Understanding membrane shape remodelling in cilia formation and function

了解纤毛形成和功能中的膜形状重塑

• 批准号: BB/X013030/1
• 负责人: Mark Van Breugel
• 金额: $ 60.58万
• 依托单位: Queen Mary University of London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX013030%2F1
• 关键词: Understanding; membrane; shape; remodelling; cilia

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.

脂质膜在生物系统中提供了边界，使细胞能够维持具有独立功能的专门内部区室（“细胞器”）。然而，脂质膜不是刚性、稳定的结构，而是动态的、不断交换物质的。为了维持细胞器的形状并发挥其功能，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，而且通常不清楚这些事件中膜曲率的精确调节有多重要。一个典型的例子是纤毛，一种对动物至关重要的毛发状细胞突起。纤毛充当运动装置和天线，细胞通过它们感知环境。它们由中心粒形成，中心粒是一种大桶形蛋白质结构，具有从其末端之一向外辐射的远端附属物 (DA)。膜重塑对于纤毛的形成至关重要。这包括小膜球（囊泡）与 DA 的对接，它们通过中心粒桶的延伸而融合和变形，以及在纤毛底部维持高度弯曲的膜区域，其中 DA（当时称为“过渡纤维”）对接中心粒桶（“基体”）。许多参与其中的球员的功能障碍会导致纤毛病，这种慢性疾病影响了大约 1:1000 人，并且没有治愈方法。此外，纤毛还​​与其他重要的人类疾病有关，例如癌症和心脏病。因此，更好地了解膜重塑和纤毛之间的联系将有助于开发纠正疾病潜在过程故障的方法。有关纤毛形成和功能中膜重塑的关键问题仍未得到解答。虽然 DA 与已知的膜成形分子相关，但尚不清楚它们如何在空间中组织和协调它们以实现复杂的纤毛形成途径。此外，DA 在此过程中与重塑的膜密切接触，这表明 DA 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）有助于膜结合和成形。没有可用的 DA 纯化方法可以解决这些问题。我们将开发一种基于中心粒碎片的方法，并使用纯化的 DA 来可视化其高分辨率结构，并在生化测定中测试其对膜的活性。这将使我们能够了解 DA 的工作原理，开发工具来进一步剖析它们在细胞中的功能，并深入了解如何纠正它们在人类疾病中的功能障碍。此外，我们将讨论 DA/过渡纤维接触的高度弯曲膜区域的精确形状对于纤毛的重要性。膜成形 DZIP1-Chibby1-FAM92 复合物与 DA 结合并定位于该膜区域。重要的是，它在纤毛形成和基底体对接中发挥作用，强烈认为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物的研究表明我们如何通过蛋白质工程改变其膜成形特性。我们将使用细胞中的工程复合物来研究改变其曲率诱导如何影响局部膜形状以及纤毛的生成及其功能。总之，这项拟议的工作将揭示动物中一个鲜为人知但基本的过程，并揭示人类纤毛病中突变的蛋白质复合物如何在其中发挥作用。所获得的见解以及我们的工具和方法将为重要的动物模型开辟新的研究途径，并促进制定缓解疾病过程功能障碍的策略。