Periodic Reporting for period 5 - CentSatRegFunc (Dissecting the function and regulation of centriolar satellites: key regulators of the centrosome/cilium complex)
Berichtszeitraum: 2022-06-01 bis 2023-05-31
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. 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. 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 centrosomes and cilia. In this project, we aimed to determine the molecular and cellular mechanisms that mediate these changes and thereby centrosome and cilia function. To this end, we investigated the structure and function of a new membrane-less organelle termed centriolar satellites, which were initially described about 50 years ago by electron microscopy studies but have remained poorly characterized for a long time. The central hypothesis of the CentSatRegFunc project was that centriolar satellites mediate timely and efficient changes in the composition, function and adaptions of centrosomes and cilia. We tested our hypothesis in three major objectives. In Objective 1, we used state-of-the art imaging, cell biology and proteomics approaches and determined the mechanisms by which centriolar satellites communicate with centrosomes and cilia. Importantly, our results described centriolar satellites as trafficking organelles critical for vital cellular functions such as cellular signaling and cell division. In Objective 2, we used biochemistry and proteomics approaches and determined the centriolar satellite proteome. By further biochemical and functional dissection of previously uncharacterized satellite residents mutated in ciliopathies, we discovered new mechanisms by which centrosomes and cilia assemble and mediate their cellular functions. Moreover, these studies provided insight into defects that underlie retinal degeneration and microcephaly. In Objective 3, we used genome editing and organelle mispositioning technologies and defined centriolar satellites as key regulators of primary cilium assembly and function. Together, the results of this project have important implications for our understanding of how cells form compartments without membranes and how membrane-less organelles communicate with each other to maintain cellular homeostasis. Importantly, results from this project also informs diagnostic and therapeutic strategies targeting centrosomes and cilia in cancer and genetic diseases.
Centriolar satellites were discovered more than 60 years ago as structures that localize and move around centrosomes. In contrast to our extensive understanding of the biology of centrosomes, centriolar satellites have remained as poorly characterized membrane-less organelles. The overall objective of the CentSatRegFunc project 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 machines 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. 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. Moreover, they also identified new components of satellites that were previously linked to genetic developmental diseases. By focusing on several of these proteins, we revealed mechanisms that contribute to retinal degeneration and microcephaly. In particular, we showed that retinal degeneration mutations lead to defects in cilium assembly and ciliary protein targeting and defects in centriole duplication and mitosis contribute to microcephaly. In Objective 3, we used genome editing and organelle mispositioning approaches to generate cells devoid of satellites and to misposition satellites to the cell periphery, respectively. Phenotypic characterization of these cells 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. Together, the results from the CentSatRegFunc project defined centriolar satellites as cellular machines where proteins are assembled into multicomponent complexes, modified, stored and/or trafficked and showed that they play critical roles during cellular processes required for development and differentiation.
CentSatRegFunc project aimed to dissect the functions and molecular mechanisms of centriolar satellites in regulating centrosomes and cilia in time and in space. This is a challenging aim in the context of these structures as their membrane-less and highly dynamic nature and low abundance in cells present challenges in their cellular and biochemical characterization. Therefore, we developed or adapted imaging, proteomic, biochemical and genetic approaches and technologies beyond the state of the art, which allowed us to address the key unknowns about centriolar satellites. By using this integrated, multidisciplinary approach, we tested the central hypothesis of the grant that proposed centriolar satellites as membrane-less trafficking machines in three main objectives. Our results identified functions for centriolar satellites in assembly, modification, trafficking and storage of their residents and supported the trafficking model. Moreover, our project generated very exciting, unexpected results regarding the biology of centriolar satellites. First, characterization of satellite-less cells in retinal and kidney epithelial cells revealed that there are differences in requirements for satellites during cilium assembly. Building on these differences, we investigated centriolar satellite properties such as composition and size in different cell types and tissues including multiciliated epithelial cells. These results advanced 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 organelles, which 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.