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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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Overexpression of NuSAP caused bundling of cytoplasmic microtubules. (A) Myc-tagged full-length NuSAP was analyzed in interphase COS1 cells 24 h after transfection. Cells were fixed in glutaraldehyde and double stained for Myc-tagged NuSAP and α-tubulin. NuSAP overexpression induces microtubule bundles that resist nocodazole treatment. (B) Time-lapse microscopy of a PtK2 cell stably expressing YFP-tagged α-tubulin and transiently expressing CFP-tagged NuSAP. NuSAP overexpression leads to a cytoplasmic pool of NuSAP with subsequent bundling of microtubules. Time is h:min. Bars: (A and B) 10 μm.
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fig5: Overexpression of NuSAP caused bundling of cytoplasmic microtubules. (A) Myc-tagged full-length NuSAP was analyzed in interphase COS1 cells 24 h after transfection. Cells were fixed in glutaraldehyde and double stained for Myc-tagged NuSAP and α-tubulin. NuSAP overexpression induces microtubule bundles that resist nocodazole treatment. (B) Time-lapse microscopy of a PtK2 cell stably expressing YFP-tagged α-tubulin and transiently expressing CFP-tagged NuSAP. NuSAP overexpression leads to a cytoplasmic pool of NuSAP with subsequent bundling of microtubules. Time is h:min. Bars: (A and B) 10 μm.

Mentions: To study the effect of gain of NuSAP function, full-length and various fragments of GFP-tagged NuSAP were overexpressed in COS1 cells, and their effects on the microtubule network were analyzed by (immuno)fluorescence microscopy. Overexpression of full-length NuSAP protein resulted in the appearance of long, curved, and unusually thick microtubule bundles in the cytoplasm, to which NuSAP colocalized. These bundled microtubules were extremely stable, and did not depolymerize in the presence of high doses of nocodazole (Fig. 5 A). Similarly, overexpression of the NuSAP fragments 243–427 and 129–367 (Table I) caused strong bundling of cytoplasmic microtubules, confirming their microtubule-binding competence.


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)

Overexpression of NuSAP caused bundling of cytoplasmic microtubules. (A) Myc-tagged full-length NuSAP was analyzed in interphase COS1 cells 24 h after transfection. Cells were fixed in glutaraldehyde and double stained for Myc-tagged NuSAP and α-tubulin. NuSAP overexpression induces microtubule bundles that resist nocodazole treatment. (B) Time-lapse microscopy of a PtK2 cell stably expressing YFP-tagged α-tubulin and transiently expressing CFP-tagged NuSAP. NuSAP overexpression leads to a cytoplasmic pool of NuSAP with subsequent bundling of microtubules. Time is h:min. Bars: (A and B) 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172854&req=5

fig5: Overexpression of NuSAP caused bundling of cytoplasmic microtubules. (A) Myc-tagged full-length NuSAP was analyzed in interphase COS1 cells 24 h after transfection. Cells were fixed in glutaraldehyde and double stained for Myc-tagged NuSAP and α-tubulin. NuSAP overexpression induces microtubule bundles that resist nocodazole treatment. (B) Time-lapse microscopy of a PtK2 cell stably expressing YFP-tagged α-tubulin and transiently expressing CFP-tagged NuSAP. NuSAP overexpression leads to a cytoplasmic pool of NuSAP with subsequent bundling of microtubules. Time is h:min. Bars: (A and B) 10 μm.
Mentions: To study the effect of gain of NuSAP function, full-length and various fragments of GFP-tagged NuSAP were overexpressed in COS1 cells, and their effects on the microtubule network were analyzed by (immuno)fluorescence microscopy. Overexpression of full-length NuSAP protein resulted in the appearance of long, curved, and unusually thick microtubule bundles in the cytoplasm, to which NuSAP colocalized. These bundled microtubules were extremely stable, and did not depolymerize in the presence of high doses of nocodazole (Fig. 5 A). Similarly, overexpression of the NuSAP fragments 243–427 and 129–367 (Table I) caused strong bundling of cytoplasmic microtubules, confirming their microtubule-binding competence.

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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