Figure 6. Association of UVRAG with CEP63 in Centrosomes is essential for Centrosome Stability.

(A) UVRAG interaction with CEP63. 293T cells were co-transfected with Flag-UVRAG and HA-CEP63. WCLs were IP with anti-Flag, followed by IB with anti-HA.

(B) Interaction between endogenous UVRAG and CEP63. WCLs of 293T cells were immunoprecipiated with control IgG, or anti-CEP63 (left panel), or anti-UVRAG (middle panel), followed by immunoblotting with anti-CEP63 or anti-UVRAG. Right panel shows the expression of endogenous UVRAG and CEP63 proteins.

(C) Colocalization between endogenous UVRAG and CEP63 at the centrosome. HeLa cells were co-stained with antibodies against UVRAG (green), CEP63 (purple), and γ-tubulin (red), followed by confocal microscopy. Arrows highlight the colocalization. Bar, 10 μm. See also Figure S5A.

(D) Confocal microscopy analysis of the colocalization of endogenous UVRAG and γ-tubulin in CEP63^+/+ and CEP63^−/− DT40 cells. Arrows denote colocalized UVRAG and γ-tubulin in CEP63^+/+, whereas insets highlight relative distribution of UVRAG with the γ-tubulin-labeled centrosomes in CEP63^−/− cells. Bars, 10 μm.

(E) UVRAG C-terminal region interacts with CEP63. 48 h posttransfection with HA-CEP63 together with Flag-UVRAG or its mutants, 293T WCLs were IP with anti-Flag followed by IB with anti-HA. WCLs were also used for IB with the indicated antibodies to show expression. See also Figure S5C.

(F) Schematic representation of UVRAG wild-type (WT) and its deletion mutants, and summary of their interactions with CEP63. Interaction was determined by coimmunoprecipitation of Flag-UVRAG with HA-CEP63 from 293T cell lysates. +, strong binding; −, no binding.

(G–H) UVRAG association with centrosomes is required for centrosome integrity and proper chromosomal segregation. (G) UVRAG^+/− ES cells reconstituted with empty vector (1^st row), Flag-UVRAG (2^nd row), or Flag-UVRAG^Δ270-442 (3^rd row) were stained with anti-γ-tubulin (red) for centrosomes, anti-α-tubulin (red) for mitotic asters, and DAPI for chromosomes (blue). Arrows denote over-duplicated centrosomes and abnormal spindle (left panel) or mis-segregated chromosomes in mitosis (right panel). Bar, 10 μm. Quantification of abnormal centrosome amplification, spindle malformation, and chromosomal missegregation from these cells is shown in (H). Data represents mean ± s.d. from three independent experiments. ***, P <0.001. See also Figure S5E–G.

(I–J) UVRAG association with centrosomes is required for chromosomal stability. The frequency of the numerical aberration of chromosomes from UVRAG^+/− ES cells reconstituted as in (G) with empty vector, Flag-UVRAG, or Flag-UVRAG^Δ270-442 was quantified (I) and reconstituted UVRAG expression was confirmed by immunoblotting (J).