Periodic Reporting for period 4 - CentSatRegFunc (Dissecting the function and regulation of centriolar satellites: key regulators of the centrosome/cilium complex)
Berichtszeitraum: 2020-12-01 bis 2022-05-31
Centrosomes are small organelles that organize microtubules in animal cells. They build the mitotic spindle in dividing cells, which is required for faithful segregation of genetic material. Although dividing cells have two centrosomes, most cancer cells have more than two centrosomes. Extra centrosomes cause by leading to cell division errors and invasive behavior. Centrosomes also form cilia, which are thread-like cellular projections. While primary cilia functions as cellular antenna to receive and transmit important developmental signals, motile cilia function in cellular and organismal motility and in driving fluid transport along epithelial cells. Defects in their structure and function causes human genetic diseases termed “ciliopathies”, which are characterized by a wide range of symptoms including blindness, kidney disease, obesity, infertility, respiratory problems and birth defects. To develop new specific diagnostic and therapeutic approaches to cancer and developmental diseases, it is essential to first determine how centrosomes and cilia assemble and function and then to reveal what goes awry in disease.
The structure and function of centrosomes and cilia are dynamically altered in response to cellular, developmental and environmental stimuli and also vary across different cell types and tissues. These dramatic changes and adaptations require timely and rapid changes in the composition of these structures. In this project, we aim to determine the molecular and cellular mechanisms that mediate these changes and thereby centrosome and cilia function. To this end, we investigate the structure and function of previously underappreciated cellular structures termed centriolar satellites, which were initially described about 50 years ago by electron microscopy studies. Centriolar satellites are specific to mammalian cells and most centrosome/cilium proteins also localize to centriolar satellites in these cells. The central hypothesis of our CentSatRegFunc project is that mammalian cells evolved centriolar satellites to mediate timely and efficient changes in the composition, function and adaptions of centrosomes and cilia. We will test our hypothesis in three major objectives. In Objective 1, we will use state-of-the art imaging, cell biology and proteomics approaches to determine how centriolar satellites communicate with centrosomes and cilia. In Objective 2, we will use biochemistry and proteomics approaches to determine the parts of centriolar satellites and how these parts come together to assemble functional structures. In Objective 3, we will use genome editing technologies to determine the cellular functions of satellites. Together, the results of this proposal will have important implications for our understanding of how cells form compartments without membranes. Importantly, they will also inform diagnostic and therapeutic strategies targeting centrosomes and cilia in cancer and genetic diseases.
The structure and function of centrosomes and cilia are dynamically altered in response to cellular, developmental and environmental stimuli and also vary across different cell types and tissues. These dramatic changes and adaptations require timely and rapid changes in the composition of these structures. In this project, we aim to determine the molecular and cellular mechanisms that mediate these changes and thereby centrosome and cilia function. To this end, we investigate the structure and function of previously underappreciated cellular structures termed centriolar satellites, which were initially described about 50 years ago by electron microscopy studies. Centriolar satellites are specific to mammalian cells and most centrosome/cilium proteins also localize to centriolar satellites in these cells. The central hypothesis of our CentSatRegFunc project is that mammalian cells evolved centriolar satellites to mediate timely and efficient changes in the composition, function and adaptions of centrosomes and cilia. We will test our hypothesis in three major objectives. In Objective 1, we will use state-of-the art imaging, cell biology and proteomics approaches to determine how centriolar satellites communicate with centrosomes and cilia. In Objective 2, we will use biochemistry and proteomics approaches to determine the parts of centriolar satellites and how these parts come together to assemble functional structures. In Objective 3, we will use genome editing technologies to determine the cellular functions of satellites. Together, the results of this proposal will have important implications for our understanding of how cells form compartments without membranes. Importantly, they will also inform diagnostic and therapeutic strategies targeting centrosomes and cilia in cancer and genetic diseases.
In the first half of the CentSatRegFunc Project, we have made progress in agreement with the proposed plans in all three objectives and have so far disseminated our results through publications, conference presentations and invited seminars. The overall objective of our proposal was to address the key unknowns about centriolar satellites, which are their components and interactions, their function and their relationship with centrosomes and cilia. As part of Objective 1, we tested the function of satellites as cellular machnies for trafficking. To this end, we developed live imaging assays and single particle tracking algorithms and determined the dynamic behavior of centriolar satellites in cells. In contrast to simple trafficking model that has been proposed, we showed that satellites combine both trafficking and storage to regulate the composition of centrosomes and cilia. Additionally, we identified the molecular mechanism of this regulation. As part of Objective 2, we combined novel proteomics approaches with purifications of satellites. The proteomics approaches rely on identifying all proteins in 20 nm proximity of the protein of interest. Using these approaches, we identified that spatial interactome for a number of known satellites proteins. This interactome informed hypothesis about the functions of these structures, which we tested in Objective 3. Moreover, they also identified new components of satellites. By focusing on one of these proteins, we revealed mechanisms that contribute to retinal degeneration. In particular, we showed that retinal degeneration mutations lead to defects in cilium assembly and ciliary protein targeting. As part of Objective 3, we used genome editing approaches, generated cells devoid of satellites and characterized the phenotypic consequences of loss of satellites. These analyses identified satellites as key regulators of cilium assembly, regulation of cilium content and tissue architecture. Unexpectedly, constitutive loss of satellites was dispensable for centrosome duplication, cell proliferation and cell cycle progression.
This project aims at determining the mechanisms that regulate assembly and function of cilia in time and in space, which is challenging in the context of these structures due to their membrane-less nature and low abundance in cells. With the three objectives we proposed to address these questions, we not only tested the initial hypothesis we proposed, but also acquired very promising and also unexpected results. Characterization of satellite-less cells in different cell types revealed that there are differences in requirements for satellites during cilium assembly. We will investigate these functions across different cell types and tissues, which will contribute to our understanding of how centrosomes and cilia adapt to tissue functions at the structure and function level. Moreover, characterization of centriolar satellite dynamics revealed new mechanisms of communication between membrane-less liquid-like structures, which will be dissecting at the mechanistic level and this will have important implications on cell compartmentalization. Finally, we have applied and developed novel proteomics approaches to generate spatial and temporal interaction maps for satellites, which provided important tools for the field and generated data for our project in forming new hypothesis.