The main findings of this study are: (i) the T. brucei genome contains homologues of all the components present in cohesin and condensin, two SMC complexes necessary for the correct segregation of chromosomes at mitosis, (ii) SCC1, the kleisin component of cohesin, is expressed from the beginning of S-phase until anaphase, (iii) knockdown of expression of SCC1 by RNAi impedes nuclear division, (iv) expression of a dominant negative mutant of SCC1 that is resistant to cleavage by separase blocks nuclear division, and (v) reduction of wild-type SCC1 activity or expression of separase-resistant SCC1 resulted in a failure to complete cell division in bloodstream forms but not in procyclic forms which divided to produce one nucleated and one anucleate daughter cell.
Cohesin is the complex that physically holds the two sister chromatids together from S-phase to anaphase.Proteolytic cleavage of the SCC1 subunit by separase at one of two specific sites is essential for sister chromatid separation at anaphase. The results from this study provide evidence that the gene Tb927.7.6900 identified as the closest homologue to yeast SCC1 is the functional orthologue; the expression pattern and nuclear phenotype of both the knockdown and dominant negative are similar to equivalent mutations in the SCC1 genes from yeast and Drosophila
(Uhlmann et al., 1999
; Vass et al., 2003
). Knockdown of TbSCC1 by RNAi in bloodstream-form trypanosomes resulted in a phenotype consistent with a delayed progression through the cell cycle and aberrant segregation of nuclear DNA. Expression of non-cleavable SCC1-mutAB produced a more severe phenotype, characterized in bloodstream-form trypanosomes by a growth arrest at 12 h post induction and a decrease of cell numbers 24 h post induction indicating cell death. During mitosis, individual T. brucei
chromosomes do not visibly condense but the movement of DNA clusters to opposite poles of the nucleus follows a defined pattern that can be observed in DAPI-stained cells (Ogbadoyi et al., 2000
). The sharp increase in the proportion of cells with 2K1N early after induction of SCC1-mutAB expression, and accumulation of cells with > 2K1N indicate a block of nuclear division early in mitosis. This phenotype is consistent with a block in sister chromatid segregation resulting in failure of anaphase A and the absence of anaphase B events including spindle elongation and correct nuclear positioning. Expression of separase-resistant SCC1 prevented both procyclic and bloodstream-form trypanosomes from partitioning nuclear DNA into two clusters of equal size and no late mitotic, anaphase B, nuclei were observed. The inducible expression of SCC1 with mutations in both separase cleavage sites (SCC1-mutAB) thus provided an experimental tool to investigate the phenotype of cells with nuclei unable to separate sister chromatids during mitosis. The conservation of function of SCC1, the kleisin subunit of cohesin and the presence of homologues for the other cohesin subunits in the genome provide evidence for a conserved mechanism of sister chromatid cohesion common to all eukaryotes. The mutational analysis of the separase cleavage sites showed that one functional cleavage site is necessary and sufficient for normal progression of T. brucei
mitosis, a similar finding was made in yeast (Uhlmann et al., 2000
). Separase cleavage of SCC1 as a trigger for anaphase was first discovered in the budding yeast Saccharomyces cerevisiae
(Uhlmann et al., 1999
) and subsequently in metazoa (Hauf et al., 2001
; Jager et al., 2001
; Wirth et al., 2006
). Our data show that the mechanism whereby separase cleaves SCC1 to release chromosome cohesion evolved early in eukaryotic evolution, before the eugleonozoan and animal, fungal and plant lineages diverged, possibly almost two billion years ago (Hedges et al., 2004
Entry into mitosis and progression to metaphase is dependent on the activity of the cyclin-dependent kinase CDK1 in all eukaryotes studied to date. Progression from metaphase to anaphase is dependent on activation of the anaphase-promoting complex or cyclosome (APC/C). The APC/C is the E3 ubiquitin ligase that indirectly activates the protease separase by targeting its inhibitory subunit securin for degradation (de Gramont and Cohen-Fix, 2005
). Separase is then responsible for cleavage of SCC1, permitting anaphase, and independently initiates the release of the CDC14 phosphatase from the nucleolus (Shou et al., 1999
), which is necessary for mitotic exit and probably reverses the CDK1 phosphorylation. This simplified pathway was elucidated in yeast and, although all steps have not been confirmed in trypanosomes, orthologues of CDK1, cyclin and the APC/C have been identified and homologues of separase (Tb927.1.3120) (Mottram et al., 2003
) and CDC14 (Tb11.01.4270) are encoded in the trypanosomatid genomes. In T. brucei,
CYC6/CRK3 are the orthologues of cyclin B/CDK1. Depletion of CYC6 by RNAi results in a mitotic block; in procyclic forms, cell division occurs in the absence of nuclear division producing a zoid daughter cell whereas in bloodstream forms there is an accumulation of cells with a single enlarged nucleus and multiple kinetoplasts (Hammarton et al., 2003
; Tu and Wang, 2004
). Similar experiments with CRK3 produced a similar phenotype (Kumar and Wang, 2005
). The different phenotypes in procyclic and bloodstream forms led to the suggestion that regulation of cell cycle progression differs in the two life cycle stages and that a mitosis to cytokinesis checkpoint only operates in bloodstream forms (Ploubidou et al., 1999
; Hammarton et al., 2003
; Tu and Wang, 2005
). The depletion of APC/C activity through RNAi knockdown of CDC27 or APC1 resulted in cell populations enriched in G2/M cells; in procyclic forms cells arrested with two kinetoplasts and a single enlarged nucleus containing a short spindle whereas bloodstream-form cells arrested with two kinetoplasts and a nucleus arrested in late anaphase (Kumar and Wang, 2005
In procyclic forms, the phenotype of cells expressing separase-resistant SCC1 was similar to cells in which CRK3 or CYC6 had been depleted with the production of zoids resulting from cell division in the absence of nuclear division. The accumulation of zoids in cultures expressing separase-resistant SCC1 (40% after 24 h) was similar both to that obtained using the microtubule assembly inhibitor rhizoxin (30% zoids after 8 h) (Robinson et al., 1995
; Ploubidou et al., 1999
) and to that obtained after RNAi depletion of CYC6 (~40% after 48 h) (Hammarton et al., 2003
). In contrast, APC/C-depleted procyclic forms accumulated as 2K1N cells, the nucleus containing a short spindle consistent with an anaphase A arrest, but did not undergo cytokinesis.
Bloodstream-form cells expressing separase-resistant SCC1 had a different phenotype to CRK3- or CYC6-depleted cells. In both cases, cells arrested with an enlarged nucleus and multiple kinetoplasts but cells expressing separase-resistant SCC1 initiated cytokinesis whereas cells depleted of CRK3 and CYC6 did not. Depletion of APC/C activity caused a late anaphase arrest with an elongated spindle. The bloodstream-form phenotype observed in our study also differed markedly from the very specific precytokinesis cell cycle arrest observed when variant surface glycoprotein (VSG) transcripts were ablated by RNAi (Sheader et al., 2005
). VSG RNAi caused a rapid accumulation of 2K2N cells with two external flagella and, in a minority of cells, additional internal flagella. No internal flagella were observed in SCC1 mutants. Clearly, not all bloodstream-form cytokinesis defects are the same and specific phenotype patterns are now emerging.
Previous RNAi studies manipulated CDK1 (CYC6/CRK3) and APC/C activity; both are regulators of the cell cycle and knockdown will have pleiotropic effects; for example, a reduction of CDK1 activity will not only affect phosphorylation of its substrates but also the substrates of downstream kinases and knockdown of the APC/C components will reduce separase activation which will prevent cleavage of SCC1 but also block the role of separase in the activation of CDC14 and mitotic exit. In contrast, SCC1 is a structural component with a single role in sister chromatid cohesion and is a substrate for cell cycle regulators. Manipulation of SCC1 was used to reveal the phenotype resulting from defects in sister chromatid cohesion. RNAi knockdown of SCC1 prevented a normal mitosis and expression of the separase-resistant SCC1 allowed an analysis of the phenotype of an early anaphase block. In both of the life cycle stages investigated cytokinesis was initiated in the presence of separase-resistant SCC1 or after SCC1 knockdown. Thus, the initiation of cytokinesis is not dependent on the completion of mitosis and there is no checkpoint capable of blocking initiation of cell division in response to incomplete mitosis in either life cycle stage. In T. brucei procyclic forms, complete cytokinesis occurred in the absence of nuclear division whereas bloodstream forms arrested with stalled cleavage furrows.
Why is cytokinesis in bloodstream-form cells incomplete after expression of separase-resistant SCC1? The trypanosome cytokinesis machinery and mechanisms of its activation are unknown (Hammarton et al., 2007
) so it is possible in bloodstream forms that furrow ingression was blocked by a checkpoint mechanism in response to a signal indicating that mitosis had failed but this would be without precedent. Alternatively, the architecture of the bloodstream trypanosome may physically prevent cytokinesis in the SCC1 mutants. In procyclic trypanosomes, the position of the anterior nucleus remains fixed, through an as yet uncharacterized anchoring system, whereas the posterior nucleus ‘moves’ into the gap between the segregated basal bodies (Robinson et al., 1995
). As the kinetoplast is physically linked to the basal body, prior to cell division the order of organelles from the anterior end is alternating kinetoplasts and nuclei – KNKN (Robinson et al., 1995
). If the posterior daughter cell receives no nucleus then a 1K0N zoid and a 1K1N* cell (N* denotes a nucleus with replicated but non-segregated DNA) are produced by cell division, as observed when separase-resistant SCC1 was expressed. Prior to cell division, the relative positions of nuclei and kinetoplasts differ between bloodstream and procyclic forms; the order is KKNN in bloodstream forms and KNKN in procyclic forms (Tyler et al., 2001
). In cells expressing separase-resistant SCC1, the close apposition of the partial cleavage furrows with the nuclear membrane evident in the electron micrographs suggests that an undivided nucleus may form a physical barrier that prevents, or slows down, further cleavage furrow ingression. Thus, the different outcomes of separase-resistant SCC1 expression in the two life cycle stages investigated can be explained by the different geometry of organelle position in the two cell types.
The difference between the separase-resistant SCC1 dominant negative phenotype and the CYC6 and CRK3 knockdown phenotypes is informative. In procyclic forms, the phenotypes were similar whereas in bloodstream forms no initiation of cleavage was reported when CYC6 or CRK3 was depleted. One possible interpretation is that CYC6/CRK3 was required for initiation of cleavage in bloodstream forms, possibly through activation of the APC/C, but not in procyclic forms. However it would be worth confirming first the precise degree of CRK3 or CYC6 knockdown and whether there was any effect on APC/C activation. The absence of cell division in procyclic cells with depleted APC/C activity cannot be explained solely by a failure to licence SCC1 cleavage but suggests the APC/C activity may be required for initiation of cell division, possibly through its role in the activation of CDC14 as occurs in yeast. In bloodstream forms, depletion of APC/C led to a late anaphase arrest whereas expression of separase-resistant SCC1 resulted in an arrest in early anaphase with no elongation of the nucleus. The easiest explanation for these contrasting observations is that the depletion of APC/C was not complete but was insufficient to activate the mitotic exit network (Sullivan and Morgan, 2007
). In yeast mutation in mitotic exit network genes results in a late anaphase arrest (Jaspersen et al., 1998
The experiments presented here provide no evidence for checkpoints that link the completion of mitosis to cytokinesis initiation in the two T. brucei life cycle stages investigated. Organelle duplication proceeds in the absence of a successful mitosis as several rounds of basal body and kinetoplast duplication occurred in bloodstream forms although not in procyclic forms. Re-initiation of nuclear S-phase was not directly determined but the nuclei were enlarged in procyclic and bloodstream forms after expression of separase-resistant SCC1 suggesting endoreduplication was occurring.
The results of the experiments above show that in the two T. brucei
life cycle stages investigated, initiation of cytokinesis is independent of sister chromatid cohesion or cohesin release following SCC1 cleavage. The nuclear division defect caused by expressing non-cleavable TbSCC1 is reminiscent of the phenotypes of yeast and human cells that express non-cleavable SCC1. In S. cerevisiae
, cytokinesis was delayed but not inhibited and progeny with abnormal DNA content were produced (Uhlmann et al., 1999
). In human cells, cytokinesis was initiated but not completed and sister chromatid separation was shown not to be required for cyclin B destruction or mitotic exit (Hauf et al., 2001
). Depletion of TbSCC1 by RNAi in bloodstream forms led to slower proliferation without cell cycle arrest and partial ingression of cleavage furrows. In yeast and higher eukaryotes a spindle assembly checkpoint inhibits APC/C and prevents anaphase onset until tension generated by sister chromatid cohesion and bipolar attachment to the spindle microtubules is sensed. With the exception of Mad2p, no homologues of known spindle checkpoint proteins were found in the trypanosome genome (Berriman et al., 2005
). Progression of the cell cycle in the absence of SCC1 could indicate that such a checkpoint is absent or, alternatively, that an arrest is only transient.