The role of ciliary Ca2+ signalling in the regulation of intraflagellar transport

纤毛和鞭毛是细胞的微小毛发状突起，在细胞运动和感知细胞环境变化中起重要作用。虽然我们熟悉纤毛在运动中的作用，但纤毛用来感知环境刺激并将信息传递给细胞其他部分的机制却不太清楚。纤毛是通过一种被称为鞭毛内运输（IFT）的过程在尖端形成的，该过程使蛋白质能够沿着纤毛移动到组装地点。研究还表明，IFT在纤毛信号传导中起着重要作用，因为许多重要的受体蛋白定位于纤毛，并通过IFT进出纤毛。由于IFT缺陷导致纤毛信号的中断可导致人类疾病和发育问题，因此了解纤毛内运输是如何调节的对我们来说很重要。我们利用活动绿藻衣藻作为模型系统研究纤毛信号，最近发现IFT可能受钙信号的调控。许多环境刺激触发细胞膜中的离子通道蛋白打开并导致钙离子（Ca2+）快速流入细胞。这导致细胞内Ca2+升高，根据刺激的性质触发各种信号级联反应。Ca2+依赖的信号传导过程是纤毛运动和感觉作用的核心，但我们对这些Ca2+升高的性质以及它们如何调节纤毛过程知之甚少。因此，Ca2+信号调节IFT的发现将纤毛中的两个非常重要的过程联系起来，并应该帮助我们更多地了解这些细胞器如何感知和响应它们的环境。我们已经使用衣藻开发了一种新的显微镜技术，使我们能够同时成像Ca2+和鞭毛中IFT颗粒的运动。衣藻是目前唯一一种可以使用这种技术的生物，这种独特的能力将使我们能够直接研究这种新型信号传导过程的机制。衣藻可以利用IFT移动鞭毛膜上的蛋白质，沿着鞭毛上的固体底物滑动。滑行是由鞭毛Ca2+信号协调的。一个鞭毛中的Ca2+升高导致IFT颗粒从鞭毛膜上分离并停止拉动细胞。因此，这种滑动过程是一个很好的模型系统，研究Ca2+信号如何调节IFT来控制鞭毛膜蛋白的运动。虽然我们知道Ca2+调节IFT，但我们还不知道这是如何发生的。这一建议旨在确定具体的细胞机制负责。首先，我们将研究Ca2+信号是如何在鞭毛衣藻中产生的，研究离子通道和限制Ca2+在单个鞭毛上升高的机制，以便在滑动运动调节过程中对IFT进行特异性控制。然后，我们将检查用于调节IFT的不同类型的Ca2+升高，使用数学模型结合实验数据来帮助我们了解鞭毛内Ca2+浓度的快速变化。最后，我们将通过识别可能与Ca2+结合并破坏这种相互作用的特定鞭毛蛋白，来研究Ca2+实际上如何导致IFT颗粒从鞭毛膜上分离。IFT的过程在真核生物中是高度保守的，Ca2+依赖性的IFT调控可能影响许多纤毛蛋白的运动，包括那些参与与人类遗传疾病相关的发育信号通路的纤毛蛋白。因此，我们对藻类的研究结果将有助于了解纤毛信号在包括哺乳动物在内的许多不同生物中是如何调节的，并揭示纤毛在感知和响应细胞环境中发挥的许多不同作用。

Cilia and flagella are tiny-hair-like projections from cells that play important roles in motility and in sensing changes in the cellular environment. Whilst we are familiar with their role in motility, the mechanisms cilia use to sense environmental stimuli and transmit this information to the rest of the cell are less clear.Cilia are built at the tip using a process known as intraflagellar transport (IFT), which enables proteins to be moved along the cilium to the site of assembly. It has also been shown that IFT plays an important role in ciliary signalling, as many important receptor proteins localise to cilia and are moved into and out of the cilium by IFT. Disruption of ciliary signalling due to defects in IFT can lead to human diseases and developmental problems, and it is therefore important for us to understand how intraflagellar transport is regulated.Using the motile green alga, Chlamydomonas, as a model system to study ciliary signalling, we recently discovered that IFT may be regulated by calcium signalling. Many environmental stimuli trigger ion channel proteins in cell membranes to open and cause a rapid influx of calcium ions (Ca2+) into cells. This results in elevated Ca2+ within the cell, which triggers various signalling cascades depending on the nature of the stimulus. Ca2+-dependent signalling processes are central to both the motile and sensory roles of cilia, but we know very little about the nature of these Ca2+ elevations and how they act to regulate ciliary processes. The discovery that Ca2+ signals are regulating IFT therefore links two very important processes in cilia and should help us understand much more about how these organelles sense and respond to their environment.We have used Chlamydomonas to develop a novel microscopy technique that allows us to simultaneously image Ca2+ and the movement of IFT particles in flagella for the first time. Chlamydomonas is currently the only organism in which this technique is possible and this unique ability will allow us to directly examine the mechanisms underlying this novel signalling process.Chlamydomonas can glide along solid substrates on its flagella by using IFT to move proteins in the flagella membrane. Gliding is coordinated by flagella Ca2+ signalling. Ca2+ elevations in one flagellum cause the IFT particles to dissociate from the flagella membrane and stop pulling the cell along. This gliding process is therefore an excellent model system in which to study how Ca2+ signalling regulates IFT to control the movement of flagella membrane proteins.Although we know that Ca2+ regulates IFT, we don't yet know how this happens. This proposal seeks to identify the specific cellular mechanisms responsible. Firstly, we will examine how Ca2+ signals are generated in Chlamydomonas flagella, looking at the ion channels responsible and at mechanisms that restrict Ca2+ elevations to individual flagella, to enable specific control of IFT during the regulation of gliding motility. We will then examine the different types of Ca2+ elevations that are used to regulate IFT, using mathematical models in combination with experimental data to help us understand the rapid changes in Ca2+ concentration inside the flagellum. Finally, we will look at how Ca2+ actually causes the IFT particles to dissociate from the flagella membrane, by identifying specific flagella proteins that may bind to Ca2+ and disrupt this interaction.The process of IFT is highly conserved amongst eukaryotes and it is likely that Ca2+-dependent regulation of IFT influences the movement of many ciliary proteins, including those involved in developmental signalling pathways relating to human genetic diseases. Therefore the results from our studies in algae will provide insight into how ciliary signalling is regulated in many different organisms, including mammals, and shed light on the many different roles cilia play in sensing and responding to the cellular environment.