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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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Rootletin interacts with itself, C-Nap1, and Nek2. (A) U2OS cells were transfected with myc-tagged rootletin (green) and were counterstained with anti–γ-tubulin antibody (red). Insets show enlargements of the centrosomes to highlight protein fibers. Bar, 5 μm. (B) U2OS cells were transfected with tagged Nek2, C-Nap1, or a COOH-terminal fragment of C-Nap1 either alone (right) or together with rootletin (left three panels), and the distribution of proteins was monitored by IF microscopy. Bars, 15 μm. (C) Yeast two-hybrid interaction of rootletin with Nek2 (top) and C-Nap1 (bottom). Transformed yeast cells were plated onto media selecting for transformants (−LW) and bait-prey interactions (−QDO), respectively. AD, activation domain; BD, binding domain; LW, leucine tryptophane; QDO, quadruple drop out.
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fig2: Rootletin interacts with itself, C-Nap1, and Nek2. (A) U2OS cells were transfected with myc-tagged rootletin (green) and were counterstained with anti–γ-tubulin antibody (red). Insets show enlargements of the centrosomes to highlight protein fibers. Bar, 5 μm. (B) U2OS cells were transfected with tagged Nek2, C-Nap1, or a COOH-terminal fragment of C-Nap1 either alone (right) or together with rootletin (left three panels), and the distribution of proteins was monitored by IF microscopy. Bars, 15 μm. (C) Yeast two-hybrid interaction of rootletin with Nek2 (top) and C-Nap1 (bottom). Transformed yeast cells were plated onto media selecting for transformants (−LW) and bait-prey interactions (−QDO), respectively. AD, activation domain; BD, binding domain; LW, leucine tryptophane; QDO, quadruple drop out.

Mentions: We next sought to determine whether rootletin was able to interact with C-Nap1 and/or Nek2, which are two proteins that were previously implicated in centrosome cohesion (Fry et al., 1998a; Mayor et al., 2000, 2002). Endogenous C-Nap1 localizes to the proximal ends of centrioles, suggesting that it might constitute docking sites for rootletin fibers, whereas Nek2 has been implicated in the regulation of C-Nap1. As a result of the low abundance of rootletin and our inability to detect endogenous rootletin by any of the available antibodies, coimmunoprecipitation experiments were not successful. Thus, we asked whether rootletin is able to interact with C-Nap1 and/or Nek2 upon coexpression in U2OS cells. The overexpression of rootletin alone led to the formation of striking filaments (Fig. 2 A), which is consistent with earlier data (Yang et al., 2002). These filaments emanated from the centrosome at low expression levels (Fig. 2 A) but filled the cytoplasm at higher levels (Fig. 2 B). The expression of GFP-Nek2 or GFP–C-Nap1 alone resulted in a diffuse distribution or in the formation of multiple globular aggregates, respectively (Fig. 2 B), as described previously (Fry et al., 1998b; Mayor et al., 2002). In stark contrast, the coexpression of either GFP-Nek2 or GFP–C-Nap1 with rootletin resulted in their recruitment to rootletin filaments (Fig. 2 B). Several proteins encompassing COOH-terminal domains of C-Nap1 were not recruited, nor was GFP–centrin-2 (Fig. 2 B and not depicted). This latter result not only provides a specificity control but also indicates that rootletin binding requires the NH2 terminus of C-Nap1.


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

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

Rootletin interacts with itself, C-Nap1, and Nek2. (A) U2OS cells were transfected with myc-tagged rootletin (green) and were counterstained with anti–γ-tubulin antibody (red). Insets show enlargements of the centrosomes to highlight protein fibers. Bar, 5 μm. (B) U2OS cells were transfected with tagged Nek2, C-Nap1, or a COOH-terminal fragment of C-Nap1 either alone (right) or together with rootletin (left three panels), and the distribution of proteins was monitored by IF microscopy. Bars, 15 μm. (C) Yeast two-hybrid interaction of rootletin with Nek2 (top) and C-Nap1 (bottom). Transformed yeast cells were plated onto media selecting for transformants (−LW) and bait-prey interactions (−QDO), respectively. AD, activation domain; BD, binding domain; LW, leucine tryptophane; QDO, quadruple drop out.
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fig2: Rootletin interacts with itself, C-Nap1, and Nek2. (A) U2OS cells were transfected with myc-tagged rootletin (green) and were counterstained with anti–γ-tubulin antibody (red). Insets show enlargements of the centrosomes to highlight protein fibers. Bar, 5 μm. (B) U2OS cells were transfected with tagged Nek2, C-Nap1, or a COOH-terminal fragment of C-Nap1 either alone (right) or together with rootletin (left three panels), and the distribution of proteins was monitored by IF microscopy. Bars, 15 μm. (C) Yeast two-hybrid interaction of rootletin with Nek2 (top) and C-Nap1 (bottom). Transformed yeast cells were plated onto media selecting for transformants (−LW) and bait-prey interactions (−QDO), respectively. AD, activation domain; BD, binding domain; LW, leucine tryptophane; QDO, quadruple drop out.
Mentions: We next sought to determine whether rootletin was able to interact with C-Nap1 and/or Nek2, which are two proteins that were previously implicated in centrosome cohesion (Fry et al., 1998a; Mayor et al., 2000, 2002). Endogenous C-Nap1 localizes to the proximal ends of centrioles, suggesting that it might constitute docking sites for rootletin fibers, whereas Nek2 has been implicated in the regulation of C-Nap1. As a result of the low abundance of rootletin and our inability to detect endogenous rootletin by any of the available antibodies, coimmunoprecipitation experiments were not successful. Thus, we asked whether rootletin is able to interact with C-Nap1 and/or Nek2 upon coexpression in U2OS cells. The overexpression of rootletin alone led to the formation of striking filaments (Fig. 2 A), which is consistent with earlier data (Yang et al., 2002). These filaments emanated from the centrosome at low expression levels (Fig. 2 A) but filled the cytoplasm at higher levels (Fig. 2 B). The expression of GFP-Nek2 or GFP–C-Nap1 alone resulted in a diffuse distribution or in the formation of multiple globular aggregates, respectively (Fig. 2 B), as described previously (Fry et al., 1998b; Mayor et al., 2002). In stark contrast, the coexpression of either GFP-Nek2 or GFP–C-Nap1 with rootletin resulted in their recruitment to rootletin filaments (Fig. 2 B). Several proteins encompassing COOH-terminal domains of C-Nap1 were not recruited, nor was GFP–centrin-2 (Fig. 2 B and not depicted). This latter result not only provides a specificity control but also indicates that rootletin binding requires the NH2 terminus of C-Nap1.

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