Using SBEM and cellular electron tomography to study the basal body/pro-basal body linker.

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 are the antenna of cells in Humans 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 great deal 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 Nagana in cattle, 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.

Basal bodies are small organelles in cells and electron microscopy is necessary to show us the structural complexity of this organelle. There have been recent developments in three dimensional microscopy which helps biologists to understand many aspects of the spatial organisation of cells. Two new techniques called serial block face scanning electron microscopy and array tomography are allowing biologists, for the first time, to reconstruct three dimensional views of individual cells and large areas of tissue such as synapse connections in the brain. Part of this grant to to work with these new techniques and develop ways in which we can gather and analyse large data sets.

1. An investigation of the ultrastructure and biogenesis of the transient connection between the mature basal body and pro-basal body, called the S-M linker will be carried out using cellular electron tomography on high pressure frozen intact cells and plunge freezing of isolated basal body complexes. The role of the cartwheel hub in this transient linker will be investigated and the spatial organisation of assembly and dissolution of the linker during the cell division cycle.

2. Development of serial block face scanning electron microscopy will include developing fixation protocols to increase contrast for auto-segmentation We will test zinc iodide/osmium tetroxide to stain selectively the nuclear envelope, endoplasmic reticulum, Golgi apparatus. Software to carry out this post-processing has been supplied on our BBSRC ALERT 13 grant. Development of a genetic tag(APEX) to locate proteins in this proposal by SBEM. Development of array tomography for use with APEX and immuno-EM using BB2 and GFP-tagged cell lines.

2. Three candidates have been chosen to further study due to their role in basal body biogenesis (PLK, SAS-4 and SAS-6). These genes are high conserved and have been shown to be essential for centriole/basal body duplication in a number of model organisms. Location and expression of each will be determined by replacement at one of the two alleles of a GFP-tagged version of each gene in order to use the endogenous promoter. Co-expression studies will be carried out to work out the temporal order of expression. Over-expression of each protein will be investigated using an inducible system which has been developed.

3. Inducible RNAi plasmids will be inserted into the cell lines above and will be assessed by studying changes in localisation and number of basal bodies during the cell division cycle. Cellular electron tomography will be used to investigate changes in the linker and SBEM to study in an individual whole cell context.

The wider scientific community will benefit from this research due to the development and use of these new technologies. We believe that serial block face scanning electron microscopy and array tomography will become relatively simple techniques for biologists to utilitise in all areas of cell and molecular biology and beyond and this project aims to demonstrate how it can be used in large scale analysis of mutant cell lines, producing quantitative three dimensional data. This new type of three dimensional microscopy is a real step change and we think it has the potential to influence future methodology in research in both academic and commercial research. Knowledge of the skills required to carry out these new techniques is low both nationally and internationally and this will give the research and technical staff on the project important research and professional skills, which will be useful for future employment within academic and commercial sectors.Knowledge of these techniques will also benefit the UK economy as it will aid our competitiveness in terms of helping to keep our scientists using and disseminating the latest cutting-edge techniques.
Beneficiaries of this research also include wider public in terms of health. Basal body and centriole research is important for our knowledge of a range of Human diseases called the ciliopathies, which includes retinal degeneration, polydactyly, cystic kidneys, Bardet-Biedl syndrome, Kartagener's syndrome. 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.