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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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Dynamic localization of NuSAP during the cell cycle. (A) Synchronized MC3T3E1 cells were fixed at specific time points, and double stained for endogenous NuSAP and nucleic acids. NuSAP is nuclear at S/G2 phases. During metaphase and early anaphase, NuSAP localizes prominently to the central spindle. Toward the end of cytokinesis and in G1 phase, NuSAP is hardly detectable. (B) Time-lapse microscopy of synchronized, live NRK cells stably expressing CFP-tagged histone 2B (H2B-CFP) and transiently expressing YFP-tagged NuSAP. In vivo images show a similar subcellular localization as with fixed cells. Arrows indicate association with condensing chromosomes at prophase. Time is h:min:s. (C) Double-stained MC3T3E1 cells (endogenous NuSAP and α-tubulin, or nucleic acids) were fixed in either glutaraldehyde or methanol. Methanol-fixed cells showed intact spindle microtubules, whereas NuSAP integrity is impaired. In contrast, cells fixed in detergent-containing glutaraldehyde solution showed more integer NuSAP, whereas microtubule integrity was affected. (D) MC3T3E1 cells at interphase were double stained for endogenous NuSAP and nucleolin. NuSAP is highly enriched in the nucleolus. (A and D) Mitotic cells were fixed in glutaraldehyde, whereas PFA was used for interphase cells. (E) Western blot of total cell lysate and cytosol and nuclear fractions of MC3T3E1 cells. The blot was probed with anti-NuSAP and anti-α-tubulin antibodies. In contrast to tubulin, NuSAP is primarily recovered in the nuclear fraction. Bars: (A and D) 10 μm; (B and C) 5 μm.
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fig3: Dynamic localization of NuSAP during the cell cycle. (A) Synchronized MC3T3E1 cells were fixed at specific time points, and double stained for endogenous NuSAP and nucleic acids. NuSAP is nuclear at S/G2 phases. During metaphase and early anaphase, NuSAP localizes prominently to the central spindle. Toward the end of cytokinesis and in G1 phase, NuSAP is hardly detectable. (B) Time-lapse microscopy of synchronized, live NRK cells stably expressing CFP-tagged histone 2B (H2B-CFP) and transiently expressing YFP-tagged NuSAP. In vivo images show a similar subcellular localization as with fixed cells. Arrows indicate association with condensing chromosomes at prophase. Time is h:min:s. (C) Double-stained MC3T3E1 cells (endogenous NuSAP and α-tubulin, or nucleic acids) were fixed in either glutaraldehyde or methanol. Methanol-fixed cells showed intact spindle microtubules, whereas NuSAP integrity is impaired. In contrast, cells fixed in detergent-containing glutaraldehyde solution showed more integer NuSAP, whereas microtubule integrity was affected. (D) MC3T3E1 cells at interphase were double stained for endogenous NuSAP and nucleolin. NuSAP is highly enriched in the nucleolus. (A and D) Mitotic cells were fixed in glutaraldehyde, whereas PFA was used for interphase cells. (E) Western blot of total cell lysate and cytosol and nuclear fractions of MC3T3E1 cells. The blot was probed with anti-NuSAP and anti-α-tubulin antibodies. In contrast to tubulin, NuSAP is primarily recovered in the nuclear fraction. Bars: (A and D) 10 μm; (B and C) 5 μm.

Mentions: The subcellular distribution of NuSAP was followed throughout the cell cycle by immunostaining and in vivo imaging. Immunofluorescence microscopy of MC3T3E1 cells revealed that during interphase, endogenous NuSAP was confined to the nucleus and concentrated in nucleoli (Fig. 3 A, S/G2; Fig. 3 D). Cell fractionation experiments confirmed this nuclear localization (Fig. 3 E). At prometaphase, NuSAP redistributed from nucleoli to the vicinity of the chromosomes, forming bundles that became progressively more defined at metaphase and early anaphase (Fig. 3 A). These NuSAP bundles colocalized with microtubules of the central spindle (Fig. 3 C). Later in anaphase, NuSAP was intensely localized to the spindle in the chromosomal area. At telophase, NuSAP localized to “spikes” around the decondensing chromosomes, which disappeared toward the end of cytokinesis (Fig. 3 A). Consistent with the reduced protein levels in G1 cells seen by Western blot (Fig. 2 D), very little NuSAP could be detected in G1 cells (Fig. 3 A), indicating a rapid degradation of NuSAP after mitosis. Interestingly, the localization of NuSAP to chromosomes in mitotic cells was not sensitive to nocodazole treatment, indicating that part of its mitotic localization may be independent of microtubules (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)

Dynamic localization of NuSAP during the cell cycle. (A) Synchronized MC3T3E1 cells were fixed at specific time points, and double stained for endogenous NuSAP and nucleic acids. NuSAP is nuclear at S/G2 phases. During metaphase and early anaphase, NuSAP localizes prominently to the central spindle. Toward the end of cytokinesis and in G1 phase, NuSAP is hardly detectable. (B) Time-lapse microscopy of synchronized, live NRK cells stably expressing CFP-tagged histone 2B (H2B-CFP) and transiently expressing YFP-tagged NuSAP. In vivo images show a similar subcellular localization as with fixed cells. Arrows indicate association with condensing chromosomes at prophase. Time is h:min:s. (C) Double-stained MC3T3E1 cells (endogenous NuSAP and α-tubulin, or nucleic acids) were fixed in either glutaraldehyde or methanol. Methanol-fixed cells showed intact spindle microtubules, whereas NuSAP integrity is impaired. In contrast, cells fixed in detergent-containing glutaraldehyde solution showed more integer NuSAP, whereas microtubule integrity was affected. (D) MC3T3E1 cells at interphase were double stained for endogenous NuSAP and nucleolin. NuSAP is highly enriched in the nucleolus. (A and D) Mitotic cells were fixed in glutaraldehyde, whereas PFA was used for interphase cells. (E) Western blot of total cell lysate and cytosol and nuclear fractions of MC3T3E1 cells. The blot was probed with anti-NuSAP and anti-α-tubulin antibodies. In contrast to tubulin, NuSAP is primarily recovered in the nuclear fraction. Bars: (A and D) 10 μm; (B and C) 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172854&req=5

fig3: Dynamic localization of NuSAP during the cell cycle. (A) Synchronized MC3T3E1 cells were fixed at specific time points, and double stained for endogenous NuSAP and nucleic acids. NuSAP is nuclear at S/G2 phases. During metaphase and early anaphase, NuSAP localizes prominently to the central spindle. Toward the end of cytokinesis and in G1 phase, NuSAP is hardly detectable. (B) Time-lapse microscopy of synchronized, live NRK cells stably expressing CFP-tagged histone 2B (H2B-CFP) and transiently expressing YFP-tagged NuSAP. In vivo images show a similar subcellular localization as with fixed cells. Arrows indicate association with condensing chromosomes at prophase. Time is h:min:s. (C) Double-stained MC3T3E1 cells (endogenous NuSAP and α-tubulin, or nucleic acids) were fixed in either glutaraldehyde or methanol. Methanol-fixed cells showed intact spindle microtubules, whereas NuSAP integrity is impaired. In contrast, cells fixed in detergent-containing glutaraldehyde solution showed more integer NuSAP, whereas microtubule integrity was affected. (D) MC3T3E1 cells at interphase were double stained for endogenous NuSAP and nucleolin. NuSAP is highly enriched in the nucleolus. (A and D) Mitotic cells were fixed in glutaraldehyde, whereas PFA was used for interphase cells. (E) Western blot of total cell lysate and cytosol and nuclear fractions of MC3T3E1 cells. The blot was probed with anti-NuSAP and anti-α-tubulin antibodies. In contrast to tubulin, NuSAP is primarily recovered in the nuclear fraction. Bars: (A and D) 10 μm; (B and C) 5 μm.
Mentions: The subcellular distribution of NuSAP was followed throughout the cell cycle by immunostaining and in vivo imaging. Immunofluorescence microscopy of MC3T3E1 cells revealed that during interphase, endogenous NuSAP was confined to the nucleus and concentrated in nucleoli (Fig. 3 A, S/G2; Fig. 3 D). Cell fractionation experiments confirmed this nuclear localization (Fig. 3 E). At prometaphase, NuSAP redistributed from nucleoli to the vicinity of the chromosomes, forming bundles that became progressively more defined at metaphase and early anaphase (Fig. 3 A). These NuSAP bundles colocalized with microtubules of the central spindle (Fig. 3 C). Later in anaphase, NuSAP was intensely localized to the spindle in the chromosomal area. At telophase, NuSAP localized to “spikes” around the decondensing chromosomes, which disappeared toward the end of cytokinesis (Fig. 3 A). Consistent with the reduced protein levels in G1 cells seen by Western blot (Fig. 2 D), very little NuSAP could be detected in G1 cells (Fig. 3 A), indicating a rapid degradation of NuSAP after mitosis. Interestingly, the localization of NuSAP to chromosomes in mitotic cells was not sensitive to nocodazole treatment, indicating that part of its mitotic localization may be independent of microtubules (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