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The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion.

Mayor T, Stierhof YD, Tanaka K, Fry AM, Nigg EA - J. Cell Biol. (2000)

Bottom Line: Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division.Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins.We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Sciences II, University of Geneva, CH-1211 Geneva, Switzerland.

ABSTRACT
Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.

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C-Nap1 antibody injection causes centrosome splitting in Hs68 cells. (A) Representative examples of Hs68 cells injected with control IgG (a and a′) or C-Ab (b and b′). Injected cells were visualized using an anti–rabbit IgG secondary antibody (a and b), and the centrosomes were stained for γ-tubulin (a′ and b′). For illustrative purposes, injected cells were surrounded by a dotted line and arrowheads in b′ point to a typical split centrosome. Bar, 10 μm. (B) Asynchronously growing Hs68 cells were injected with control or anti–C-Nap1 antibodies and analyzed 16 h later by immunofluorescence microscopy, as described above. The histogram indicates the percent of cells with split centrosomes. A total of 167 cells were injected with C-Ab and 158 cells with control IgG. Cells were scored as having split centrosomes whenever the distance between the two γ-tubulin dots exceeded 2 microns, i.e., two times the diameter of these dots. Results were averaged from two independent experiments. (C) Representative examples of asynchronously growing Hs68 cells injected with control IgG (a and a′) or C-Ab (b and b′). Injected cells were visualized using an anti–rabbit IgG secondary antibody (a and b), and the centrioles were stained with GT335, a mAb specific for polyglutamylated tubulin (a′ and b′). Bar, 10 μm. (D) Total protein from Hs68 cells was separated by SDS-PAGE and probed by immunoblotting with preimmune serum (PI) or affinity purified antibody against the COOH-terminal domain of C-Nap1 (C-Ab). The positions of molecular weight markers are indicated (in kD); the arrowhead marks C-Nap1.
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Figure 1: C-Nap1 antibody injection causes centrosome splitting in Hs68 cells. (A) Representative examples of Hs68 cells injected with control IgG (a and a′) or C-Ab (b and b′). Injected cells were visualized using an anti–rabbit IgG secondary antibody (a and b), and the centrosomes were stained for γ-tubulin (a′ and b′). For illustrative purposes, injected cells were surrounded by a dotted line and arrowheads in b′ point to a typical split centrosome. Bar, 10 μm. (B) Asynchronously growing Hs68 cells were injected with control or anti–C-Nap1 antibodies and analyzed 16 h later by immunofluorescence microscopy, as described above. The histogram indicates the percent of cells with split centrosomes. A total of 167 cells were injected with C-Ab and 158 cells with control IgG. Cells were scored as having split centrosomes whenever the distance between the two γ-tubulin dots exceeded 2 microns, i.e., two times the diameter of these dots. Results were averaged from two independent experiments. (C) Representative examples of asynchronously growing Hs68 cells injected with control IgG (a and a′) or C-Ab (b and b′). Injected cells were visualized using an anti–rabbit IgG secondary antibody (a and b), and the centrioles were stained with GT335, a mAb specific for polyglutamylated tubulin (a′ and b′). Bar, 10 μm. (D) Total protein from Hs68 cells was separated by SDS-PAGE and probed by immunoblotting with preimmune serum (PI) or affinity purified antibody against the COOH-terminal domain of C-Nap1 (C-Ab). The positions of molecular weight markers are indicated (in kD); the arrowhead marks C-Nap1.

Mentions: To explore a possible involvement of C-Nap1 in a cohesive structure linking centrioles, a highly specific, affinity-purified anti–C-Nap1 antibody (C-Ab) was microinjected into nonimmortalized human fibroblasts (Hs68 cells). This antibody, hereafter referred to as C-Ab, was raised against the COOH-terminal domain of C-Nap1 (aa 1,986–2,442), and has been described previously (R63; Fry et al. 1998b). As shown by Western blotting, C-Ab recognized a single band of the expected molecular mass in Hs68 whole-cell extracts, while the corresponding preimmune serum showed no reactivity (Fig. 1 D). Asynchronously growing Hs68 cells were injected with either C-Ab or nonspecific rabbit IgG for control, fixed with methanol 16 h later, and stained with a mAb anti–γ-tubulin (Fig. 1 A, a′ and b′). To identify injected cells, cultures were counterstained with anti-IgG antibodies (Fig. 1 A, a and b). Whereas the vast majority of the uninjected or control-injected cells showed the expected staining of centrosomes as closely spaced doublets (Fig. 1 A, a′), most cells injected with C-Ab displayed two widely separated γ-tubulin–positive dots (Fig. 1 A, b′). To confirm that these dots contained centrioles (as opposed to merely constituting fragments of PCM), identical experiments were performed using GT335, a mAb that reacts with polyglutamylated tubulin and constitutes a convenient reagent for staining centrioles (Wolff et al. 1992; Bobinnec et al. 1998). As shown in Fig. 1 C (a′ and b′), the split centrosomes clearly contained GT335-positive centrioles. Quantitative analyses revealed that >70% of the cells injected with anti–C-Nap1 antibodies displayed split centrosomes, whereas only 7% was observed in control-injected cells (Fig. 1 B). These results show that antibody-mediated interference with C-Nap1 function causes a disruption of cohesion between centrioles, supporting the hypothesis that C-Nap1 is part of a structure that links centrioles together.


The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion.

Mayor T, Stierhof YD, Tanaka K, Fry AM, Nigg EA - J. Cell Biol. (2000)

C-Nap1 antibody injection causes centrosome splitting in Hs68 cells. (A) Representative examples of Hs68 cells injected with control IgG (a and a′) or C-Ab (b and b′). Injected cells were visualized using an anti–rabbit IgG secondary antibody (a and b), and the centrosomes were stained for γ-tubulin (a′ and b′). For illustrative purposes, injected cells were surrounded by a dotted line and arrowheads in b′ point to a typical split centrosome. Bar, 10 μm. (B) Asynchronously growing Hs68 cells were injected with control or anti–C-Nap1 antibodies and analyzed 16 h later by immunofluorescence microscopy, as described above. The histogram indicates the percent of cells with split centrosomes. A total of 167 cells were injected with C-Ab and 158 cells with control IgG. Cells were scored as having split centrosomes whenever the distance between the two γ-tubulin dots exceeded 2 microns, i.e., two times the diameter of these dots. Results were averaged from two independent experiments. (C) Representative examples of asynchronously growing Hs68 cells injected with control IgG (a and a′) or C-Ab (b and b′). Injected cells were visualized using an anti–rabbit IgG secondary antibody (a and b), and the centrioles were stained with GT335, a mAb specific for polyglutamylated tubulin (a′ and b′). Bar, 10 μm. (D) Total protein from Hs68 cells was separated by SDS-PAGE and probed by immunoblotting with preimmune serum (PI) or affinity purified antibody against the COOH-terminal domain of C-Nap1 (C-Ab). The positions of molecular weight markers are indicated (in kD); the arrowhead marks C-Nap1.
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Related In: Results  -  Collection

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Figure 1: C-Nap1 antibody injection causes centrosome splitting in Hs68 cells. (A) Representative examples of Hs68 cells injected with control IgG (a and a′) or C-Ab (b and b′). Injected cells were visualized using an anti–rabbit IgG secondary antibody (a and b), and the centrosomes were stained for γ-tubulin (a′ and b′). For illustrative purposes, injected cells were surrounded by a dotted line and arrowheads in b′ point to a typical split centrosome. Bar, 10 μm. (B) Asynchronously growing Hs68 cells were injected with control or anti–C-Nap1 antibodies and analyzed 16 h later by immunofluorescence microscopy, as described above. The histogram indicates the percent of cells with split centrosomes. A total of 167 cells were injected with C-Ab and 158 cells with control IgG. Cells were scored as having split centrosomes whenever the distance between the two γ-tubulin dots exceeded 2 microns, i.e., two times the diameter of these dots. Results were averaged from two independent experiments. (C) Representative examples of asynchronously growing Hs68 cells injected with control IgG (a and a′) or C-Ab (b and b′). Injected cells were visualized using an anti–rabbit IgG secondary antibody (a and b), and the centrioles were stained with GT335, a mAb specific for polyglutamylated tubulin (a′ and b′). Bar, 10 μm. (D) Total protein from Hs68 cells was separated by SDS-PAGE and probed by immunoblotting with preimmune serum (PI) or affinity purified antibody against the COOH-terminal domain of C-Nap1 (C-Ab). The positions of molecular weight markers are indicated (in kD); the arrowhead marks C-Nap1.
Mentions: To explore a possible involvement of C-Nap1 in a cohesive structure linking centrioles, a highly specific, affinity-purified anti–C-Nap1 antibody (C-Ab) was microinjected into nonimmortalized human fibroblasts (Hs68 cells). This antibody, hereafter referred to as C-Ab, was raised against the COOH-terminal domain of C-Nap1 (aa 1,986–2,442), and has been described previously (R63; Fry et al. 1998b). As shown by Western blotting, C-Ab recognized a single band of the expected molecular mass in Hs68 whole-cell extracts, while the corresponding preimmune serum showed no reactivity (Fig. 1 D). Asynchronously growing Hs68 cells were injected with either C-Ab or nonspecific rabbit IgG for control, fixed with methanol 16 h later, and stained with a mAb anti–γ-tubulin (Fig. 1 A, a′ and b′). To identify injected cells, cultures were counterstained with anti-IgG antibodies (Fig. 1 A, a and b). Whereas the vast majority of the uninjected or control-injected cells showed the expected staining of centrosomes as closely spaced doublets (Fig. 1 A, a′), most cells injected with C-Ab displayed two widely separated γ-tubulin–positive dots (Fig. 1 A, b′). To confirm that these dots contained centrioles (as opposed to merely constituting fragments of PCM), identical experiments were performed using GT335, a mAb that reacts with polyglutamylated tubulin and constitutes a convenient reagent for staining centrioles (Wolff et al. 1992; Bobinnec et al. 1998). As shown in Fig. 1 C (a′ and b′), the split centrosomes clearly contained GT335-positive centrioles. Quantitative analyses revealed that >70% of the cells injected with anti–C-Nap1 antibodies displayed split centrosomes, whereas only 7% was observed in control-injected cells (Fig. 1 B). These results show that antibody-mediated interference with C-Nap1 function causes a disruption of cohesion between centrioles, supporting the hypothesis that C-Nap1 is part of a structure that links centrioles together.

Bottom Line: Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division.Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins.We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Sciences II, University of Geneva, CH-1211 Geneva, Switzerland.

ABSTRACT
Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.

Show MeSH
Related in: MedlinePlus