May, Sophie Frances
Research on regulation of cytokinesis in Trypanosoma brucei.
PhD thesis, University of Glasgow.
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Trypanosoma brucei is a protozoan parasite, and the causative agent of Human African Trypanosomiasis and Nagana in cattle. The life cycle and cell cycle of the parasite is complex and unusual. In particular, cytokinesis regulation in T. brucei is divergent, and significantly does not involve the formation of an actomyosin contractile ring; instead, a furrow ingresses longitudinally along the cell following the axis of the subpellicular microtubules which form a cytoskeletal cage around the cell body. As the organelles are positioned longitudinally in the posterior half of the cell, and in different positions according to life cycle stage, the cleavage axis is therefore subject to a number of constraints which must be overcome for symmetrical allocation of organelles to the daughter cells. It is highly likely that the subpellicular microtubule cytoskeleton plays important roles in cytokinesis furrow ingression. Presumably this process must involve microtubule and membrane remodelling at the site of the furrow apex to form the two discrete daughter cell bodies, and this is likely to require changes in microtubule dynamics, at least locally. Additionally, the cytoskeleton could influence the position of the cleavage plane by denoting polarity, or the timing of furrow initiation through mechanosensing.
The divergent nature of T. brucei cytokinesis implies that regulators of this process could be exploited as a source of potential novel drug targets. The Polo-like kinase, PLK, has previously been shown to be required for furrow ingression during cytokinesis in bloodstream form T. brucei. This study aimed to further our understanding of the regulation of cytokinesis by PLK by investigating how its activity is regulated in vitro and in vivo. In other organisms, PLK is known to influence microtubule dynamics, and given the likelihood that microtubule dynamics are important for furrow ingression in T. brucei, this study also aimed to investigate the role of the cytoskeleton in cytokinesis. An orthologue of a microtubule-associated protein required for cytokinesis in plants, AIR9, was functionally characterised in T. brucei, and the role of subpellicular microtubules in cytokinesis was investigated via the use of microtubule inhibitors.
Soluble and active recombinant PLK was purified from E. coli as a 6X Histidine fusion protein (6XHis:PLK). 6XHis:PLK autophosphorylated prolifically, and removal of these phosphorylations with lambda protein phosphatase significantly reduced the ability of 6XHis:PLK to transphosphorylate generic kinase substrates. Further, the importance of a conserved threonine residue (T198) in the T-loop of T. brucei PLK, which is a major site for regulation by upstream kinases in other organisms, was investigated. Substitution of T198 with a non-polar (alanine or valine) or a phosphomimetic (aspartic acid) residue revealed that this residue was important for PLK activity in vitro. However, expression of T198 variants in vivo showed T198V and T198D to be functional, suggesting that PLK activity is not regulated in vivo by phosphorylation at this site. The role of the polo box domain (PBD) of PLK in regulating PLK activity was also investigated. In PLKs from other organisms, the PBD autoinhibits PLK activity. Here, while recombinant 6XHis:PBD could pull down full length ty:PLK from T. brucei lysates, kinase assays indicated that the PBD did not inhibit the activity of full length PLK. Thus, regulation of the activity of PLK in T. brucei appears to be divergent.
Functional characterisation of T. brucei AIR9 by RNAi mediated depletion revealed roles for this protein in the relative positions of organelles and the position of the cleavage plane in both life cycle stages. However, the phenotypes observed during RNAi experiments differed between procyclic and bloodstream form parasites. For procyclic form parasites, the defective cytokinesis resulting in non-equivalent progeny seemed to occur in cells with organelle positioning defects suggesting that problems with cytokinesis were secondary to the major organelle positioning defect. Abnormal organelle positioning was far less frequent in bloodstream form parasites, which none the less seemed impaired in the accurate positioning of the cleavage axis.
AIR9 was shown to localise to the cytoskeleton of bloodstream and procyclic trypanosomes using two different epitope tagging approaches, and following induction of AIR9 RNAi, AIR9 was preferentially depleted from the posterior end of the cell, supporting a designation of AIR9 as a microtubule-associated protein. However, data do not support a role for AIR9 in cytoskeletal stability, since transmission electron microscopy showed the structure of cytoskeletal microtubules was not affected following depletion of AIR9. Hence, I suggest that AIR9 is more likely to be a scaffold protein potentially involved in the integration of signalling pathways to control cell polarity and cytokinesis.
This study also demonstrated through the application of inhibitors of microtubule dynamics that microtubule dynamics are important for cytokinesis in T. brucei. Vinca alkaloid treatment of bloodstream form parasites arrested furrow ingression, while taxol inhibited initiation of cytokinesis in bloodstream form parasites and affected cleavage plane positioning in procyclic cells. In addition, organelle positioning was inhibited through the application of the vinca alkaloid, vinblastine, to procyclic form cells. Thus, although these studies indicate that there are differences in the cytoskeleton makeup of bloodstream and procyclic trypanosomes, the data obtained are consistent with microtubule dynamics playing crucial roles in cell polarity and cytokinesis in both life cycle stages.
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