CILIA MEDIATED MECHANISM OF OTOLITH MINERALIZATION

Cilia are finger-like cell surface protrusions that function in the detection and processing of an extraordinary variety of signals. In humans and other vertebrates, cilia are present on the surface of nearly every cell. They are intimately involved in vision, olfaction, early embryonic development, morphogenesis and physiology of duct epithelia, cell motility and metabolism. Cilia defects result in abnormalities ranging from limb malformation to blindness, lack of the sense of smell, obesity, infertility and lung infections.

In humans and other vertebrates alike, the sense of balance is mediated by mineralized round stone-like structures, known as otoliths or otoconia. These structures are intimately associated with cilia of mechanosensitive cells in the ear. We have recently found that mutations in an important ciliary protein lead to otolith mineralization defects, so that otoliths mineralize in an abnormal way, forming aberrant crystal structure. We also found that protein content of otoliths is abnormal in mutant animals. These defects lead to a hyperactive behaviour. Similar human cilia defects may lead to balance disorders and the sensation of "ringing in the ear", medically known as "tinnitus". Such abnormalities are especially common in older people.

This project has several goals. First, we will determine morphological and structural defects of cilia that may account for their abnormal function in otolith crystalization. In parallel, we will search for related cilia genes that may affect otoliths. Since otoliths form outside tissues and cells in the fluid-filled chambers of the ear, a question that needs to be solved is how cilia, which are cell surface features, affect such an extracellular structure. This is likely to involve protein secretion from cells into the ear lumen. We will thus determine which cells in the ear produce and secrete otolithic proteins. We will also establish using biochemical tests how proteins that contribute to otolith structure interact with one another. Finally, we have evidence that otolith defects become more severe with age. We will characterize these age-related changes by studying older animals.

These studies will reveal an essential cilia-dependent mineralization mechanism, determine its molecular components, and evaluate its malfunction in the course of ageing. This mechanism is likely to deteriorate in human population among older people, leading to balance disorders and associated injuries. Our studies will potentially reveal ways to alleviate age-related malfunction of otolith biomineralization.

In humans and other vertebrates, the sense of balance is mediated by mineralized round stone-like structures, which form in the ear lumen and are known as otoliths or otoconia. Physically, otoliths form in the close proximity of cilia, suggesting that cilia may play a role in otolith formation. Here we combine genetic and biochemical approaches to investigate the mechanism of cilia involvement in otolith biomineralization.

Our experiments will be performed using an animal model, the zebrafish. We will use CRISPR-mediated mutagenesis to generate mutations in genes that affect otolith biomineralization and to engineer knock-in tags in some of these genes. The tagging of genes and consequently proteins expressed by these genes will allow us to determine where these proteins localize in the ear on the cellular and subcellular level. This will suggest how they affect secretion into the ear lumen. Some of the proteins that we will study are themselves secreted into the ear lumen where they contribute to otolith mineralization. We will determine in which cells and in which subcellular compartment these proteins are expressed.

In parallel, we will use biochemical approaches, such as crosslinking followed by mass spectrometry, to determine how otolithic proteins interact with one another. This will advance the understanding of how the otolithic matrix forms and how it regulates crystallization of inorganic salts, such as calcium carbonate, that constitute the major component of the otolith structure.

Finally, our observations suggest that otolith abnormalities become more severe with age. We will evaluate this by monitoring otolith formation in ageing animals. Combined together, these studies will reveal a key secretory pathway that functions to assemble proteinaceous matrix necessary for biomineralization of otoliths and perhaps other hard tissues.

- Advances in Basic Science
This proposal aims at increasing the fundamental understanding of mechanisms of cilia function. Cilia are now recognised to play a central role in many processes in vertebrate organs, including signal transduction and regulating fluid flow. Research outlined in this project will focus on novel roles of cilia in biomineralization and protein secretion. We will advance the understanding of these hitherto poorly researched cilia-mediated processes that may be nonetheless of key importance for development and function of many organs.

Primary beneficiaries will be researchers with interests in cilia formation and function, currently a very productive area of research that receives a lot of attention in the scientific press, and involves a broad range of scientists, including cell biologists, human and animal geneticists, biochemists, and biophysicists. Our studies will also benefit cell biologists focusing on secretory mechanisms and bioengineers interested in the use of inorganic compounds for the purpose of engineering implants and for tissue reconstruction.

- Medical Impact
Aging of populations is an acute problem in the western world. Our research will help the medical community to diagnose and treat balance disorders that are frequently associated with older age. It will advance the understanding of age-associated changes in human vestibular system and lead to the improvement of methods that are used to alleviate them, thus potentially extending human healthspan and improving the ability of older people to stay in the work force and to lead productive lives. As ageing-associated degenerative changes affect everyone, this aspect of our research will have a particularly broad impact.

Balance and hearing disorders are not restricted to older people. Our studies will thus also impact the understanding of these abnormalities in young people and children, thus improving their quality of life and preventing accidents, such as falls, due to vestibular system defects.

- Commercial opportunities
The improved understanding of age-related changes in the vestibular system will enhance the development of treatment methods by pharmaceutical industry. For example, our studies will reveal secretory mechanisms that may be manipulated pharmacologically to affect otolith biomineralization. The analysis of otolith matrix components that we propose to perform is likely to find industrial applications as it may be used to enhance the quality of scaffolds for neural and dermal tissue engineering as well as bone and cartilage reconstruction.

- Training Opportunities and Collaborations
Postdoctoral scientists, masters and doctoral students, and undergraduate summer students in our laboratory will benefit from research opportunities generated by this project. They will acquire expertise in the use of genetic, proteomic, and imaging approaches, and will gain technical knowledge that will advance their professional development. The broader research community in Sheffield, with interests ranging from basic cell biology to engineering, will benefit from the development of new reagents and intellectual interactions with our research group. Our laboratory has developed a number of collaborations in Sheffield, Yorkshire, and internationally. The funding of this project will allow these collaborations to flourish.

- Public engagement
We will interact with the public to improve the awareness of the most pressing biomedical problems of our time, such as ageing of the human population. We will highlight the importance of biomedical research in trying to address these problems through the use of parallel approaches, such as proteomic analysis, imaging, and studies of animal models.