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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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Mutual dependency of rootletin and C-Nap1 localization and models for centrosome cohesion. (A) U2OS cells were transfected for 48 or 72 h with control (GL2) or rootletin-specific siRNA duplexes and were stained for C-Nap1 (green) and γ-tubulin (red). Bars, 5 μm. (B) U2OS cells were depleted of C-Nap1 by siRNA and were subjected to immuno-EM labeling of rootletin (antibody R145). Bars, 250 nm. (C) U2OS cells were transfected for 48 h with a C-Nap1–specific siRNA duplex and were stained for rootletin (red) and γ-tubulin (green). Rootletin disappeared altogether (I) and formed elongated fibers that either appeared to connect split centrosomes (II) or protruded away from split centrosomes (III). Bars, 5 μm. (A and C) Insets show enlargements of the centrosome area to highlight centrosome splitting. (D) Quantitation of the observed effects of C-Nap1 RNA interference on rootletin localization (I–III; compare with C). Error bars represent SEM. (E) Schematic illustration of possible models for centrosome cohesion. (a) Centrioles (rectangles) embedded in the PCM (speckled). (b) Centrioles connected by a continuous proteinaceous linker. (c) Centrioles connected by entangling filaments. C, C-Nap1; L, hypothetical continuous linker; R, rootletin; X, additional linker proteins that are yet to be identified. Parental centrioles (dark gray) are depicted as having associated nascent procentrioles (dotted lines).
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fig5: Mutual dependency of rootletin and C-Nap1 localization and models for centrosome cohesion. (A) U2OS cells were transfected for 48 or 72 h with control (GL2) or rootletin-specific siRNA duplexes and were stained for C-Nap1 (green) and γ-tubulin (red). Bars, 5 μm. (B) U2OS cells were depleted of C-Nap1 by siRNA and were subjected to immuno-EM labeling of rootletin (antibody R145). Bars, 250 nm. (C) U2OS cells were transfected for 48 h with a C-Nap1–specific siRNA duplex and were stained for rootletin (red) and γ-tubulin (green). Rootletin disappeared altogether (I) and formed elongated fibers that either appeared to connect split centrosomes (II) or protruded away from split centrosomes (III). Bars, 5 μm. (A and C) Insets show enlargements of the centrosome area to highlight centrosome splitting. (D) Quantitation of the observed effects of C-Nap1 RNA interference on rootletin localization (I–III; compare with C). Error bars represent SEM. (E) Schematic illustration of possible models for centrosome cohesion. (a) Centrioles (rectangles) embedded in the PCM (speckled). (b) Centrioles connected by a continuous proteinaceous linker. (c) Centrioles connected by entangling filaments. C, C-Nap1; L, hypothetical continuous linker; R, rootletin; X, additional linker proteins that are yet to be identified. Parental centrioles (dark gray) are depicted as having associated nascent procentrioles (dotted lines).

Mentions: We next asked whether the depletion of rootletin would impair the localization of C-Nap1 or vice versa. The depletion of rootletin did not detectably influence the association of C-Nap1 with centrosomes (Fig. 5 A), but the depletion of C-Nap1 clearly affected the localization of rootletin (Fig. 5, B and C). In siRNA experiments targeting C-Nap1, the protein was completely depleted in ∼20% of cells and was strongly depleted in the remainder (unpublished data). In line with this observation, rootletin was no longer detectable at centrosomes in 21% of C-Nap1–depleted cells (Fig. 5, C and D, I). In the other cells, rootletin could be seen to form fibers (Fig. 5 C, II and III) that were fewer and often more than twice as long as those seen in control cells (Fig. 1 A). These fibers either pointed in various directions (50% of cells; Fig. 5, C and D, III) or appeared to span the entire distance between the two parental centrioles (29% of cells; Fig. 5, C and D, II). The observation that rootletin was not lost from all centrioles upon C-Nap1 depletion probably reflects residual levels of C-Nap1, although interactions with other centriolar proteins cannot be excluded. If one assumes that C-Nap1 docking sites and preexisting rootletin fibers compete for the binding of rootletin subunits, a reduced number of docking sites would be expected to result in the anchoring of fewer but longer rootletin fibers exactly as seen in Fig. 5 (B and C).


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

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

Mutual dependency of rootletin and C-Nap1 localization and models for centrosome cohesion. (A) U2OS cells were transfected for 48 or 72 h with control (GL2) or rootletin-specific siRNA duplexes and were stained for C-Nap1 (green) and γ-tubulin (red). Bars, 5 μm. (B) U2OS cells were depleted of C-Nap1 by siRNA and were subjected to immuno-EM labeling of rootletin (antibody R145). Bars, 250 nm. (C) U2OS cells were transfected for 48 h with a C-Nap1–specific siRNA duplex and were stained for rootletin (red) and γ-tubulin (green). Rootletin disappeared altogether (I) and formed elongated fibers that either appeared to connect split centrosomes (II) or protruded away from split centrosomes (III). Bars, 5 μm. (A and C) Insets show enlargements of the centrosome area to highlight centrosome splitting. (D) Quantitation of the observed effects of C-Nap1 RNA interference on rootletin localization (I–III; compare with C). Error bars represent SEM. (E) Schematic illustration of possible models for centrosome cohesion. (a) Centrioles (rectangles) embedded in the PCM (speckled). (b) Centrioles connected by a continuous proteinaceous linker. (c) Centrioles connected by entangling filaments. C, C-Nap1; L, hypothetical continuous linker; R, rootletin; X, additional linker proteins that are yet to be identified. Parental centrioles (dark gray) are depicted as having associated nascent procentrioles (dotted lines).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171225&req=5

fig5: Mutual dependency of rootletin and C-Nap1 localization and models for centrosome cohesion. (A) U2OS cells were transfected for 48 or 72 h with control (GL2) or rootletin-specific siRNA duplexes and were stained for C-Nap1 (green) and γ-tubulin (red). Bars, 5 μm. (B) U2OS cells were depleted of C-Nap1 by siRNA and were subjected to immuno-EM labeling of rootletin (antibody R145). Bars, 250 nm. (C) U2OS cells were transfected for 48 h with a C-Nap1–specific siRNA duplex and were stained for rootletin (red) and γ-tubulin (green). Rootletin disappeared altogether (I) and formed elongated fibers that either appeared to connect split centrosomes (II) or protruded away from split centrosomes (III). Bars, 5 μm. (A and C) Insets show enlargements of the centrosome area to highlight centrosome splitting. (D) Quantitation of the observed effects of C-Nap1 RNA interference on rootletin localization (I–III; compare with C). Error bars represent SEM. (E) Schematic illustration of possible models for centrosome cohesion. (a) Centrioles (rectangles) embedded in the PCM (speckled). (b) Centrioles connected by a continuous proteinaceous linker. (c) Centrioles connected by entangling filaments. C, C-Nap1; L, hypothetical continuous linker; R, rootletin; X, additional linker proteins that are yet to be identified. Parental centrioles (dark gray) are depicted as having associated nascent procentrioles (dotted lines).
Mentions: We next asked whether the depletion of rootletin would impair the localization of C-Nap1 or vice versa. The depletion of rootletin did not detectably influence the association of C-Nap1 with centrosomes (Fig. 5 A), but the depletion of C-Nap1 clearly affected the localization of rootletin (Fig. 5, B and C). In siRNA experiments targeting C-Nap1, the protein was completely depleted in ∼20% of cells and was strongly depleted in the remainder (unpublished data). In line with this observation, rootletin was no longer detectable at centrosomes in 21% of C-Nap1–depleted cells (Fig. 5, C and D, I). In the other cells, rootletin could be seen to form fibers (Fig. 5 C, II and III) that were fewer and often more than twice as long as those seen in control cells (Fig. 1 A). These fibers either pointed in various directions (50% of cells; Fig. 5, C and D, III) or appeared to span the entire distance between the two parental centrioles (29% of cells; Fig. 5, C and D, II). The observation that rootletin was not lost from all centrioles upon C-Nap1 depletion probably reflects residual levels of C-Nap1, although interactions with other centriolar proteins cannot be excluded. If one assumes that C-Nap1 docking sites and preexisting rootletin fibers compete for the binding of rootletin subunits, a reduced number of docking sites would be expected to result in the anchoring of fewer but longer rootletin fibers exactly as seen in Fig. 5 (B and C).

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