Coordination of cell cycle and cytokinesis in Trypanosoma brucei

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Coordination of cell cycle and cytokinesis in Trypanosoma brucei Paul G McKean In common with all eukaryotic cells, trypanosomes must coordinate a complex series of morphogenetic events both temporally and spatially during the cell cycle. The structural and molecular cues that synchronise these events in trypanosomes have started to be elucidated, and intriguingly although similarities to cell cycle events in other eukaryotes can be identified, trypanosomes have also evolved novel solutions to the common challenges faced by dividing eukaryotic cells. Although cellular morphology is clearly pivotal for successful progression through the trypanosome cell cycle, most cytological studies to date have focused exclusively on procyclic form trypanosomes. These studies provide an excellent framework for understanding cell cycle events in trypanosomes, however recent data indicates that profound differences might exist between different life cycle stages in relation to the regulation of cell cycle and cytokinesis. Addresses Department of Biological Sciences, The Lancaster Environment Centre, Lancaster University, Lancaster, Lancashire LA1 4YQ, UK e-mail: [email protected]

Current Opinion in Microbiology 2003, 6:600–607 This review comes from a themed issue on Growth and development Edited by Claudio Scazzocchio and Jeff Errington 1369-5274/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2003.10.010

Abbreviations FAZ flagellum attachment zone

novel cell cycle control mechanisms exist to co-ordinate these events [6]. Drug inhibition studies certainly indicate that key eukaryotic checkpoints are absent in trypanosomes as T. brucei can undergo cytokinesis without completing mitosis [7–9]. As an alternative, Keith Gull has proposed that predominant trypanosome checkpoints regulate entry into cytokinesis by monitoring the status of kinetoplast division and segregation [9]. In this review, I consider recent progress towards identifying regulatory proteins and structures involved in coordinating cell cycle events and cytokinesis in T. brucei. These studies confirm that in trypanosomes dependency relationships exist in which cell morphology and cytoskeletal architecture are critically important. However, these recent studies also indicate that cell cycle regulatory mechanisms may vary between trypanosome life cycle stages.

The trypanosome cell – a primer The life cycle of T. brucei involves cyclical transmission between a mammalian host and an insect vector. Within each of these distinct environments the trypanosome undergoes a series of differentiation events characterised by morphological restructuring, changes in surface coat and biochemical adaptation (Figure 1). Life cycle progression is intimately linked to cell cycle regulation with replicating forms alternating with cell cycle arrested forms pre-adapted for transmission between the different hosts (Figure 1). Upon transmission to a new host the cell cycle block is released and trypanosomes resume division. Although clear morphological differences exist between trypanosomes at distinct stages of the life cycle, the procyclic trypanosome cell illustrated in Figure 2 acts as a paradigm for the basic architecture of T. brucei [10].

Introduction African trypanosomes are among the most ancient eukaryotic organisms known [1], an ancestry reflected on occasion in unusual biology [2]. Although trypanosomes contain familiar organelles such as a nucleus and flagellum, they also contain distinctive structures such as glycosomes [3], along with organelles such as the mitochondrion, which although conserved in function exhibit unique features [4,5]. For instance, the mitochondrial DNA of trypanosomes is organised into a large disc-like structure (the kinetoplast) residing within the matrix of the single mitochondrion. In addition to the discrete nuclear events characteristic of a normal eukaryotic cell cycle, the mitochondrial genome in the kinetoplast atypically exhibits a discrete DNA synthesis phase (see below). The requirement to replicate and segregate a ‘two-unit’ genome in trypanosomes has led to the suggestion that Current Opinion in Microbiology 2003, 6:600–607

Cell shape is defined by a cage of microtubules, organised in a helical arrangement along the longitudinal axis of the cell. The single flagellum exits the cell near the posterior pole and extends towards the anterior following a defined left-handed helical path. For most of its length the flagellum is physically attached to the cell body via the flagellum attachment zone (FAZ). The FAZ consists of a series of filaments in association with four specialised microtubules and defines a specific site within the subpellicular cortex of the cell body [6]. At the beginning of the cell cycle trypanosomes possess a single flagellum, nucleus, kinetoplast and mitochondrion (Figure 2) and must ensure the faithful duplication and segregation of all these organelles to produce two viable daughters. The order and timing of cell cycle events in T. brucei have been extensively studied, although it should be noted that this work has been largely undertaken in procyclic form cells www.current-opinion.com

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Figure 1

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Variant surface glycoprotein (VSG) surface coat Current Opinion in Microbiology

Schematic of the T. brucei life cycle. Profound changes in morphology, surface coat and biochemistry (as exemplified by mitochondrial function) occur during the trypanosome life cycle. The coordinated control of cell cycle and differentiation events are essential to life cycle progression. Metacyclic and short stumpy trypanosomes are non-replicating forms arrested in the G0/G1 phase of the cell cycle and are pre-adapted for survival in the ensuing host. Adapted from [42,43].

Figure 2

[11,12] and it is now becoming clear that subtle differences may exist in different life cycle stages. Flagellum

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Cell cycle events The first morphological event in the T. brucei cell cycle is the elongation and maturation of the pro-basal body and the nucleation of a new flagellum, an event dependent upon the de novo recruitment of g-tubulin [13].

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Schematic representation of procyclic T. brucei on the basis of electron microscopy studies. The relative positions of organelles such as the nucleus (dark blue), kinetoplast (light blue), mitochondrion (green), basal and probasal bodies (red and yellow respectively) and the flagellum are shown. Reproduced from [10] with permission. Copyright 2003 The Company of Biologists Ltd. www.current-opinion.com

Both the nuclear and kinetoplast genomes of trypanosomes exhibit a discrete S phase [12]. Kinetoplast S phase (Sk) initiates immediately before the onset of the nuclear S phase (Sn), but is considerably shorter and kinetoplast segregation (D) is completed before the onset of mitosis (M). This has practical implications, as labelling of asynchronous cultures of trypanosomes with nucleic acid stains such as DAPI can position trypanosomes into clearly defined stages of the cell cycle (Figure 3). Early in the G2 phase of the nuclear cycle, basal bodies separate in a microtubule-mediated process [14]. A Current Opinion in Microbiology 2003, 6:600–607

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Diagram showing the major morphological events of the procyclic T. brucei cell cycle on the basis of data in [11,12] (a) The trypanosome cell cycle is separated into nuclear and kinetoplast components. Cell cycle duration for exponentially growing procyclic trypanosomes is 8.5 h. Kinetoplast replication (S) initiates before nuclear S phase, but is considerably shorter and consequently kinetoplast segregation (D) occurs before the onset of nuclear mitosis (M). The phase annotated on the kinetoplast cycle as ‘A’ refers to the ‘apportioning’ phase during which basal bodies continue to move apart. (b) Schematic representations of trypanosome cells taken from various time points through the cell cycle. The black arrow indicates the direction and position of the cleavage furrow.

tripartite attachment complex (TAC) connects the kinetoplast to the proximal end of the basal body, ensuring that basal body movements also result in the segregation of replicated kDNA [15]. If the TAC is incorrectly formed, kDNA networks might become asymmetrical apportioned to daughter cells [16]. In trypanosomes, mitosis is a ‘closed’ process with an intranuclear spindle being formed without disruption of the nuclear envelope [17]. Although both kinetochore and pole–pole microtubules are formed during mitosis, they are insufficient in number to allow for individual microtubule–kinetochore interactions to be made, and an unusual ‘lateral stacking’ mechanism has been proposed to facilitate chromosomal segregation [17,18]. The relevance of this unusual mechanism to mitotic checkpoint controls has not yet been determined.

The flagellum and cytokinesis Defining the plane of cleavage is a fundamental problem to cells undergoing division. In trypanosomes, a cleavage furrow forms along the entire longitudinal axis of the dividing trypanosome, although ingression occurs uniCurrent Opinion in Microbiology 2003, 6:600–607

directionally from anterior to posterior passing between the two flagella to form daughter cells. It has been proposed that the FAZ may provide the structural information required to position the cleavage furrow [8]. Trypanosomes with structural defects in the FAZ have problems in cytokinesis, and either fail to initiate cytokinesis [19], or generate abnormal cellular forms such as multinucleate and anucleate cells (zoids) (Keith Gull, personal communication), see also Update. As the flagellum defines the positioning of the FAZ [20] outgrowth of the new flagellum can be viewed as a pivotal event in trypanosome morphogenesis. Recent studies from the Gull laboratory reveal that in procyclic trypanosomes the distal tip of the new flagellum is physically tethered to the side of the old flagellum by a novel structure termed the flagella connector or FC [21]. The physical connection afforded by the FC is suggested to ensure that the new flagellum (and consequently the FAZ) trace the same helical path along the cell as the old flagellum thus implicating the epigenetic phenomenon of cytotaxis in trypanosome morphogenesis. Paradoxically, despite the apparent importance of the FC in www.current-opinion.com

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procyclic trypanosomes, conclusive evidence for the existence of this structure in other life cycle forms has not yet been forthcoming. The T. brucei glycoprotein FLA1 has a critical role in attaching the flagellum to the trypanosome cell body as the RNAi knockdown of FLA1 expression results in flagella detachment [21,22,23]. Ablation of FLA1 expression also prevents cytokinesis; cells are unable to divide but undergo multiple rounds of mitosis resulting in an increase in multinucleated cells. These results are consistent with the proposal that coupled flagellum and FAZ growth is important in the placement of the cleavage furrow [6,21]. However, recent experiments from the Donelson laboratory [23] now appear to call this model into question. FLA1 is the T. brucei orthologue of the T. cruzi protein GP72; a protein also responsible for flagellum attachment in this related trypanosomatid [24]. Heterologous expression of GP72 in T. brucei results in flagella detachment but cells remain competent for cytokinesis (albeit with a reduced growth rate). At present it is difficult to reconcile these results with the proposal that flagellum attachment is critical for cell division. However, it should be noted that GP72 overexpression does not result in stable cell lines as trypanosomes with attached flagella eventually outgrow cells with detached flagella, implying that flagella attachment may be required for efficient cell division. Further detailed cell biology studies are needed to resolve this issue and in particular meticulous attention needs to be paid to the fate of individual trypanosomes with detached flagella. There is also a general point to make here regarding the veracity of many current trypanosome cell biology studies. It is becoming customary to describe trypanosome cells with unusual combinations of flagella, kinetoplasts and nuclei simply as ‘monsters’. This generic description makes it impossible to develop any real insight into the cytokinetic deficiencies in these trypanosomes. However ‘monster-like’ these cells appear at first, it is essential that more rigorous morphological categorisation is provided in future along with a lineage analysis of the cells generated after the induction of RNAi.

and/or in coordinating the replication and segregation of the ‘two-unit’ genome of trypanosomes. To address specific questions regarding cell cycle regulation and checkpoint controls in trypanosomes, the laboratories of CC Wang and J Mottram independently initiated the functional characterisation of T. brucei cyclin genes using RNAi [27,28]. Although the RNAi knockdown of many T. brucei cyclin genes gave an equivocal or no phenotype [27], two cyclin genes did produce phenotypes indicative of defects in cell cycle progression. Both groups demonstrated that the RNAi mediated ablation of a mitotic B-type cyclin CYC6 (designated CycB2 by the Wang laboratory), blocked mitosis in procyclic trypanosomes but not cytokinesis, resulting in the generation of 1K1N cells (with a 4C nuclear DNA content) and 1K0N zoids [27,28] (See Figure 4). This result mirrors the previously reported anti-mitotic effects of the drug rhizoxin [9] and confirms that procyclic trypanosomes lack the mitosis to cytokinesis checkpoint. However, subsequent experiments by the Mottram laboratory revealed fundamental differences in cell cycle regulation in bloodstream forms, as CYC6 depletion inhibited both mitosis and cytokinesis but not kinetoplast S phase in this life cycle stage [28]. Moreover, as these cells were multi-kinetoplastic and multi-flagellated it suggests that both the proximal (TAC formation) and distal (flagellum nucleation) functions of the basal body were unaffected. Although the positioning of the FAZ was unfortunately not recorded in this study it does suggest that in bloodstream form cells kinetoplast segregation and flagellum growth were not sufficient to promote cytokinesis. Although this could indicate the existence of an operational mitosis/cytokinesis checkpoint in bloodstream forms, it has also been suggested by the Mottram laboratory that this phenotype may reflect differences in morphology between the two life cycle forms [26]. In bloodstream trypanosomes for instance, the kinetoplasts are positioned at the posterior of the cell throughout division and so the positioning event that places one kinetoplast between the two nuclei (found in the procyclic form) is absent [29] (See Figure 4). However, precisely how such structural differences influence cleavage furrow positioning and cell cycle progression still remains to be determined.

Cell cycle regulation in T. brucei Regulatory pathways controlling the eukaryotic cell cycle have been studied in considerable detail in yeast and other eukaryotes [25] and shown to involve regulatory proteins such as cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors (CKIs). To date eight cyclins (CYC2-9) and six CDK homologues (CRK1-4 and TbCRK6-7) have been identified in T. brucei, an unusually large number for a unicellular organism (reviewed in [26]). This might indicate that these proteins have distinct roles in the different life cycle stages (see below) www.current-opinion.com

A cell cycle phenotype also resulted following the RNAi knockdown of a PH080-like cyclin, CycE1 [27] (designated CYC2 by the Mottram laboratory [30]). In yeast, PHO80 cyclins are involved in phosphate signalling but have no apparent function in cell cycle control [31]. However, unusually the trypanosome CycE1/CYC2 cyclin may regulate transition through G1/S phase, as cells replicate and segregate kDNA but appear blocked in nuclear S phase. However these cells still divide, confirming that progression through cytokinesis is not dependent upon Current Opinion in Microbiology 2003, 6:600–607

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A schematic illustration of the morphological differences between bloodstream and procyclic cells during normal division and following CYC6 RNAi on the basis of figures in [26]. Wild type, note the differences in positioning of kinetoplast and nucleus between procyclic and bloodstream forms (see text for more details). CYC6 RNAi, phenotypes generated following RNAi mediated knockdown of the mitotic cyclin CYC6. In procyclic cells CYC6 knockdown prevents mitosis but not cytokinesis, whereas in bloodstream cells CYC6 knockdown prevents cytokinesis and mitosis but not kinetoplast S phase/segregation resulting in the generation of multi-kinetoplast and multi-flagellated cells. Note: as no data has been provided on the positioning of the FAZ following CYC6 RNAi knockdown this structure has been omitted from these figures.

mitosis in procyclics [27]. The effect of RNAi knockdown of CycE1/CYC2 in bloodstream forms was not undertaken in this study, although this would clearly be an interesting question. The importance of trypanosome cellular architecture to cell cycle progression and cytokinesis has important implications when studying phenotypes generated by RNAi as the depletion of proteins not directly involved in cell cycle regulation may generate cells with cell cycle phenotypes by invoking a block in cytokinesis [28]. In bloodstream form trypanosomes for instance, RNAi mediated knockdown of GPI8 (a catalytic subunit of the enzyme adding GPI anchors onto nascent polypeptides) results in multinucleate, multikinetoplast and multiflagellated cells [32]. It has been suggested that the block in cytokinesis resulting from GPI8 depletion could be caused by inefficient delivery of GPI-anchored proteins involved in cell division, although procyclic cells (that also express GPI anchored proteins on their surface) remain viable upon targeted deletion of the GPI8 gene.

Differentiation and cell cycle progression Differentiation from a non-dividing bloodstream stumpy form to a proliferative procyclic form (Figure 1) involves progression through a normal cell cycle; S phase followed by kinetoplast separation, mitosis and cytokinesis. HowCurrent Opinion in Microbiology 2003, 6:600–607

ever, superimposed upon this first cell cycle is a significant reorganisation of cellular architecture and repositioning of organelles [33]. During differentiation and coincident with S phase, the kinetoplast is repositioned from the posterior pole of the cell to a position adjacent to the nucleus as a result of microtubule outgrowth from the posterior end of the cell. Two novel CCCH zinc finger proteins (TbZFP1 and TbZFP2) have been implicated in controlling this remodelling event during differentiation [34]. Further kinetoplast repositioning occurs in the epimastigote form with the kinetoplast moving to occupy a site anterior to the nucleus (Figure 1). Although the rationale for these kinetoplast-repositioning events remains uncertain, recent experiments suggest the existence of a differentiation checkpoint dependent upon the kinetoplast [35]. Even though the requirement for kDNA in bloodstream forms is controversial [5] naturally occurring dyskinetoplastic bloodstream trypanosomes can be isolated. When these dyskinetoplastic bloodstream forms are induced to differentiate into procyclic forms they become arrested at a specific point in the differentiation process [35]. This arrest point occurs after differentiation is initiated but before trypanosomes re-enter the cell cycle, indicating that dyskinetoplastic trypanosomes are competent to receive differentiation signals but cannot progress through the differentiation pathway. www.current-opinion.com

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Intracellular signalling pathways coordinating differentiation events and cell cycle progression in trypanosomes remain largely unknown [36]. However advances are being made, with two recently identified protein kinase genes appearing to have roles in regulating cell cycle progression.

studies are providing strong evidence that regulation of the trypanosome cell cycle also differs in key respects. This uniqueness is of course to be welcomed from a practical perspective as these pathways might yet provide novel targets for chemotherapeutic intervention.

The first of these two genes, a mitogen activated protein kinase (TbMAPK2) has been shown to have a role in promoting cell cycle progression specifically in procyclic forms [37]. Deletion mutants of TbMAPK2 can be generated in bloodstream forms with no phenotypic effect, but upon differentiation to procyclics, TbMAPK2 null mutants undergo cell cycle arrest. Although one of the major functions of MAP kinases in other eukaryotic systems is to release cells from a cell cycle arrested state, TbMAPK2 null mutants differentiate normally, early kinetoplast repositioning events are unaffected and mutants progress efficiently through S-phase of the first cell cycle. However, TbMAPK2 null mutants subsequently undergo cell cycle arrest (in all phases of the cell cycle) indicating that the role of TbMAPK2 may be to promote continued cell cycle progression in procyclics. The stage specificity exhibited by TbMAPK2 in promoting procyclic cell cycle progression indicates that the complex life cycle of trypanosomes is also reflected in distinct (and life cycle specific) regulatory signalling pathways.

Update

In other eukaryotic organisms, a specific family of protein kinases has been implicated in coordinating morphology and division. The fission yeast protein kinase Orb6p regulates cell shape and division; reduced levels of Orb6 result in loss of polarised cell morphology and mitotic advance, whereas increases in Orb6p maintain polarised growth but delay mitosis [38,39]. A functional T. brucei homologue of Orb6 (TBPK50) has now been identified [40]; TBPK50 is expressed only in proliferative trypanosome forms and totally absent from quiescent stages suggesting this protein might regulate life cycle progression and cell division.

Recent work from the Bastin laboratory [41] confirms that the flagellum attachment zone (FAZ) plays a key role in trypanosome morphogenesis. Using RNAi, Bastin and colleagues ablated expression of key proteins involved in intra-flagellar transport (IFT); an evolutionarily conserved mechanism involved in flagellum formation. Ablation of IFT in T. brucei not only prevents the formation of a new flagellum but also results in the formation of an abnormally short FAZ. Although such cells can undergo cytokinesis, the cleavage furrow initiates from a position defined by the anterior end of the new shortened FAZ. Consequently, the progeny of this division are asymmetric; consisting of a small non-flagellated daughter cell and a larger daughter cell characterised by an excessively long posterior end. Non-flagellated cells are unable to divide further, and although the flagellated daughter cell can reiterate this abnormal division process 2–3 times they too also ultimately fail to undergo cytokinesis. These studies demonstrate a pivotal role for the FAZ and flagellum in defining cell size, shape, polarity and cytokinetic processes in T. brucei.

Acknowledgements I would like to gratefully acknowledge the financial support of the BBSRC for the work in my laboratory. I would also like to thank Keith Gull for critical reading of the manuscript and Tansy Hammarton for many useful discussions regarding trypanosome cyclins.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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Conclusions It is clear that in T. brucei there is an intimate association between cellular morphology, differentiation and cell cycle progression. The introduction of RNAi as an experimental tool in trypanosome research is now providing intriguing insights into these processes. Recent evidence indicates that cell cycle regulation appears to differ between distinct trypanosome life cycle stages and although cellular morphology clearly exerts an influence, our understanding of this interplay between morphology and cell cycle remains inadequate. This particularly applies to the final events of cytokinesis, but also to the pivotal roles played by the basal body and the significance of kinetoplast repositioning events during the life cycle. Although fundamental similarities to higher eukaryotic cell cycle regulatory mechanisms are apparent, RNAi www.current-opinion.com

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results in flagellum detachment but cells are able to undergo cytokinesis. Although the study is incomplete, as it does not provide a detailed study of organellar positioning in such cells, it raises important questions regarding the role of the flagellum in cytokinesis.

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25. Nurse P: Cyclin dependent kinases and cell cycle. Chembiochem 2002, 3:596-603. 26. Hammarton TC, Mottram JC, Doerig C: The cell cycle of parasitic protozoa: potential for chemotherapeutic exploitation. Prog Cell Cycle Res 2003, 5:91-101. 27. Li Z, Wang CC: A PHO80-like cyclin and a B-type cyclin control  the cell cycle of the procyclic form of Trypanosoma brucei. J Biol Chem 2003, 278:20652-20658. The first of two recent publications investigating cyclin function(s) during the trypanosome cell cycle (see also [28]). In this study, Li and Wang undertake the systematic ablation of seven different cyclins using RNAi in procyclic form trypanosomes and provide evidence for the involvement of two different cyclins in progression through G1/S and M phase in T. brucei. 28. Hammarton TC, Clark J, Douglas F, Boshart M, Mottram JC: Stage specific differences in cell cycle control in Trypanosoma brucei revealed by RNAi of a mitotic cyclin. J Biol Chem 2003, 278:22877-22886. This important paper demonstrates that different phenotypes are generated in procylic and bloodstream form trypanosomes in response to the RNAi mediated knockdown of the cyclin CYC6. These results suggest that fundamental differences exist in cell cycle regulatory mechanisms between trypanosome life-cycle stages, thus revealing a hitherto unappreciated complexity to the regulation of the cell cycle in trypanosomes. 29. Tyler KM, Matthews KR, Gull K: Anisomorphic cell division of African trypanosomes. Protist 2001, 152:367-378. 30. Van Hellemond JJ, Neuville P, Schwarz RT, Matthews KR, Mottram JC: Isolation of Trypanosoma brucei CYC2 and CYC3 cyclin genes by rescue of a yeast G(1) cyclin mutant. Functional characterization of CYC2. J Biol Chem 2000, 275:8315-8323. 31. Carroll AS, O’Shea EK: Pho85 and signalling environmental conditions. Trends Biochem Sci 2002, 27:87-93. 32. Lillico S, Field MC, Blundell P, Coombs GH, Mottram JC: Essential roles for GPI-anchored proteins in African trypanosomes revealed using mutants deficient in GPI8. Mol Biol Cell 2003, 14:1182-1194. 33. Matthews KR, Sherwin T, Gull K: Mitochondrial genome repositioning during the differentiation of the African trypanosome between life cycle forms is microtubule mediated. J Cell Sci 1995, 108:2231-2239. 34. Hendriks EF, Robinson DR, Hinkins M, Matthews KR: A novel CCCH protein which modulates differentiation of Trypanosoma brucei to its procyclic form. EMBO J 2001, 20:6700-6711. 35. Timms MW, van Deursen FJ, Hendriks EF, Matthews KR:  Mitochondrial development during life cycle differentiation of African trypanosomes: evidence for a kinetoplast-dependent differentiation control point. Mol Biol Cell 2002, 13:3747-3759. In this paper the Matthews laboratory provide convincing evidence for the existence of a stumpy, procyclic trypanosome differentiation checkpoint dependent upon the presence of a kinetoplast. 36. Parsons M, Ruben L: Pathways involved in environmental sensing in trypanosomatids. Parasitol Today 2000, 16:56-62. 37. Muller IB, Domenicali-Pfister D, Roditi I, Vassella E: Stage-specific  requirement of a mitogen-activated protein kinase by Trypanosoma brucei. Mol Biol Cell 2002, 13:3787-3799. This paper describes the identification and preliminary characterisation of a mitogen activated protein (MAP) kinase that appears to regulate progression through the cell cycle. Although deletion mutants can be generated in bloodstream forms with no phenotypic effect, upon differentiation to procyclic forms the null mutants undergo cell cycle arrest. This indicates that the complexity of the trypanosome life cycle is likely to be reflected in distinct and life-cycle specific signalling pathways. 38. Verde F, Wiley DJ, Nurse P: Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic www.current-opinion.com

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dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle. Proc Natl Acad Sci USA 1998, 95:7526-7531. 39. Hou MC, Wiley DJ, Verde F, McCollum D: Mob2p interacts with the protein kinase Orb6p to promote coordination of cell polarity with cell cycle progression. J Cell Sci 2003, 116:125-135. 40. Garcia-Salcedo JA, Nolan DP, Gijon P, Gomez-Rodriguez J,  Pays E: A protein kinase specifically associated with proliferative forms of Trypanosoma brucei is functionally related to a yeast kinase involved in the co-ordination of cell shape and division. Mol Microbiol 2002, 45:307-319. This paper reports the identification and characterisation of the T. brucei homologue of a conserved protein kinase, Orb6p, which is involved in

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coordinating cell shape and division in fission yeast. The T. brucei homolog of Orb6p designated TBPK50 is expressed only in proliferative trypanosomes and totally absent from quiescent stages. This suggests that TBPK50 might regulate life cycle progression and cell division in trypanosomes. 41. Kohl L, Robinson D, Bastin P: Novel roles for the flagellum in cell morphogenesis and cytokinesis of trypanosomes. EMBO J 2003, 22:5336-5346. 42. Vickerman K: Developmental cycles and biology of pathogenic trypanosomes. Br Med Bull 1985, 41:105-114. 43. El-Sayed NM, Hegde P, Quackenbush J, Melville SE, Donelson JE: The African trypanosome genome. Int J Parasitol 2000, 30:329-345.

Current Opinion in Microbiology 2003, 6:600–607