The spatial organisation of Ca2+ signalling mechanisms in eukaryote flagella

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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.