Cep44 functions in centrosome cohesion by stabilizing rootletin

Abstract

The centrosome linker serves to hold the duplicated centrosomes together until they separate in late G2/early mitosis. Precisely how the linker is assembled remains an open question. In this study, we identify Cep44 as a novel component of the linker in human cells. Cep44 localizes to the proximal end of centrioles, including mother and daughter centrioles, and its ablation leads to loss of centrosome cohesion. Cep44 does not impinge on the stability of C-Nap1 (also known as CEP250), LRRC45 or Cep215 (also known as CDK5RAP2), and vice versa, and these proteins are independently recruited to the centrosome. Rather, Cep44 associates with rootletin and regulates its stability and localization to the centrosome. Our findings reveal a role of the previously uncharacterized protein Cep44 for centrosome cohesion and linker assembly.

Keywords: Centrosome; Cep44; Cohesion; Rootletin; Splitting.

Fig. 1.

Cep44 is a proximal end centriolar protein whose level changes during the cell cycle. (A) U2OS cells or RPE-1 G0 cells were stained with the indicated antibodies with or without DAPI. GT335, glutamylated tubulin. Scale bar: 10 μm. (B) U2OS cells were stained with the indicated antibodies and images were acquired with 3D-SIM. M, mother centriole; D, daughter centriole. Scale bar: 200 µm. (C) RPE-1 G0 cells expressing GFP–Cep44 were stained with the indicated antibodies. Scale bar: 10 μm. (D) RPE-1 cells were stained with DAPI and the indicated antibodies. Scale bar: 10 μm. (E) Quantification of Cep44 fluorescence intensity at the centrosome. Results are mean±s.e.m. based on three independent experiments with 20 cells per experiment (n=60). **P<0.01 (one-way ANOVA). (F) RPE-1 cell lysates were western blotted with antibodies against Cep44, C-Nap1 or rootletin. α-tubulin was used as loading control. AS, asynchronous. A representative experiment is shown. (G) Quantification of Cep44 level relative to α-tubulin. Results are mean±s.e.m. based on five independent experiments (n=5). *P<0.05 (one-way ANOVA).

Fig. 2.

Cep44 is required for centrosome cohesion. (A) U2OS cells transfected with non-specific (NS) or Cep44 siRNAs (oligo 1 or 2) were stained with the indicated antibodies. Scale bar: 10 µm. (B) The percentage of U2OS cells with separated γ-tubulin dots is presented. (C) The percentage of GFP-positive U2OS cells with separated γ-tubulin dots is presented. (D) FACS analysis of the cell cycle states of U2OS cells transfected with NS or Cep44 siRNAs. For B,C, three independent experiments were performed, and each experiment involved counting >100 cells for each condition.

Fig. 3.

Cep44 is required for the localization of rootletin to the centrosome. (A,B) Quantification of the distance between two centrin dots in U2OS or RPE-1 cells. G1 cells with the best knockdown were chosen for quantification. (C,D) Quantification of rootletin fluorescence intensity at the centrosome in U2OS or RPE-1 cells. (E,F) Quantification of rootletin fluorescence intensity at the centrosome in U2OS or RPE-1 cells. GFP-positive cells were chosen for quantification. (G,H) Quantification of Cep68 fluorescence intensity at the centrosome in U2OS or RPE-1 cells. For A–H, results are mean+s.e.m. based on three independent experiments with 20 cells per experiment (n=60). **P<0.01 (two-tailed Student's t-test).

Fig. 4.

Cep44 specifically binds to and stabilizes rootletin, and its localization is independent of known linker proteins. (A) U2OS cells transfected with non-specific (NS), C-Nap1, LRRC45, rootletin, Cep215 or Cep68 siRNAs were stained with the indicated antibodies. Scale bar: 10 µm. (B) U2OS cells were transfected with NS or Cep44 siRNAs (oligo 1 or 2). Lysates were western blotted with the indicated antibodies. α-tubulin was used as loading control. (C) U2OS cells were transfected with NS, C-Nap1, LRRC45, rootletin, Cep215 or Cep68 siRNAs. Lysates were western blotted with the indicated antibodies. α-tubulin was used as loading control. (D) Western blotting of endogenous Cep44, rootletin and C-Nap1 after immunoprecipitation from U2OS lysates with control, anti-Cep44 or anti-rootletin antibodies. IN, input (5 %); IP, immunoprecipitation. (E) U2OS cells were transfected with NS or Cep44 siRNAs and treated with vehicle, MG132 or chloroquine. Lysates were western blotted with the indicated antibodies. α-tubulin was used as loading control.