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Rootletin forms centriole-associated filaments and functions in centrosome cohesion.

Bahe S, Stierhof YD, Wilkinson CJ, Leiss F, Nigg EA - J. Cell Biol. (2005)

Bottom Line: Similar to C-Nap1, rootletin is phosphorylated by Nek2 kinase and is displaced from centrosomes at the onset of mitosis.Whereas the overexpression of rootletin results in the formation of extensive fibers, small interfering RNA-mediated depletion of either rootletin or C-Nap1 causes centrosome splitting, suggesting that both proteins contribute to maintaining centrosome cohesion.The ability of rootletin to form centriole-associated fibers suggests a dynamic model for centrosome cohesion based on entangling filaments rather than continuous polymeric linkers.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Max-Planck-Institute for Biochemistry, D-82152 Martinsried, Germany.

ABSTRACT
After duplication of the centriole pair during S phase, the centrosome functions as a single microtubule-organizing center until the onset of mitosis, when the duplicated centrosomes separate for bipolar spindle formation. The mechanisms regulating centrosome cohesion and separation during the cell cycle are not well understood. In this study, we analyze the protein rootletin as a candidate centrosome linker component. As shown by immunoelectron microscopy, endogenous rootletin forms striking fibers emanating from the proximal ends of centrioles. Moreover, rootletin interacts with C-Nap1, a protein previously implicated in centrosome cohesion. Similar to C-Nap1, rootletin is phosphorylated by Nek2 kinase and is displaced from centrosomes at the onset of mitosis. Whereas the overexpression of rootletin results in the formation of extensive fibers, small interfering RNA-mediated depletion of either rootletin or C-Nap1 causes centrosome splitting, suggesting that both proteins contribute to maintaining centrosome cohesion. The ability of rootletin to form centriole-associated fibers suggests a dynamic model for centrosome cohesion based on entangling filaments rather than continuous polymeric linkers.

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Nek2 phosphorylates rootletin. (A) U2OS cells were transfected with wild-type (wt) or catalytically inactive (K37R) GFP-Nek2, and endogenous rootletin was stained with antibody. Bars, 5 μm. (B–D) After the coexpression of GFP-Nek2 with full-length myc-rootletin (B) or myc-rootletin fragments (D, arrowheads), total 293T cell extracts were probed by Western blotting with anti-myc antibody. (C) Schematic illustrating rootletin fragments (black bars indicate predicted coiled-coil). (E) In vitro kinase assay using wild-type or K37R Nek2 on His-rootletin NH2- and COOH-terminal fragments. Proteins were resolved by SDS-PAGE, and gels were subjected to autoradiography (left) and Coomassie blue staining (right). Arrowheads indicate the migration of His-rootletin fragments; the arrow points to autophosphorylated Nek2.
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fig3: Nek2 phosphorylates rootletin. (A) U2OS cells were transfected with wild-type (wt) or catalytically inactive (K37R) GFP-Nek2, and endogenous rootletin was stained with antibody. Bars, 5 μm. (B–D) After the coexpression of GFP-Nek2 with full-length myc-rootletin (B) or myc-rootletin fragments (D, arrowheads), total 293T cell extracts were probed by Western blotting with anti-myc antibody. (C) Schematic illustrating rootletin fragments (black bars indicate predicted coiled-coil). (E) In vitro kinase assay using wild-type or K37R Nek2 on His-rootletin NH2- and COOH-terminal fragments. Proteins were resolved by SDS-PAGE, and gels were subjected to autoradiography (left) and Coomassie blue staining (right). Arrowheads indicate the migration of His-rootletin fragments; the arrow points to autophosphorylated Nek2.

Mentions: C-Nap1 was identified as a substrate of Nek2 (Fry et al., 1998a). To determine whether rootletin might also be regulated by Nek2, we transfected U2OS cells with wild-type or catalytically inactive Nek2 and monitored the localization of endogenous rootletin by IF microscopy. Whereas the overexpression of inactive Nek2 did not detectably affect rootletin localization, wild-type kinase caused the dissociation of rootletin from centrosomes (Fig. 3 A). In addition, wild-type Nek2 caused centrosome splitting as expected (Fry et al., 1998b). Active Nek2, but not the catalytically inactive mutant, also retarded the mobility of rootletin (Fig. 3 B). This mobility shift was sensitive to phosphatase treatment (unpublished data), indicating that rootletin was phosphorylated by Nek2 either directly or indirectly. When different rootletin fragments (Fig. 3 C) were coexpressed with active Nek2, they were all upshifted (Fig. 3 D), suggesting the existence of multiple phosphorylation sites. To determine whether rootletin was a direct substrate of Nek2, in vitro kinase assays were performed. Upon the incubation of NH2- and COOH-terminal fragments with Nek2 in the presence of γ-[32P]ATP, both were readily phosphorylated by active but not inactive Nek2 (Fig. 3 E), which is consistent with the aforementioned results. All samples containing active Nek2 showed an additional phosphorylated band at ∼50 kD (Fig. 3 E), reflecting autophosphorylation (Fry et al., 1995).


Rootletin forms centriole-associated filaments and functions in centrosome cohesion.

Bahe S, Stierhof YD, Wilkinson CJ, Leiss F, Nigg EA - J. Cell Biol. (2005)

Nek2 phosphorylates rootletin. (A) U2OS cells were transfected with wild-type (wt) or catalytically inactive (K37R) GFP-Nek2, and endogenous rootletin was stained with antibody. Bars, 5 μm. (B–D) After the coexpression of GFP-Nek2 with full-length myc-rootletin (B) or myc-rootletin fragments (D, arrowheads), total 293T cell extracts were probed by Western blotting with anti-myc antibody. (C) Schematic illustrating rootletin fragments (black bars indicate predicted coiled-coil). (E) In vitro kinase assay using wild-type or K37R Nek2 on His-rootletin NH2- and COOH-terminal fragments. Proteins were resolved by SDS-PAGE, and gels were subjected to autoradiography (left) and Coomassie blue staining (right). Arrowheads indicate the migration of His-rootletin fragments; the arrow points to autophosphorylated Nek2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171225&req=5

fig3: Nek2 phosphorylates rootletin. (A) U2OS cells were transfected with wild-type (wt) or catalytically inactive (K37R) GFP-Nek2, and endogenous rootletin was stained with antibody. Bars, 5 μm. (B–D) After the coexpression of GFP-Nek2 with full-length myc-rootletin (B) or myc-rootletin fragments (D, arrowheads), total 293T cell extracts were probed by Western blotting with anti-myc antibody. (C) Schematic illustrating rootletin fragments (black bars indicate predicted coiled-coil). (E) In vitro kinase assay using wild-type or K37R Nek2 on His-rootletin NH2- and COOH-terminal fragments. Proteins were resolved by SDS-PAGE, and gels were subjected to autoradiography (left) and Coomassie blue staining (right). Arrowheads indicate the migration of His-rootletin fragments; the arrow points to autophosphorylated Nek2.
Mentions: C-Nap1 was identified as a substrate of Nek2 (Fry et al., 1998a). To determine whether rootletin might also be regulated by Nek2, we transfected U2OS cells with wild-type or catalytically inactive Nek2 and monitored the localization of endogenous rootletin by IF microscopy. Whereas the overexpression of inactive Nek2 did not detectably affect rootletin localization, wild-type kinase caused the dissociation of rootletin from centrosomes (Fig. 3 A). In addition, wild-type Nek2 caused centrosome splitting as expected (Fry et al., 1998b). Active Nek2, but not the catalytically inactive mutant, also retarded the mobility of rootletin (Fig. 3 B). This mobility shift was sensitive to phosphatase treatment (unpublished data), indicating that rootletin was phosphorylated by Nek2 either directly or indirectly. When different rootletin fragments (Fig. 3 C) were coexpressed with active Nek2, they were all upshifted (Fig. 3 D), suggesting the existence of multiple phosphorylation sites. To determine whether rootletin was a direct substrate of Nek2, in vitro kinase assays were performed. Upon the incubation of NH2- and COOH-terminal fragments with Nek2 in the presence of γ-[32P]ATP, both were readily phosphorylated by active but not inactive Nek2 (Fig. 3 E), which is consistent with the aforementioned results. All samples containing active Nek2 showed an additional phosphorylated band at ∼50 kD (Fig. 3 E), reflecting autophosphorylation (Fry et al., 1995).

Bottom Line: Similar to C-Nap1, rootletin is phosphorylated by Nek2 kinase and is displaced from centrosomes at the onset of mitosis.Whereas the overexpression of rootletin results in the formation of extensive fibers, small interfering RNA-mediated depletion of either rootletin or C-Nap1 causes centrosome splitting, suggesting that both proteins contribute to maintaining centrosome cohesion.The ability of rootletin to form centriole-associated fibers suggests a dynamic model for centrosome cohesion based on entangling filaments rather than continuous polymeric linkers.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Max-Planck-Institute for Biochemistry, D-82152 Martinsried, Germany.

ABSTRACT
After duplication of the centriole pair during S phase, the centrosome functions as a single microtubule-organizing center until the onset of mitosis, when the duplicated centrosomes separate for bipolar spindle formation. The mechanisms regulating centrosome cohesion and separation during the cell cycle are not well understood. In this study, we analyze the protein rootletin as a candidate centrosome linker component. As shown by immunoelectron microscopy, endogenous rootletin forms striking fibers emanating from the proximal ends of centrioles. Moreover, rootletin interacts with C-Nap1, a protein previously implicated in centrosome cohesion. Similar to C-Nap1, rootletin is phosphorylated by Nek2 kinase and is displaced from centrosomes at the onset of mitosis. Whereas the overexpression of rootletin results in the formation of extensive fibers, small interfering RNA-mediated depletion of either rootletin or C-Nap1 causes centrosome splitting, suggesting that both proteins contribute to maintaining centrosome cohesion. The ability of rootletin to form centriole-associated fibers suggests a dynamic model for centrosome cohesion based on entangling filaments rather than continuous polymeric linkers.

Show MeSH
Related in: MedlinePlus