Chromosome segregation machinery in a basal eukaryotic system

When cells grow and divide they must partition their genetic material equally between the two new daughter cells. This is essential if the new cells are to inherit the genes necessary to grow and divide. In non-bacterial organisms, division is more complex because the genes are packaged up onto several discrete chromosomes and one copy of each chromosome must be inherited by each of the cell's progeny. This requires an intricate mechanism whereby chromosomes are attached to a specific division apparatus which then separates copies into the daughter cells. Attachment of the chromosomes to the division apparatus occurs via a specialised region of DNA, around which a complex molecular machine is built. Much work has been done in a few 'model' organisms to describe the components of this machine and understand how it works. As a result, a good paradigm exists for the workings of division in simple yeast cells, and some of the features of this machinery are the same or similar in human cells. However, for many important non-bacterial organisms, this model of cell division does not work - either their division looks different from the paradigm or key components are missing from the system. This implies that we do not yet fully understand the fundamentals of the system or the way in which the system evolved.
African trypanosomes are single-celled parasites of the blood which are spread by the bite of tsetse flies. The parasite causes a tropical disease known as "sleeping sickness", which kills ~50 000 people annually in sub-Saharan regions. A related disease of cattle, n'gana, also has a major detrimental impact on the economy of rural areas. African trypanosomes are unusual parasites, in that they multiply in the blood in full view of the body's defence systems. They do this by periodically changing their cell surface to escape the host's immune response. This process is so important to the parasite that they dedicate a large proportion of their genome to it, including very many small specialised chromosomes that exist solely for this process. When the parasite divides, one copy of each of these chromosomes has to be partitioned into each of the two daughter cells, but very little is known about how this is achieved. What is clear is that it must occur in a manner different from our current models of chromosome segregation - and that many key components seen in other systems are not present in trypanosomes.
African trypanosomes and their relatives are also unusual in another way: their lineage branched from the rest of non-bacterial life very early in evolution and they may even have evolved from some of the first non-bacterial life ever to have existed. This means that studying chromosome segregation in these organisms not only illuminates a novel mechanism for the process, but tells us about how the system evolved in organisms such as humans. I aim to identify the proteins that make up the machinery that is necessary for the stable inheritance of chromosomes in African trypanosomes. Because trypanosomes are so different from human and cattle cells, this information might be exploitable in the future for the design of drugs. Conversely, the parts of this machinery that are shared between trypanosomes and other cells must be extremely ancient and will inform us about some of the fundamental processes/components which are necessary to make a non-bacterial cell.

At mitosis, eukaryotic genomes are partitioned equally between daughter cells by attachment of individual chromosomes to the mitotic spindle. Extensive work in a few model systems has led to a paradigm wherein the centromere of each chromosome forms the base for a complex multi-protein machine, the kinetochore, into which microtubule ends are embedded. However, not all eukaryotes conform to this paradigm and kinetochore components are not ubiquitously conserved.
The protozoan parasite Trypanosoma brucei is a member of the Euglenozoa, a lineage that diverged very early in eukaryotic evolution (perhaps even at the very first branch). The organism parasitises the bloodstream in an exclusively extracellular form - a feat that is achieved by manipulation of the humoral response through the expression of a series of immunologically-distinct cell surface coats. ~20% of the genome is dedicated to this process. This includes >100 small specialised chromosomes that are intimately linked to antigenic variation. In spite of being so numerous, the small chromosomes are segregated with great fidelity at mitosis. However, the small chromosomes exist in such abundance that their interaction with the spindle cannot be a canonical one. Moreover, almost none of the kinetochore components that are essential in model systems are encoded in the trypanosome genome.
This project aims to identify the machinery necessary for the mitotic segregation of trypanosome chromosomes. Complementary approaches will be used to identify components of the machinery on the basis of: 1) deep homology to models, 2) interactions with known components or chromosomes, and 3) screening of libraries of RNAi mutants. Putative components will be validated by localisation and RNAi and used to isolate other components in the same pathway. Finally, identified components will be used in comparative genomic analyses to investigate the evolution of the system across eukaryotes.

The work in this project will identify the molecular components of a fundamental biological process in a parasitic organism that breaks the system paradigms. The research is pure - concentrating on discovery of the basic principles of the system - rather than focused on a specific downstream application. Nonetheless, the work is predicted to have a strong potential for wide and significant impact and a large number of potential beneficiaries.
The anticipated impact can be divided into two general strands. First, the work will identify protein components of an essential system in an organism of both medical and veterinary importance. It is already clear that these components will be very different from those of the host organism, and disrupting the system is known to be lethal. Therefore, there is a potential in the work to discover parasite-specific proteins that might be druggable. Any data regarding such molecules will be passed directly to the TDR Target Database pipeline for further exploitation. Such discoveries could lead to great impact on the health and economic development of some of the poorest regions in the world in sub-Saharan Africa, where diseases in man and cattle caused by the parasite are prevalent. Moreover, since large parts of the system are expected to be conserved in related parasites, such as Trypanosoma evansi, Trypanosoma cruzi, and Leishmania spp., and possibly also in other protozoa such as Giardia lamblia, there is potential for results from this project to positively impact work on a number of other important disease-causing eukaryotes.
Second, there is potential for a much more general advance in our understanding of the evolution of a fundamental eukaryotic process as a result of the work. Directly or indirectly this would impact on large swathes of cell biological research. If, as is expected, components of the system of mitosis in trypanosomes are conserved in other systems which have yet to be fully described, this would directly inform and stimulate research into these other systems, which obviously include many organisms of medical, veterinary, and/or economic relevance. Alternatively, should the system of trypanosome mitosis be entirely restricted to this lineage, this would lend support to hypothesis that this lineage is the product of the very first branch of the tree of extant eukaryotic life.
As with much pure research, the immediate beneficiaries of both impact strands of this project are likely to be in academic settings, or at the academic/commercial boundary. The impact on this community will be within or just following the lifetime of the project. However, in the longer term it is envisioned that a wider group will benefit from the work. Although it is difficult to predict precisely who these beneficiaries will be before the work is completed, it can readily be seen that a greater understanding of the evolution of mitosis across eukaryotes has potential beneficiaries outside of academia, particularly in industrial or agricultural settings where the division of algae or protozoa are important to productivity.