Bilateral BBSRC-SFI: Structure-function relationships in the ciliary transition zone

Cilia are small 'antennae-like' structures which protrude from the surface of most animal cells. Like antennae, they receive and transduce signals from other cells and their surroundings in order to coordinate appropriate cell behaviours. This is especially important in development, and defects in cilia lead to a range of human developmental diseases called "ciliopathies". These conditions range from severe, lethal conditions that involve complex defects in multiple organs including the brain, to relatively mild and organ-specific conditions such as retinitis pigmentosa, which is a form of hereditary, progressive sight loss. Scientists still do not fully understand how cilia help to control brain and retina development. Although these conditions are individually rare, collectively they are a common cause of morbidity and mortality in babies and young children but remain difficult to diagnose and treat.

This research proposes to identify genes that contribute individually, or within groups (pathways), to the formation of a sub-structure of the cilium called the transition zone. The transition zone at the base of the cilium is thought to function as a type of 'gate', controlling which signal transduction molecules are allowed to enter and exit the cilium. Many of the genes that cause ciliopathies are thought to function in the transition zone and regulate its 'gating' function. To achieve our aims, we will take advantage of recent exciting advances in genetic technology that allow us to evaluate the contribution of every gene to cilia and transition zone formation ("reverse genetics screen"). We are uniquely placed to do this work and the team of investigators have a proven track record of success in this field: we have formed excellent research partnerships with other workers in the field to participate in gene identification studies; we have the appropriate state-of-the-art technology, image analysis tools and experience; and we have already produced and validated large data-sets from existing work that we now wish to exploit more extensively in the present research proposal. We will study key genes ("screen hits") and their contributions to cilia and transition zone formation, and, in particular we will use specialised cell model systems in combination with a versatile animal model (a nematode roundworm).

The identification and characterisation of new genes required for the structure and function of the cilium and the transition zone 'gate' provides a number of major benefits. Firstly, important and often unexpected scientific insights are made into disease processes and into the normal function of the disease gene that can lead to new treatments. Secondly, new ciliary genes often enable accurate genetic testing for patients and families with ciliopathies, which improve diagnosis and genetic counselling. Thirdly, our proposed work has a wider biological relevance because cilia have recently been shown to control metabolism, which may link to neurodegenerative diseases and metabolic disorders such as insulin resistance. Thus, a better understanding of cilium biology may provide opportunities for developing drugs or new treatments to prevent the progression of more common ailments (e.g., diabetes. obesity) that present in some ciliopathy patients. Finally, we expect that our work will provide new insights into how cilia disease proteins are arranged relative to one another within the transition zone, and how this molecular organisation facilitates the gating function of this discrete region of the cilium. This new knowledge will have important implications for molecular 'gates' that exist elsewhere in the cell, and serve to expand the impact of our work to scientists working on related questions.

This research programme builds on the applicants' combined expertise in reverse genetics screening and in vivo modelling of transition zone (TZ) and ciliary function in cell and C. elegans animal models. The proposal impetus, and an example of our collaboration, was our 2015 study on TMEM107. Using high-throughput siRNA technology in mammalian cells (Workpackage 1; WP1) and C. elegans mutant alleles (WP2), we will identify novel candidate TZ genes. The resulting functional genomics data-sets will be integrated and common 'hits' validated and functionally interrogated in both systems (including knockout cell lines) to define new TZ proteins (WP3). Specifically, we will dissect their roles in cilium-based signalling, transport, composition, 'gating', and relationship to known or novel functional modules. In WP4, both models will integrate new and known TZ proteins into the emerging architectural framework of the ciliary gate, and provide new insights into structure-function relationships within the ciliary apparatus such as the TZ permeability barrier. These experiments will involve advanced confocal imaging to visualise protein-protein interactions in living cells (FRET; BioID; BiDC) and worms (BiFC), and complementary super-resolution microscopy approaches (STED, dSTORM), combined with multiplexed imaging using artificial binding proteins ("Adhirons"), to enable unprecedented spatial and temporal resolution of TZ proteins. We provide preliminary data on exemplar projects to show the exciting discoveries that rapidly arise from functional genomics to guide hypothesis generation. In vitro high content imaging and in vivo genetic complementation assays is a cost and time effective approach to complex combinatorial and multiplexed experimentation, compared to more conventional approaches. The research will significantly improve the functional annotation of genes contributing to TZ formation and ciliogenesis, and will enhance our understanding of basic cilium biology.

Although ciliopathies are a heterogenous group of rare inherited conditions, collectively they are a common cause of perinatal morbidity and mortality (1 in 500 births) and remain difficult to diagnose and treat. In the UK, about 4000 patients require renal replacement therapy (dialysis and transplantation) as a result of cystic kidney disease leading to kidney failure. The identification of new disease gene functions and pathways related to the ciliary transition zone (TZ) may enable the future rational design of preventative treatments or new therapeutic interventions for cystic kidney disease, retinal degeneration or ciliopathy disease progression, or approaches to improve the long-term outlook of patients with these conditions. For example, by understanding how the ciliary TZ and associated ciliopathy proteins regulate signalling via diffusion barriers, our work could lead to the design of small molecules that can advantageously increase or decrease permeability at the ciliary base, and thereby alleviate some of the progressive post-natal ciliopathy pathologies. Also, since these conditions result from absence of normal TZ proteins, they could in principle be corrected by gene replacement, therapeutic approaches now undergoing Phase III clinical trials.

It is likely that new TZ genes that we discover in this project will subsequently link to ciliary disease. Patients in whom new mutations are found can therefore be given a clearer prognosis and prioritised for any emerging new treatments, making inherited disease a top priority for further characterization. Recent advances have improved the molecular diagnosis of children with a known or suspected ciliopathy, and reduced the burden and disruption of a "diagnostic odyssey" for families. For clinicians, new gene identification can also reduce misdiagnosis or late diagnosis, inform the establishment of proper care pathways for ciliopathy patients, and prioritize patients that can most benefit from future targeted therapies. In families with a ciliopathy, the identification of a novel disease gene immediately permits genetic testing to at-risk relatives. Carrier tests and prenatal diagnosis can also encourage informed reproductive choices for families.

Recently, cilia have been shown to be essential for normal autophagic processes and have a broader role in metabolic control through mTOR signalling. This has wider medical relevance because autophagy is implicated in many human pathological processes, including neurodegenerative diseases and human metabolic disorders. The understanding of these pathophysiological processes poses some of the most significant challenges to modern biomedical research, and the regulatory relationships that control cilia function could therefore provide a new appreciation of their potential medical relevance in older age. The identification of new ciliary genes and pathways provides insights into normal human physiology and development, as well as the pathophysiology of common conditions (such as such as insulin resistance, obesity and cystic kidney disease) thus benefiting the wider population.

To understand ciliary TZ structure/function regulation, we will use functional genomics strategies to evaluate the contribution of large data-sets of genes. This is a proven strategy for identifying new and unexpected disease genes, mechanisms and pathways. We will integrate functional genomics with advanced cell biology methodologies in complementary physiologically-relevant cell and animal mutant models to derive cutting edge knowledge of the TZ system. Our work also aims to demonstrate the clinical utility and validity of integrated "systems medicine" annotation and its ability to make relevant predictions about ciliopathy disease causality. The development of a strong interface between gene discovery, functional annotation and diagnostic or translational development work is therefore an indirect but important added benefit of this research.