanias. Mol Biochem Parasitol 2007;156:93–101. [26] Sheader K, Vaughan S, Minchin J, Hughes K, Gull K, Rudenko G. Variant surface glycoprotein RNA interference triggers a precytokinesis cell cycle arrest in African trypanosomes. Proc Natl Acad Sci USA 2005;102:8716–21. [27] Colasante C, Alibu VP, Kirchberger S, Tjaden J, Clayton C, Voncken F. Characterisation and developmentally regulated localisation of the mitochondrial carrier protein homologue MCP6 from Trypanosoma brucei. Eukaryot Cell 2006;5:1194–205.

[28] Hermand D. F-box proteins: more than baits for the SCF? Cell Div 2006;1:30. [29] Winston JT, Koepp DM, Zhu C, Elledge SJ, Harper JW. A family of mammalian F-box proteins. Curr Biol 1999;9:1180–2. [30] Deshaies RJ. SCF and Cullin/Ring H2-based ubiquitin ligases. Annu Rev Cell Dev Biol 1999;15:435–67. [31] Zielke N, Querings S, Grosskortenhaus R, Reis T, Sprenger F. Molecular dissection of the APC/C inhibitor Rca1 shows a novel F-box-dependent function. EMBO Rep 2006;7:1266–72.