Understanding membrane shape remodelling in cilia formation and function

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.

Cilia are hair-like cell projections that are indispensable for vertebrate development and tissue homeostasis through their functions in motility, signalling and sensing. Membrane shaping is crucial for cilia formation and the distal appendages (DAs) of mother centrioles play a central role in this process. DAs recruit small vesicles that are remodelled and fused into a larger ciliary vesicle that becomes deformed by the onset of axoneme formation before fusing with the cell membrane to externalise the cilium. DAs ("transition fibres") then keep the centriole ("basal body") docked at a region of high positive membrane curvature at the boundary between plasma and ciliary membrane. Due to the paucity of structural and biochemical data, it is poorly understood how DAs spatially organise the associated membrane binding and remodelling complexes and facilitate these events. Furthermore, it is unclear how important an exact membrane curvature regulation by the DA-associated complexes is for ciliogenesis and cilia function. We will address these questions through: A) Establishing a purification method for DAs that will allow us to determine their membrane binding and remodelling activity in vitro and to begin their structural characterisation to understand how they function. B) Using structure-guided protein-engineering of the DA-associated membrane shaping DZIP1-Chibby1-FAM92 complex to establish whether and how changing its curvature induction properties alters the membrane shape at the ciliary base and how this affects cilia formation and function. Our study will provide deep insights into protein complexes mutated in human ciliopathies and shed light on the mechanistic links between DAs, local membrane shaping events and cilia. Our purification protocols for DAs will open new research avenues to better understand a critical but understudied membrane-contacting substructure of basal bodies and to learn how to rectify its dysfunction in disease.