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。它们在细胞生物学中扮演着许多重要的角色，从运动到感觉。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疾病有关，如支气管炎、不孕症和多囊肾病。鞭毛含有丰富的信号机制，尽管其中许多机制尚不清楚。这一建议旨在确定这些机制是如何协调调节复杂的运动鞭毛反应。为了对环境做出快速反应，细胞必须产生胞内信号来响应刺激。细胞利用细胞膜中的离子通道蛋白在细胞质中产生Ca2+升高。Ca2+在细胞质中得到很好的缓冲，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Ca2+依赖的信号传导过程对鞭毛的运动和感觉作用都很重要。然而，我们对鞭毛中Ca2+升高的性质知之甚少，例如它们持续多长时间，发生在哪里以及负责它们产生的Ca2+通道的类型。许多关于纤毛和鞭毛功能的研究都是在一种小的单细胞绿藻衣藻中进行的。衣藻的许多特性使其成为研究鞭毛功能的一个很好的模型系统。然而，直到最近，我们还不能成像Ca2+动力学在鞭毛衣瘤。我们开发了一种新技术，使我们能够将荧光染料引入衣藻，该染料对不同的Ca2+浓度做出反应，并使用高分辨率显微镜首次在这种藻类的鞭毛中成像Ca2+。肌醇三磷酸受体（IP3Rs）是哺乳动物细胞中一种众所周知的Ca2+可渗透通道，尽管它们在非动物生物体中的作用（甚至它们的存在）还远远不清楚。我们已经发现哺乳动物IP3Rs的同源物在调节衣藻鞭毛运动中起重要作用。该提案将使用衣藻中CrIP3R的详细检查，以告知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Ca2+信号机制的空间组织。首先，我们将确定衣藻IP3R是否具有与其哺乳动物同源物相似的特性，以及它在细胞内的位置。我们有初步的证据表明它是一种丰富的鞭毛蛋白，表明它可能直接在鞭毛Ca2+信号传导中起作用。其次，我们将研究鞭毛衣藻Ca2+升高的空间分布，并测试不同刺激诱导的Ca2+升高是否局限于鞭毛的特定区域。我们已经发现了衣藻细胞在基因敲低CrIP3R后的运动性缺陷，我们将探索Ca2+信号缺陷是否与此有关。我们将使用数学建模方法来研究Ca2+升高如何在空间受限的鞭毛内空间内发展和扩散。第三，我们使用新的高分辨率显微镜技术来检查鞭毛Ca2+在鞭毛跳动和鞭毛波形变化过程中的空间组织。最后，我们将检查鞭毛的结构完整性，看看基因敲低CrIP3R是否也可能对鞭毛的组装产生影响。这项研究将告诉我们细胞在空间上不同的区域之间协调信号的机制。这些发现与鞭毛细胞从配子受精到由纤毛功能障碍引起的许多人类遗传疾病的许多不同过程有关。

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.