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 identification and characterization of proteins that regulate T. brucei cell cycle progression have been the main focus of our recent studies. Among the limited number of cyclin homologs and cdc2-related protein kinases (CRKs) 1 identified in the trypanosome genome data base, we found CycB2/CYC6 to be the only cyclin and CRK3 the sole protein kinase involved in regulating the G 2 /M transition in both the procyclic and bloodstream forms of T. brucei (3,4). This unusually simple scheme of G 2 /M passage regulation prompted us to look further into it with the expectation of establishing a simple model for in-depth understanding of the mechanisms of G 2 /M checkpoint control.
The detailed features of mitosis in T. brucei have not yet been well delineated because of technical difficulties in synchronizing the cell growth. Consequently, important discrete stages occurring during mitosis such as the prophase, metaphase, anaphase, telophase, etc., have not yet been positively identified in this organism. It is known that the function of an E3 ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C), is closely associated with mitotic exit in eukaryotes. It is a multisubunit complex that promotes degradation of proteins by forming a tripartite complex along with the target protein and the ubiquitin-conjugating enzyme E2 (5). It polyubiquitinates important cell cycle regulatory proteins such as cyclin B and Pds1/securin, targets them for proteolysis by the 26 S proteasome, and promotes chromosome segregation, transition from metaphase to anaphase, and exit from mitosis (6,7). APC/C has at least two very important roles during the metaphase to anaphase transition in a metazoan. It activates the cysteine-protease separase by ubiquitinating its inhibitor Pds1/securin. The activated separase then destroys the cohesion between sister chromatins and enables the mitotic spindle to pull them to opposite spindle poles (8,9). Secondly, APC/C ubiquitinates mitotic cyclins and thereby inactivates cyclin-dependent kinase 1 (Cdk1, designated Cdc28 in budding yeast), which is required for exit from mitosis and initiation of cytokinesis. Disruption of APC/C functions by knocking down expression of its subunits leads to cell cycle arrest at the metaphase to anaphase transition (5). The failure in chromosome segregation arrests the cells at the G 2 /M boundary with metaphaselike mitotic spindles. There are 13 core subunits of APC/C in Saccharomyces cerevisiae (6,7,10), which form a core of the particle and remain tightly associated throughout the cell cycle. There are also two APC/C activator proteins, Cdc20 and Cdh1, and an E2 protein, Ubc4, which associate with APC/C at specific points during the cell cycle for polyubiquitination of specific proteins destined for proteasome degradation (11). Cdc20 activates APC/C specifically for promoting the anaphase, whereas Cdh1 activates the complex after the mitotic exit (11). In the current study, we identified an APC/C equivalent in T. brucei and seven core subunit homologs in the genome data base. The expression of six of them was ablated from both the procyclic and bloodstream forms of cells with RNA interference (RNAi), and the cells were examined for mitotic arrest and accumulation in the metaphase. The results indicated that only the loss of the APC1 or the CDC27 homolog led to mitotic arrest. The procyclic form was apparently blocked in the metaphase, whereas the bloodstream form was more likely stopped in the late anaphase. Thus, the APC/C machinery appears to perform distinctive functions at different stages of development in T. brucei.

MATERIALS AND METHODS
Cell Culture-The procyclic-form T. brucei strain 29-13 (12) was cultured in Cunningham's medium at 26°C supplemented with 10% fetal bovine serum (Atlanta Biological). G418 (15 g/ml) and hygromycin B (50 g/ml) were added to the culture medium to maintain the T7 RNA polymerase and tetracycline repressor gene constructs within the cells.
RNAi Interference-RNAi experiments were designed for six of the APC/C core subunit homologs (see "Results"). The sequences used for RNAi were as follows: nucleotides 1723-2174 for APC1, 623-961 for APC2, 1-246 for APC11, 562-933 for CDC16, 832-1311 for CDC23, and 906 -1353 for CDC27. The corresponding 250 -500-bp DNA fragments were amplified by PCR using a pair of primers with added XhoI and HindIII linkers to the ends and subcloned into the tetracycline-inducible pZJM vector by replacing the ␣-tubulin fragment in the vector (14). The resulting RNAi constructs, with the inserts between two opposing T7 promoters and two opposing tetracycline operators, were linearized with NotI and introduced into T. brucei cells by electroporation for integration into the ribosomal DNA spacer region in the chromosome. Transfection of the procyclic-form T. brucei was performed essentially according to a procedure described previously (15,16). After phleomycin selection, single transfected cells were cloned on semisolid agarose plates, cultivated under phleomycin, and induced by tetracycline to synthesize the double-stranded RNA encoded by the cDNA insert. The double-stranded RNA degrades the corresponding mRNA in T. brucei and thus blocks the expression of the encoding gene (3).
The bloodstream-form T. brucei was transfected by a procedure described previously, with some modifications (17). Briefly, a sample of 1 ϫ 10 7 log-phase cells were harvested, washed once, and suspended in 0.5 ml of Cytomix buffer (18) containing 100 g of the linearized pZJM DNA construct. Electroporation was carried out in a 4-mm cuvette using the Gene Pulser (Bio-Rad) with parameters set as follows: 1.7-kV voltage, 400-ohm resistance, and 25-microfarad capacitance. The cells were transferred to a 24-well plate in HMI9 medium immediately after electroporation and incubated at 37°C for 24 h. The transfectants were then selected with the addition of 2.5 g/ml phleomycin, and a single cell was cloned by limiting dilution on soft agarose plates. For induction of RNAi, the cloned transfectants were cultured in the presence of 1.0 g/ml tetracycline. Cell numbers in time samples were counted with a microscope using a hemocytometer.
Semiquantitative Reverse Transcription-PCR-To monitor the effect of tetracycline-induced RNAi on gene expression, semiquantitative reverse transcription-PCR was conducted by a procedure described previously on cellular RNA from cells 3 days after tetracycline induction (3). Total RNA was extracted from T. brucei cells using the TRIzol reagent (Amersham Biosciences). First-strand cDNA was generated from DNase-I-treated RNA samples using an oligo(dT) 15 primer and Moloney murine leukemia virus-reverse transcriptase (Promega). PCR was then performed using the first-strand cDNA and a pair of genespecific primers that differs from the primer pair used in generating the original RNAi construct. 2 Fluorescence-activated Cell Sorting (FACS) Analysis-Cell samples for FACS analysis were prepared as described previously (18), and FACS analysis was conducted by a reported procedure (3). The propidium iodidestained cell samples from the analysis were also examined with a Nikon phase-contrast and fluorescence microscope for a tabulation of numbers of nuclei and kinetoplasts in individual cells and a count of cells with different morphologies from a population of 200 cells.
Complementation of S. cerevisiae Deletion Mutants-Full-length genes encoding yeast Cdc27 (yCdc27), yeast APC1 (yApc1), T. brucei CDC27 (tCdc27), and T. brucei APC1 (tApc1) were each amplified by PCR and subcloned into the vector pGADT7 (Invitrogen). This vector was modified by adding an additional NdeI site at position 1480 and removing fragment 1480 -1964 by NdeI digestion. Constructs containing yCDC27 and tCDC27 were each introduced into the yeast Tet promoter Hughes (yTHC) strain YSC1180-7428818 (obtained from Open Biosystem, Huntsville, AL) (19) cells grown in yeast extract/ peptone/dextrose medium with 10 g/ml doxycycline and incubated for 3 days at 37°C. yAPC1 and tAPC1 were each transfected into a MAT␣ apc1-1 yeast strain kindly provided by Dr. W. Zachariae of the Max Planck Institute (20). Cells were grown in yeast extract/peptone/dextrose medium at 37°C for 3 days.
Labeling of Mitotic Spindles with KMX-1-Mitotic spindles in T. brucei were labeled with mouse monoclonal antibody KMX-1 (a kind gift from Dr. Keith Gull, Oxford University) (21), which preferentially recognizes ␤-tubulin in the mitotic spindle. The immunofluorescence protocol was followed exactly as described previously (22). Slides were mounted in Vectashield in the presence of 1 g of 4,6-diamino-2-phenylindole/ml and examined with a fluorescence microscope.

Identification of Homologs of APC/C Subunits in the Genome
Data Base of T. brucei-The 10 core subunits of APC/C in S. cerevisiae include Apc1, Apc2, Apc4, Apc5, Apc9, Cdc16, Cdc23, Cdc26, Cdc27, and Doc1 (6,7,10). They and the two APC/C activator proteins, Cdc20 and Cdh1, plus the E2 enzyme Ubc4 were each used as a query to screen the Sanger trypanosome 2 Sequences available upon request.  Yeast CDC27 (ycdc27) and APC1 (yapc1) mutants were transfected with the full-length genes of T. brucei CDC27 (tcdc27) and APC1 (tApc1), respectively. The transfected cells were plated in various dilutions on yeast extract/peptone/dextrose plates and incubated for 3 days under different conditions. A, Cdc27 complementation was assayed in the presence of 50 g/ml doxycycline. B, Apc1 complementation was monitored by incubation at 37°C. Ts, temperature-sensitive. genome data base, which resulted in identification of seven core subunit homologs (APC1, APC2, APC11, CDC16, CDC23, CDC27, and DOC1), one activator protein (CDC20), and a UBC4 homolog (Table I). The presence of two APC/C hallmark proteins (Apc2 and Apc11) (20,23), the three tetratricopeptide repeat (TPR) proteins (Cdc16, Cdc23, Cdc27) (24), and Doc1, a subunit promoting substrate binding and polyubiquitination (25), suggests a functioning, albeit simpler, APC/C in T. brucei. This is further supported by the presence of a Cdc20 and a Ubc4 homolog, which is consistent with APC/C functioning as an anaphase-promoting complex in the trypanosome (11).

CDC27 and APC1 Are the Only Subunits Required for T. brucei
Growth-Six of the APC/C core subunit homologs in T. brucei were chosen for RNAi analysis (APC1, APC2, APC11, CDC16, CDC23, and CDC27). We did not knock down DOC1 because it is a nonessential subunit in budding yeast, and the deletion mutant of Doc1 in yeast can grow normally (25). Apc1 is the largest subunit of APC/C with its motif sharing similarity with Rpn1 and Rpn2 of the 19 S cap of proteasome (26 -30). Tetrad analysis of the S. cerevisiae apc1-1 temperature-sensitive mutant showed that, at non-permissive temperature, it failed to exit from mitosis and was arrested at the G 2 /M phase (28). Apc2 carries a cullin domain and is associated with Apc11 and plays a major role in recruiting the E2 enzyme (31)(32)(33). Apc11 is the smallest subunit in APC/C. It carries a ring H2 finger domain, mediating proteinprotein interactions and interacting with the cullin domain of Apc2 (34). Tetrad analysis indicated that both APC11 and APC2 are also essential genes in S. cerevisiae (31,35). Cdc16, Cdc23, and Cdc27 are related TPR proteins with molecular masses ranging from 70 to 100 kDa and 9 -10 copies of TPR (24,32). Temperature-sensitive mutants of these genes in S. cerevisiae blocked the cell cycle at the G 2 /M phase with large budded cells that failed to proceed from metaphase to anaphase at 37°C (36). Thus, based on this information from yeast, a knockdown of each of the six chosen APC/C core subunit homologs in T. brucei was expected to result in a growth arrest in the G 2 /M phase.
The RNAi experiments were performed on both the procyclic and bloodstream forms of T. brucei. Semiquantitative reverse transcription-PCR was carried out on cellular RNA samples 3 days after tetracycline induction (3). The results, presented among the insets in Fig. 1 show that, in every instance, the level of mRNA was significantly diminished after a 3-day RNAi when compared with that of ␣-tubulin mRNA included as a sampling control. The RNAi technique thus successfully down-regulated the expression of specific mRNAs in both forms of T. brucei.
The potential effect from knocking down the expression of each of the six APC/C core subunit genes on the proliferation of T. brucei was examined (Fig. 1). Only the knock-downs of CDC27 (Fig. 1A) and APC1 (Fig. 1B) were followed by significant growth inhibition, whereas decreased expression of APC2 (Fig. 1C), APC11 (Fig. 1D), CDC16 (Fig. 1E), and CDC23 (Fig.  1F) all failed to register any detectable effect on the growth of both forms of T. brucei. Quantitative analysis of cell numbers demonstrated that the growth of the CDC27-depleted procyclic form was completely stopped 2 days into RNAi induction, whereas the CDC27-depleted bloodstream form assumed a growth rate about 1% of the uninduced control (Fig. 1A). For APC1 depletion, the procyclic form grew at a 10-fold reduced rate, whereas the bloodstream form grew at ϳ1% of the control (Fig. 1B).

Role of APC/C Subunits in Cell Cycle Regulation of T. brucei
Assuming that the cause of inhibited growth was attributed to a compromised APC/C function in T. brucei, CDC27 and APC1 may be then playing essential roles in the complex, whereas APC2, APC11, CDC16, and CDC23 could be either dispensable or not included in the complex. A third possibility could be that their expressions need to be ablated together for a phenotype. We chose to learn first whether T. brucei CDC27 and APC1 can complement the functions of their counterparts in S. cerevisiae and to find out if the growth arrest caused by CDC27 and APC1 depletions in T. brucei is the same or similar to that observed after APC/C depletion in yeast and mammalian cells (24,28).

T. brucei CDC27 and APC1 Do Not Exhibit Complementary Functions in S. cerevisiae-T. brucei CDC27 and APC1
have sequence identities of 20 and 17% with the Cdc27 and Apc1 in S. cerevisiae, respectively (Table I). These relatively low degrees of sequence homology cast some doubt on whether they could complement the Cdc27 and Apc1 functions in yeast. Thus, the complementation studies were first carried out in S. cerevisiae strain YSC1180-7428818 with CDC27 expression under doxycycline regulation. Addition of doxycycline turned off the expression of the endogenous CDC27 gene and made the cell growth dependent on the ectopically introduced one. When the yeast CDC27 gene was introduced, the cells were able to grow normally under doxycycline ( Fig. 2A). However, when the T. brucei CDC27 gene was introduced into these cells, they failed to grow under the drug pressure ( Fig. 2A), indicating that T. brucei CDC27 cannot complement the function of the yeast CDC27 gene.
Similar results were observed when the T. brucei APC1 gene was used to rescue the yeast temperature-sensitive apc1-1 mutant (MAT␣, apc1). APC1 of T. brucei failed to complement the function of yeast APC1 at the restrictive temperature, whereas the exogenously introduced yeast APC1 could (Fig.  2B). Thus, neither CDC27 nor APC1 from T. brucei can replace their counterparts in yeast. Although somewhat anticipated from the poor sequence alignment, the negative results raised some concern over the real functions of the CDC27 and APC1 homologs in T. brucei, which, we hope, could be answered by further characterizing the phenotypes of CDC27 and APC1 RNAi knockdown mutants of T. brucei.
The CDC27 and APC1-depleted Cells Are Arrested in the G 2 /M Phase-Time samples were collected during the RNAi experiments, stained with propidium iodide, and analyzed with FACScan (Fig. 3). The control cells containing an empty pZJM vector had 52-55% of the cells remaining in the G 1 phase, 25-26% in the S phase, and 20 -23% in G 2 /M phase during the ensuing 3 days of incubation. The CDC27-depleted procyclic form had a 15% increase in G 2 /M cells and a corresponding decrease in G 1 cells without much change in S-phase cells, whereas the CDC27-depleted bloodstream form exhibited 25% enhancement in G 2 /M, 35% reduction in G 1 , and 10% increase in S-phase cells (Fig. 3A, left-hand panel). For the APC1-depleted procyclic form, there was about a 10% increase in G 2 /M, a similar decrease in G 1 , and a 5% decrease in S-phase cells. The APC1-depleted bloodstream form had ϳ15% increase in G 2 /M, 15% reduction in G 1 , and virtually no change in S-phase cells (Fig. 3B, left-hand panel). The histograms in the righthand panels of Fig. 3 present the raw data from FACScan and demonstrate an unambiguous shift from G 1 to the G 2 /M phase during the 3-day RNAi induction. This enrichment of G 2 /M cells in both forms of T. brucei depleted of either CDC27 or APC1 suggests that the two proteins could function as subunits of an APC/C-like complex in T. brucei.
Distinctive Morphological Changes between the Procyclic and Bloodstream Forms Depleted of CDC27 and APC1-The CDC27-and APC1-depleted cells stained with propidium iodide were examined under a fluorescence microscope for aberration in intracellular numbers of nuclei and kinetoplasts (Fig.  4). There was a significant drop in the cells with a single nucleus and a single kinetoplast (1N1K) reflecting a reduced population of G 1 cells. Cells with one nucleus and two kinetoplasts (1N2K) were slightly increased, suggesting an enrichment of G 2 /M cells.
In the procyclic form, the population of two nuclei-two kinetoplasts (2N2K) cells remained unchanged by the knockdowns, and the two nuclei were always well separated from each other (Fig. 4, A and C). There was, however, an emergence of anucleate cells (0N1K, zoids) up to 10 to 15% of the population. This was anticipated from our previous observation that for the procyclic form arrested in G 2 /M through a knockdown of CycB2/CYC6 or CRK3, a substantial number of the cells could still proceed into cytokinesis and cell division resulting in the formation of zoids (3,4). The appearance of zoids in the present study could be regarded as an additional proof that the cells are arrested in G 2 /M by depleting CDC27 or APC1.
The bloodstream form arrested in G 2 /M by cyclin or kinase knockdown does not produce zoids but instead accumulates cells with one nuclear aggregate and multiple kinetoplasts (3,4). Such a population increase was, however, not obvious in the CDC27-or APC1-depleted bloodstream form. Instead, a sharp rise in the population of 2N2K cells became apparent (Fig. 4, B  and D). This increase could reach 35-40% of the total population. Upon closer examination, however, the two nuclei in each cell were found always closely associated with each other with the two closely associated kinetoplasts located at a distance toward the posterior end (Fig. 4, B and D), suggesting that they have not quite yet exited from mitosis (see below).
The Mitotic Spindles in CDC27-or APC1-depleted Cells Differ between Procyclic and Bloodstream Forms-To resolve this mystery, we stained the cells with the KMX-1 monoclonal antibody known to stain ␤-tubulin preferentially in mitotic spindles (22). We observed in the Cdc27-and APC1-depleted procyclic form an increase of cells with short mitotic spindles from 10% to 19 and 17%, respectively (Fig. 5, A and B, and Fig. 6). These cells were likely arrested in the metaphase, and their populations were enhanced as a consequence of CDC27 (Fig.  6A) or APC1 (Fig. 6B) depletion. In Fig. 5C, there is a zoid in the process of being generated. The mother cell is arrested in the metaphase by the depletion of CDC27, but a zoid is formed nevertheless, illustrating that cytokinesis and cell division are not stopped by the metaphase arrest of procyclic-form T. brucei.
For the CDC27-and APC1-depleted bloodstream form, KMX-1 staining revealed that the two nuclei in the 2N2K cells are not completely separated but are linked by a bundle of microtubules, which could be the elongated spindles one observes in the late anaphase (Fig. 7A). This is illustrated in greater detail in Fig. 7B, in which two apparent nuclear DNA clusters are connected by a bundle of microtubules, whereas the two kinetoplasts are both located at the posterior end of the cell. Thus, deficiency of APC/C function in the bloodstream form may have arrested the cells in late anaphase instead of metaphase.

DISCUSSION
In this study, we have selectively down-regulated the expression of six core APC/C subunit homologs by RNAi in both the procyclic and bloodstream forms of T. brucei to investigate whether they play essential roles in APC/C-mediated cell cycle progression. Only depletion of the CDC27 or APC1 homolog led to a G 2 /M arrest. The failure of these two homologs in complementing their counterparts in yeast further suggested that T. brucei may have a structurally unique APC/C with its CDC27 and APC1 subunits playing essential functions but incapable of replacing their counterparts in yeast because of structural distinctions. The lack of a phenotype from depleting the other APC/C subunits in T. brucei could mean that some of them may have overlapping structural and functional roles. They may require a combined knockdown, e.g. APC2 with APC11 or CDC16 with CDC23, to register an effect. A search for the remainder of the core subunits in this complex, if any, using perhaps the yeast two-hybrid assay (37) and tandem affinity protein tag affinity chromatography (38), may help our future understanding of the composition of this protein complex. To the best of our knowledge, there has not yet been any APC/C identified among eukaryotic microorganisms other than budding and fission yeast. A dissec- tion of the T. brucei APC/C could cast some light on the structural as well as functional evolution of this complex.
Meanwhile, the identification of a CDC20 homolog with a 25% sequence identity and 37% similarity with the yeast Cdc20 and the apparent absence of a CDH1 homolog from T. brucei (see Table I) suggest that the APC/C in this protist could have the sole function of promoting anaphase without involving the CDH1-activated function beyond mitotic exit (11). The presence of a UBC4 homolog in T. brucei (see Table I) with 71% identity and 84% similarity with yeast Ubc4 (see Table I) suggests a highly conserved E2 ubiquitin-conjugating enzyme performing the function of a donor of ubiquitin for T. brucei APC/C. This well conserved E2 also suggests structural conservation of APC/C in interacting with UBC4 (39).
Depletion of CDC27 and APC1 from the procyclic form of T. brucei resulted in G 2 /M cells that possessed primarily a single nucleus with a short metaphase-like mitotic spindle. The spindles were short, rhomboid, and pointed toward the ends with DNA compressed more at the spindle consistent with the formation of a metaphase plate. Spindle poles were sharp and pointed. These results are consistent with those observed in S. cerevisiae (24,28,40) and human (Homo sapiens) Cdc27 mutants (41,42) or the Apc1 conditional mutants (26,28) in which the cells are specifically arrested at the metaphase to anaphase transition.
For the bloodstream form depleted of CDC27 or APC1, however, the G 2 /M cells were enriched in an apparent 2N2K morphology. The mitotic spindles increased in size and became stretched (Fig. 7). Two closely associated nuclei were visible and apparently moving toward the opposite poles, whereas the newly formed kinetoplast was located at the posterior end of the cell (Fig. 4, B and D). The spindles formed microtubule bundles at the middle of the two separated nuclei present on the opposite poles. This was apparently the final stage of the mitosis after which two daughter cells should have separated from each other to complete the mitosis. But the eventual separation never occurred. The arrested state could likely represent the late anaphase.
In eukaryotic cells, expression of non-degradable cyclin B inhibits CDK inactivation, spindle disassembly, and cytokinesis without affecting chromosome segregation, which results in late anaphase-like arrest (43,44). Thus it appears that the function of APC/C in the bloodstream form of T. brucei is required only for degrading mitotic cyclin for mitotic exit but not for Pds1/securin degradation for chromosome segregation. The two forms of T. brucei have been shown to have distinct cell cycle regulations. Although depletion of CycB2/CYC6 or CRK3 leads to G 2 /M arrest in both forms of T. brucei (3,45), the procyclic form is arrested at the G 2 /M phase with one somewhat enlarged nucleus, whereas the bloodstream form continues to enter the G 1 phase for another round of the nuclear cycle resulting in a multinuclear aggregate. Thus, a lowered mitotic CDK activity through knocking down either CycB2/CYC6 or CRK3 is relatively ineffective in placing the bloodstream form under mitotic arrest, whereas an apparent overexpression of mitotic CDK through APC/C knockdown stops it in the late anaphase. These differences in regulation of mitosis in the bloodstream and procyclic forms of T. brucei will be the subject of future experiments.