The spatial organisation of Ca2+ signalling mechanisms in eukaryote flagella

Cilia and flagella are whip-like appendages present on the surface of many cells. They play many important roles in cell biology from motility to sensory roles. However, we are only just beginning to understand the importance of this organelle. For example, we are becoming increasingly aware that defects in human cilia are associated with a whole range of genetic disorders such as bronchitis, infertility and polycystic kidney disease. Flagella contain an abundance of signalling mechanisms, although many of these mechanisms are poorly understood. This proposal aims to determine how these mechanisms are coordinated in the regulation of complex motile flagella responses. In order to respond rapidly to its environment, a cell must generate intracellular signals in response to a stimulus. Cells use ion channel proteins in their cellular membranes to generate Ca2+ elevations in the cytosol. Ca2+ is well buffered in the cytosol and diffuses slowly, allowing a single messenger to regulate many spatially distinct processes within the cell. We know that Ca2+-dependent signalling processes are central to both the motile and sensory roles of flagella. However, we know very little about the nature of Ca2+ elevations in flagella, e.g. how long they last, where they occur and the types of Ca2+ channel responsible for their generation. Much of the research into the function of cilia and flagella has been performed in a small unicellular green alga, Chlamydomonas. Chlamydomonas has many attributes which make it an excellent model system to study flagella function. However, until recently we were not able to image Ca2+ dynamics in Chlamydomomas flagella. We have developed a novel technique which allows us to introduce fluorescent dyes, which respond to differing Ca2+ concentrations, into Chlamydomonas and, using high resolution microscopy, image Ca2+ in the flagella of this alga for the first time. Inositol triphosphate receptors (IP3Rs) are a very well known type of Ca2+-permeable channel in mammalian cells, although their role (and even their presence) in non-animal organisms is far from clear. We have discovered that a homologue of mammalian IP3Rs plays an important role in regulating flagella motility in Chlamydomonas. This proposal will use a detailed examination of CrIP3R in Chlamydomonas, to inform us on the how signals are coordinated between the flagella and the cytosol and the spatial organisation of Ca2+ signalling mechanisms within flagella itself. Firstly, we will determine whether Chlamydomonas IP3R has similar properties to its mammalian homologues and also where it is found within the cell. We have preliminary evidence to indicate it is an abundant flagellar protein, suggesting that it may function directly in flagella Ca2+ signalling Secondly, we will examine the spatial distribution of Ca2+ elevations in Chlamydomonas flagella and test whether Ca2+ elevations induced by different stimuli are restricted to specific regions of the flagella. We have found motility defects in Chlamydomonas cells following gene knockdown of CrIP3R and we will explore whether defects in Ca2+ signalling are responsible. We will use mathematical modelling approaches to examine how Ca2+ elevations develop and diffuse within the spatially restricted intraflagellar space. Thirdly, we use novel high resolution microscopy techniques to examine the spatial organisation of flagellar Ca2+ during changes in flagellar beat and shifts in flagellar waveform. Finally, we will examine the structural integrity of the flagella to see whether gene knockdown of CrIP3R may also have effects on the assembly of flagella. The research will inform us on the mechanisms cells use to coordinate signalling between spatially distinct regions. The findings are of relevance to many different processes involving flagellated cells from gamete fertilisation through to the many human genetic disorders resulting from ciliary dysfunction.

Cilia and flagella play essential roles in many eukaryotes and defects in human cilia are associated with multiple disease states. Ca2+ dependent signalling mechanisms are central to cilia and flagella function and regulate both the motile and the sensory roles of this organelle. There is increasing evidence to suggest that a number of Ca2+ channels in flagella exhibit a non-uniform distribution, suggesting spatial specificity in flagella Ca2+ signalling. The genetic and biochemical approaches available in the model alga, Chlamydomonas, have enabled much progress to be made into the structure and function of flagella. We have developed novel imaging techniques to visualise flagella Ca2+ in the previously intractable Chlamydomonas system, at very high levels of spatial and temporal resolution. Furthermore, by using RNAi gene knock down we have determined that an inositol triphosphate receptor (IP3R) homologue, which localises to both the flagella and the cytosol, plays an important role in regulating Ca2+-dependent flagella motility in Chlamydomonas. CrIP3R RNAi strains exhibit reduced swimming speed and defects in cytosolic Ca2+ homeostasis. As CrIP3R exhibits a non-uniform distribution in the flagella, we will use a detailed study of the role of CrIP3R in flagella function to improve our understanding of the spatial organisation of flagella Ca2+ signalling and its interaction with the cytosol. We will determine whether CrIP3R acts to modulate cytosolic Ca2+ or flagella Ca2+ in the control of flagella beat frequency and will spatially map Ca2+ elevations in Chlamydomonas flagella to determine the functional signifcance of spatial specifcity. We will also determine whether CrIP3R plays a wider role in flagellar function through the regulation of flagella assembly. The research will provide a significant advance into our understanding of flagella Ca2+ signalling which has significant implications for both the motile and sensory roles of this organelle.

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 may be significant for understanding human genetic disorders relating to ciliary dysfunction. These disorders include infertility, defects in embryonic development and polycystic kidney disease where abnormal fluid sensing by kidney epithelial cells leads to proliferation and cyst formation in the kidneys. Polycystic kidney disiease 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. The research will clearly also inform us on the motility of flagellated organisms. As many organisms rely on motile gametes for their reproductive strategies, 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 on agriculture and aquaculture industries on factors associated with reproductive success. The results will also inform us on the diffusive behaviour of flagellated algae. This may have a number of applications for end-users, such as informing marine industries on the spread of harmful algal blooms in our coastal waters and informing scientists constructing algal bioreactors on the consequences of algal motility. Dissemination The research will be disseminated in a wide variety of media. Dissemination to the scientific community will be performed through publication in broad interest, high impact scientific journals and through presentation of the findings at national and international conferences The MBA is actively involved in the Oceans 2025 Knowledge Exchange Programme, involving the six partners of the UK Oceans 2025 Programme. This supports a range of activities ranging from scientific workshops to public outreach events and exhibitions. The proposed project will be represented at such events. In addition, the MBA hosts the internationally renowned workshops on 'Microelectrode Techniques for Cell Physiology', Optical Techniques for Cell Physiology and Developmental Biology' and 'Environmental Statistics (PRIMER)' in addition to a number of specialist Workshops on specific topics. These workshops provide an excellent venue for knowledge exchange between established scientists and young researchers. Wider dissemination of the project findings will occur through several mechanisms. Both organisations have active policies aimed at delivering science to a wide audience. For example, the MBA delivers regular updates on its science to all members of the MBA Society. The MBA has approximately 1200 international members, including academics, postgraduate and undergraduate students and interested members of the public. Further dissemination to the general public occurs through activities in the MBA's Public Understanding of Science programme including participation in the annual National Science Week exhibition of current research activities.