Three dimensional organisation and duplication of the eukaryotic basal body

Nearly all cells in the human body contain an organelle called a centriole or basal body which has multiple functions. It is important in organising the mitotic spindle that segregates chromosomes when cells divide. It assembles a structure called a cilium or flagellum that allow cells such as sperm cells to move and cilia are found in cells in the lining of the lungs where they aid the movement of liquids. Cilia and flagella also acts as the cells antenna to sense the external cellular environment. Most organisms contain cilia or flagella and they are important for locomotion in many diverse single celled organisms as well as allowing cells to attach to surfaces. There is a lot of interest in basal bodies because defects in the assembly of flagella and cilia and defects in duplication of basal bodies have been implicated in a range of human diseases that are collectively called the ciliopathies. These diseases include polycystic kidney disease, retinal degeneration, Kartagener's syndrome, Bardet-Biedl syndrome. These latter two diseases, for example are associated with a broad spectrum of pathologies, such as chronic bronchitis, male sterility, obesity and diabetes.This bewildering array of pathologies is due, in part, to the many functions of basal bodies in the human body. Parasitic organisms such as Trypanosoma brucei which causes African sleeping sickness and Trypanosoma cruzi, which causes Chagas disease in South America, as well as the malaria parasite Plasmodium all rely on the assembly and function of a flagellum and is a major virulence factor that is being investigated by researchers. It is clear that basal bodies are crucial for many different cells and our work is aimed at understanding how this organelle duplicates. Electron microscopy has shown us the structural complexity of this organelle. A technique called cellular electron tomography is now allowing us to visualise the three dimensional organisation of cells and this is really enhancing our understanding of many aspects of cell biology. Using this technique and other microscopy approaches we aim firstly to understand the three dimensional organisation of basal bodies in cells; secondly to map the order with which each component of the basal body duplicates and, thirdly investigate role of genes that are involved in basal body duplication in order to gain insights into how this duplication process is ordered and regulated.

1) We will produce a ultrastructural 'map' of basal body duplication, basal body re-orientation and segregation using cellular electron tomography and serial thin section analysis of high pressure frozen Trypanosoma brucei cells. 2) Three candidate genes have been chosen to further our knowledge of the regulatory factors in basal body biogenesis. A. Epsilon tubulin - Our preliminary data demonstrates that ablation by RNAi of epsilon tubulin results in defects in basal body biogenesis. We will use cellular electron tomography, serial section electron microscopy to determine precisely what ultrastructure has been assembled and how this relates to assembly of a wild-type basal body. Are there any dependency relationships? Have the partially assembed basal bodies re-orientated? Are there any connections assembled? Do they segregate? In collaboration with Prof. K Gull we will produce endogenous GFP tagging of epsilon tubulin and use a range of fluorescently labelled antibodies that we have previously used to provide cell cycle timings of basal body biogenesis. B. TbLRTP - Overexpression of TbLRTP prevents basal body duplication. We will determine the severity of the defect compared to epsilon tubulin RNAi cell line to discover any regulatory differences, depending on differences in assembly defects. RNAi ablation of TbLRTP leads to excess basal bodies that will be used to discover the maturation status of the excess basal bodies using the microscopy techniques above. How are they connected? How are they orientated? We will provide immuno-EM localisation. C. Centrin 1 & 2 - Connections between the basal bodies are poorly understood in both molecular terms and ultrastructural terms. Centrins are implicated in the organisation of some of these connections. We will study the ultrastructural defects in the RNAi cell lines and provide immuno-EM localisation to look for differences in localisation.

The beneficiaries of this research are the wider public in terms of health. Abnormalities in flagella and cilia are now implicated in a number of Human diseases called the ciliopathies, which includes retinal degeneration, polydactyly, cystic kidneys, Bardet-Biedl syndrome, Kartagener's syndrome. Knowledge of basal body biogenesis and assembly of a eukaryotic cilium or flagellum is critical to understanding the basis of these diseases. There is a need to understand the precise heirchary of basal body and cilium assembly in order to further our knowledge of how defects in these structures contribute to this wide spectrum of diseases. The model organism we are using to study basal body biogenesis is the protozoan parasite Trypanosoma brucei, which causes African sleeping sickness in Humans and Nagana in cattle. Over 70 million people in sub-Saharan Africa live where disease transmission can take place. The disease also affects domestic animals, particularly cattle and is recognised as a major obstacle to the economic development of the rural areas affected in Africa. The flagellum of this parasite is now recognised as a major virulence factor in the maintenance and spread of the parasite. Knowledge of how the flagellum is assembled, maintained and regulated as cells divide and differentiate through the life cycle is important to fully understanding how this parasite spreads. There are a number of ways in which we will engage with users and beneficiaries of this research. These include interacting with my collaborator who has contacts with clinicians working on ciliopathies. Send results of research to The Sanger Centre, Cambridge where curation of parasite genomes takes place; attending relevant national and international conferences to disseminate results and publishing in a timely manner. Interacting with the wider public using the excellent links that have been established at Oxford Brookes, including regular events such as 'How science works', the National Science and Engineering Week, and demonstrating the uses of electron microscopy at various outreach programmes. The PDRA working on the proposed project will receive full and relevant training. The technique of cellular electron tomography is now recognised as an important technique in the field of structural cell biology, but the number of trained staff nationally and internationally is still relatively low. The Principle Investigator has received extensive training from an internationally recognised centre of excellence at the US National Centre for Research Resources, Boulder laboratory for 3D electron microscopy at the Unversity of Colorado, USA. I will disseminate this knowledge to the PDRA working on the project, to collaborators and staff at Oxford Brookes.