PHOSPHOINOSITIDES IN CILIA FUNCTION AND CILIOPATHIES

Lead Research Organisation: University of Sheffield
Department Name: Biomedical Science

Abstract

Phosphoinositides (hereafter PIPs) are fat-related compounds that reside in cell membranes and function as signals in intracellular processes. For example, PIP-mediated signaling directs transport of proteins within the cell. It also contributes to changes in cell shape, directs cell migration, and mediates the detection of environmental signals by cells. We are particularly interested in PIPs which function in cell surface protrusions known as cilia. One of the main functions of these protrusions is to detect environmental stimuli, such as light, chemicals, and mechanical pressure. The role of PIPs in cilia is poorly understood.

The purpose of this project is to determine the identity and function of PIPs in cilia. As the first step, we will determine which PIPs are present in cilia by using molecules that detect PIPs inside the cell. We call these molecules PIP sensors. There are seven PIP species in the cell, and we would like to determine which of these are enriched in cilia.

Once we know which PIPs are found in cilia, we will determine their function by removing specific PIPs from cilia and monitoring consequences of their absence. Alternatively, we will test the function of specific PIPs by increasing their content in cilia. Again, we will monitor the consequences of such an increase. To monitor the function of ciliary PIPs in as many tissues as possible, we will perform these tests in intact embryos of zebrafish. We chose the zebrafish for this purpose as this organism is easy to manipulate genetically. Its embryos are also easy to observe as they develop externally outside the maternal organism.

We expect that loss or excess of PIPs may have profound consequences on embryonic development. This is because the absence of some PIP metabolizing enzymes causes dramatic abnormalities, such as the absence of eyes, the appearance additional fingers or toes, and brain malformations. Some of these abnormalities are found in human diseases known as ciliopathies. To determine the causes of these diseases, we will use our sensors to monitor PIP changes in embryos mutant for disease genes. We will also attempt to rescue mutant embryo defects by compensating for changes of ciliary PIP content. This may prove to be a viable strategy as a cure for human ciliopathies.

Finally, to improve PIP detection in cells, we will construct a new kind of PIP sensors. Cilia are small compared to other parts of the cell and detecting PIPs in their membranes is difficult. The use of a new sensor design will improve sensitivity of PIP detection and will make is possible to monitor small changes in PIP content. Combined together, these studies will generate important insights into the mechanisms of cilia function in processes as diverse as vision, olfaction, embryonic development, and metabolism. Our studies will also help to understand how ciliary PIP changes contribute to human disease.

Technical Summary

To determine phosphoinositide (hereafter PIP) content in ciliary and periciliary membranes, we will express PIP sensors in living zebrafish embryos. These sensors consist of PIP binding pleckstrin homology (PH) domains fused to a fluorescent tag, such as GFP. We chose PH domains because they are small and their binding specificities are well defined. Although such sensors have been used in tissue culture, their uses in intact organisms have limited history. By imaging the embryo, we will determine PIP content in physiologically relevant conditions at several stages of embryogenesis in a broad range of tissues.

In parallel, we will test the function of several PIPs by either down- or up-regulating their ciliary content. To do that, we will take advantage of the ability to efficiently and specifically target exogenous proteins to cilia. Using this approach, we will target to cilia PIP kinases and phosphatases of well-characterized specificities. These enzymes will either decrease or increase the concentration of specific PIPs in the cilium.

Because cilia are small, imaging PIPs in cilia requires particularly sensitive tools. To improve sensitivity of PIP detection in cilia, we designed a sensor that is initially actively targeted to cilia via small molecule-mediated dimerization with a ciliary protein. Once the dimerising agent is removed, sensor retention rate in cilia is indicative of PIP content.

Numerous human ciliopathy genes are likely to affect ciliary PIP content. We will use PIP sensors to determine PIP content alterations in zebrafish mutants that model human ciliopathies. Moreover, we will manipulate ciliary PIP content to alleviate pathologies in such mutants.

This project will enhance our understanding of ciliary function in normal development and physiology and reveal pathological processes that contribute to human disease. We will also generate tools broadly applicable to study cell biology of many cilia-related processes.

Planned Impact

Mutations in ciliary phosphoinositide-related genes cause human disorders. Abnormalities associated with these ciliopathies include blindness, cystic kidneys, polydactyly, infertility, obesity and mental retardation, as is the case in the Bardet-Biedl syndrome. These pathologies have dramatically negative effects on the quality of life. The primary beneficiaries of this project will be the diseased individuals affected by phosphoinositide-related ciliopathies. For some of them, new treatment approaches will improve life quality while in other cases new therapies may be life-saving. The benefits of this project will not materialize overnight as the path from understanding the function of a disease-causing gene to a treatment takes, in the best case, several years and frequently much longer. We do n o t have, however, a more reliable way to combat diseases than to determine their causes at the molecular level.

Research into molecular mechanisms underlying human disorders has already produced enormous benefits in the treatment of such diseases as cystic fibrosis, cancer, and recently certain forms of blindness. In the case of cystic fibrosis, information about the molecular nature of the CFTR channel was of paramount importance for the development of therapeutics, such as Ivacaftor, which benefit patients with specific CFTR mutations. A therapy of this type would not be possible without detailed knowledge of the molecular mechanism that is affected by this disease. Similarly, the studies of ciliary phosphoinositides outlined in this proposal will make it possible to devise therapies targeted towards specific proteins or pathways, phosphoinositide metabolism in cilia in particular.

Insights into the mechanisms of ciliary diseases that we will generate will benefit pharmaceutical companies as these will be able to capitalize on our findings to develop new therapies. We will promote our findings, via the commercialization team at the UoS, to the pharmaceutical industry to ensure effective applications of our work. As new therapies are frequently very profitable, this will contribute to the development of the pharmaceutical industry, create new jobs and contribute to the material prosperity of the society.

Research on ciliary diseases will train postdoctoral fellows and students, who will become aware of the importance of cilia-related mechanisms in the treatment of human disorders and will develop technical and conceptual skills necessary to advance this discipline and associated practical applications to the new level. Some of them will continue to study ciliogenesis as independent investigators, while others will join pharmaceutical companies and contribute to the development of therapies for ciliopathies and other genetic disorders. The researchers will also gain transferrable skills such as project management, problem solving, written and oral communication. They will also be aware of the benefits of the use of non-mammalian animal models in research and will be able to communicate this to the wider community. During the course of the project they will become better equipped to participate in science outreach activities.

Finally, our research will generate conceptual advances that will benefit researchers with interests in cilia formation and function, currently a very broad and productive area of biology that receives a lot of attention in high impact scientific journals, and involves a wide range of scientists, including cell and developmental biologists, human and animal geneticists, biochemists, imaging experts, structural biologists, and chemists.
 
Title transgenic lines 
Description We generted a number of transgenic lines (>10) to manipulate phosphoinositide content in cilia. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact revealed new functions of phosphoinositides