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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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Related in: MedlinePlus

NuSAP expression is up-regulated in proliferating cells during G2/M phase of the cell cycle. (A–D) Northern and Western blot analysis for NuSAP expression in synchronized MC3T3E1 cells. RNA or protein was isolated at the indicated time points (h). (A and B) Cells were arrested in their growth by serum starvation, and subsequently released by addition of complete medium. (C and D) Cells were synchronized using a double-thymidine block. (D) DNA content of thymidine-synchronized cells was determined by FACS® analysis at time points I–III. (A and C) Northern blots were also probed for histone H4 expression, an S phase marker. NuSAP transcript sizes (kb) are indicated. As a loading control, blots were probed for 18S or β-actin expression.
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fig2: NuSAP expression is up-regulated in proliferating cells during G2/M phase of the cell cycle. (A–D) Northern and Western blot analysis for NuSAP expression in synchronized MC3T3E1 cells. RNA or protein was isolated at the indicated time points (h). (A and B) Cells were arrested in their growth by serum starvation, and subsequently released by addition of complete medium. (C and D) Cells were synchronized using a double-thymidine block. (D) DNA content of thymidine-synchronized cells was determined by FACS® analysis at time points I–III. (A and C) Northern blots were also probed for histone H4 expression, an S phase marker. NuSAP transcript sizes (kb) are indicated. As a loading control, blots were probed for 18S or β-actin expression.

Mentions: To confirm the initial observation that NuSAP expression is proliferation related, MC3T3E1 cells were specifically arrested in their growth by serum withdrawal and analyzed for NuSAP expression by Northern blot analysis. An ∼2.4-kb band was identified as the major transcript, and as expected, NuSAP RNA levels were reduced more than 10-fold. When quiescent cells were resupplemented with serum, NuSAP RNA levels increased again, displaying an expression pattern similar to the growth marker histone H4 (Stein et al., 1990b; Fig. 2 A). At the protein level, NuSAP expression was reduced more than threefold, 48 h after serum withdrawal, and increased again in cells that subsequently resumed growth (Fig. 2 B).


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)

NuSAP expression is up-regulated in proliferating cells during G2/M phase of the cell cycle. (A–D) Northern and Western blot analysis for NuSAP expression in synchronized MC3T3E1 cells. RNA or protein was isolated at the indicated time points (h). (A and B) Cells were arrested in their growth by serum starvation, and subsequently released by addition of complete medium. (C and D) Cells were synchronized using a double-thymidine block. (D) DNA content of thymidine-synchronized cells was determined by FACS® analysis at time points I–III. (A and C) Northern blots were also probed for histone H4 expression, an S phase marker. NuSAP transcript sizes (kb) are indicated. As a loading control, blots were probed for 18S or β-actin expression.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: NuSAP expression is up-regulated in proliferating cells during G2/M phase of the cell cycle. (A–D) Northern and Western blot analysis for NuSAP expression in synchronized MC3T3E1 cells. RNA or protein was isolated at the indicated time points (h). (A and B) Cells were arrested in their growth by serum starvation, and subsequently released by addition of complete medium. (C and D) Cells were synchronized using a double-thymidine block. (D) DNA content of thymidine-synchronized cells was determined by FACS® analysis at time points I–III. (A and C) Northern blots were also probed for histone H4 expression, an S phase marker. NuSAP transcript sizes (kb) are indicated. As a loading control, blots were probed for 18S or β-actin expression.
Mentions: To confirm the initial observation that NuSAP expression is proliferation related, MC3T3E1 cells were specifically arrested in their growth by serum withdrawal and analyzed for NuSAP expression by Northern blot analysis. An ∼2.4-kb band was identified as the major transcript, and as expected, NuSAP RNA levels were reduced more than 10-fold. When quiescent cells were resupplemented with serum, NuSAP RNA levels increased again, displaying an expression pattern similar to the growth marker histone H4 (Stein et al., 1990b; Fig. 2 A). At the protein level, NuSAP expression was reduced more than threefold, 48 h after serum withdrawal, and increased again in cells that subsequently resumed growth (Fig. 2 B).

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