The spatial organisation of Ca2+ signalling mechanisms in eukaryote flagella

真核生物鞭毛中Ca2信号传导机制的空间组织

• 批准号: BB/H013814/1
• 负责人: Colin Brownlee
• 金额: $ 64.31万
• 依托单位: Marine Biological Association of the United Kingdom
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH013814%2F1
• 关键词: spatial; organisation; Ca2+; signalling; mechanisms

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.

纤毛和鞭毛是许多细胞表面的鞭状附属物。它们在细胞生物学中起着许多重要作用，从运动到感觉作用。然而，我们才刚刚开始了解这个细胞器的重要性。例如，我们越来越意识到，人类纤毛的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传导机制，尽管其中许多机制知之甚少。这项建议的目的是确定这些机制是如何协调复杂的能动鞭毛反应的调节。为了对其环境迅速作出反应，细胞必须产生细胞内信号以响应刺激。细胞利用其细胞膜中的离子通道蛋白在胞质溶胶中产生Ca2+升高。Ca2+在胞质溶胶中得到很好的缓冲，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是中央的鞭毛的运动和感觉的作用。然而，我们对鞭毛中Ca2+升高的性质知之甚少，例如它们持续多久，它们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型的单细胞绿色藻类衣原体中进行的。衣原体具有许多特性，使其成为研究鞭毛功能的极好模型系统。然而，直到最近，我们还不能在衣原体鞭毛中成像Ca2+动态。我们已经开发出一种新的技术，使我们能够引入荧光染料，响应不同的Ca2+浓度，到衣原体，并使用高分辨率显微镜，图像Ca2+在鞭毛的这一鞭毛的第一次。肌醇三磷酸受体（IP3R）是哺乳动物细胞中非常公知类型的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）远不清楚。我们发现哺乳动物IP3Rs的同源物在调节衣原体鞭毛运动中起着重要作用。该提案将使用衣原体中CrIP3R的详细检查，以告知我们鞭毛和胞质溶胶之间的信号是如何协调的，以及鞭毛本身内Ca2+信号传导机制的空间组织。首先，我们将确定衣原体IP3R是否与其哺乳动物同源物具有相似的特性，以及它在细胞内的位置。我们有初步的证据表明，它是一个丰富的鞭毛蛋白，这表明它可能直接在鞭毛Ca 2+信号的功能。其次，我们将研究的空间分布的Ca 2+海拔在衣原体鞭毛和测试是否由不同的刺激引起的Ca 2+海拔仅限于特定区域的鞭毛。我们已经发现了运动缺陷的衣原体细胞基因敲低CrIP3R，我们将探讨是否在Ca2+信号缺陷负责。我们将使用数学建模的方法来研究如何Ca2+海拔发展和扩散的空间限制内鞭毛空间。第三，我们使用新的高分辨率显微镜技术来研究鞭毛Ca2+的空间组织在鞭毛节拍和鞭毛波形的变化。最后，我们将检查鞭毛的结构完整性，看看CrIP3R的基因敲除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用于协调空间不同区域之间信号传导的机制。这些发现与涉及鞭毛细胞的许多不同过程有关，从配子受精到由纤毛功能障碍引起的许多人类遗传疾病。