Microtubule asters anchored by FSD1 control axoneme assembly and ciliogenesis

Abstract

Defective ciliogenesis causes human developmental diseases termed ciliopathies. Microtubule (MT) asters originating from centrosomes in mitosis ensure the fidelity of cell division by positioning the spindle apparatus. However, the function of microtubule asters in interphase remains largely unknown. Here, we reveal an essential role of MT asters in transition zone (TZ) assembly during ciliogenesis. We demonstrate that the centrosome protein FSD1, whose biological function is largely unknown, anchors MT asters to interphase centrosomes by binding to microtubules. FSD1 knockdown causes defective ciliogenesis and affects embryonic development in vertebrates. We further show that disruption of MT aster anchorage by depleting FSD1 or other known anchoring proteins delocalizes the TZ assembly factor Cep290 from centriolar satellites, and causes TZ assembly defects. Thus, our study establishes FSD1 as a MT aster anchorage protein and reveals an important function of MT asters anchored by FSD1 in TZ assembly during ciliogenesis.

Fig. 1

FSD1 is required for ciliogenesis in human cells and zebrafish. a RPE-1 cells were transfected with the indicated siRNA and serum-starved for 48 h. Cells were stained with the indicated antibodies and examined by immunofluorescence microscopy. Scale bar, 5 μm. b Effects of FSD1 depletion on cilia formation in cycling (+Serum) or quiescent (−Serum) RPE-1 cells. Acetylated α-tubulin (Ac-tubulin) is ciliary marker. c Cilia defects induced by FSD1 knockdown in RPE-1 cells were rescued by expressing a GFP-tagged, RNAi-resistant form of FSD1. Data are presented as mean ± s.d. of three independent experiments. n number of cells. d, e Fsd1 morphants (aMO and sMO) displayed curved body and pericardial edema at 72 hpf. The arrows mark curved body and arrowheads mark pericardial edema. The fsd1-misMO were used as control. Scale bars, 1 mm. Data are presented as the mean ± s.d. of three independent experiments. f, g Fsd1 MOs (aMO and sMO) caused left-right asymmetry defects. The spaw probe was used to label the left lateral plate mesoderm in the whole-mount in situ hybridization at 18-somite stage. Scale bar, 150 μm. n number of fishes. h–j Fsd1 MOs (aMO and sMO) impaired ciliogenesis in Kupffer’s vesicle at 10-somite stage (10 s). Bars indicate the median. Scale bar, 10 μm. k Fsd1 knockout impaired ciliogenesis in Kupffer’s vesicle at 10 s. Bars indicate the median. Scale bar, 10 μm. In all panels, statistical comparisons between two groups were carried out by two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Loss of FSD1 blocks ciliogenesis at the stage of transition zone assembly. a A table summarizing the centrosome localization of the tested proteins in FSD1-depleted RPE-1 cells. CV ciliary vesicle. b–e RPE-1 cells were treated with control or FSD1 siRNAs followed by serum starvation for 48 h, then subjected to staining with indicated antibodies. Scale bars, 1 μm. b The Cep164 localization was not affected by FSD1 depletion. c FSD1 depletion did not affect the disruption of CP110 at the mother centriole. d, e Loss of FSD1 affected the localization of TMEM67 and NPHP8. f Depletion of FSD1 caused ciliary transition zone assembly defects. Representative electron micrographs of basal bodies in RPE-1 cells transfected with control (left) or FSD1 (right) siRNA, followed by serum starvation. Arrows indicate small membrane vesicles connected to FSD1-deficient centrioles through the distal appendage. Schematic diagrams summarizing the phenotype are shown. Scale bars, 200 nm. Data are presented as mean ± s.d. of three independent experiments. n number of cells. In all panels, statistical comparisons between two groups were carried out by two-tailed t-test. NS not significant, ***P < 0.001

Fig. 3

FSD1 forms a ring structure at the subdistal region of both centrioles. a Schematic of centrosome structure. The centrosome is composed of a pair of centrioles, and each centriole is divided into three portions, including proximal end (red), middle region (green), and distal end (blue). The older centriole of the pair (the mother) is often decorated with distal appendages and subdistal appendages. During ciliogenesis, the transition zone extends from the distal end of the mother centriole. b FSD1 was visualized with centriolar proximal (C-Nap1), distal (Cep162, Centrin2, and IFT88), subdistal appendage (ODF2), and distal appendage (Cep164) markers. ODF2, IFT88, and Cep164 localize to mother centrioles. Scale bar, 500 nm. c The centriolar localization of FSD1 was observed in ciliated and non-ciliated cells stained with indicated antibodies. Cep162, the centriole distal end marker. ARL13B, the primary cilia marker. Scale bars, 5 μm (main image) and 500 nm (magnified region). d The centriolar localization of FSD1 was observed in ciliated cells stained with indicated antibodies. Cep162, the centriole distal end marker. MKS1 and NPHP8, the ciliary transition zone markers. Scale bar, 500 nm. e RPE-1 cells were transfected with mCherry-Centrin2 plasmids and stained with the indicated antibodies. The samples were then imaged using the stimulated emission depletion microscopy (STED). Cep164 and FBF1 localize to the distal appendage of mother centrioles. Scale bars, 500 nm. f Schematic representation of the centrosome illustrates that FSD1 forms a ring structure at the middle region encircling both centrioles

Fig. 4

The maintenance of Cep290 by FSD1 at CS is required for TZ assembly. a Non-ciliated (+Serum) or ciliated (−Serum) RPE-1 cells were stained with indicated antibodies as shown. Scale bars, 5 μm (main image) and 1 μm (magnified region). b Effects of FSD1 depletion on the localization of Cep290 at centriolar satellites in cycling cells. RPE-1 cells transfected with control or FSD1 siRNAs were stained with anti-Cep290 (green) and anti-Centrin2 (red) antibodies. Scale bars, 5 μm (main image) and 1 μm (magnified region). c Effects of FSD1 depletion on the PCM1 localization at centriolar satellites in cycling RPE-1 cells. RPE-1 cells transfected with control or FSD1 siRNAs were stained with indicated antibodies. Scale bars, 5 μm (main image) and 1 μm (magnified region). d Effects of FSD1 or PCM1 depletion on the Cep290 localization at centriolar satellites in cycling RPE-1 cells. e, f Effects of FSD1 or PCM1 depletion on the relative intensity of the transition zone components TMEM67 (e) and NPHP8 (f) at mother centrioles in quiescent cells. g Model accounting for the TZ assembly defects caused by FSD1 depletion due to the disruption of CS Cep290. TZ transition zone, CS centriolar satellite. Data are presented as mean ± s.d. of three independent experiments. n number of cells. In all panels, statistical comparisons between two groups were carried out by two-tailed t-test. **P < 0.01, ***P < 0.001

Fig. 5

MT aster formation is required for Cep290 localization at CS and TZ assembly. a–c Percentage of cells with CS Cep290 after treatment with DMSO, 20 μM nocodazole (a), 1 μM taxol (b), or 10 μM ciliobrevin D (c) for 30 min. d RPE-1 cells transfected with control or FSD1 siRNA were subjected to nocodazole treatment for 2 h and released at indicated time points. Next, cells were fixed with 4% paraformaldehyde in PBS and stained with antibodies to α-tubulin to visualize microtubules (green) and Centrin2 to mark centrosomes (red). Percentage of cells with different microtubule regrowth statuses at indicated time points was quantified on the right panel. Scale bars, 5 μm (main image) and 1 μm (magnified region). e RPE-1 cells were transfected with indicated siRNA were subjected to nocodazole treatment for 2 h and released at indicated time points. Cells were then fixed and permeabilized with cold methanol and stained with antibodies to Cep290 (red), α-tubulin (green), and Centrin2 (purple). Percentage of cells with CS Cep290 after nocodazole release for 20 min was quantified on the right panel. Scale bars, 5 μm (main image) and 1 μm (magnified region). f Effects of FSD1, Ninein, or Kif3a depletion on the Cep290 localization at centriolar satellites in cycling RPE-1 cells. g, h Effects of FSD1, Ninein, or Kif3a depletion on the relative intensity of the transition zone components TMEM67 (g) and NPHP8 (h) at mother centrioles in quiescent cells. Data are presented as mean ± s.d. of three independent experiments. n number of cells. In all panels, statistical comparisons between two groups were carried out by two-tailed t-test. NS not significant, **P < 0.01, ***P < 0.001

Fig. 6

MT asters are anchored by FSD1 at mother centrioles. a RPE-1 cells transfected with control, FSD1, or Kif3a siRNAs were stained with anti-Ninein (green), anti-C-Nap1 (red), and anti-Ac-tubulin (purple) antibodies. Scale bar, 1 μm. b RPE-1 cells stained with indicated antibody were visualized using the stimulated emission depletion microscopy (STED). Schematic representation of the precise FSD1 localization on mother centrioles. Scale bar, 500 nm. c RPE-1 cells subjected to a microtubule regrowth assay were fixed and permeabilized with cold methanol and stained with antibodies against FSD1 and α-tubulin. The sample was then imaged through super-resolution 3D-SIM. Scale bars, 5 μm (main image) and 500 nm (magnified region). d Ectopically expressed full-length FSD1 co-localized with microtubule asters regrown after nocodazole release 10 min. Magnified centrioles are shown in the insets. Scale bars, 5 μm (main image) and 1 μm (magnified region). e Schematic diagram illustrates the different domains of FSD1 for its microtubule (MT) binding and centriolar (CEN) localization. f Endogenous FSD1 binds to microtubules. RPE-1 cell extracts were incubated with increasing amounts of Taxol-stabilized microtubules and then centrifuged (100,000 × g for 40 min) to form supernatant and pellet fractions. Samples from both fractions were probed with the indicated antibodies. The p38 and γ-tubulin were used as negative and positive controls for microtubule binding, respectively. g The FSD1-SPRY domain could directly bind to microtubules. The different purified MBP-tagged FSD1 truncations were incubated in the presence (+) or absence (−) of Taxol-stabilized microtubules (MTs) and were separated into supernatant (S) and pellet (P) fractions after high-speed centrifugation. Coomassie blue staining of both tubulins and FSD1 is indicated. h FSD1 could bind to microtubules (MTs), as shown by TIRF microscopy. GMPCPP seed MTs (with Alexa-647 and biotin labeled tubulin) were attached to a neutravidin-coated coverslip. Unpolymerized tubulin labeled with Alexa 561 was added and allowed to polymerize at room temperature for 5 min, then, 10 nM Flag-GFP-Vector or Flag-GFP-FSD1 (green) was added to the solution. The sample was imaged after reaction setup for 5 min through TIRF microscopy. Scale bars, 5 μm (main image) and 2 μm (magnified region)

Fig. 7

CAP350-FOP complex are essential for FSD1 centrosome localization. a Effects of depletion of known 12 anchoring proteins on the FSD1 localization at centrosomes. RPE-1 cells were transfected with indicated siRNAs and stained with antibodies to FSD1 (green) and γ-tubulin (red). Bars indicate the median. Significance between two groups was determined by two-tailed t-test. ***P < 0.001. Scale bar, 500 nm. b Immunofluorescence analysis of the relationship between FSD1 and the CAP350-FOP complex. RPE-1 cells transfected with the indicated siRNAs were stained with antibodies to CEP19, CAP350, or FOP (green), and to Centrin2 (red). Scale bar, 500 nm. c Immunoprecipitation and immunoblot analysis of the interaction of Flag-FSD1 with endogenous FOP, CAP350, and CEP19 in HEK293T cells. HEK293T cells were transfected with Flag-tagged FSD1; lysates immunoprecipitated with anti-Flag, as well as input lysates, were analyzed by immunoblot with indicated antibodies. d The interaction between endogenous FSD1 and CAP350, FOP in RPE-1 cells. Immunoblot analysis of lysates of RPE-1 cells immunoprecipitated with anti-FSD1, anti-CAP350, anti-FOP, or control IgG. e Model depicting the interactions between FSD1 and the CAP350-FOP complex. The size of arrowhead represents the affected strength on corresponding centrosome localization

Fig. 8

The microtubule-binding activity of FSD1 is essential for ciliogenesis. a A representative sequence of amino-acid alignment of the FSD1-SPRY domain was shown. The dotted box indicates six conserved arginine residues. b Expression of GFP-FSD1 WT, but not FSD1 3RE (R332E, R335E, and R337E) mutant, induced microtubule bundles. Scale bars, 5 μm (main image) and 1 μm (magnified region). c Mutation of three conserved arginine residues to glutamic acid (R332E, R335E, and R337E) abolishes the microtubule-binding activity of FSD1-SPRY domain. Purified FSD1-SPRY-WT, but not FSD1-SPRY-3RE, was co-pelleted with Taxol-stabilized microtubules. d Expression of GFP-FSD1 WT, but not 3RE mutant, rescued MT aster formation defects caused by FSD1 depletion. RPE-1 cells transfected with indicated siRNA and plasmids were subjected to a microtubule regrowth assay after nocodazole release 20 min. e Expression of GFP-FSD1 WT, but not 3RE mutant, rescued ciliogenesis defects caused by FSD1 depletion in quiescent cells. f Expression of GFP-FSD1 WT, but not 3RE mutant, rescued Cep290 localization at centriolar satellites in cycling cells. g Expression of GFP-FSD1 WT, but not 3RE mutant, rescued TMEM67 localization at centrioles in quiescent cells. h Expression of GFP-FSD1 WT, but not 3RE mutant, rescued NPHP8 localization at centrioles in quiescent cells. i Model of MT aster promoting ciliary transition zone assembly. FSD1 and other subdistal appendage proteins, such as Ninein, Kif3a, facilitate the formation of MT asters by anchoring MTs at the mother centriole. MT asters keep Cep290 at the centriolar satellites and promote transition zone assembly during ciliogenesis. MC mother centriole, MT microtubule, TZ transition zone, CV ciliary vesicle. Data are presented as mean ± s.d. of three independent experiments. n number of cells. In all panels, statistical comparisons between two groups were carried out by two-tailed t-test. **P < 0.01, ***P < 0.001