lower levels of microtubule acetylation modulating microtubule severing, remains to be seen. Glutamylation and tyrosination have been detected on both a and b tubulin [11,55]. Microtubules become detyrosinated over time, and in procyclic cells, there are cell cycle differences in the tyrosination status of microtubules, with cells in early stages of the cell cycle displaying the most tyrosinated microtubules, and cells around mitosis displaying the least [51]. However, little is currently known about other potential post-translational modifications of microtubules, such as cell cycle dependent phosphorylation, which could also play a role in regulating microtubule severing. A number of trypanosome cytoskeleton or microtubule-associated proteins (MAPs) [48–50,56–62] have been identified, and in the case of CAP15 and CAP17, shown to increase the stability of microtubules [48]. Depletion of the MAPs CAP5.5 and WCB leads to aberrant cytokinesis [50,56] due to resultant defects in cytoskeleton stability, structure or polarity and their downstream consequences; whether decreased cytoskeleton stability results directly from the loss of the MAP or indirectly due to the loss of the MAP increasing the availability of sites on the microtubules that can be utilised by microtubule severing enzymes is not clear at present. The apparent role of KAT80 in promoting furrow ingression is intriguing given that p80 katanins do not have any microtubule severing activity and are involved in microtubule severing only via their interactions with p60 katanin subunits. However, to date, despite repeated attempts, we have been unable to detect an interaction between KAT80 and KAT60b or KAT60c either in non-synchronised or hydroxyurea-synchronised cytokinesis cell extracts (Fig. S5 and data not shown). We cannot rule out that KAT80 interacts with KAT60b or KAT60c, perhaps very transiently during cytokinesis, or that it interacts with KAT60a, as tagged KAT60a proteins remained below the detection limit of Western blotting, precluding analysis of their interactions with KAT80. The function of the KAT60 proteins early in cytokinesis may occur independently of KAT80, which appears to be required later during furrow ingression, since katanin p60 subunits are known to be enzymatically active and functional in the absence of p80 katanin [24]. However, interaction of the KAT60 proteins with KAT80 later in cytokinesis could alter their function, since binding of the C. elegans p80 subunit, MEI-2, to p60 katanin (MEI- 1) was shown to dramatically change its microtubule binding properties [38]. A role for katanins in cytokinesis has not been widely reported. Studies of KAT80 and KAT60a in the related kinetoplastid, Leishmania major, suggest they may play a role in cytokinesis, since they relocalise to the cleavage furrow during cytokinesis [47], but to date, the only other evidence linking katanins with cytokinesis comes from studies of the LAPSER1 candidate tumour suppressor gene for prostate cancer in a rat cell line [63,64]. Overexpression of LAPSER1 results in defects in central spindle formation during cytokinesis that lead to the formation of binucleated cells. LAPSER1 binds to p80 katanin, inhibiting p60 katanin microtubule severing activity and p80 translocates in a LAPSER1-dependent manner from the centrosomes to the midbody during cytokinesis [63], and thus, the observed defects following LAPSER1 overexpression may be mediated, at least in part, through the inhibition of katanin. However, trypanosome mitosis and cytokinesis are highly divergent compared to mammalian cell division. For example, mitosis is closed, DNA does not condense, trypanosomes do not have centrosomes and as discussed earlier, the mechanics of cytokinesis are very different [5,6,14]. Additionally, while trypanosome daughter cells may remain joined at their posterior ends for some time Katanins and Spastin in Trypanosome Cytokinesis prior to abscission, a midbody-like structure has not been described. Thus, while it is unlikely that trypanosome katanins operate in an analogous manner to mammalian katanin, it appears that that T. brucei has exploited the microtubule severing activities of katanins to achieve efficient cytoskeleton cleavage during cell division. Katanins play a variety of other roles in other organisms, being required for regulating the length of meiotic and mitotic spindles [37,65,66], assembly and disassembly of flagella and cilia [35,52,67], and neuronal cell morphogenesis [40,68–70]. No role was noted for any of the katanins in mitosis or flagellar physiology in bloodstream trypanosomes in this study. An accumulation of 2N2K cells following RNAi of the katanins and lack of obvious defects in nucleus morphology, and the absence of spindles by TEM, suggests mitosis was unaffected by their depletion, although without conditional katanin knockout cell lines, a role for these proteins in mitosis cannot be completely excluded. It is also possible that the katanins are required for mitosis, but, unlike for their cytokinesis functions, there is a degree of redundancy between the proteins. However, our data suggest that mitosis in bloodstream T. brucei may not be regulated by the action of katanins. This is not without precedence, since katanins are not present in the mitotic spindle of Caenorhabditis elegans [71,72]. It is also known that the kinesin, TbKif13-1, regulates mitotic spindle length in T. brucei; its depletion results in the formation of abnormally long spindles during mitosis, while its overexpression leads to an early mitotic block and lack of spindle formation [73,74], and two other kinesin-like proteins, KINA and KINB also appear to be important for the establishment of the mitotic spindle [75], and hence, katanins may not be required for mitotic spindle formation. Detailed analyses of flagella were not performed during this study, but no defects in flagellar motility were observed, and all flagella cross- sections viewed by TEM for KAT80 and SPA RNAi cell lines appeared normal (Figs. S2, S4 and data not shown), suggesting that depletion of the katanins was unlikely to have caused gross structural defects, such as the loss of the central pair microtubules, which occurs in flagella following mutation of p80 katanin in Chlamydomonas [67] or in cilia after deletion of p60 katanin in Tetrahymena [52]. Our data also identify a role for spastin in abscission in bloodstream T. brucei, consistent with the known function of the human spastin 60 kDa isoform at the midbody restructuring microtubules in the intercellular bridge to help bring about abscission [42,45]. Despite being unable to localise spastin in this life cycle stage, its RNAi phenotype suggests that it is required for the final severing of microtubules at the posterior ends of the daughter cells. Spastin may play a different role in procyclic T. brucei since ectopically expressed GFP-tagged spastin localised to the nucleus and affected nuclear morphology, possibly suggesting a role in mitosis in this life cycle stage [47], although mislocalisation due to ectopic expression or the fluorescent tag cannot completely be ruled out. However, life cycle-specific functions for cell cycle regulators are common in T. brucei, and particularly in this case, may reflect differences in the composition of the cytoskeleton, based on differential expression of MAPs [48–50], and, as discussed above, potential differences in post-translational modi- fications, which may affect the binding of microtubule severing proteins to the cytoskeletal microtubules [27–29]. In summary, cytokinesis requires a family of katanins and spastin in bloodstream T. brucei, while mitosis may not. While the presence of multiple p60 katanin proteins is not without precedence (Drosophila, for example, has three: CG10229, which is most similar to human p60 and is essential for microtubule PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e30367