The spatial organisation of Ca2+ signalling mechanisms in eukaryote flagella

纤毛和鞭毛是存在于许多细胞表面的鞭状附属物。从运动到感觉，它们在细胞生物学中扮演着许多重要的角色。然而，我们才刚刚开始了解这种细胞器的重要性。例如，我们越来越意识到，人类纤毛缺陷与一系列遗传性疾病有关，如支气管炎、不孕症和多囊肾病。鞭毛包含丰富的信号机制，尽管其中许多机制鲜为人知。这项建议旨在确定如何协调这些机制来调节复杂的运动性鞭毛反应。为了对环境做出快速反应，细胞必须对刺激产生细胞内信号。细胞利用细胞膜中的离子通道蛋白在胞浆中产生钙离子升高。钙离子在胞浆中缓冲良好，扩散缓慢，允许单个信使调节细胞内许多空间上不同的过程。我们知道，依赖于钙离子的信号过程对鞭毛的运动和感觉作用都是至关重要的。然而，我们对鞭毛中钙离子升高的性质知之甚少，例如它们持续多长时间，它们发生在哪里，以及导致它们产生的钙通道类型。对纤毛和鞭毛功能的大部分研究都是在一种小型单细胞绿藻衣藻中进行的。衣藻具有多种属性，是研究鞭毛功能的良好模型系统。然而，直到最近，我们还不能对鞭毛衣藻的钙动态进行成像。我们开发了一种新的技术，可以将对不同钙离子浓度响应的荧光染料引入衣藻，并首次使用高分辨率显微镜对这种藻类的鞭毛中的钙离子进行成像。三磷酸肌醇受体(IP3Rs)在哺乳动物细胞中是一种众所周知的钙离子通透通道，尽管它们在非动物生物中的作用(甚至它们的存在)还远不清楚。我们发现，哺乳动物IP3Rs的同源物在衣藻的鞭毛运动调节中起着重要作用。这项建议将使用衣藻中CrIP3R的详细研究，来告诉我们鞭毛和细胞质之间的信号是如何协调的，以及鞭毛内部钙离子信号机制的空间组织。首先，我们将确定衣藻单胞菌IP3R是否具有与其哺乳动物同源物相似的性质，以及它在细胞内的位置。我们有初步证据表明它是一种丰富的鞭毛蛋白，提示它可能直接作用于鞭毛的钙信号转导。其次，我们将研究衣藻鞭毛中钙离子升高的空间分布，并测试不同刺激诱导的钙升高是否局限于鞭毛的特定区域。我们在衣藻细胞中发现了CrIP3R基因敲除后的运动性缺陷，我们将探索钙信号通路的缺陷是否与此有关。我们将使用数学建模方法来研究在空间受限的鞭毛内空间内，钙离子高度是如何发展和扩散的。第三，我们使用新的高分辨率显微镜技术来研究鞭毛节拍和鞭毛波形变化过程中鞭毛钙离子的空间组织。最后，我们将检查鞭毛的结构完整性，看看CrIP3R基因敲除是否也会对鞭毛的组装产生影响。这项研究将告诉我们细胞用来协调空间不同区域之间的信号传递的机制。这一发现与鞭毛细胞的许多不同过程有关，从配子受精到纤毛功能障碍导致的许多人类遗传病。

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.