Understanding membrane shape remodelling in cilia formation and function

脂质膜在生物系统中提供边界，使细胞能够维持具有独立功能的专门内部室（“细胞器”）。然而，脂质膜不是刚性的、稳定的结构，而是动态的、不断交换的物质。为了保持细胞器的形状并使其发挥作用，细胞使用一整套蛋白质来重塑脂质膜。这种情况发生的机制尚不完全清楚，而且通常不清楚在这些事件中膜曲率的确切调节有多重要。一个恰当的例子是纤毛，一种对动物至关重要的毛发状细胞突起。纤毛是细胞感知周围环境的运动装置和天线。它们是由中心粒组成的，中心粒是一个大的桶状蛋白质结构，末端附属物（DAs）从中心粒的一端向外辐射。膜重构对纤毛的形成至关重要。这包括小膜球（囊泡）与DAs的对接，它们通过中心粒桶的延伸融合和变形，以及在纤毛基部高度弯曲的膜区域的维持，DAs（当时被称为“过渡纤维”）与中心粒桶（“基底”）对接。许多参与者的功能障碍导致纤毛病，这是一种慢性疾病，影响约1:1000的人，而且无法治愈。此外，纤毛还与癌症和心脏病等其他重要的人类疾病有关。因此，更好地了解膜重构和纤毛之间的联系将有助于纠正疾病中潜在过程的功能障碍。关于纤毛形成和功能中的膜重塑的关键问题仍然没有答案。虽然DAs与已知的膜形成分子有关，但尚不清楚它们如何在空间上组织和协调这些分子，从而带来复杂的纤毛形成途径。此外，在这个过程中，DAs与重塑的膜密切接触，这表明DAs不仅仅是其他蛋白质的临时招募平台，然后完成这项工作。然而，尚不清楚DA成分本身是否（以及如何）参与膜结合和成型。目前还没有针对DAs的纯化方法可以解决这些问题。我们将开发这样一种基于中心粒碎片的方法，并使用纯化的DAs来可视化它们的高分辨率结构，并在生化分析中测试它们对膜的活性。这将使我们能够了解da是如何工作的，开发工具来进一步分析它们在细胞中的功能，并深入了解它们在人类疾病中的功能障碍是如何纠正的。此外，我们将讨论DAs/过渡纤维接触的高度弯曲膜区域的精确形状对纤毛的重要性。形成膜的DZIP1-Chibby1-FAM92复合体与DAs结合并定位于该膜区。重要的是，它在纤毛形成和基底体对接中都起作用，这强烈表明它是维持或感知局部膜形状的积极参与者。我们最近对这种复合体的研究表明，我们可以通过蛋白质工程改变它的膜成型特性。我们将在细胞中使用工程复合体来研究改变其曲率诱导如何影响局部膜形状以及纤毛的产生及其功能。总之，拟议的工作将阐明一个不被理解但基本的动物过程，并揭示人类纤毛病中突变的蛋白质复合物如何在其中起作用。获得的见解以及我们的工具和方法将在重要的动物模型中开辟新的研究途径，并促进减轻疾病中这一过程功能障碍的策略的发展。

Lipid membranes provide boundaries in biological systems that enable cells to maintain specialised internal compartments ("cell organelles") with separate functions. However, lipid membranes are not rigid, stable structures, but are dynamic and continuously exchange material. To maintain the shapes of their organelles and allow their function, cells use a whole battery of proteins that remodel lipid membranes. The mechanisms of how this happens are incompletely understood and it is often unclear how important the exact regulation of membrane curvature in these events is.A case in point are cilia, hair-like cell projections that are crucial for animals. Cilia serve as motility devices and antennas through which cells sense their environment. They are formed by centrioles, large barrel-shaped, protein structures with distal appendages (DAs) that radiate outwards from one of their ends. Membrane remodelling is critical for cilia formation. This includes the docking of small membrane spheres (vesicles) against the DAs, their fusion and deformation by the extension of the centriole barrel as well as the maintenance of a highly curved membrane region at the base of the cilium where the DAs (then called "transition fibres") dock the centriole barrel ("basal body"). Dysfunction of many of the involved players results in ciliopathies, chronic diseases that affect an estimated 1:1000 people and have no curative treatment. Additionally, cilia are also implicated in other important human disorders such as cancer and heart diseases. Thus, a better understanding of the link between membrane remodelling and cilia will help the development of approaches to rectify malfunctions of the underlying processes in disease.Crucial questions concerning membrane remodelling in cilia formation and function remain unanswered. While DAs associate with known membrane shaping molecules, it is unclear how they organise and coordinate them in space to bring about the complex cilia formation pathway. Furthermore, DAs make intimate contact to the remodelled membranes in this process, which argues that DAs are more than just temporary recruitment platforms for other proteins that then do the job. However, it is not known whether (and how) the DA components on their own contribute to membrane binding and shaping. There are no purification methods available for DAs that would allow to address these questions. We will develop such a method based on centriole fragmentation and use the purified DAs to visualise their high-resolution architecture and test their activity towards membranes in biochemical assays. This will enable us to understand how DAs work, to develop tools to further dissect their function in cells and to gain insights into how their dysfunction in human disease might be rectified. Furthermore, we will address how important the precise shape of the highly curved membrane region contacted by the DAs/transition fibres is for cilia. The membrane shaping DZIP1-Chibby1-FAM92 complex associates with DAs and localises to this membrane region. Importantly, it functions in both cilia formation and basal body docking strongly arguing that it is an active player in maintaining or sensing the local membrane shape there. Our recent work on this complex shows how we can change its membrane shaping properties through protein engineering. We will use the engineered complex in cells to study how altering its curvature induction affects the local membrane shape as well as the generation of cilia and their function. Together, the proposed work will shed light on an ill-understood but fundamental process in animals and reveal how protein complexes mutated in human ciliopathies function in it. The gained insights, as well as our tools and approaches will open new research avenues in important animal models and facilitate the development of strategies for alleviating dysfunctions of this process in disease.