Ccdc61 controls centrosomal localization of Cep170 and is required for spindle assembly and symmetry

FIGURE 1:. Ccdc61 localizes MT-dependent at centrosomes and in their close proximity. (A) Costaining of endogenous Ccdc61 (green) with pericentrin or PCM1 (red) in U2OS and RPE1 cells. Chromatin is visualized by Hoechst33258 staining (blue). Magnified panels (magn.) show enlarged views of the boxed regions. Bar, 10 µm. (B) Graph shows the percentage of colocalization of endogenous Ccdc61 with either PCM1 or pericentrin in U2OS cells. Therefore, signals of PCM1 or pericentrin were used as a mask to measure the corresponding Ccdc61 signal intensity. n = 100 cells for each colocalization. Data represent mean value ± SD. (C) Endogenous Ccdc61 signal was quantified in a 3-µm2 circle around the centrosome in interphase or mitotic U2OS cells. Interphase Ccdc61 signal was normalized to 1.0. Interphase n = 54, mitosis = 40 centrosomes. Data represent mean value ± SD. (D) HeLa cells were released from a thymidine block and samples taken at indicated time points to localize endogenous Ccdc61 (green) at centrosomes by costaining pericentrin (red). Bar, 4 µm. (E) RPE1 cells were subjected to nocodazole (noco) to depolymerize MTs. After nocodazole washout, cells were fixed at the indicated time points…

FIGURE 2:. Ccdc61 is required for proper timing of mitosis, chromatin alignment and spindle asembly. (A) U2OS cells stably expressing tagged α-tubulin (green) and H2B (red) were imaged every 5 min for 15–19 h 72 h after siRNA transfection (ct: control; O2: Oligo2; O2R: Oligo2 + FLAG-Ccdc61 siRNA-resistant). Representative images from the movies were cropped out from the original time series (time in min). Bar, 10 µm. Quantification of the indicated treatments show the mitotic duration from nuclear envelope breakdown (start of chromosome condensation and movement of centrosomes) until telophase (decondensation of chromosomes) (top panel) and duration from bipolar spindle formation to anaphase (bottom panel). Data represent mean ± SD from four independent experiments, n > 167 cells. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired Student’s t test). (B) IF analysis of mitotic figures in U2OS cells indicated treatments. Mitotic spindles and centrosomes are visualized by antibody staining directed against α-tubulin (green) and pericentrin (red), costained with Hoechst33258 (DNA; blue). Bar, 10 µm. (C) U2OS cells stably expressing tagged α-tubulin and H2B were imaged every 5…

FIGURE 3:. Ccdc61 promotes MT nucleation. (A) U2OS cells were treated as indicated and subjected to nocodazole (noco) for 2 h to depolymerize MTs. After nocodazole washout, cells were harvested at the indicated time points and stained with antibodies directed against α-tubulin (green) and pericentrin (red). DNA was costained with Hoechst33258 (blue). Cells without nocodazole exposure were used as a control (ct). Bar, 10 µm. Quantification indicates the MT aster size formation 4 min after nocodazole release compared with the control (without nocodazole treatment) (normalized to 1.0). Data represent mean ± SD from three independent experiments, n > 80 cells. ****p < 0.0001 (unpaired Student’s t test). (B) HeLa S/A Ccdc61 miRNA Oligo2 tet on cells were used to perform a MT regrowth assay. α-tubulin was used to visualize mitotic spindles (green), pericentrin (red) used to stain the centrosomes, and Hoechst33258 to stain DNA (blue). Ccdc61 miRNA O2 expression was induced by the addition of 1 µg/ml doxycycline for 72 h (+Dox) prior cold treatment. For MT depolymerization cells with or without Dox addition were incubated at 4°C for 45 min, and then reincubated at 37°C for the indicated time…

FIGURE 4:. MT EB3-tracking measurements reveal a significant decrease in spindle symmetry in the absence of Ccdc61. (A–C) HeLa cells stably expressing EGFP-EB3 were used for EB3-tracking experiments. Cells were either treated with control (ct), Ccdc61 O2 siRNA (O2), or a combination of Ccdc61 O2 siRNA together with FLAG-Ccdc61 siRNA-resistant construct (O2R). To study mitotic properties, like directionality and orientation, the individual EB3 tips were imaged every 0.13 s and analyzed via an automated process. (A) Example images of a single time frame (top row) and a time projection from 125 s recordings (bottom row) of HeLa cells treated as indicated. Western blot shows the efficiency of indicated Ccdc61 Oligo that was tested by its ability to deplete transiently transfected FLAG-Ccdc61 or siRNA-resistant FLAG-Ccdc61 (O2R). α-tubulin served as a loading control. (B) EGFP-EB3 tracks from HeLa cells were analyzed as described in the Materials and Methods section and as published in the preprint (Jafarpour and Lorenz, 2017). The table represents velocity (µm/min), displacement (µm), duration (s) (mean ± SD.) and the total number of analyzed tracks (of 10 independent cells). The significance…

FIGURE 5:. Ccdc61 binds to and colocalizes with Cep170. (A) Immunoprecipitation (IP) experiments using HEK293T cells expressing GFP-Ccdc61 or FLAG-Ccdc61 as bait. Western blot analysis shows total cell extract (TCL) as input control and corresponding GFP- and FLAG-IP fraction. GFP or FLAG antibody was used to visualize the overexpressed Ccdc61 and Cep170 antibody (mouse and rabbit) was used for detection of endogenous Cep170. (B) Schematic representation of Cep170 full length (FL) and Cep170 fragments: Cep170 missing the FHA domain (ΔFHA), Cep170 N-terminus (N), Cep170 C-terminus, Cep170 first half of C-terminus (CI) and Cep170 second half of C-terminus (CII) (Guarguaglini et al., 2005). (C) GFP-IP using HEK293T cells expressing GFP-Cep170 fragments as bait and FLAG-Ccdc61. Western blot shows TCL as input control and GFP-IP fraction. GFP antibody was used to visualize overexpressed Cep170 and FLAG antibody to visualize overexpressed Ccdc61. (D) Costaining of Ccdc61 (green) together with either (left) Cep170 or (right) ninein (both in red) antibodies in interphase U2OS and RPE1 cells. Chromatin was visualized by costaining with Hoechst33258 (blue). Magnified panels (magn.) show enlarged…

FIGURE 6:. Ccdc61 regulates the centrosomal anchorage of the MT binding protein Cep170. (A) U2OS cells were transfected with the indicated siRNAs or siRNA/plasmid combinations and the localization of Cep170 analyzed. Representative images show staining with Cep170 (green) and γ-tubulin (red); DNA was costained with Hoechst33258 (blue). Scattered dot plot shows the Cep170 intensity quantified in a 25-µm2 circle around the centrosome normalized to the control (ct). Data represent mean ± SD from three independent experiments, n > 77 cells. ****p < 0.0001 (unpaired Student’s t test). (B) Centrosomal localization of Cep170 in interphase U2OS cells after indicated treatments was determined by visual inspection of 120–150 cells per treatment and experiment. Data represent mean ± SD from three independent experiments, ***p < 0.001 (unpaired Student’s t test). (C) Mitotic U2OS cells were transfected with the indicated siRNAs or siRNA/plasmid combinations. γ-tubulin was used to visualize mitotic centrosomes (green), Cep170 visualized in red, and DNA in blue (Hoechst33258). Magnified panels (magn.) show enlarged views of the boxed regions. Bar, 10 µm. (D, E) U2OS cells were transfected with the…

FIGURE 7:. Ccdc61 promotes the complex formation of Cep170 with TBK1. (A) IF analysis of U2OS and RPE1 cells in interphase. Images show endogenous localization of Ccdc61 (green) and phospho-TBK1 (pTBK1) in red visualized with specific antibodies. Chromatin was visualized by Hoechst33258 staining (blue). Bar, 10 µm. (B) Endogenous immunoprecipitation experiment using HeLa S/A Ccdc61 miRNA Oligo2 tet on cells. Cells were either untreated (–) or Ccdc61 miRNA O2 expression was induced via incubation with 1 µg/ml doxycycline for 72 h (+). Immunoprecipitation was performed using normal mouse IgG or mouse anti-Cep170 antibody coupled to ProteinG beads. Western blot shows TCL as input control and IP fractions. Mouse anti-Cep170 antibody was used to visualize endogenous Cep170 and anti-TBK1 antibody was used for detection of endogenous TBK1. Quantification shows the normalized ratio of TBK1/Cep170 intensity. Data represent mean ± SD from three independent experiments, *p < 0.05 (unpaired Student’s t test).