Fig 1.
Overexpression of CSAP promotes the formation of centrosome-free MT asters on mitotic spindles.
(A) U2OS cells transiently expressing GFP-CSAP (a) or GFP-centrin (mitosis, b-d; interphase, e). Cells were stained for α-tubulin (Red) and DNA (Blue). GFP-CSAP localizes to centrosomes; cells overexpressing GFP-CSAP show multiple GFP foci on the mitotic spindles. Scale bar, 5 μm. Over-expression is indicated by the graph on the left side. (B, C) Proportion of U2OS (B) or HeLa cells (C) transiently expressing GFP-centrin or GFP-CSAP with the number of GFP foci on mitotic spindles. Data represent the mean ± SD of three experiments. (D) U2OS cells transiently expressing TrAP (a) or TrAP-CSAP (b, c). Cells were stained for α-tubulin (red), γ-tubulin (green), and DNA (blue). Microtubule asters with γ-tubulin (arrowheads) and without γ-tubulin (arrows) are indicated. (E) Proportion of U2OS cells transiently expressing TrAP or TrAP-CSAP with the number of γ-tubulin foci on mitotic spindles. Data represent the mean ± SD of three experiments. (F) U2OS cells transiently expressing GFP-CSAP. The cells were stained forα-tubulin (red), γ-tubulin (green), and DNA (blue). Microtubule asters with γ-tubulin (arrowheads) and without γ-tubulin (arrows) are indicated. (G) Proportion of U2OS cells transiently expressing GFP-CSAP with the number of GFP-CSAP foci vs. γ-tubulin foci on mitotic spindles. (H, I) Comparison of α-tubulin (H) and γ-tubulin (I) on mitotic spindles in cells transiently expressing control (blue) or TrAP-CSAP (red). In (I), two types of γ-tubulin foci at centrosomes and centrosome-free MT asters are separately shown. Data represent the mean ± SD relative intensity. *** indicates p < 0.005 (n = ~40).
Fig 2.
Time-lapse analysis of CSAP-overexpressing cells during mitosis.
Panels summarize time-lapse recordings of control U2OS (A, S1 Movie) and TrAP-CSAP-overexpressing cells (B, S2 Movie) expressing GFP–α-tubulin (upper) and H2B-mRFP (lower). Times are minutes after NEBD (0’).
Fig 3.
Pericentrosomal material composition of centrosome-free MT asters.
(A, C) Mitotic U2OS cells transiently expressing control (a) and TrAP-CSAP (b). Cells were stained for α-tubulin (Red), DNA (Blue), and CDK5RAP2 (A; Green) or Aurora A (C; Green). (B, D, E) Mitotic U2OS cells transiently expressing GFP-CSAP (Green). Cells were stained for α-tubulin (White), DNA (Blue), and CDK5RAP2 (B; Red), Aurora A (D; Red), or pericentrin (E; Red). (F) Mitotic U2OS cells transiently expressing control (a) and TrAP-CSAP (b). The cells were stained for γ-tubulin (Red), DNA (Blue), and Aurora A. Scale bar, 5 μm. (G, H) Comparison of CDK5RAP2 (G) and Aurora A (H) at mitotic spindle poles in cells transiently expressing control (blue) or TrAP-CSAP (red). In (H), two types of Aurora A foci at centrosomes and centrosome-free MT asters are separately shown. Data represent the mean ± SD relative intensity. *** indicates p < 0.005 (n = ~30).
Fig 4.
Increasing NuMA and polyglutamylation on mitotic spindles containing centrosome-free MT asters.
(A, D) Mitotic U2OS cells transiently expressing control (a) and TrAP-CSAP (b). Cells were stained for α-tubulin (Red), DNA (Blue), and NuMA (Green). (B) Mitotic U2OS cells transiently expressing GFP-CSAP (A; Green) or polyglutamylation (D). Cells were stained for NuMA (Red), α-tubulin (White), and DNA (Blue). (C) Mitotic U2OS cells transiently expressing GFP-CSAP (Green). Cells were stained for polyglutamylation (Red), CDK5RAP2 (White), and DNA (Blue). Scale bar, 5 μm. (E, F) Comparison of NuMA (E) and polyglutamylation (F) on mitotic spindles in cells transiently expressing control (blue) or TrAP-CSAP (red). (G, H) Comparison of α-tubulin vs. NuMA (G) or polyglutamylation (H) on the spindles around the centrosomes in transiently expressing control (blue) or on centrosome-free MT asters in cells transiently expressing TrAP-CSAP (red). Data represent the mean ± SD relative intensity. *** indicates p < 0.005 (n = ~30).
Fig 5.
CSAP depletion causes NuMA mislocalization during mitosis.
(A) At 72 h after transfection with control or pooled siRNAs for CSAP, U2OS cells were lysed and immunoblotted with anti-CSAP antibody. (B) Proportion of control or CSAP siRNA-transfected U2OS cells with γ-tubulin foci on mitotic spindles. Data represent the mean ± SD of three experiments. (C, D, E) Control (a) or CSAP (b, c) siRNA-transfected U2OS cells were fixed and immunostained for α-tubulin (Red), DNA (Blue), and γ-tubulin (C; Green), Aurora A (D; Green), or NuMA (E; Green). (F) Control (a) and CSAP (b) siRNA-transfected U2OS cells were fixed and immunostained for polyglutamylation (Green), CDK5RAP2 (Red), α-tubulin (white), and DNA (Blue). Scale bar, 5 μm. (G) Comparison of α-tubulin, γ-tubulin, Aurora A, NuMA, and polyglutamylation staining intensity on mitotic spindles in control (blue) or CSAP-depleted (red) cells. (H, I) Comparison of α-tubulin vs. NuMA (H) or polyglutamylation (I) immunostaining on the spindles around the centrosomes in control (blue) or CSAP-depleted (red) cells. Data represent the mean ± SD relative intensity. ** and *** indicate p < 0.01 and p < 0.005, respectively. (n = ~30).
Fig 6.
MT regrowth assay in CSAP over-expressing cells.
(A, B, C) MT regrowth assay of mitotic U2OS cells transiently over-expressing TrAP-centrin (a) or GFP-CSAP (b). After recovery from cold treatment for 0 min (A), 3 min (B), and 25 min (C), the cells were stained for α-tubulin (Red), γ-tubulin (Green), and DNA (Blue). (c) Quantitative comparisons of α-tubulin are shown. Data represent the mean ± SD relative intensity. ** and *** indicate p < 0.01 and p < 0.005, respectively. (D) Mitotic U2OS cells transiently over-expressing TrAP-CSAP after cold treatment. Cells were stained for TrAP-CSAP (Green), γ-tubulin (Red), and DNA (Blue). (E) Images of mitotic U2OS cells transiently over-expressing GFP-Centrin (a) or GFP-CSAP (b) after 1 h of treatment with nocodazole before fixation. The cells were stained for α-tubulin (Red) and DNA (Blue). (F, G) Mitotic U2OS cells transiently over-expressing GFP-Centrin (a; Green) or GFP-CSAP (b) with nocodazole (F) or monastrol (G) for 12 h. Cells were stained for α-tubulin (Red) and DNA (Blue). Scale bar, 5 μm. (H, I) Quantitative comparisons of α-tubulin in cells treated with nocodazole (H) or monastrol (I) for 1 h. Data represent the mean ± SD relative intensity. *** indicates p < 0.005 (n = ~30).