Elucidating the Cep135 - CPAP- STIL protein interaction network behind primary microcephaly and centriole formation

Primary microcephaly is a hereditary disease characterised by reduced brain size from birth and mental retardation. It occurs in ~1 in 10,000 individuals in some populations, but, importantly, it is one of few diseases where we can directly trace the effects of single mutations to brain development and cognitive functions. As a result primary microcephaly cases have proved instructive in identifying crucial brain development factors for which no backup systems exist.

Centrosomes, are small organelles in human cells that organise a network of thin filaments (known as microtubules) that are essential for cells to grow, duplicate, move and sense their surroundings. Defects in centrosomes have been implicated in microcephaly. Five out of nine genes known to cause the disease correspond to centrosome components, and these include three proteins known to be essential for the formation of these organelles. In addition to microcephaly, centrosomal defects are causative agents for multiple human medical conditions, including male sterility, ciliopathies and possibly cancer. Thus, understanding how centrosomes form is an important biological question with direct medical relevance.

Over the last few years our group, and others, have shown how a single protein, SAS-6, forms the initial framework onto which centrosomes are build. Crucial to this understanding was a combination of biophysical, structural and cell biology tools that allowed us to analyse the shape of essential proteins, envision how such proteins might join to form molecular machines, and test these insights in human cells. Here, we propose to build upon our understanding of the initial centrosomal framework by studying three protein components (Cep135, CPAP and STIL) that link to it. We have selected these components because they are essential for centrosomes, they appear to be connected to one another and to SAS-6, and importantly, they are all directly implicated in primary microcephaly. We believe that understanding the role of these three proteins will also inform us on how centrosomes are formed in normal cells and how defects in them cause severe diseases. We expect that these results will underpin future efforts on how to treat such diseases.

Our group has long experience in the biophysical and structural biology methods necessary for the pursuit of this project. However, we do not rely on our core competencies alone. Our goal of understanding the centrosome structure is shared with internationally acclaimed groups in Oxford and abroad, with whom we collaborate. Our network of laboratories provides the broadest possible base of technical expertise and, thus, the best hope for determining how centrosomes form.

Centrosomes are cell organelles necessary for division, motility and signalling in many species including humans. They organize the mitotic spindle, the microtubule network, cilia and flagella, and defects in their formation are linked to ciliopathies, male sterility or even cancer. Crucially, centrosomes are implicated in hereditary primary microcephaly, a bona fide genetic neurodevelopmental disorder. There, abnormalities in the timing and outcome of cell divisions by neuronal progenitor cells lead to reduced cerebral cortex size.

Centrosomes directly control crucial aspects of the cell division process, thus understanding how centrosomes, and their components, centrioles, form has attracted world-wide interest. Structural and functional studies have shown that a single protein, SAS-6, oligomerises into an initial assembly that defines the overall centriolar symmetry. However, we have relatively little mechanistic information on what follows SAS-6 oligomerisation. Here, we propose to study the essential proteins Cep135, CPAP and STIL. These proteins form an interaction network upstream of SAS-6, link to the initial SAS-6 framework and are implicated in primary microcephaly.

Our approach initially utilises protein dissection to resolve high-resolution structures (by crystallography or NMR) of individual domains and protein complexes. We then employ biophysical methods (including crosslinking assays and EM) to place the domain and complexes in the context of full-length proteins and of the whole interaction network. The structural and mechanistic insights gained in this manner will then be tested in human cells by collaborating laboratories, thereby checking the functional significance of our models.

We expect our activities in this project to have significant societal and economic impact in three distinct areas: (1) through direct relevance to medical conditions and improvements in human health, (2) by enhancing the education, training and career prospects of people and (3) by improving the public engagement with research.

1) Centrioles perform multiple functions in human cells including organizing the sperm flagellum, cilia in lungs, the sensory primary cilium and the mitotic spindle for all cell types. Primary microcephaly is emblematic of human diseases caused by defects in centriole formation, which also include male sterility, possibly cancer and ciliopathies. The later are an emerging class of genetic multi-symptom diseases that can affect the liver, kidneys, gut and the respiratory track. Our understanding of the molecular basis of these diseases is limited, since our knowledge of centriole formation is incomplete. Here, we aim to understand at a mechanistic level the function of centriolar proteins directly relevant to primary microcephaly, and centriole formation as a whole. We expect that the information gained could, in the future, be used by medical practitioners, for example in judging the severity of mutations identified in early embryos, and to industries aiming to provide pharmaceuticals for such diseases. Where relevant, IP protection will be sought via ISIS Innovation (a wholly owned subsidiary of the University of Oxford) to protect potentially sensitive information.

2) Basic research improves the career prospects of students and postdocs engaged in it through training and the acquisition of transferable skills. These are not limited to learning new techniques and a multi-disciplinary way of thinking; rather, they extend to mentoring people towards independence, allowing self-management and encouraging interaction and communication activities. Thus trained, researchers can engage productively in the wider knowledge economy, transfer their skills to sectors beyond academia, and assume leadership positions in their fields.

3) Increased public understanding of science is an important benefit to society as a whole. We recognize the responsibility of researchers to engage with the public and explain how expenditure in basic research underpins the economic advancement and well-being of society as a whole. In addition to creating publicly-accessible websites and authoring in blogs, our first target audience will be undergraduate students, the majority of whom radiate from academia to other sectors. By engaging undergraduates directly in our research as in point (2), and by using current research examples during teaching, we can increase their awareness, understanding and appreciation for science and its societal and economic impacts.