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The role of ciliary Ca2+ signalling in the regulation of intraflagellar transport

Reference	BB/M02508X/1
Principal Investigator / Supervisor	Dr Glen Wheeler
Co-Investigators / Co-Supervisors	Professor Colin Brownlee
Institution	Marine Biological Association
Department	Marine Biology
Funding type	Research
Value (£)	416,779
Status	Completed
Type	Research Grant
Start date	01/10/2015
End date	30/09/2019
Duration	48 months

Abstract

Rationale: Eukaryote cilia play essential roles in cell biology, contributing to key developmental, reproductive and physiological pathways. The process of intraflagellar transport (IFT) directs the movement of ciliary proteins and is central to ciliary function. Whilst the importance of IFT is now becoming clear, the mechanisms regulating IFT are not well known. This must be addressed if we are to better understand the role of cilia in cell biology and human health. Approach: Using high resolution imaging techniques in the model alga Chlamydomonas, we have demonstrated for the first time that IFT is directly regulated by ciliary Ca2+ signalling. Chlamydomonas is currently the only system in which ciliary Ca2+ and IFT can be measured simultaneously. We propose to characterise the cellular mechanisms responsible in order to understand how Ca2+ acts to direct the movement of ciliary proteins. We will examine the nature of the ciliary Ca2+ elevations, to determine the spatiotemporal properties that enable regulation of IFT and the ion channels responsible. We will also identify ciliary components that are responsible for the Ca2+-sensitive interaction between the IFT particle and ciliary membrane proteins. Outputs: The research will provide mechanistic insight into a novel signalling process in eukaryotes that is likely to play a major role in ciliary function. The proposed research will also provide broader insight into how Ca2+ signalling may interact with microtubule motor proteins.

Summary

Cilia and flagella are tiny-hair-like projections from cells that play important roles in motility and in sensing changes in the cellular environment. Whilst we are familiar with their role in motility, the mechanisms cilia use to sense environmental stimuli and transmit this information to the rest of the cell are less clear. Cilia are built at the tip using a process known as intraflagellar transport (IFT), which enables proteins to be moved along the cilium to the site of assembly. It has also been shown that IFT plays an important role in ciliary signalling, as many important receptor proteins localise to cilia and are moved into and out of the cilium by IFT. Disruption of ciliary signalling due to defects in IFT can lead to human diseases and developmental problems, and it is therefore important for us to understand how intraflagellar transport is regulated. Using the motile green alga, Chlamydomonas, as a model system to study ciliary signalling, we recently discovered that IFT may be regulated by calcium signalling. Many environmental stimuli trigger ion channel proteins in cell membranes to open and cause a rapid influx of calcium ions (Ca2+) into cells. This results in elevated Ca2+ within the cell, which triggers various signalling cascades depending on the nature of the stimulus. Ca2+-dependent signalling processes are central to both the motile and sensory roles of cilia, but we know very little about the nature of these Ca2+ elevations and how they act to regulate ciliary processes. The discovery that Ca2+ signals are regulating IFT therefore links two very important processes in cilia and should help us understand much more about how these organelles sense and respond to their environment. We have used Chlamydomonas to develop a novel microscopy technique that allows us to simultaneously image Ca2+ and the movement of IFT particles in flagella for the first time. Chlamydomonas is currently the only organism in which this technique is possible and this unique ability will allow us to directly examine the mechanisms underlying this novel signalling process. Chlamydomonas can glide along solid substrates on its flagella by using IFT to move proteins in the flagella membrane. Gliding is coordinated by flagella Ca2+ signalling. Ca2+ elevations in one flagellum cause the IFT particles to dissociate from the flagella membrane and stop pulling the cell along. This gliding process is therefore an excellent model system in which to study how Ca2+ signalling regulates IFT to control the movement of flagella membrane proteins. Although we know that Ca2+ regulates IFT, we don't yet know how this happens. This proposal seeks to identify the specific cellular mechanisms responsible. Firstly, we will examine how Ca2+ signals are generated in Chlamydomonas flagella, looking at the ion channels responsible and at mechanisms that restrict Ca2+ elevations to individual flagella, to enable specific control of IFT during the regulation of gliding motility. We will then examine the different types of Ca2+ elevations that are used to regulate IFT, using mathematical models in combination with experimental data to help us understand the rapid changes in Ca2+ concentration inside the flagellum. Finally, we will look at how Ca2+ actually causes the IFT particles to dissociate from the flagella membrane, by identifying specific flagella proteins that may bind to Ca2+ and disrupt this interaction. The process of IFT is highly conserved amongst eukaryotes and it is likely that Ca2+-dependent regulation of IFT influences the movement of many ciliary proteins, including those involved in developmental signalling pathways relating to human genetic diseases. Therefore the results from our studies in algae will provide insight into how ciliary signalling is regulated in many different organisms, including mammals, and shed light on the many different roles cilia play in sensing and responding to the cellular environment.

Impact Summary

Economic and societal beneficiaries The research will be of significant benefit to the wider community as a whole. The biology of cilia and flagella is an exciting area of research and we are only just beginning to understand the complexity of these organelles. Moreover their presence on virtually every cell in the human body means the findings are likely to be significant for understanding human genetic disorders relating to ciliary dysfunction. Dysfunctional cilia are known to underlie a number of often chronically disabling and sometimes life-threatening genetic conditions. They affect multiple systems, causing blindness, deafness, chronic respiratory infections, kidney disease, heart disease, infertility, obesity and diabetes. Polycystic kidney disease is one the most prevalent monogenic human genetic disorders and the major cause of end stage renal failure. Moreover, one the most common genes associated with polycystic kidney disease is PKD2, a cilia localised Ca2+ channel, which highlights how important the proposed research will be to human health. Cilia and flagella are remarkably conserved allowing cross-species comparisons to be made. Indeed, discovery of homologue of a Chlamydomonas gene in mice, led to an understanding of how defects in ciliogenesis results in polycystic kidney disease. The identification of specific genes in Chlamydomonas may allow biotechnology and pharmaceutical companies to design drugs targeted to their human homologues which are aimed at restoring ciliary function. More widely, a broader understanding of ciliary signalling will provide insight into the biology of eukaryote organisms. Many organisms rely on motile gametes and ciliary signalling for their reproductive strategies and the findings are therefore relevant to researchers and industries associated with organisms as diverse as mammals and seaweeds. For example, the results may ultimately inform agriculture and aquaculture industries on factors associated with reproductive success. The findings may even contribute to methodologies for controlling reproduction of specific organisms by inhibiting ciliary signalling. In terms of humans, this could include novel contraceptives to disrupt sperm flagella signalling, which could play an important role in attempts to control world's current population explosion. End users Fundamental research such as this may ultimately be of benefit to many end-users beyond the academic beneficiaries. The most immediate end-users of this research are likely to be charities and patient support groups associated with cilia-related diseases (ciliopathies). In the UK, these include Alström Syndrome UK, Eurordis, Laurence-Moon-Bardet-Biedl Society, Polycystic Kidney Disease Charity, Primary Ciliary Dyskinesia Family Support Group, RP Fighting Blindness, Sense and there are more internationally. A registered charity has been formed, named the Ciliopathy Alliance, to bring together these UK patient support groups with researchers, doctors and health professionals working on ciliary disease. It is a major aim of this proposal to build a link between researchers such as ourselves working on fundamental aspects on ciliary research using model organisms with these more medically-orientated research groups and end users (described in Pathways to Impact). Development of UK skill base The staff involved in this project will be trained to high level in both experimental and computational techniques. These clearly are of benefit to the academic research sector, but if these researchers do not choose to follow an academic career path, these skills will contribute significantly to the UK skill base within both the private and public sectors. Recent alumini from our laboratory are using skills relating to this research area in both the public sector (e.g. Knowledge Exchange for a research institute), and the private sector (e.g. development of educational software and in the microscopy industry).

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Microbiology, Systems Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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