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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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siRNA depletion of rootletin causes centrosome splitting. (A) U2OS cells were transfected for 48 or 72 h with control (GL2) or different rootletin-specific siRNA duplexes and were costained for rootletin and γ-tubulin. Arrows and arrowheads mark paired centrioles and split centrosomes, respectively. Bars, 10 μm. (B) Quantitation of centrosome splitting in control (GL2) or rootletin siRNA-treated cells (oligonucleotide 222). Centrosomes were counted as split when the distance between centrioles was >2 μm. Error bars represent SEM.
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fig4: siRNA depletion of rootletin causes centrosome splitting. (A) U2OS cells were transfected for 48 or 72 h with control (GL2) or different rootletin-specific siRNA duplexes and were costained for rootletin and γ-tubulin. Arrows and arrowheads mark paired centrioles and split centrosomes, respectively. Bars, 10 μm. (B) Quantitation of centrosome splitting in control (GL2) or rootletin siRNA-treated cells (oligonucleotide 222). Centrosomes were counted as split when the distance between centrioles was >2 μm. Error bars represent SEM.

Mentions: To test more directly whether rootletin was involved in centrosome cohesion, we used siRNA. Three different rootletin-specific duplex oligonucleotides caused the near complete loss of rootletin from the centrosome (Fig. 4 A) and, concomitantly, extensive centrosome splitting (Fig. 4). In quantitative terms, >85% of cells that were treated with the most efficient siRNA duplex displayed split centrosomes, whereas <11% of cells showed a similar phenotype in GL2 control–treated cells (Fig. 4 B). Rootletin depletion caused centrosome splitting not only in U2OS but also in hTERT-RPE1 and A549 cells (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200504107/DC1). Centrosome splitting was observed upon the siRNA-mediated depletion of C-Nap1 (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200504107/DC1), which is a protein that was previously implicated in centrosome cohesion (Mayor et al., 2000), but not upon the depletion of Cep170, centrin-2, BBS4, PCM-1, or ninein. These results indicate that centrosome splitting is not a general response to the depletion of centrosomal proteins, supporting the hypothesis that both rootletin and C-Nap1 contribute to confer centrosome cohesion. Additional proteins that are involved in this process certainly await identification, as indicated by the recent demonstration that the depletion of dynamin-2 also causes centrosome splitting (Thompson et al., 2004).


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

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

siRNA depletion of rootletin causes centrosome splitting. (A) U2OS cells were transfected for 48 or 72 h with control (GL2) or different rootletin-specific siRNA duplexes and were costained for rootletin and γ-tubulin. Arrows and arrowheads mark paired centrioles and split centrosomes, respectively. Bars, 10 μm. (B) Quantitation of centrosome splitting in control (GL2) or rootletin siRNA-treated cells (oligonucleotide 222). Centrosomes were counted as split when the distance between centrioles was >2 μm. Error bars represent SEM.
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Related In: Results  -  Collection

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fig4: siRNA depletion of rootletin causes centrosome splitting. (A) U2OS cells were transfected for 48 or 72 h with control (GL2) or different rootletin-specific siRNA duplexes and were costained for rootletin and γ-tubulin. Arrows and arrowheads mark paired centrioles and split centrosomes, respectively. Bars, 10 μm. (B) Quantitation of centrosome splitting in control (GL2) or rootletin siRNA-treated cells (oligonucleotide 222). Centrosomes were counted as split when the distance between centrioles was >2 μm. Error bars represent SEM.
Mentions: To test more directly whether rootletin was involved in centrosome cohesion, we used siRNA. Three different rootletin-specific duplex oligonucleotides caused the near complete loss of rootletin from the centrosome (Fig. 4 A) and, concomitantly, extensive centrosome splitting (Fig. 4). In quantitative terms, >85% of cells that were treated with the most efficient siRNA duplex displayed split centrosomes, whereas <11% of cells showed a similar phenotype in GL2 control–treated cells (Fig. 4 B). Rootletin depletion caused centrosome splitting not only in U2OS but also in hTERT-RPE1 and A549 cells (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200504107/DC1). Centrosome splitting was observed upon the siRNA-mediated depletion of C-Nap1 (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200504107/DC1), which is a protein that was previously implicated in centrosome cohesion (Mayor et al., 2000), but not upon the depletion of Cep170, centrin-2, BBS4, PCM-1, or ninein. These results indicate that centrosome splitting is not a general response to the depletion of centrosomal proteins, supporting the hypothesis that both rootletin and C-Nap1 contribute to confer centrosome cohesion. Additional proteins that are involved in this process certainly await identification, as indicated by the recent demonstration that the depletion of dynamin-2 also causes centrosome splitting (Thompson et al., 2004).

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