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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 by siRNA causes delayed entry into mitosis. (A–E) HeLa cells were analyzed at specific time points after transfection with either control or NuSAP-specific siRNA duplexes. (A) Western blot of total cell lysates (harvested 24 h after transfection) was probed with antibodies against NuSAP and β-actin (loading control). (B) Time course of change in cellular DNA content after transfection. Quantification also includes the percentage of cells with a larger than tetraploid (4n) DNA content. Data are derived from three independent experiments. (C) Interphase cells 20 h after transfection were fixed in methanol and double stained for α-tubulin (green) and nucleic acids (blue). Abnormally shaped nuclei were observed in NuSAP-suppressed cells. (D) Prometaphase and metaphase spindle appearance in synchronized cells 30 h after transfection (first mitosis with manifest NuSAP suppression). Cells were fixed in methanol and double stained for α-tubulin and nucleic acids. (E) Quantification of the observed abnormal spindle and nuclear phenotypes. For spindles, 100 cells in prometaphase or metaphase from each of three independent experiments were scored for normal or disorganized phenotype. For nuclei, 200 interphase cells from each of three independent experiments were scored for normal or malformed phenotype. Bars: (C and D) 10 μm.
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fig6: Suppression of NuSAP by siRNA causes delayed entry into mitosis. (A–E) HeLa cells were analyzed at specific time points after transfection with either control or NuSAP-specific siRNA duplexes. (A) Western blot of total cell lysates (harvested 24 h after transfection) was probed with antibodies against NuSAP and β-actin (loading control). (B) Time course of change in cellular DNA content after transfection. Quantification also includes the percentage of cells with a larger than tetraploid (4n) DNA content. Data are derived from three independent experiments. (C) Interphase cells 20 h after transfection were fixed in methanol and double stained for α-tubulin (green) and nucleic acids (blue). Abnormally shaped nuclei were observed in NuSAP-suppressed cells. (D) Prometaphase and metaphase spindle appearance in synchronized cells 30 h after transfection (first mitosis with manifest NuSAP suppression). Cells were fixed in methanol and double stained for α-tubulin and nucleic acids. (E) Quantification of the observed abnormal spindle and nuclear phenotypes. For spindles, 100 cells in prometaphase or metaphase from each of three independent experiments were scored for normal or disorganized phenotype. For nuclei, 200 interphase cells from each of three independent experiments were scored for normal or malformed phenotype. Bars: (C and D) 10 μm.

Mentions: To study the effect of loss of NuSAP function, NuSAP protein levels were suppressed in HeLa cells using siRNA duplexes (Elbashir et al., 2001). Immunoblot and immunofluorescence analysis showed that at 24 h after transfection, the level of endogenous NuSAP could be reduced by more than ninefold in cells transfected with duplexes directed to NuSAP compared with control cells, which were transfected with the nonspecific duplex (transfection efficiencies were typically ∼80%; Fig. 6 A; 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 by siRNA causes delayed entry into mitosis. (A–E) HeLa cells were analyzed at specific time points after transfection with either control or NuSAP-specific siRNA duplexes. (A) Western blot of total cell lysates (harvested 24 h after transfection) was probed with antibodies against NuSAP and β-actin (loading control). (B) Time course of change in cellular DNA content after transfection. Quantification also includes the percentage of cells with a larger than tetraploid (4n) DNA content. Data are derived from three independent experiments. (C) Interphase cells 20 h after transfection were fixed in methanol and double stained for α-tubulin (green) and nucleic acids (blue). Abnormally shaped nuclei were observed in NuSAP-suppressed cells. (D) Prometaphase and metaphase spindle appearance in synchronized cells 30 h after transfection (first mitosis with manifest NuSAP suppression). Cells were fixed in methanol and double stained for α-tubulin and nucleic acids. (E) Quantification of the observed abnormal spindle and nuclear phenotypes. For spindles, 100 cells in prometaphase or metaphase from each of three independent experiments were scored for normal or disorganized phenotype. For nuclei, 200 interphase cells from each of three independent experiments were scored for normal or malformed phenotype. Bars: (C and D) 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172854&req=5

fig6: Suppression of NuSAP by siRNA causes delayed entry into mitosis. (A–E) HeLa cells were analyzed at specific time points after transfection with either control or NuSAP-specific siRNA duplexes. (A) Western blot of total cell lysates (harvested 24 h after transfection) was probed with antibodies against NuSAP and β-actin (loading control). (B) Time course of change in cellular DNA content after transfection. Quantification also includes the percentage of cells with a larger than tetraploid (4n) DNA content. Data are derived from three independent experiments. (C) Interphase cells 20 h after transfection were fixed in methanol and double stained for α-tubulin (green) and nucleic acids (blue). Abnormally shaped nuclei were observed in NuSAP-suppressed cells. (D) Prometaphase and metaphase spindle appearance in synchronized cells 30 h after transfection (first mitosis with manifest NuSAP suppression). Cells were fixed in methanol and double stained for α-tubulin and nucleic acids. (E) Quantification of the observed abnormal spindle and nuclear phenotypes. For spindles, 100 cells in prometaphase or metaphase from each of three independent experiments were scored for normal or disorganized phenotype. For nuclei, 200 interphase cells from each of three independent experiments were scored for normal or malformed phenotype. Bars: (C and D) 10 μm.
Mentions: To study the effect of loss of NuSAP function, NuSAP protein levels were suppressed in HeLa cells using siRNA duplexes (Elbashir et al., 2001). Immunoblot and immunofluorescence analysis showed that at 24 h after transfection, the level of endogenous NuSAP could be reduced by more than ninefold in cells transfected with duplexes directed to NuSAP compared with control cells, which were transfected with the nonspecific duplex (transfection efficiencies were typically ∼80%; Fig. 6 A; 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