The Trypanosoma brucei cyclin, CYC2, is required for cell cycle progression through G1 phase and for maintenance of procyclic form cell morphology

CYC2 is an essential PHO80-like cyclin that forms a complex with the cdc2-related kinase CRK3 in Trypanosoma brucei. In both procyclic and bloodstream form T. brucei, knock-down of CYC2 by RNA interference (RNAi) led to an accumulation of cells in G(1) phase. Additionally, in procyclic cells, but not in bloodstream form cells, CYC2 RNAi induced a specific cell elongation at the posterior end. The G(1) block, as well as the posterior end elongation in the procyclic form, was irreversible once established. Staining for tyrosinated alpha-tubulin and morphometric analyses showed that the posterior end elongation occurred through active microtubule extension, with no repositioning of the kinetoplast. Hence, these cells can be classified as exhibiting the "nozzle" phenotype as has been described for cells that ectopically express TbZFP2, a zinc finger protein that is involved in the differentiation of the bloodstream form to procyclic form. Within the tsetse fly, procyclic trypanosomes differentiate to elongated mesocyclic cells. However, although mesocyclic trypanosomes isolated from tsetse flies also show active microtubule extension at the posterior end, the kinetoplast is coincidentally repositioned such that it always lies approximately midway between the nucleus and posterior end of the cell. Thus, in the procyclic form CYC2 has dual functionality and is required for both cell cycle progression through G(1) and for the maintenance of correct cell morphology, whereas in the bloodstream form only a role for CYC2 in G(1) progression is evident.     

The involvement of two cdc2-related kinases (CRKs) in Trypanosoma brucei cell cycle regulation and the distinctive stage-specific phenotypes caused by CRK3 depletion

Cyclin-dependent protein kinases are among the key regulators of eukaryotic cell cycle progression. Potential functions of the five cdc2-related kinases (CRK) in Trypanosoma brucei were analyzed using the RNA interference (RNA(i)) technique. In both the procyclic and bloodstream forms of T. brucei, CRK1 is apparently involved in controlling the G(1)/S transition, whereas CRK3 plays an important role in catalyzing cells across the G(2)/M junction. A knockdown of CRK1 caused accumulation of cells in the G(1) phase without apparent phenotypic change, whereas depletion of CRK3 enriched cells of both forms in the G(2)/M phase. However, two distinctive phenotypes were observed between the CRK3-deficient procyclic and bloodstream forms. The procyclic form has a majority of the cells containing a single enlarged nucleus plus one kinetoplast. There is also an enhanced population of anucleated cells, each containing a single kinetoplast known as the zoids (0N1K). The CRK3-depleted bloodstream form has an increased number of one nucleus-two kinetoplast cells (1N2K) and a small population containing aggregated multiple nuclei and multiple kinetoplasts. Apparently, these two forms have different mechanisms in cell cycle regulation. Although the procyclic form can be driven into cytokinesis and cell division by kinetoplast segregation without a completed mitosis, the bloodstream form cannot enter cytokinesis under the same condition. Instead, it keeps going through another G(1) phase and enters a new S phase resulting in an aggregate of multiple nuclei and multiple kinetoplasts in an undivided cell. The different leakiness in cell cycle regulation between two stage-specific forms of an organism provides an interesting and useful model for further understanding the evolution of cell cycle control among the eukaryotes.     

A PHO80-like cyclin and a B-type cyclin control the cell cycle of the procyclic form of Trypanosoma brucei

Cyclins bind and activate cyclin-dependent kinases that regulate cell cycle progression in eukaryotes. Cell cycle control in Trypanosoma brucei was analyzed in the present study. Genes encoding four PHO80 cyclin homologues and three B-type cyclin homologues but no G1 cyclin homologues were identified in this organism. Through knocking down expression of the seven cyclin genes with the RNA interference technique in the procyclic form of T. brucei, we demonstrated that one PHO80 homologue (CycE1/CYC2) and a B-type cyclin homologue (CycB2) are the essential cyclins regulating G1/S and G2/M transitions, respectively. This lack of overlapping cyclin function differs significantly from that observed in the other eukaryotes. Also, PHO80 cyclin is known for its involvement only in phosphate signaling in yeast with no known function in cell cycle control. Both observations thus suggest the presence of simple and novel cell cycle regulators in trypanosomes. T. brucei cells deficient in CycE1/CYC2 displayed a long slender morphology, whereas those lacking CycB2 assumed a fat stumpy form. These cells apparently still can undergo cytokinesis generating small numbers of anucleated daughter cells, each containing a single kinetoplast known as a zoid. Two different types of zoids were identified, the slender zoid derived from reduced CycE1/CYC2 expression and the stumpy zoid from CycB2 deficiency. This observation indicates an uncoupling between the kinetoplast and the nuclear cycle, resulting in cell division driven by kinetoplast segregation with neither a priori S phase nor mitosis in the trypanosome.     

Changing roles of aurora-B kinase in two life cycle stages of Trypanosoma brucei

Aurora-B kinase is a chromosomal passenger protein essential for chromosome segregation and cytokinesis. In the procyclic form of Trypanosoma brucei, depletion of an aurora-B kinase homologue TbAUK1 inhibited spindle formation, mitosis, cytokinesis, and organelle replication without altering cell morphology. In the present study, an RNA interference knockdown of TbAUK1 or overexpression of inactive mutant TbAUK1-K58R in the bloodstream form also resulted in defects in spindle formation, chromosome segregation, and cytokinesis but allowed multiple rounds of nuclear DNA synthesis, nucleolus multiplication, and continuous replication of kinetoplast, basal body, and flagellum. The typical trypanosome morphology was lost to an enlarged round shape filled with microtubules. It is thus apparent that there are distinctive mechanisms of action of TbAUK1 in regulating cell division between the two developmental stages of trypanosome. While it exerts a tight control on mitosis, organelle replication, and cytokinesis in the procyclic form, it regulates cytokinesis without rigid control over either nuclear DNA synthesis or organelle replication in the bloodstream form. The molecular basis underlining these discrepancies remains to be explored.     

Polo-like kinase is expressed in S/G2/M phase and associated with the flagellum attachment zone in both procyclic and bloodstream forms of Trypanosoma brucei

Trypanosoma brucei, the etiologic agent of African sleeping sickness, divides into insect (procyclic) and bloodstream forms. These two forms are subject to distinct cell cycle regulations, with cytokinesis controlled primarily by basal body/kinetoplast segregation in the procyclic form but by mitosis in the bloodstream form. Polo-like kinases (PLKs), known to play essential roles in regulating both mitosis and cytokinesis among eukaryotes, have a homologue in T. brucei, TbPLK, which regulates only cytokinesis. In our previous study, overexpressed triply hemagglutinin-tagged TbPLK (TbPLK-3HA) in the procyclic form localized to a mid-dorsal point and the anterior tip of the cell along the flagellum attachment zone (FAZ). In our current study, TbPLK-3HA expressed at the endogenous level was identified at the same dorsal location of both procyclic and bloodstream forms, albeit it was no longer detectable at the anterior tip of the cell. Endogenously expressed TbPLK fused with an enhanced yellow fluorescent protein (EYFP) localized to the same dorsal location along the FAZs in living procyclic and bloodstream cells. Fluorescence-activated cell sorter analysis of hydroxyurea-synchronized procyclic cells revealed that TbPLK-EYFP emerges during S phase, persists through G(2)/M phase, and vanishes in G(1) phase. An indicated TbPLK-EYFP association with the FAZs of G(2)/M cells may thus represent a timely localization to a potential initiation site of cytokinesis, which agrees with the recognized role of TbPLK in cytokinetic initiation.     

An aurora kinase homologue is involved in regulating both mitosis and cytokinesis in Trypanosoma brucei

The chromosomal passenger protein aurora kinases have been implicated in regulating chromosome segregation and cell division. Three aurora kinase homologues were identified (TbAUK1, -2 and -3) in the Trypanosome Genomic Data Base, and their expressions in the procyclic form of Trypanosoma brucei were knocked down individually by using the RNA interference technique. Only a knockdown of TbAUK1 arrested the cells in G(2)/M phase with each cell showing an extended posterior end, two kinetoplasts, and an enlarged nucleus, apparently the result of an inhibited kinetoplast multiplication and a failed mitosis. There is no mitotic spindle structure in the TbAUK1-depleted cell. The two kinetoplasts moved apart from each other but stopped just before cytokinesis, suggesting that cytokinesis was blocked in its early phase. Overexpression of TbAUK1 in the cells resulted in little change in cell growth. By immunofluorescence, TbAUK1 was primarily localized to the nucleus in interphase and to the mitotic spindle during apparent metaphase and anaphase. Thus, differing from other eukaryotes, TbAUK1 has an apparent triple function in coupling mitosis and kinetoplast replication with cytokinesis in T. brucei. T. brucei polo-like kinase, previously identified as the initiator of cytokinesis without apparent involvement in mitosis in the trypanosome, was either depleted or overexpressed in the TbAUK1-deficient cells. A dominant TbAUK1-depleted phenotype was demonstrated in both cases, suggesting that TbAUK1 plays an essential role in cytokinesis that cannot be affected by changes in the level of T. brucei polo-like kinase. To our knowledge, this is the first time that the function of an aurora B-like kinase is a prerequisite for polo-like kinase action in initiating cytokinesis. TbAUK1 is also the first identified protein that couples both mitosis and kinetoplast replication with cytokinesis in the trypanosome.     

Trypanosoma brucei MOB1 is required for accurate and efficient cytokinesis but not for exit from mitosis

Two MOB1 genes, MOB1-A and MOB1-B, were identified in Trypanosoma brucei. MOB1-A of T. brucei was shown to form a complex with TbPK50, a functional homologue of the Schizosaccharomyces pombe protein kinase Orb6, and immune precipitated MOB1-A exhibited histone H1 protein kinase activity. MOB1-A and TbPK50 were also shown to bind p12cks1, a cyclin-dependent kinase accessory protein. Immune fluorescence of epitope-tagged MOB1-A and MOB1-B in bloodstream form trypanosomes showed they had a punctate distribution all through the cell cytoplasm and were excluded from the nucleus throughout the cell cycle. Using RNA interference (RNAi), MOB1 was shown to be essential in both bloodstream and procyclic life cycle stages. In the bloodstream form, RNAi of MOB1 resulted, after 8 h, in a significant increase in post-mitotic cells, the majority of which had a visible cleavage furrow. This was followed by the appearance of cells with abnormal complements of nuclei and kinetoplasts, often with the number of nuclei exceeding the number of kinetoplasts. Thus, downregulation of MOB1 in the bloodstream form results in a delay in cytokinesis, and leads to a deregulation of the cell cycle, possibly through an inhibitory effect on kinetoplast replication. In contrast, downregulation of MOB1 in the procyclic form appears to impede the accuracy of cytokinesis, by allowing mispositioning of the cleavage furrow and inappropriate cytokinesis. Unlike its counterpart in budding yeast, T. brucei MOB1 does not appear to be required for mitotic exit.     

Trypanosoma brucei Polo-like kinase is essential for basal body duplication, kDNA segregation and cytokinesis

Polo-like kinases (PLKs) are conserved eukaryotic cell cycle regulators, which play multiple roles, particularly during mitosis. The function of Trypanosoma brucei PLK was investigated in procyclic and bloodstream-form parasites. In procyclic trypanosomes, RNA interference (RNAi) of PLK, or overexpression of TY1-epitope-tagged PLK (PLKty), but not overexpression of a kinase-dead variant, resulted in the accumulation of cells that had divided their nucleus but not their kinetoplast (2N1K cells). Analysis of basal bodies and flagella in these cells suggested the defect in kinetoplast division arose because of an inhibition of basal body duplication, which occurred when PLK expression levels were altered. Additionally, a defect in kDNA replication was observed in the 2N1K cells. However, the 2N1K cells obtained by each approach were not equivalent. Following PLK depletion, the single kinetoplast was predominantly located between the two divided nuclei, while in cells overexpressing PLKty, the kinetoplast was mainly found at the posterior end of the cell, suggesting a role for PLK kinase activity in basal body and kinetoplast migration. PLK RNAi in bloodstream trypanosomes also delayed kinetoplast division, and was further observed to inhibit furrow ingression during cytokinesis. Notably, no additional roles were detected for trypanosome PLK in mitosis, setting this protein kinase apart from its counterparts in other eukaryotes.     

Distinct Roles of a Mitogen-Activated Protein Kinase in Cytokinesis between Different Life Cycle Forms of Trypanosoma brucei

Mitogen-activated protein kinase (MAPK) modules are evolutionarily conserved signaling cascades that function in response to the environment and play crucial roles in intracellular signal transduction in eukaryotes. The involvement of a MAP kinase in regulating cytokinesis in yeast, animals, and plants has been reported, but the requirement for a MAP kinase for cytokinesis in the early-branching protozoa is not documented. Here, we show that a MAP kinase homolog (TbMAPK6) from Trypanosoma brucei plays distinct roles in cytokinesis in two life cycle forms of T. brucei. TbMAPK6 is distributed throughout the cytosol in the procyclic form but is localized in both the cytosol and the nucleus in the bloodstream form. RNA interference (RNAi) of TbMAPK6 results in moderate growth inhibition in the procyclic form but severe growth defects and rapid cell death in the bloodstream form. Moreover, TbMAPK6 appears to be implicated in furrow ingression and cytokinesis completion in the procyclic form but is essential for cytokinesis initiation in the bloodstream form. Despite the distinct defects in cytokinesis in the two forms, RNAi of TbMAPK6 also caused defective basal body duplication/segregation in a small cell population in both life cycle forms. Altogether, our results demonstrate the involvement of the TbMAPK6-mediated pathway in regulating cytokinesis in trypanosomes and suggest distinct roles of TbMAPK6 in cytokinesis between different life cycle stages of T. brucei.     

Mitochondrial development during life cycle differentiation of African trypanosomes: evidence for a kinetoplast-dependent differentiation control point

Life cycle differentiation of African trypanosomes entails developmental regulation of mitochondrial activity. This requires regulation of the nuclear genome and the kinetoplast, the trypanosome's unusual mitochondrial genome. To investigate the potential cross talk between the nuclear and mitochondrial genome during the events of differentiation, we have 1) disrupted expression of a nuclear-encoded component of the cytochrome oxidase (COX) complex; and 2) generated dyskinetoplastid cells, which lack a mitochondrial genome. Using RNA interference (RNAi) and by disrupting the nuclear COX VI gene, we demonstrate independent regulation of COX component mRNAs encoded in the nucleus and kinetoplast. However, two independent approaches (acriflavine treatment and RNA interference ablation of mitochondrial topoisomerase II) failed to establish clonal lines of dyskinetoplastid bloodstream forms. Nevertheless, dyskinetoplastid forms generated in vivo could undergo two life cycle differentiation events: transition from bloodstream slender to stumpy forms and the initiation of transformation to procyclic forms. However, they subsequently arrested at a specific point in this developmental program before cell cycle reentry. These results provide strong evidence for a requirement for kinetoplast DNA in the bloodstream and for a kinetoplast-dependent control point during differentiation to procyclic forms.     

Trypanosoma brucei FLA1 is required for flagellum attachment and cytokinesis

The single flagellum of the protozoan parasite Trypanosoma brucei is attached along the length of the cell body by a complex structure that requires the FLA1 protein. We show here that inhibition of FLA1 expression by RNA interference in procyclic trypanosomes causes flagellar detachment and prevents cytokinesis. Despite being unable to divide, these cells undergo mitosis and develop a multinucleated phenotype. The Trypanosoma cruzi FLA1 homolog, GP72, is unable to complement either the flagellar detachment or cytokinesis defects in procyclic T. brucei that have been depleted of FLA1 by RNA interference. Instead, GP72 itself caused flagellar detachment when expressed in T. brucei. In contrast to T. brucei cells depleted of FLA1, procyclic T. brucei expressing GP72 continued to divide despite having detached flagella, demonstrating that flagellar attachment is not absolutely necessary for cytokinesis. We have also identified a FLA1-related gene (FLA2) whose sequence is similar but not identical to FLA1. Inhibition of FLA1 and FLA2 expression in bloodstream T. brucei caused flagellar detachment and blocked cytokinesis but did not inhibit mitosis. These experiments demonstrate that the FLA proteins are essential and suggest that in procyclic T. brucei, the FLA1 protein has separable functions in flagellar attachment and cytokinesis.     

Dissociation of cytokinesis initiation from mitotic control in a eukaryote

Cytokinesis is initiated only after mitotic exit in eukaryotes. However, in the insect (procyclic) form of an ancient protist, Trypanosoma brucei, a blockade at the G2/M checkpoint results in an enrichment of anucleate cells (zoids), suggesting separated regulations between mitosis and cytokinesis (X. Tu and C. C. Wang, J. Biol. Chem. 279:20519-20528, 2004). Polo-like kinases (Plks) are known to play critical roles in controlling both mitosis and cytokinesis. A single Plk homologue in T. brucei, TbPLK, was found to be capable of complementing the Plk (Cdc5) functions in Saccharomyces cerevisiae, thus raising the question of how it may function in the trypanosome with cytokinesis dissociated from mitosis. Depletion of TbPLK in the procyclic form of T. brucei by RNA interference resulted in growth arrest with accumulation of multiple nuclei, kinetoplasts, basal bodies, and flagella in approximately equal numbers among individual cells. There were, however, few zoids detectable, indicating inhibited cytokinesis with unblocked mitosis and kinetoplast segregation. TbPLK is thus apparently involved only in initiating cytokinesis in T. brucei. Overexpression of TbPLK in the trypanosome did not affect cell growth, but 13% of the resulting population was in the zoid form, suggesting runaway cytokinesis. An immunofluorescence assay indicated that TbPLK was localized in a chain of likely flagellum attachment zones in the cytoskeleton. In a dividing cell, a new line of such zones appeared closely paralleling the existing one, which could constitute the cleavage furrow. An exposed region of TbPLK at the anterior tip of the cell may provide the trigger of cytokinesis. Taken together, our results revealed a novel mechanism of cytokinesis initiation in the trypanosome that may serve as a useful model for further in-depth investigations.     

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

The anaphase-promoting complex or cyclosome (APC/C) is a multiprotein subunit E3 ubiquitin ligase complex that controls segregation of chromosomes and exit from mitosis in eukaryotes. It triggers elimination of key cell cycle regulators such as securin and mitotic cyclins during mitosis by polyubiquitinating them for proteasome degradation. Seven core subunit homologs of APC/C (APC1, APC2, APC11, CDC16, CDC23, CDC27, and DOC1) were identified in the Trypanosoma brucei genome data base. Expression of six of them was individually ablated by RNA interference in both the procyclic and bloodstream forms of T. brucei. Only the CDC27- and APC1-depleted cells were enriched in the G2/M phase with inhibited growth. Further studies indicated that T. brucei APC1 and CDC27 failed to complement the corresponding deletion mutants of budding yeast. However, their depletion from procyclic-form T. brucei enriched cells with two kinetoplasts and an enlarged nucleus possessing short metaphase-like mitotic spindles, suggesting that APC1 and CDC27 may play essential roles in promoting anaphase in the procyclic form. Their depletion from the bloodstream form, however, enriched cells with two kinetoplasts and two nuclei connected through a microtubule bundle, suggesting a late anaphase arrest. This is the first time functional APC/C subunit homologs were identified in T. brucei. The apparent differential activities of this putative APC/C in two distinct developmental stages suggest an unusual function. The apparent lack of functional involvement of some of the other individual structural subunit homologs of APC/C may indicate the structural uniqueness of T. brucei APC/C.     

The Chromosomal Passenger Complex and a Mitotic Kinesin Interact with the Tousled-Like Kinase in Trypanosomes to Regulate Mitosis and Cytokinesis

Aurora B kinase plays essential roles in mitosis and cytokinesis in eukaryotes. In the procyclic form of Trypanosoma brucei, the Aurora B homolog TbAUK1 regulates mitosis and cytokinesis, phosphorylates the Tousled-like kinase TbTLK1, interacts with two mitotic kinesins TbKIN-A and TbKIN-B and forms a novel chromosomal passenger complex (CPC) with two novel proteins TbCPC1 and TbCPC2. Here we show with time-lapse video microscopy the time course of CPC trans-localization from the spindle midzone in late anaphase to the dorsal side of the cell where the anterior end of daughter cell is tethered, and followed by a glide toward the posterior end to divide the cell, representing a novel mode of cytokinesis in eukaryotes. The three subunits of CPC, TbKIN-B and TbTLK1 interact with one another suggesting a close association among the five proteins. An ablation of TbTLK1 inhibited the subsequent trans-localization of CPC and TbKIN-B, whereas a knockdown of CPC or TbKIN-B disrupted the spindle pole localization of TbTLK1 during mitosis. In the bloodstream form of T. brucei, the five proteins also play essential roles in chromosome segregation and cytokinesis and display subcellular localization patterns similar to that in the procyclic form. The CPC in bloodstream form also undergoes a trans-localization during cytokinesis similar to that in the procyclic form. All together, our results indicate that the five-protein complex CPC-TbTLK1-TbKIN-B plays key roles in regulating chromosome segregation in the early phase of mitosis and that the highly unusual mode of cytokinesis mediated by CPC occurs in both forms of trypanosomes.     

Microtubule polarity and dynamics in the control of organelle positioning,segregation, and cytokinesis in the trypanosome cell cycle

Trypanosoma brucei has a precisely ordered microtubule cytoskeleton whose morphogenesis is central to cell cycle events such as organelle positioning, segregation, mitosis, and cytokinesis. We have defined microtubule polarity and show the + ends of the cortical microtubules to be at the posterior end of the cell. Measurements of organelle positions through the cell cycle reveal a high degree of coordinate movement and a relationship with overall cell extension. Quantitative analysis of the segregation of the replicated mitochondrial genome (the kinetoplast) by the flagellar basal bodies identifies a new G2 cell cycle event marker. The subsequent mitosis then positions one "daughter" nucleus into the gap between the segregated basal bodies/kinetoplasts. The anterior daughter nucleus maintains its position relative to the anterior of the cell, suggesting an effective yet cryptic nuclear positioning mechanism. Inhibition of microtubule dynamics by rhizoxin results in a phenomenon whereby cells, which have segregated their kinetoplasts yet are compromised in mitosis, cleave into a nucleated portion and a flagellated, anucleate, cytoplast. We term these cytoplasts "zoids" and show that they contain the posterior (new) flagellum and associated basal-body/kinetoplast complex. Examination of zoids suggests a role for the flagellum attachment zone (FAZ) in defining the position for the axis of cleavage in trypanosomes. Progression through cytokinesis, (zoid formation) while mitosis is compromised, suggests that the dependency relationships leading to the classical cell cycle check points may be altered in trypanosomes, to take account of the need to segregate two unit genomes (nuclear and mitochondrial) in this cell.     

KMP-11, a basal body and flagellar protein, is required for cell division in Trypanosoma brucei

Kinetoplastid membrane protein 11 (KMP-11) has been identified as a flagellar protein and is conserved among kinetoplastid parasites, but its potential function remains unknown. In a recent study, we identified KMP-11 as a microtubule-bound protein localizing to the flagellum as well as the basal body in both procyclic and bloodstream forms of Trypanosoma brucei (Z. Li, J. H. Lee, F. Chu, A. L. Burlingame, A. Gunzl, and C. C. Wang, PLoS One 3:e2354, 2008). Silencing of KMP-11 by RNA interference inhibited basal body segregation and cytokinesis in both forms and resulted in multiple nuclei of various sizes, indicating a continuous, albeit somewhat defective, nuclear division while cell division was blocked. KMP-11 knockdown in the procyclic form led to severely compromised formation of the new flagellum attachment zone (FAZ) and detachment of the newly synthesized flagellum. However, a similar phenotype was not observed in the bloodstream form depleted of KMP-11. Thus, KMP-11 is a flagellar protein playing critical roles in regulating cytokinesis in both forms of the trypanosomes. Its distinct roles in regulating FAZ formation in the two forms may provide a clue to the different mechanisms of cytokinetic initiation in procyclic and bloodstream trypanosomes.     

Functional characterization of cohesin subunit SCC1 in Trypanosoma brucei and dissection of mutant phenotypes in two life cycle stages

In yeast and metazoa, structural maintenance of chromosome (SMC) complexes play key roles in chromosome segregation, architecture and DNA repair. The main function of the cohesin complex is to hold replicated sister chromatids together until segregation at anaphase, which is dependent on proteolytic cleavage of the cohesin subunit SCC1. Analysis of trypanosomatid genomes showed that the core cohesin and condensin complexes are conserved, but SMC5/6 is absent. To investigate the functional conservation of cohesin in eukaryotes distantly related to yeast and metazoa, we characterized the Trypanosoma brucei SCC1 orthologue. TbSCC1 is expressed prior to DNA synthesis at late G1, remains in the nucleus throughout S- and G2-phases of the cell cycle and disappears at anaphase. Depletion of SCC1 by RNAi or expression of a non-cleavable SCC1 resulted in karyokinesis failure. Using the dominant negative phenotype of non-cleavable SCC1 we investigated checkpoint regulation of cytokinesis in response to mitosis failure at anaphase. In the absence of chromosome segregation, procyclic trypanosomes progressed through cytokinesis to produce one nucleated and one anucleate cell (zoid). In contrast, cytokinesis was incomplete in bloodstream forms, where cleavage was initiated but cells failed to progress to abscission. Kinetoplast duplication was uninterrupted resulting in cells with multiple kinetoplasts and flagella.     

Essential roles for GPI-anchored proteins in African trypanosomes revealed using mutants deficient in GPI8

The survival of Trypanosoma brucei, the causative agent of Sleeping Sickness and Nagana, is facilitated by the expression of a dense surface coat of glycosylphosphatidylinositol (GPI)-anchored proteins in both its mammalian and tsetse fly hosts. We have characterized T. brucei GPI8, the gene encoding the catalytic subunit of the GPI:protein transamidase complex that adds preformed GPI anchors onto nascent polypeptides. Deletion of GPI8 (to give Deltagpi8) resulted in the absence of GPI-anchored proteins from the cell surface of procyclic form trypanosomes and accumulation of a pool of non-protein-linked GPI molecules, some of which are surface located. Procyclic Deltagpi8, while viable in culture, were unable to establish infections in the tsetse midgut, confirming that GPI-anchored proteins are essential for insect-parasite interactions. Applying specific inducible GPI8 RNAi with bloodstream form parasites resulted in accumulation of unanchored variant surface glycoprotein and cell death with a defined multinuclear, multikinetoplast, and multiflagellar phenotype indicative of a block in cytokinesis. These data show that GPI-anchored proteins are essential for the viability of bloodstream form trypanosomes even in the absence of immune challenge and imply that GPI8 is important for proper cell cycle progression.     

Pairwise knockdowns of cdc2-related kinases (CRKs) in Trypanosoma brucei identified the CRKs for G1/S and G2/M transitions and demonstrated distinctive cytokinetic regulations between two developmental stages of the organism

Expression of the cdc2-related kinase 3 (CRK3) together with expression of CRK1, -2, -4, or -6, were knocked down in pairs in the procyclic and bloodstream forms of Trypanosoma brucei, using the RNA interference technique. Double knockdowns of CRK3 and CRK2, CRK4, or CRK6 exerted significant growth inhibition and enriched the cells in G2/M phase, whereas a CRK3 plus CRK1 (CRK3 + CRK1) knockdown arrested cells in both G1/S and G2/M transitions. Thus, CRK1 and CRK3 are apparently the kinases regulating the G1/S and G2/M checkpoint passages, respectively, whereas the other CRKs are probably playing only minor roles in cell cycle regulation. A CRK1 + CRK2 knockdown in the procyclic form was found to cause aberrant posterior cytoskeletal morphogenesis (X. M. Tu and C. C. Wang, Mol. Biol. Cell 16:97-105, 2005). A CRK3 + CRK2 knockdown, however, did not lead to such a change, suggesting that CRK2 depletion can lead to the abnormal morphogenesis only when procyclic-form cells are arrested in the G1 phase. The G2/M-arrested procyclic form produces up to 20% stumpy anucleated cells (zoids) in the population, suggesting that cytokinesis and cell division are not blocked by mitotic arrest but are apparently driven to completion by the kinetoplast cycle. In the bloodstream form, however, G2/M arrest resulted in little zoid formation but, instead, enriched a population of cells each containing multiple kinetoplasts, basal bodies, and flagella and an aggregate of multiple nuclei, indicating failure in entering cytokinesis. The two different cytokinetic regulations between two distinct stage-specific forms of the same organism may provide an interesting and useful model for further understanding the evolution of cytokinetic control among eukaryotes.