TonEBP inhibits ciliogenesis by controlling aurora kinase A and regulating centriolar satellite integrity

Abstract

Background: Primary cilia on the surface of eukaryotic cells serve as sensory antennas for the reception and transmission in various cell signaling pathways. They are dynamic organelles that rapidly form during differentiation and cell cycle exit. Defects in these organelles cause a group of wide-ranging disorders called ciliopathies. Tonicity-responsive enhancer-binding protein (TonEBP) is a pleiotropic stress protein that mediates various physiological and pathological cellular responses. TonEBP is well-known for its role in adaptation to a hypertonic environment, to which primary cilia have been reported to contribute. Furthermore, TonEBP is involved in a wide variety of other signaling pathways, such as Sonic Hedgehog and WNT signaling, that promote primary ciliogenesis, suggesting a possible regulatory role. However, the functional relationship between TonEBP and primary ciliary formation remains unclear.

Methods: TonEBP siRNAs and TonEBP-mCherry plasmids were used to examine their effects on cell ciliation rates, assembly and disassembly processes, and regulators. Serum starvation was used as a condition to induce ciliogenesis.

Results: We identified a novel pericentriolar localization for TonEBP. The results showed that TonEBP depletion facilitates the formation of primary cilia, whereas its overexpression results in fewer ciliated cells. Moreover, TonEBP controlled the expression and activity of aurora kinase A, a major negative regulator of ciliogenesis. Additionally, TonEBP overexpression inhibited the loss of CP110 from the mother centrioles during the early stages of primary cilia assembly. Finally, TonEBP regulated the localization of PCM1 and AZI1, which are necessary for primary cilia formation.

Conclusions: This study proposes a novel role for TonEBP as a pericentriolar protein that regulates the integrity of centriolar satellite components. This regulation has shown to have a negative effect on ciliogenesis. Investigations into cilium assembly and disassembly processes suggest that TonEBP acts upstream of the aurora kinase A - histone deacetylase 6 signaling pathway and affects basal body formation to control ciliogenesis. Taken together, our data proposes previously uncharacterized regulation of primary cilia assembly by TonEBP.

Keywords: Aurora kinase A; Centriolar satellite; Ciliogenesis; TonEBP; Γ-tubulin.

Fig. 1

TonEBP is enriched at the pericentriolar area. a. RPE1 cells were immunostained for TonEBP (green) and γ-tubulin (red). Scale bar, 10 μm. Zoom scale bar, 2 μm; b. Cells were serum staved for 24 h and immunostained for TonEBP (green), acetylated tubulin (red), and γ-tubulin (magenta). Scale bar, 10 μm. Zoom scale bar, 2 μm; c. Cells were transfected with TonEBP-mCherry plasmid for 24 h and immunostained for TonEBP (green), mCherry (red) and γ-tubulin (magenta). Scale bar, 10 μm

Fig. 2

Depletion of TonEBP increases the percentage of ciliated cells in a dose-dependent manner. a. RPE1 cells were transfected with TonEBP siRNA for 24 h and used to measure protein expression by Western blot; b. Cells were transfected with siRNAs against TonEBP for 24 h, followed by 24 h of serum starvation, and immunostained for ARL13b (green) and γ-tubulin (red). Scale bar, 25 μm; c. Quantification of ciliated cell percentage as shown in (B). Data is represented as mean ± SD (n = 3 experiments). Two hundred cells were scored per condition per experiment; *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test; d. Quantification of average primary cilia length as shown in (B). Data is represented as mean ± SD (n = 3 experiments). Two hundred cells were scored per condition per experiment; NS > 0.05, Student’s t-test; e. Cells were transfected with siRNAs against TonEBP for 24 h, followed by 24 h of serum starvation, and immunostained for TonEBP (green), acetylated tubulin (red), and γ-tubulin (magenta). Scale bar, 10 μm. Zoom scale bar, 2 μm; f. Cells were transfected with different concentrations of TonEBP siRNA for 24 h and subjected to Western blot. They were used to quantify the percentage of ciliated cells. Data is represented as mean ± SD (n = 3 experiments). Two hundred cells were scored per condition per experiment; **P < 0.01, ***P < 0.001, Student’s t-test

Fig. 3

Overexpression of TonEBP markedly suppresses ciliogenesis. a. RPE1 cells were transfected with TonEBP-mCherry plasmid for 24 h and then subjected to Western blot; b. Cells were transfected with TonEBP-mCherry plasmid for 24 h, followed by serum starvation, and immunostained for ARL13b (green), mCherry (red), and γ-tubulin (magenta). Scale bar, 10 μm; c. Quantification of ciliated cell percentage as shown in (B). Data is represented as mean ± SD (n = 3 experiments). One hundred fifty cells were scored per condition per experiment; ***P < 0.001, Student’s t-test

Fig. 4

TonEBP regulates the expression and activity of aurora A kinase. a. RPE1 cells were serum-starved for 24 h, transfected with TonEBP siRNA for 24 h, followed by serum stimulation, and harvested at different time points, as indicated; b. Cells were transfected with siRNAs against TonEBP for 24 h, followed by 24 h of serum starvation, and immunostained for ARL13b (green) and γ-tubulin (red). Scale bar, 25 μm; c. Quantification of ciliated cell percentage as shown in (B). Data is represented as mean ± SD (n = 3 experiments). Two hundred cells were scored per condition per experiment; **P < 0.01, ***P < 0.001, Student’s t-test; d. Cells were transfected with TonEBP siRNA for 24 h, serum-starved, followed by serum restimulation. Then, cells were harvested at different time points and subjected to Western blot and qPCR. qPCR data is represented as mean ± SD (n = 3 experiments). ***P < 0.001, Student’s t-test; e. Cells were transfected with TonEBP-mCherry plasmid for 24 h, followed by serum starvation, and subjected to Western blot and qPCR. qPCR data is represented as mean ± SD (n = 3 experiments). *P < 0.05, Student’s t-test

Fig. 5

Overexpression of TonEBP inhibits the CP110 removal from mother centrioles to suppress ciliogenesis. a. Cells were transfected with TonEBP-mCherry for 24 h, followed by serum starvation 24 h, and stained with antibodies against Rab11 (green), mCherry (red) and γ-tubulin (magenta). Scale bar, 25 μm; b. Cells were transfected with TonEBP-mCherry for 24 h, followed by serum starvation 24 h, and stained with antibodies against EHD1 (green), mCherry (red) and γ-tubulin (magenta). Scale bar, 25 μm. Arrows indicate colocalization of EHD1 and y-tubulin; c. Cells were transfected with TonEBP-mCherry for 24 h, followed by serum starvation 24 h, and stained with antibodies against CP110 (green), mCherry (red) and γ-tubulin (magenta). Scale bar, 10 μm. Zoom scale bar, 2 μm; d. Quantification of the percentage of cells with two CP110 puncta at the centrioles shown in (C). Data is represented as mean ± SD (n = 3 experiments). One hundred fifty cells were scored per condition per experiment; ***P < 0.001, Student’s t-test

Fig. 6

TonEBP regulates PCM1 and AZI1 localization. a. RPE1 cells were transfected with TonEBP-mCherry for 24 h, followed by serum starvation 24 h, and immunostained for PCM1 (green), mCherry (red) and γ-tubulin (magenta). Scale bar, 20 μm; b. Quantification of the percentage of PCM1 positive cells as shown in (A). Data is represented as mean ± SD (n = 3 experiments). Two hundred cells were scored per condition per experiment; ***P < 0.001, Student’s t-test; c. Cells were transfected with TonEBP-mCherry for 24 h, followed by serum starvation 24 h, and immunostained for AZI1 (green), mCherry (red) and γ-tubulin (magenta). Scale bar, 20 μm; d. Quantification of the percentage of AZI1 positive cells as shown in (A). Data is represented as mean ± SD (n = 3 experiments). Two hundred were scored per condition per experiment; ***P < 0.001, Student’s t-test; e. Cells were transfected with TonEBP siRNA for 24 h and immunostained for PCM1 (green) and γ-tubulin (red). Scale bar, 20 μm; f. Cells were transfected with TonEBP siRNA for 24 h and immunostained for AZI1 (green) and γ-tubulin (red). Scale bar, 20 μm

Fig. 7

A conceptual model proposing the regulatory influence of TonEBP on ciliogenesis