Spatial and proteomic profiling reveals centrosome-independent features of centriolar satellites.
ABSTRACT: Centriolar satellites are small electron-dense granules that cluster in the vicinity of centrosomes. Satellites have been implicated in multiple critical cellular functions including centriole duplication, centrosome maturation, and ciliogenesis, but their precise composition and assembly properties have remained poorly explored. Here, we perform in vivo proximity-dependent biotin identification (BioID) on 22 human satellite proteins, to identify 2,113 high-confidence interactions among 660 unique polypeptides. Mining this network, we validate six additional satellite components. Analysis of the satellite interactome, combined with subdiffraction imaging, reveals the existence of multiple unique microscopically resolvable satellite populations that display distinct protein interaction profiles. We further show that loss of satellites in PCM1-depleted cells results in a dramatic change in the satellite interaction landscape. Finally, we demonstrate that satellite composition is largely unaffected by centriole depletion or disruption of microtubules, indicating that satellite assembly is centrosome-independent. Together, our work offers the first systematic spatial and proteomic profiling of human centriolar satellites and paves the way for future studies aimed at better understanding the biogenesis and function(s) of these enigmatic structures.
Project description:Centriolar satellites are an array of membrane-less granules that localize and move around the vertebrate centrosome/cilium complex. They have recently emerged as key regulators of the biogenesis and function of the centrosome/cilium-complex and their mutations are linked to ciliopathies. Although centriolar satellites are ubiquitous structures of the vertebrate cells, their precise function and molecular mechanism of action in different cell types remain poorly understood. Here, we generated kidney and retinal epithelial cells that lack centriolar satellites by genetically ablating their scaffolding protein PCM1 and investigated the cellular and molecular consequences of satellite loss in cells. We showed that centriolar satellites are required for cilium assembly, regulation of ciliary content, timely response to Hedgehog signals and three- dimensional epithelial cell organization, but not for cell proliferation, cell cycle progression and centriole duplication. Importantly, the requirement for centriolar satellites in cilium assembly varied between retinal and kidney epithelial cells and we identified the differences in the efficiency of targeting key ciliogenesis factors to the centrosome including Mib1 and Talpid3 as the likely molecular basis for this phenotypic variability. Quantitative global transcriptomic and proteomic profiling of satellite-less cells showed that loss of centriolar satellites does not lead to a major transcriptional response, but leads to a significant rearrangement of the global proteome. Together, our findings identify important roles for centriolar satellites in key cilium-related cellular processes through regulating the proteostasis and centrosomal/ciliary targeting of proteins and provide insight into the disease mechanisms of ciliopathies. Overall design: We performed global transcriptome analysis of wild type (WT) and PCM1 knock-out cells by using two different cells: human RPE1, and mouse IMCD3 cells. Deep sequencing technique was utilized via illumina HiSeq4000 platfrom in two biological replicates for each condition.
Project description:Autophagy maintains cellular health and homeostasis during stress by delivering cytosolic material captured by autophagosomes to lysosomes for degradation. Autophagosome formation is complex: initiated by the recruitment of autophagy (Atg) proteins to the formation site, it is sustained by activation of Atg proteins to allow growth and closure of the autophagosome. How Atg proteins are translocated to the forming autophagosome is not fully understood. Transport of the ATG8 family member GABARAP from the centrosome occurs during starvation-induced autophagosome biogenesis, but how centrosomal proteins regulate GABARAP localization is unknown. We show that the centriolar satellite protein PCM1 regulates the recruitment of GABARAP to the pericentriolar material. In addition to residing on the pericentriolar material, GABARAP marks a subtype of PCM1-positive centriolar satellites. GABARAP, but not another ATG8 family member LC3B, binds directly to PCM1 through a canonical LIR motif. Loss of PCM1 results in destabilization of GABARAP, but not LC3B, through proteasomal degradation. GABARAP instability is mediated through the centriolar satellite E3 ligase Mib1, which interacts with GABARAP through its substrate-binding region and promotes K48-linked ubiquitination of GABARAP. Ubiquitination of GABARAP occurs in the N terminus, a domain associated with ATG8-family-specific functions during autophagosome formation, on residues absent in the LC3 family. Furthermore, PCM1-GABARAP-positive centriolar satellites colocalize with forming autophagosomes. PCM1 enhances GABARAP/WIPI2/p62-positive autophagosome formation and flux but has no significant effect on LC3B-positive autophagosome formation. These data suggest a mechanism for how centriolar satellites can specifically regulate an ATG8 ortholog, the centrosomal GABARAP reservoir, and centrosome-autophagosome crosstalk.
Project description:Centriolar satellites are ubiquitous in vertebrate cells. They have recently emerged as key regulators of centrosome/cilium biogenesis, and their mutations are linked to ciliopathies. However, their precise functions and mechanisms of action remain poorly understood. Here, we generated a kidney epithelial cell line (IMCD3) lacking satellites by CRISPR/Cas9-mediated PCM1 deletion and investigated the cellular and molecular consequences of satellite loss. Cells lacking satellites still formed full-length cilia but at significantly lower numbers, with changes in the centrosomal and cellular levels of key ciliogenesis factors. Using these cells, we identified new ciliary functions of satellites such as regulation of ciliary content, Hedgehog signaling, and epithelial cell organization in three-dimensional cultures. However, other functions of satellites, namely proliferation, cell cycle progression, and centriole duplication, were unaffected in these cells. Quantitative transcriptomic and proteomic profiling revealed that loss of satellites affects transcription scarcely, but significantly alters the proteome. Importantly, the centrosome proteome mostly remains unaltered in the cells lacking satellites. Together, our findings identify centriolar satellites as regulators of efficient cilium assembly and function and provide insight into disease mechanisms of ciliopathies.
Project description:Centrioles are core structural elements of both centrosomes and cilia. Although cytoplasmic granules called centriolar satellites have been observed around these structures, lack of a comprehensive inventory of satellite proteins impedes our understanding of their ancestry. To address this, we performed mass spectrometry (MS)-based proteome profiling of centriolar satellites obtained by affinity purification of their key constituent, PCM1, from sucrose gradient fractions. We defined an interactome consisting of 223 proteins, which showed striking enrichment in centrosome components. The proteome also contained new structural and regulatory factors with roles in ciliogenesis. Quantitative MS on whole-cell and centriolar satellite proteomes of acentriolar cells was performed to reveal dependencies of satellite composition on intact centrosomes. Although most components remained associated with PCM1 in acentriolar cells, reduced cytoplasmic and satellite levels were observed for a subset of centrosomal proteins. These results demonstrate that centriolar satellites and centrosomes form independently but share a substantial fraction of their proteomes. Dynamic exchange of proteins between these organelles could facilitate their adaptation to changing cellular environments during development, stress response and tissue homeostasis.
Project description:Centrioles are the major constituents of the animal centrosome, in which Plk4 kinase serves as a master regulator of the duplication cycle. Many eukaryotes also contain numerous peripheral particles known as centriolar satellites. While centriolar satellites aid centriole assembly and primary cilium formation, it is unknown whether Plk4 plays any regulatory roles in centriolar satellite integrity. Here we show that Plk4 is a critical determinant of centriolar satellite organisation. Plk4 depletion leads to the dispersion of centriolar satellites and perturbed ciliogenesis. Plk4 interacts with the satellite component PCM1, and its kinase activity is required for phosphorylation of the conserved S372. The nonphosphorylatable PCM1 mutant recapitulates phenotypes of Plk4 depletion, while the phosphomimetic mutant partially rescues the dispersed centriolar satellite patterns and ciliogenesis in cells depleted of PCM1. We show that S372 phosphorylation occurs during the G1 phase of the cell cycle and is important for PCM1 dimerisation and interaction with other satellite components. Our findings reveal that Plk4 is required for centriolar satellite function, which may underlie the ciliogenesis defects caused by Plk4 dysfunction.
Project description:Primary microcephaly (MCPH) associated proteins CDK5RAP2, CEP152, WDR62 and CEP63 colocalize at the centrosome. We found that they interact to promote centriole duplication and form a hierarchy in which each is required to localize another to the centrosome, with CDK5RAP2 at the apex, and CEP152, WDR62 and CEP63 at sequentially lower positions. MCPH proteins interact with distinct centriolar satellite proteins; CDK5RAP2 interacts with SPAG5 and CEP72, CEP152 with CEP131, WDR62 with MOONRAKER, and CEP63 with CEP90 and CCDC14. These satellite proteins localize their cognate MCPH interactors to centrosomes and also promote centriole duplication. Consistent with a role for satellites in microcephaly, homozygous mutations in one satellite gene, CEP90, may cause MCPH. The satellite proteins, with the exception of CCDC14, and MCPH proteins promote centriole duplication by recruiting CDK2 to the centrosome. Thus, centriolar satellites build a MCPH complex critical for human neurodevelopment that promotes CDK2 centrosomal localization and centriole duplication.
Project description:The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.
Project description:Centriolar satellites are proteinaceous granules that are often clustered around the centrosome. Although centriolar satellites have been implicated in protein trafficking in relation to the centrosome and cilium, the details of their function and composition remain unknown. FOP (FGFR1 Oncogene Partner) is a known centrosome protein with homology to the centriolar satellite proteins FOR20 and OFD1. We find that FOP partially co-localizes with the satellite component PCM1 in a cell cycle-dependent manner, similarly to the satellite and cilium component BBS4. As for BBS4, FOP localization to satellites is cell cycle dependent, with few satellites labeled in G1, when FOP protein levels are lowest, and most labeled in G2. FOP-FGFR1, an oncogenic fusion that causes a form of leukemia called myeloproliferative neoplasm, also localizes to centriolar satellites where it increases tyrosine phosphorylation. Depletion of FOP strongly inhibits primary cilium formation in human RPE-1 cells. These results suggest that FOP is a centriolar satellite cargo protein and, as for several other satellite-associated proteins, is involved in ciliogenesis. Localization of the FOP-FGFR1 fusion kinase to centriolar satellites may be relevant to myeloproliferative neoplasm disease progression.
Project description:Centriolar satellites are numerous electron-dense granules dispersed around the centrosome. Mutations in their components are linked to various human diseases, but their molecular roles remain elusive. In particular, the significance of spatial communication between centriolar satellites and the centrosome is unknown. hMsd1/SSX2IP localizes to both the centrosome and centriolar satellites and is required for tethering microtubules to the centrosome. Here we show that hMsd1/SSX2IP-mediated microtubule anchoring is essential for proper centriole assembly and duplication. On hMsd1/SSX2IP knockdown, the centriolar satellites become stuck at the microtubule minus end near the centrosome. Intriguingly, these satellites contain many proteins that normally localize to the centrosome. Of importance, microtubule structures, albeit not being anchored properly, are still required for the emergence of abnormal satellites, as complete microtubule depolymerization results in the disappearance of these aggregates from the vicinity of the centrosome. We highlighted, using superresolution and electron microscopy, that under these conditions, centriole structures are faulty. Remarkably, these cells are insensitive to Plk4 overproduction-induced ectopic centriole formation, yet they accelerate centrosome reduplication upon hydroxyurea arrest. Finally, the appearance of satellite aggregates is cancer cell specific. Together our findings provide novel insights into the mechanism of centriole assembly and microtubule anchoring.
Project description:Centriolar satellites are membrane-less structures that localize and move around the centrosome and cilium complex in a microtubule-dependent manner. They play important roles in centrosome- and cilium-related processes, including protein trafficking to the centrosome and cilium complex, and ciliogenesis, and they are implicated in ciliopathies. Despite the important regulatory roles of centriolar satellites in the assembly and function of the centrosome and cilium complex, the molecular mechanisms of their functions remain poorly understood. To dissect the mechanism for their regulatory roles during ciliogenesis, we performed an analysis to determine the proteins that localize in close proximity to the satellite protein CEP72, among which was the retinal degeneration gene product CCDC66. We identified CCDC66 as a microtubule-associated protein that dynamically localizes to the centrosome, centriolar satellites and the primary cilium throughout the cell cycle. Like the BBSome component BBS4, CCDC66 distributes between satellites and the primary cilium during ciliogenesis. CCDC66 has extensive proximity interactions with centrosome and centriolar satellite proteins, and co-immunoprecipitation experiments revealed interactions between CCDC66, CEP290 and PCM1. Ciliogenesis, ciliary recruitment of BBS4 and centriolar satellite organization are impaired in cells depleted for CCDC66. Taken together, our findings identify CCDC66 as a targeting factor for centrosome and cilium proteins.