Structural mechanisms of centriole assembly during cell duplication

From embryo to adulthood and every day of our lives we rely on the normal growth and division of millions of cells. For each cell division our genetic material, packed in chromosomes, must also properly divide. In humans and animals this is achieved by an elaborate structure, called the nuclear spindle, organized by copies of the centrosome at each end of the cell. After division, each new cell has only one centrosome; therefore it must duplicate in order for further cell divisions to occur.
Centrosome duplication is a process for which we have relatively little information. We know that any abnormalities in this process can cause diseases or even cancer; however we do not know how this process is regulated, what is the role of each individual centrosomal component or how they assemble together. If we could understand how centrosome duplication takes place and how abnormalities can occur, then we could possibly devise treatments or new drugs. Therefore, there is much interest in understanding this process at the most detailed level possible.
We propose to use a combination of approaches, including cell biology, microscopy and biophysics to answer specific questions about the centrosome duplication process. We are interested in how their elaborate 9-fold symmetric shape is defined, and whether this is achieved by the same method in all organisms or whether there are significant differences. We would like to study how the cells control the start of the duplication process to ensure that it only happens once per cell division. Finally, we aim to find the correct place in the assembly for the various centrosomal components, similar to pieces in a puzzle.
The centrosome is a molecular machine, composed of many large proteins. To be able to study these components in sufficient detail, we first aim to find smaller, functional subunits, which we can analyze with biophysical tools. We can then make predictions for the properties of whole components based on these individual fragments, and test these predictions using electron microscopy and cell biology. Some important aspects of this work require expertise not present in our core group. Therefore we collaborate with internationally recognized groups in the UK and abroad to access the broadest base of expertise possible.

Centrioles are structures in eukaryotic cells with almost universal 9-fold symmetry. They are large, typically 400 nm by 150 nm in size, and contain symmetric sets of microtubules. Centrioles serve primarily two roles: near the cell membrane they organize flagella and cilia, and in animal cells a pair of centrioles forms the centrosome, the primary microtubule organizing centre. To maintain their numbers centrioles must duplicate once per cell cycle. Defects in this process are linked to medical conditions such as ciliopathies, male sterility or even cancer. As a result, there is widespread interest in understanding how centrioles duplicate, how this process is controlled and what determines their unique architecture.
A relatively small number of proteins, including SAS-6, SAS-5 and SAS-4 in C. elegans and their equivalents in higher organisms, was identified by genetic analysis as critical for the initial steps of the duplication process. The most conserved member of this group, SAS-6, was recently studied by biophysical and cell biology methods, and shown to form oligomers similar to early ultrastructures in centriole duplication. Here, we propose to study how SAS-6 is regulated, how it interacts with other centriolar components, and how these complexes recruit microtubules to the growing centriole.
We plan to use a dissection approach, whereby centriolar components will be initially studied at the level of individual domains. We will use biophysical methods (CD, fluorescence, AUC and ITC) to characterize these domains and their interactions, and crystallography and NMR to obtain high-resolution structures. Information from individual components will be used to create models of higher-order assemblies, which will be studied through EM. The functional significance of complexes observed will then be examined in C. elegans embryos or human cell lines through residue substitutions designed to interfere with the assembly process.

We expect our activities to have societal and economic impact through three distinct pathways: direct relevance to medical conditions, education and training of people, and outreach into the community.
Centrioles are important for human cells as they organize flagella, cilia, and the mitotic spindle during cell division. Aberrations in centriole structure or numbers can affect human health in a number of ways. Abnormal cilia formation can disrupt cell migration during development, giving rise to genetic diseases collectively known as ciliopathies. This is an emerging class of multi-sympton conditions that can affect liver, kidneys, gut and the respiratory track. In adults, epithelial cells and sperm also use cilia and flagella for function, thus defects in centriole duplication can directly lead to male sterility or ectopic pregnancies. In addition, centrioles, in the guise of the centrosome, organize the mitotic spindle and are thus necessary for correct chromosome segregation across cell divisions.
Our current understanding of the origins of many of these conditions is limited, since our knowledge of centriole structure is incomplete. Our research aims to provide a mechanistic view of how centrioles are put together, what are the structural and functional roles of their components, and how they are regulated. Eventually, our research may yield diagnostics and tools to control centriole duplication and structure. We may achieve this kind of control in a not too-distant timeframe: already our work on the structural role of SAS-6 in centriole assembly suggests a possible avenue of inhibiting centriole duplication by limiting oligomerization of SAS-6.
In addition to long-term considerations, academic basic research provides benefits through education and training. The PDRAs involved in this project will be trained in new techniques that will enhance their skills. Our collaboration with cell biologists and electron microscopists, as well as the multi-disciplinary nature of the project ensures their exposure to new ideas. In addition, they will be mentored towards independence, by giving them the chance of designing as well as implementing research, by allowing self-management and by encouraging their exposure to large audiences through presentations and conferences. Whether they choose to remain in academia or move to industry, this course should allow them to assume leadership positions in the future.
The PI is also keen to recruit postgraduate and undergraduate students to participate in this project, and he and the PDRAs will provide appropriate personal training and mentoring. For students, this project can offer exposure to a wide range of experimental methods and a corresponding increase in skills. At the same time care will be taken that the students develop their ability to frame scientific questions and pursue answers with all tools available, as well as troubleshoot when problems arise. In collaboration with other departments, Oxford Biochemistry offers a number of Ph.D. studentships funded by BBSRC, EPSRC and the Wellcome Trust. Furthermore, the department requires undergraduate students to complete a 6-month research project at their final year. The PI is active in student recruitment from both of these pools.
Finally, we recognize that we have a responsibility to reach out to the community and explain our goals and prospects. We will engage in outreach activities, such as open days and scientific blogs, aimed at making our research accessible to the public, explaining why this research is important, how it can contribute to healthcare, and why they should support basic research.