Fig 2: The centrosome and the Golgi MTOC associate with the ZIKV viroplasm, (a) IF staining for the centrosome protein CEP192 (red) in mock- and ZIKV (MR766)-infected SNB19 cells 24 h p.i. Viroplasms are marked by Env (green), and MTs are white. Insets show zoomed area where the centrosome is present, and arrowheads point to the centrosome; (b) quantification of the number of cells with 4 or fewer centrioles, measured by the number of centrin foci in mock- and ZIKV-infected cells 24 h p.i. T-test comparing centrosome number for the following conditions from four independent experiments (30–61 infected cells/experiment), mock vs. ZIKV (p = 0.67, n.s.); (c–e) representative images of the centrosomal proteins (red) CEP152 (c), centrin (d), and gamma-tubulin complex protein 2 (GCP2) (e) staining in mock- and ZIKV-infected cells 24 h p.i. Viroplasms are marked by Env (green); (f) normalized total intensity of centrosome proteins CEP152, centrin, and gamma-tubulin complex protein 2 (GCP2) in mock- and ZIKV-infected cells 24 h p.i. T-tests comparing mock- and ZIKV-infected intensity at the centrosome for the following centrosomal proteins from eight independent experiments (23–84 infected cells/experiment), CEP152: mock vs. ZIKV (p-value < 0.001, ***), centrin: mock vs. ZIKV (p-value < 0.01, **), GCP2: mock vs. ZIKV (p-value = 0.70, n.s.); (g,g’) IF staining of the Golgi protein GM130 (red) and MTs (white) in mock (g) and ZIKV-infected (g’) cells 24 h p.i. Viroplasms are marked by Env (green). Yellow arrowheads point to the Golgi at the core, and white arrowheads point to the Golgi surrounding the viroplasm; (h) MT regrowth assay. Noc-treated cells were imaged immediately (top) and after 1.5 min of regrowth (bottom) of mock- and ZIKV-infected cells. IF staining of Env (green), MTs (red), and GM130 (white). For the regrowth (bottom) panel, representative images of mock- and ZIKV-infected cells from two independent experiments that show MT regrowth (1–1.5 min) from the centrosome and the Golgi MTOC (mock, n = 15 imaged cells; ZIKV, n = 27 imaged cells). The right panel for regrowth shows zoomed insets of the centrosome and the Golgi MT regrowth; (i) illustration of ZIKV-infected cells depicting the localization of the centrosome, the Golgi, and the organization of MTs at the viroplasm; for (b,f), the mean percentage of cells with 4 or fewer centrioles (b) and mean centrosomal protein intensities (f) from each image were normalized to mock-infected combined means; significance was assessed using an unpaired two-tailed Student’s t-test. Error bars are means ± s.e.m.; for all IF staining, DAPI labels the nucleus (blue). Scale bars: 5 μm.

Fig 3: PPP1R35 is critical for centrosome duplication.(a) Sequence of PPP1R35 with the regions targeted by the siRNA used in this study. (b) Schematic representing the timing of the siRNA treatments used in the PPP1R35 knockdown experiments. (c) PPP1R35 protein levels were reduced in U2OS cells by treatment with siRNAs targeting either an exon (red bars) or the 3’ untranslated region (UTR; orange bars) of the mRNA. The percentage of cells with greater than one CEP152-labeled centrosome, a protein recruited in the later stages of centrosome duplication, is plotted. In this and following figures, the grey bar represents U2OS cells treated with a control scrambled siRNA strand; red bar, U2OS cells treated with a siRNA targeting an exon of PPP1R35; orange bar, U2OS cells treated with a siRNA targeting the 3’ UTR of endogenous PPP1R35; the green bar represents U2OS cells treated with the 3’UTR-targeting siRNA with exogenously expressed GFP-PPP1R35. (d) Representative micrographs showing decreased numbers of CEP152 positive centrioles in cells treated with PPP1R35 siRNA. Note that CETN1 was not severely perturbed upon treatment with PPP1R35 siRNA. High magnifications of the centrosome for each image are shown in the bottom left corner. Scale bars, 2 µm. (e) U2OS cells treated with either an exon (red bars) or the 3’UTR (orange bars) targeting siRNA (grey bars- control siRNA treated) for the different components of the centriole duplication pathway. The corresponding step of centriole duplication to which the different components belong is depicted with arrows below the plot, with the impacted steps highlighted with a red box. (f) PPP1R35 protein levels were reduced in U2OS cells by treatment with either of the two PPP1R35-targeting siRNA strands, for time points ranging from 24 to 144 hr and subsequently stained for CEP152. The timeline depicted indicate the timing of the siRNA treatments and subsequent time points when cells were fixed and stained for immunofluorescence. (g) 3DSIM micrographs of scrambled- and PPP1R35-siRNA treated cells stained for SAS6 (red) and CEP152 (green). Cells in which two engaged centrosomes were present were analyzed for the presence of a SAS6 cartwheel. Fractions of cells falling into each category are indicated in the top left corner of each micrograph. All scale bars, 250 nm. All error bars show the standard deviation of at least three replicate experiments. Statistics for significance were determined using Barnard’s test. All statistical values can be found in Supplementary file 4.10.7554/eLife.37846.019Figure 3—source data 1.Source data for Figure 3c and d (blue tabs) (PPP1R35 siRNA phenotype) and 3f (siRNA time-course, labeled with antibodies against CEP152).The blue tabs are also the source data for Figure 3—figure supplement 4.

Fig 4: Cep152 is an inhibitor of mitotic spindle assembly and forms a complex with Cep57. (A) HEK293 FlpIn T-Rex cell lines expressing eGFP–Cep152WT (WT) or eGFP–Cep152DBK (DBK) were treated as shown in Fig. 2A and stained against the indicated proteins. Microtubules were labelled using an α-tubulin antibody and DNA was stained with DAPI. (B) The fluorescence intensity of tubulin in cells from A was measured in the whole cell and normalized against the eGFP–Cep152WT cell line (left). N cells=78 (WT), 77 (DBK). The fluorescence intensity of PCNT at the centrosome in cells from A was measured as indicated in Fig. 1B and normalized against the eGFP–Cep152WT cell line (right). N cells=158 (WT), 154 (DBK). ****P<0.0001 (simple one-way ANOVA with Dunnett's multiple comparison test). (C) Setup of experiments to understand microtubule stability and nucleation. Cells were collected at the indicated time points. (D) HEK293 FlpIn T-Rex cell lines expressing eGFP–Cep152WT or eGFP–Cep152DBK were treated according to the schedule in C and fixed to assess microtubule stability. Cells were stained to detect microtubules (anti-α-tubulin) and acetylated microtubules (antibody against acetylated Lys40 of α-tubulin), and DNA was labelled with DAPI (left). The fluorescence intensity of acetylated microtubules was measured in the whole cell and normalized to unmodified microtubules (right). N cells=19 (WT) and 23 (DBK). n.s., not significant (Mann–Whitney U-test). (E) HEK293 FlpIn T-Rex cell lines expressing eGFP–Cep152WT or eGFP–Cep152DBK were treated according to the schedule in C and fixed to assess microtubule nucleation. Cells were fixed and stained against tubulin and DNA was labelled with DAPI (left). The fluorescence intensity of tubulin was measured in the whole cell at 30 min after shifting cells back to 37°C and normalized against the eGFP–Cep152WT cell line (right). N cells=61 (WT), 66 (DBK). ****P<0.0001 (simple one-way ANOVA with Dunnett's multiple comparison test). (F) Cells from E were analysed for their spindle morphology. Data are the mean±s.d. percentages of cells with each spindle phenotype from three experiments. **P=0.0038 (two-tailed paired Student's t-test). Box plots in B,D,E show the median (line), 25–75% range (box) and 5–95% range (whiskers). Scale bars: 5 µm.

Fig 5: CCDC66 co-localizes and interacts with PCM proteins and spindle/midbody MAPs.(A) mNG-CCDC66 localizes to the centrosome throughout the cell cycle. U2OS::mNG-CCDC66 cells were fixed with 4% PFA and stained for gamma-tubulin and DAPI. Still deconvolved confocal images represent centrosomal co-localization of mNG-CCDC66 with gamma-tubulin during different stages of cell division. Scale bar: 5 μm, insets show 4× magnifications of the centrosomes. (B) Localization of mNG-CCDC66 in U2OS cells relative to PCM proteins. U2OS::mNG-CCDC66 cells were fixed with 4% PFA and stained for CEP192, CEP152, or CDK5RAP2 and DAPI. Deconvolved confocal images represent their relative localization at the centrosomes during metaphase. Scale bar: 5 μm, insets show 4× magnifications of the centrosomes. (C) Effect of microtubule depolymerization on CCDC66 localization at the centrosome. U2OS::mNG-CCDC66 cells were treated with 0.1% DMSO or 5 μg/ml nocodazole for 1 h. Cells were then fixed with 4% PFA and stained for gamma-tubulin or CDK5RAP2 and DAPI. Scale bar: 5 μm, insets show 4× magnifications of the centrosomes. (D) Localization of mNG-CCDC66 in RPE1 cells relative to midbody proteins. RPE1::mNG-CCDC66 cells were fixed with methanol and stained for mNeonGreen and PRC1, Cep55, Kif23 (MKLP1), or pAurora A/B/C and DAPI. Graphs show the plot profiles to assess co-localization with the indicated marker. Using ImageJ, a straight line was drawn on the midbody and intensity along the distance was plotted on Graphpad Prism. (E) Co-immunoprecipitation of Flag-miniTurbo-CCDC66 or Flag-miniTurbo with PCM proteins from HEK293T cells. Flag-miniTurbo-CCDC66 was co-transfected with myc-BirA-Cep192, myc-BirA-Cep152, and myc-BirA-CDK5RAP2 in HEK293T cells. Flag-miniTurbo-CCDC66 and Flag-miniTurbo were precipitated with Flag beads and input and eluates (IP) were blotted with Flag and myc antibodies to assess the interaction. HEK293T cells were also co-transfected with Flag-miniTurbo-CCDC66 and Ruby-Gamma-tubulin-T2A-mNG-EB1 fusion construct. Flag-miniTurbo-CCDC66 was precipitated with Flag beads and Input and eluates (IP) were blotted with Flag, EB1, and gamma-tubulin antibodies. (F) Co-immunoprecipitation of Flag-CCDC66 with midbody proteins from HEK293T cells. Flag-CCDC66 was co-transfected with GFP, GFP-Cep55, or GFP-PRC1 in HEK293T cells. Flag-CCDC66 was precipitated with Flag beads and Input and eluates (IP) were blotted with Flag and GFP antibodies to assess the interaction. The data underlying the graphs showing the plot profiles in the figure can be found in S1 Data. CCDC66, coiled-coil domain-containing protein 66; CDK5RAP2, CDK5 regulatory subunit-associated protein 2; Cep55, centrosomal protein of 55 kDa; CEP192, centrosomal protein of 192 kDa; CEP152, centrosomal protein of 152 kDa; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; GFP, green fluorescent protein; Kif23, kinesin-like protein KIF23 (MKLP1, mitotic kinesin-like protein 1); pAurora A/B/C, phospho-AuroraA/B/C; PCM, pericentriolar material; PFA, paraformaldehyde; PRC1; protein regulator of cytokinesis 1.