A new cellular stress response that triggers centriolar satellite reorganization and ciliogenesis

Abstract

Centriolar satellites are small, granular structures that cluster around centrosomes, but whose biological function and regulation are poorly understood. We show that centriolar satellites undergo striking reorganization in response to cellular stresses such as UV radiation, heat shock, and transcription blocks, invoking acute and selective displacement of the factors AZI1/CEP131, PCM1, and CEP290 from this compartment triggered by activation of the stress-responsive kinase p38/MAPK14. We demonstrate that the E3 ubiquitin ligase MIB1 is a new component of centriolar satellites, which interacts with and ubiquitylates AZI1 and PCM1 and suppresses primary cilium formation. In response to cell stress, MIB1 is abruptly inactivated in a p38-independent manner, leading to loss of AZI1, PCM1, and CEP290 ubiquitylation and concomitant stimulation of ciliogenesis, even in proliferating cells. Collectively, our findings uncover a new two-pronged signalling response, which by coupling p38-dependent phosphorylation with MIB1-catalysed ubiquitylation of ciliogenesis-promoting factors plays an important role in controlling centriolar satellite status and key centrosomal functions in a cell stress-regulated manner.

Figure 1

Rapid displacement of AZI1, PCM1, and CEP290 from centriolar satellites in response to UV radiation. (A) U2OS cells were mock treated or exposed to UV or IR, fixed 1 h later and co-immunostained with AZI1 and γ-tubulin antibodies. Scale bar, 10 μm. (B) U2OS cells subjected to UV or not and treated as in (A) were co-immunostained with PCM1 and γ-tubulin antibodies. Scale bar, 10 μm. (C) As in (B), except that cells were co-immunostained with CEP290 and γ-tubulin antibodies. Scale bar, 10 μm. (D) U2OS cells were exposed or not to UV, collected 1 h later and separated into subcellular fractions. Cytoplasm- and cytoskeleton-enriched fractions were immunoblotted with indicated antibodies. (E) U2OS cells were left untreated, transfected with siRNAs against AZI1 or PCM1, or exposed to UV and subsequently prepared for electron microscopy combined with PCM1 immunogold labelling (black dots). Centrioles (rod shaped) are indicated by arrows. Scale bars, 1000, nm. (F) As in (B), except that cells were co-immunostained with OFD1 and γ-tubulin antibodies. Scale bar, 10 μm. High-magnification images are shown in Supplementary Figure S2. (G) Quantification of centriolar satellite localization of PCM1 and OFD1, analysed as in (B) and (F). At least 100 cells per condition were counted. Results depict the mean (±s.d.) of three independent experiments.

Figure 2

The stress-responsive kinase p38 is required for dispersal of centriolar satellite factors in response to a range of cell stresses. (A) U2OS cells were exposed to UV, heat shock, or the transcriptional inhibitors DRB or Actinomycin D, fixed and immunostained with AZI1 antibody. Scale bar, 10 μm. (B) U2OS cells were incubated with p38 inhibitor (p38i) for 1 h or transfected with p38 siRNA for 72 h. Subsequently, cells were irradiated or not with UV, fixed 1 h later and stained with AZI1 antibody. Scale bar, 10 μm. (C) U2OS cells treated as in (B) were stained with AZI1 and γ-tubulin antibodies. Scale bar, 10 μm. (D) U2OS cells treated as in (B) were immunoblotted with indicated antibodies. (E) Cytoskeletal and cytoplasmic fractions of cells treated with p38 inhibitor and/or UV for 1 h prior to harvest were analysed with the indicated antibodies. (F) U2OS cells transfected with WT or kinase-dead (K82A) versions of FLAG-tagged MKK6 were fixed and immunostained with AZI1 and FLAG antibodies. Scale bars, 10 μm.

Figure 3

The E3 ubiquitin ligase MIB1 is a centriolar satellite-associated protein that interacts with AZI1 and PCM1. (A) Mass spectrometry (MS)-based analysis of proteins interacting with GFP-AZI1. U2OS cells grown in light (L) or heavy (H) SILAC media were transfected with empty vector or GFP-AZI1 plasmid, respectively. GFP-AZI1 and its associated proteins were enriched using GFP-Trap resin and analysed by MS. Selected proteins with high SILAC (H/L) ratios and/or sequence coverage are indicated. Asterisk indicates the bait protein. (B) Interaction between AZI1 and MIB1 was analysed by immunoblotting AZI1 immunoprecipitates (IPs) from whole-cell extracts (WCEs) of U2OS cells transfected with AZI1 or control (−) siRNAs. (C) As in (A), except that cells labelled with H medium were transfected with GFP-MIB1 plasmid. (D) Extracts of U2OS cells transfected with indicated combinations of GFP-MIB1 plasmid and AZI1 siRNA were subjected to GFP IP followed by immunoblotting with PCM1, AZI1, and GFP antibodies. (E) U2OS cells were transfected with constructs encoding Strep-HA-tagged AZI1 fragments spanning the indicated amino acids. Interactions between AZI1 and MIB1 or PCM1 were analysed by immunoblotting Strep-Tactin pull-downs with MIB1 and PCM1 antibodies. (F) U2OS cells stably expressing GFP-MIB1 were mock treated or transfected with AZI1 siRNA for 48 h, exposed to UV or not and fixed 1 h later. Cells were immunostained with AZI1 antibody. Scale bars, 10 μm.

Figure 4

MIB1 promotes ubiquitylation of centriolar satellite factors and is inactivated by cell stress. (A) SILAC-labelled U2OS/Strep-HA-ubiquitin cells were mock treated (Light (L) medium) or subjected to UV radiation (25 J/m²) or ionizing radiation (IR, 10 Gy) (Heavy (H) medium) and collected 1 h later. Ubiquitylated proteins were purified on Strep-Tactin resin under denaturing conditions and analysed by mass spectrometry (MS) for determination of SILAC ratios for individual proteins (Supplementary Figure S4A; Supplementary Table S1). SILAC ratios (H/L), indicating relative DNA damage-induced change in ubiquitylation level, for selected proteins are shown. (B) U2OS/Strep-HA-ubiquitin cells left untreated or exposed to UV were collected 1 h later. Where indicated, p38 inhibitor (p38i) was added to the medium 30 min before UV radiation. Cell extracts were subjected to Strep-Tactin pull-down followed by immunoblotting with MIB1 antibody. (C) U2OS or U2OS/Strep-HA-ubiquitin cells were mock treated or subjected to UV, collected 1 h later, and lysed under denaturing conditions. Ubiquitylation of centriolar satellite proteins was analysed by immunoblotting Strep-Tactin pull-downs from whole-cell extracts (WCEs) with AZI1, PCM1, and CEP290 antibodies. (D) U2OS or U2OS/Strep-HA-ubiquitin cells were transfected for 72 h with control siRNA or independent siRNAs targeting MIB1. Ubiquitylation of AZI1 and PCM1 in WCEs was analysed by immunoblotting Strep-Tactin pull-downs with the indicated antibodies. (E) U2OS or U2OS/Strep-HA-ubiquitin cells were transfected for 24 h with WT or catalytically inactive (CI) versions of GFP-MIB1 and treated or not with UV. Ubiquitylation of AZI1 and PCM1 was analysed as in (C). (F) Extracts of U2OS cells transfected with indicated combinations of plasmids were analysed for ubiquitylation of AZI1(F1) (amino acids 1–268) by immunoblotting S-tag pull-downs with HA antibody.

Figure 5

MIB1 ubiquitin ligase activity suppresses primary cilium formation. (A) Human RPE1 cells were serum starved by culturing in medium containing 0.25% FCS, fixed and immunostained with antibodies against AZI1 and acetylated tubulin (Ac-tubulin). Scale bar, 10 μm. (B) Effect of AZI1 or PCM1 knockdown on primary cilium formation in cells treated as in (A) was analysed by immunostaining with antibodies against pericentrin and acetylated tubulin (left). Data from three independent experiments were quantified (right). At least 300 cells were counted in each experiment. Scale bar, 10 μm. (C) RPE1 cells were transfected with GFP-MIB1 WT or CI for 24 h and serum starved for 16 h. Cells were then fixed, immunostained with antibody to acetylated tubulin, and the fraction of GFP-positive cells with a cilium was determined. The experiment was performed in triplicates and at least 150 GFP-positive cells were counted for each condition. (D) RPE1 cells were transfected with control (CTRL) or MIB1 siRNAs for 72 h and cultured in normal medium containing 10% serum (10% FCS). Spontaneous cilium formation was visualized and quantified as in (B). Scale bar, 10 μm. Data in (B–D) were analysed by pairwise two-tailed t-test. **P<0.01, ***P<0.001. (E) GFP-MIB1 autoubiquitylation was analysed by transfecting RPE1 cells with GFP-MIB1 and S-Myc-ubiquitin plasmids. Cells were then grown in medium containing high (10% FCS) or low (0.25% FCS) serum concentrations for 24 h and collected. Whole-cell extracts (WCEs) were subjected to pull-down with S-tag agarose and immunoblotted with GFP antibody.

Figure 6

Cell stresses induce p38-dependent primary cilium formation in proliferating cells. (A) RPE1 cells were exposed or not to UV (10 J/m²), kept at normal serum concentrations (10% FCS), and fixed 18 h later. Spontaneous cilium formation was analysed by immunostaining with antibodies against pericentrin and acetylated tubulin (left). Data from three independent experiments were quantified (right). At least 300 cells were counted in each experiment. Scale bars, 10 μm. (B) As in (A), except that cells were mock treated or subjected to heat shock for 30 min and fixed 18 h later. (C) Exponentially growing RPE1 cells were exposed to UV (10 J/m²) or subjected to heat shock. Where indicated, p38 inhibitor (p38i) was added to the cultures 30 min before treatment. After an additional 16 h, cells were fixed, co-immunostained with antibodies to acetylated tubulin and pericentrin, and the proportion of cilium-positive cells was determined. The experiment was performed in triplicates and at least 300 cells were counted for each condition. (D) RPE1 cells were transfected with control (−) or MIB1 siRNAs for 48 h, exposed to UV (10 J/m²) and fixed 16 h later. Cells were then immunostained and analysed as in (C). Data in (A–D) were analysed by pairwise two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001. (E) Model for stress-induced reorganization of centriolar satellites and ciliogenesis. During unperturbed cell proliferation, AZI1, PCM1, and CEP290 are associated with centriolar satellites and ubiquitylated by the MIB1 E3 ubiquitin ligase, which is itself a component of centriolar satellites. In response to cell stresses such as UV radiation, heat shock, and transcription blocks, activation of the p38 kinase triggers acute dissociation of these proteins from centriolar satellites, leading to enhanced AZI1–PCM1 interaction. In parallel, cell stress also causes p38-independent inactivation of MIB1 and concomitant loss of AZI1, PCM1, and CEP290 ubiquitylation. Cooperatively, these stress-induced pathways cooperate to relieve inhibitory constraints to primary cilium formation in proliferating cells.