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NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization.

Raemaekers T, Ribbeck K, Beaudouin J, Annaert W, Van Camp M, Stockmans I, Smets N, Bouillon R, Ellenberg J, Carmeliet G - J. Cell Biol. (2003)

Bottom Line: Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain.In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis.These results suggest a crucial role for NuSAP in spindle microtubule organization.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium.

ABSTRACT
Here, we report on the identification of nucleolar spindle-associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.

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Suppression of NuSAP results in defective anaphase and cytokinesis. (A–E) HeLa cells were analyzed at specific time points after transfection with siRNA duplexes. (A) Anaphase and cytokinesis appearance in synchronized cells 30 h after transfection. Cells were fixed in methanol and double stained for α-tubulin and nucleic acids. (B) Quantification of the observed abnormal anaphase spindles. The percentage of normal and abnormal anaphase spindles is depicted with the total anaphase fraction set as 100% (three independent experiments). (C). Relative number of binucleated cells 10 and 40 h after transfection; 300 interphase cells from each of three independent experiments were scored for normal or binucleate phenotype. (D) Cells were fixed in methanol and stained for γ-tubulin, centrin, and nucleic acids 48 h after transfection. Some centrin foci are not clearly visible, as the image was taken at a specific z-axis section. Insets show a higher magnigfication of γ-tubulin and centrin foci. (E) Quantification of the multipolar spindle phenotype in synchronized cells at the indicated time points corresponding to successive mitosis; 100 mitotic cells from each of three independent experiments were scored for the presence of multipolar spindles. Bars: (A, D, and E) 10 μm.
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fig7: Suppression of NuSAP results in defective anaphase and cytokinesis. (A–E) HeLa cells were analyzed at specific time points after transfection with siRNA duplexes. (A) Anaphase and cytokinesis appearance in synchronized cells 30 h after transfection. Cells were fixed in methanol and double stained for α-tubulin and nucleic acids. (B) Quantification of the observed abnormal anaphase spindles. The percentage of normal and abnormal anaphase spindles is depicted with the total anaphase fraction set as 100% (three independent experiments). (C). Relative number of binucleated cells 10 and 40 h after transfection; 300 interphase cells from each of three independent experiments were scored for normal or binucleate phenotype. (D) Cells were fixed in methanol and stained for γ-tubulin, centrin, and nucleic acids 48 h after transfection. Some centrin foci are not clearly visible, as the image was taken at a specific z-axis section. Insets show a higher magnigfication of γ-tubulin and centrin foci. (E) Quantification of the multipolar spindle phenotype in synchronized cells at the indicated time points corresponding to successive mitosis; 100 mitotic cells from each of three independent experiments were scored for the presence of multipolar spindles. Bars: (A, D, and E) 10 μm.

Mentions: Some of the NuSAP-depleted mitotic cells with highly disorganized spindles appeared to be able to proceed to chromosome segregation. A more than threefold increase in abnormal anaphase cells was observed (n = 100, three independent experiments; Fig. 7, A and B). Again, these cells showed a less dense and disorganized microtubule array, often with aberrant segregation of less condensed chromosomes. At later stages, cells often displayed defective cytokinesis with both chromosome sets in one daughter cell (Fig. 7 A). Consistent with this, we observed a threefold higher number of binucleated cells 40 h after transfection relative to control cells (n = 300, three independent experiments; Fig. 7 C). These cells frequently contained four centrosomes, each containing a pair of centrioles as determined by centrin staining (Fig. 7 D). 50 h after transfection, a time sufficient for cells to complete two cell cycles with reduced levels of NuSAP, we observed a more than threefold increase in the number of multipolar spindles compared with control cells (n = 100, three independent experiments; Fig. 7 E). This agrees well with the increase in binucleated cells at 40 h, and was confirmed by DNA content analysis, which showed a more than threefold increase in cells with a larger than tetraploid DNA content (particularly 6N and 8N DNA content) at both 48 and 72 h after transfection (Fig. 6 B; unpublished data). 72 h after transfection, we could also detect an increase of hypodiploid cells, indicating that NuSAP-depleted cells eventually became apoptotic (unpublished data).


NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization.

Raemaekers T, Ribbeck K, Beaudouin J, Annaert W, Van Camp M, Stockmans I, Smets N, Bouillon R, Ellenberg J, Carmeliet G - J. Cell Biol. (2003)

Suppression of NuSAP results in defective anaphase and cytokinesis. (A–E) HeLa cells were analyzed at specific time points after transfection with siRNA duplexes. (A) Anaphase and cytokinesis appearance in synchronized cells 30 h after transfection. Cells were fixed in methanol and double stained for α-tubulin and nucleic acids. (B) Quantification of the observed abnormal anaphase spindles. The percentage of normal and abnormal anaphase spindles is depicted with the total anaphase fraction set as 100% (three independent experiments). (C). Relative number of binucleated cells 10 and 40 h after transfection; 300 interphase cells from each of three independent experiments were scored for normal or binucleate phenotype. (D) Cells were fixed in methanol and stained for γ-tubulin, centrin, and nucleic acids 48 h after transfection. Some centrin foci are not clearly visible, as the image was taken at a specific z-axis section. Insets show a higher magnigfication of γ-tubulin and centrin foci. (E) Quantification of the multipolar spindle phenotype in synchronized cells at the indicated time points corresponding to successive mitosis; 100 mitotic cells from each of three independent experiments were scored for the presence of multipolar spindles. Bars: (A, D, and E) 10 μm.
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Related In: Results  -  Collection

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fig7: Suppression of NuSAP results in defective anaphase and cytokinesis. (A–E) HeLa cells were analyzed at specific time points after transfection with siRNA duplexes. (A) Anaphase and cytokinesis appearance in synchronized cells 30 h after transfection. Cells were fixed in methanol and double stained for α-tubulin and nucleic acids. (B) Quantification of the observed abnormal anaphase spindles. The percentage of normal and abnormal anaphase spindles is depicted with the total anaphase fraction set as 100% (three independent experiments). (C). Relative number of binucleated cells 10 and 40 h after transfection; 300 interphase cells from each of three independent experiments were scored for normal or binucleate phenotype. (D) Cells were fixed in methanol and stained for γ-tubulin, centrin, and nucleic acids 48 h after transfection. Some centrin foci are not clearly visible, as the image was taken at a specific z-axis section. Insets show a higher magnigfication of γ-tubulin and centrin foci. (E) Quantification of the multipolar spindle phenotype in synchronized cells at the indicated time points corresponding to successive mitosis; 100 mitotic cells from each of three independent experiments were scored for the presence of multipolar spindles. Bars: (A, D, and E) 10 μm.
Mentions: Some of the NuSAP-depleted mitotic cells with highly disorganized spindles appeared to be able to proceed to chromosome segregation. A more than threefold increase in abnormal anaphase cells was observed (n = 100, three independent experiments; Fig. 7, A and B). Again, these cells showed a less dense and disorganized microtubule array, often with aberrant segregation of less condensed chromosomes. At later stages, cells often displayed defective cytokinesis with both chromosome sets in one daughter cell (Fig. 7 A). Consistent with this, we observed a threefold higher number of binucleated cells 40 h after transfection relative to control cells (n = 300, three independent experiments; Fig. 7 C). These cells frequently contained four centrosomes, each containing a pair of centrioles as determined by centrin staining (Fig. 7 D). 50 h after transfection, a time sufficient for cells to complete two cell cycles with reduced levels of NuSAP, we observed a more than threefold increase in the number of multipolar spindles compared with control cells (n = 100, three independent experiments; Fig. 7 E). This agrees well with the increase in binucleated cells at 40 h, and was confirmed by DNA content analysis, which showed a more than threefold increase in cells with a larger than tetraploid DNA content (particularly 6N and 8N DNA content) at both 48 and 72 h after transfection (Fig. 6 B; unpublished data). 72 h after transfection, we could also detect an increase of hypodiploid cells, indicating that NuSAP-depleted cells eventually became apoptotic (unpublished data).

Bottom Line: Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain.In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis.These results suggest a crucial role for NuSAP in spindle microtubule organization.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium.

ABSTRACT
Here, we report on the identification of nucleolar spindle-associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.

Show MeSH
Related in: MedlinePlus