Acute inhibition of centriolar satellite function and positioning reveals their functions at the primary cilium

Abstract

Centriolar satellites are dynamic, membraneless granules composed of over 200 proteins. They store, modify, and traffic centrosome and primary cilium proteins, and help to regulate both the biogenesis and some functions of centrosomes and cilium. In most cell types, satellites cluster around the perinuclear centrosome, but their integrity and cellular distribution are dynamically remodeled in response to different stimuli, such as cell cycle cues. Dissecting the specific and temporal functions and mechanisms of satellites and how these are influenced by their cellular positioning and dynamics has been challenging using genetic approaches, particularly in ciliated and proliferating cells. To address this, we developed a chemical-based trafficking assay to rapidly and efficiently redistribute satellites to either the cell periphery or center, and fuse them into stable clusters in a temporally controlled way. Induced satellite clustering at either the periphery or center resulted in antagonistic changes in the pericentrosomal levels of a subset of proteins, revealing a direct and selective role for their positioning in protein targeting and sequestration. Systematic analysis of the interactome of peripheral satellite clusters revealed enrichment of proteins implicated in cilium biogenesis and mitosis. Importantly, induction of peripheral satellite targeting in ciliated cells revealed a function for satellites not just for efficient cilium assembly but also in the maintenance of steady-state cilia and in cilia disassembly by regulating the structural integrity of the ciliary axoneme. Finally, perturbing satellite distribution and dynamics inhibited their mitotic dissolution, and mitotic progression was perturbed only in cells with centrosomal satellite clustering. Collectively, our results for the first time showed a direct link between satellite functions and their pericentrosomal clustering, suggested new mechanisms underlying satellite functions during cilium assembly, and provided a new tool for probing temporal satellite functions in different contexts

Aurora Kinase A proximity interactome reveals centriolar satellites as regulators of its function during primary cilium biogenesis

“…In agreement with previous reports, PCM1 depletion resulted in a significant decrease in the percentage of ciliated cells relative to control cells and the cilia that formed in these cells were significantly shorter ( Fig. 7B,C ) (siControl: 79.80 ± 6.14%, siPCM1: 31.97 ± 2.79%, p<0.001; siControl: 5.33μm ± 0.14μm; siPCM1:3.89 μm ± 0.15 μm, p<0.0001) (Aydin et al, 2020, Kim et al, 2008, Stowe et al, 2012. While MLN8237-and DMSO-treated cells ciliated with similar efficiencies (p>0.1), AURKA inhibition partially restored the ciliogenesis defect in PCM1-depleted cells (siPCM1+DMSO: 31.97 ± 2.79%; siPCM1+MLN8237: 49.70 ± 2.14%, p<0.01; siControl+DMSO: 79.80 ± 6.14%, siControl+MLN8237: 49.70 ± 7.6%, p=0.94) ( Fig 7C).…”

“…Having established the link between satellites and AURKA regulation in quiescent cells, we next investigated the functional consequences of this regulatory relationship. Recent studies showed that satellites function as trafficking and storage sites for key ciliogenesis factors and regulate primary cilium assembly, maintenance and disassembly (Aydin et al, 2020, Odabasi et al, 2019, Wang et al, 2016). However, the proteins regulated by satellites during cilium biogenesis have not been fully characterized.…”

“…Recent studies showed that satellites function as trafficking and storage sites for key ciliogenesis factors and regulate primary cilium assembly, maintenance and disassembly (Aydin et al, 2020, Odabasi et al, 2019. However, the proteins regulated by satellites during cilium biogenesis have not been fully characterized.…”

A Proximity Mapping Journey into the Biology of the Mammalian Centrosome/Cilium Complex

“…These lines of evidence confirm the tight regulatory and functional relationship of satellites with centrosomes and cilia. Consistently, satellites have emerged as key regulators of cilium assembly and maintenance, Hedgehog signaling and mitotic progression [ 3 , 85 , 86 , 87 ]. Recent studies aimed at dissecting satellite composition, dynamics and functions have unraveled regulation of centrosomal and ciliary protein abundance as the major mechanism by which satellites mediate their functions [ 2 , 3 ].…”

“…Upon mitotic exit, DYRK3 is degraded following its APC/C-mediated ubiquitination and thereby satellites reassemble and cluster around centrosomes [ 89 ]. Although the functional significance of the mitotic dissolution of satellites is not known, recent studies suggest that it might contribute to satellite functions during mitotic progression [ 85 ]. Future studies are required to elucidate the mechanisms that regulate satellite assembly, remodeling and disassembly as well as their number control.…”