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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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Human rootletin localizes to centrosomes. (A) U2OS cells were costained with antirootletin (green) and anti–γ-tubulin (red) antibodies. (a) Interphase cells; insets show enlargements of centrosomes. (b–d) Mitotic cells showing prophase (b), metaphase (c), and telophase (d). DNA was stained with DAPI. Bars, 5 μm. (B) Western blots. (left) Antirootletin antibody R145 on total extracts from myc-rootletin–transfected (lane 1) or untransfected (lane 2) 293T cells. (right) Preimmune (P, lane 3) and antirootletin (I, lane 4) serum on centrosomes isolated from KE37 cells. Arrowheads point to human rootletin. (C) U2OS cells were subjected to preembedding immunogold-labeling EM. Cells were labeled with either antirootletin antibody R145 followed by Nanogold-coupled secondary antibody (a and c), secondary antibody alone (b), or antibodies directed against the NH2 terminus of rootletin (R146; d). Brackets (d) emphasize the distance of gold particles from the centriole surface with R146 labeling. Bars, 250 nm.
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fig1: Human rootletin localizes to centrosomes. (A) U2OS cells were costained with antirootletin (green) and anti–γ-tubulin (red) antibodies. (a) Interphase cells; insets show enlargements of centrosomes. (b–d) Mitotic cells showing prophase (b), metaphase (c), and telophase (d). DNA was stained with DAPI. Bars, 5 μm. (B) Western blots. (left) Antirootletin antibody R145 on total extracts from myc-rootletin–transfected (lane 1) or untransfected (lane 2) 293T cells. (right) Preimmune (P, lane 3) and antirootletin (I, lane 4) serum on centrosomes isolated from KE37 cells. Arrowheads point to human rootletin. (C) U2OS cells were subjected to preembedding immunogold-labeling EM. Cells were labeled with either antirootletin antibody R145 followed by Nanogold-coupled secondary antibody (a and c), secondary antibody alone (b), or antibodies directed against the NH2 terminus of rootletin (R146; d). Brackets (d) emphasize the distance of gold particles from the centriole surface with R146 labeling. Bars, 250 nm.

Mentions: We asked whether rootletin might play a general role in centrosome cohesion. Several antibodies that were raised against recombinant human rootletin recognized a protein of the expected molecular mass (228 kD) in purified centrosomes, whereas the corresponding preimmune serum showed no reactivity (Fig. 1 B). The antibodies also recognized overexpressed rootletin in 293T cells (Fig. 1 B), but no signal representing endogenous rootletin could be detected in any cell line that was tested, including 293T, HeLa S3, and U2OS (Fig. 1 B, Fig. S1 B, available at http://www.jcb.org/cgi/content/full/jcb.200504107/DC1; and not depicted), confirming that rootletin is a low abundance protein in cells that lack a rootlet system (Yang et al., 2002). As shown by immunofluorescence (IF) microscopy, endogenous human rootletin stained thin fibers protruding away from the two centrioles (Fig. 1 A, a). Rootletin staining was uniform throughout interphase, but at least threefold diminished in mitotic cells (Fig. 1 A, b–d). Staining dropped at the onset of prophase (Fig. 1 A, b) and remained low until after telophase (Fig. 1 A, c and d). This suggests that rootletin dissociates from centrosomes as cells go through division, which is highly reminiscent of C-Nap1 (Fry et al., 1998a; Mayor et al., 2000, 2002).


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

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

Human rootletin localizes to centrosomes. (A) U2OS cells were costained with antirootletin (green) and anti–γ-tubulin (red) antibodies. (a) Interphase cells; insets show enlargements of centrosomes. (b–d) Mitotic cells showing prophase (b), metaphase (c), and telophase (d). DNA was stained with DAPI. Bars, 5 μm. (B) Western blots. (left) Antirootletin antibody R145 on total extracts from myc-rootletin–transfected (lane 1) or untransfected (lane 2) 293T cells. (right) Preimmune (P, lane 3) and antirootletin (I, lane 4) serum on centrosomes isolated from KE37 cells. Arrowheads point to human rootletin. (C) U2OS cells were subjected to preembedding immunogold-labeling EM. Cells were labeled with either antirootletin antibody R145 followed by Nanogold-coupled secondary antibody (a and c), secondary antibody alone (b), or antibodies directed against the NH2 terminus of rootletin (R146; d). Brackets (d) emphasize the distance of gold particles from the centriole surface with R146 labeling. Bars, 250 nm.
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Related In: Results  -  Collection

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fig1: Human rootletin localizes to centrosomes. (A) U2OS cells were costained with antirootletin (green) and anti–γ-tubulin (red) antibodies. (a) Interphase cells; insets show enlargements of centrosomes. (b–d) Mitotic cells showing prophase (b), metaphase (c), and telophase (d). DNA was stained with DAPI. Bars, 5 μm. (B) Western blots. (left) Antirootletin antibody R145 on total extracts from myc-rootletin–transfected (lane 1) or untransfected (lane 2) 293T cells. (right) Preimmune (P, lane 3) and antirootletin (I, lane 4) serum on centrosomes isolated from KE37 cells. Arrowheads point to human rootletin. (C) U2OS cells were subjected to preembedding immunogold-labeling EM. Cells were labeled with either antirootletin antibody R145 followed by Nanogold-coupled secondary antibody (a and c), secondary antibody alone (b), or antibodies directed against the NH2 terminus of rootletin (R146; d). Brackets (d) emphasize the distance of gold particles from the centriole surface with R146 labeling. Bars, 250 nm.
Mentions: We asked whether rootletin might play a general role in centrosome cohesion. Several antibodies that were raised against recombinant human rootletin recognized a protein of the expected molecular mass (228 kD) in purified centrosomes, whereas the corresponding preimmune serum showed no reactivity (Fig. 1 B). The antibodies also recognized overexpressed rootletin in 293T cells (Fig. 1 B), but no signal representing endogenous rootletin could be detected in any cell line that was tested, including 293T, HeLa S3, and U2OS (Fig. 1 B, Fig. S1 B, available at http://www.jcb.org/cgi/content/full/jcb.200504107/DC1; and not depicted), confirming that rootletin is a low abundance protein in cells that lack a rootlet system (Yang et al., 2002). As shown by immunofluorescence (IF) microscopy, endogenous human rootletin stained thin fibers protruding away from the two centrioles (Fig. 1 A, a). Rootletin staining was uniform throughout interphase, but at least threefold diminished in mitotic cells (Fig. 1 A, b–d). Staining dropped at the onset of prophase (Fig. 1 A, b) and remained low until after telophase (Fig. 1 A, c and d). This suggests that rootletin dissociates from centrosomes as cells go through division, which is highly reminiscent of C-Nap1 (Fry et al., 1998a; Mayor et al., 2000, 2002).

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