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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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NuSAP binds to microtubules in vitro and in vivo. (A and B) Microtubule sedimentation assay of in vitro produced NuSAP with pure prepolymerized microtubules. (A) The assay was performed using crude in vitro translation product in the presence (+) or absence (−) of microtubules. Recovery of NuSAP in the microtubule pellet (P) fraction, as opposed to the soluble supernatant (SN) fraction, was determined by immunoblotting with anti-NuSAP and anti-α-tubulin antibodies. As a negative control, the assay was performed using reaction product with no template DNA. Included on the blot is an in vitro transcription and translation product of NuSAP (TNT) and total cell lysate prepared from MC3T3E1 cells (L). (B) Affinity of NuSAP for microtubules was determined by plotting bound NuSAP versus the tubulin concentration yielding a dissociation constant (Kd) of ∼1 μM. (C) Microtubule sedimentation assay with purified recombinant NuSAP (detected by Western blot) and pure prepolymerized microtubules (Coomassie). (D) Interphase MC3T3E1 cells were briefly permeabilized before PFA fixation, and double stained for α-tubulin and endogenous NuSAP or NuMA. NuSAP (but not NuMA) colocalizes to perinuclear microtubules. (E and F) Full-length and various GFP-tagged NuSAP fragments were analyzed in transfected COS1 cells for subcellular localization and microtubule-binding potential. In the latter assay, cells were permeabilized (perm.) before glutaraldehyde fixation. The microtubule-binding domain of NuSAP lies toward the COOH terminus, as fragments 243–427 and 129–367, like full-length NuSAP, associates with microtubules. For subcellular localization studies, nonpermeabilized cells (nonperm.) were fixed in PFA. Bars: (D and F) 10 μm.
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fig4: NuSAP binds to microtubules in vitro and in vivo. (A and B) Microtubule sedimentation assay of in vitro produced NuSAP with pure prepolymerized microtubules. (A) The assay was performed using crude in vitro translation product in the presence (+) or absence (−) of microtubules. Recovery of NuSAP in the microtubule pellet (P) fraction, as opposed to the soluble supernatant (SN) fraction, was determined by immunoblotting with anti-NuSAP and anti-α-tubulin antibodies. As a negative control, the assay was performed using reaction product with no template DNA. Included on the blot is an in vitro transcription and translation product of NuSAP (TNT) and total cell lysate prepared from MC3T3E1 cells (L). (B) Affinity of NuSAP for microtubules was determined by plotting bound NuSAP versus the tubulin concentration yielding a dissociation constant (Kd) of ∼1 μM. (C) Microtubule sedimentation assay with purified recombinant NuSAP (detected by Western blot) and pure prepolymerized microtubules (Coomassie). (D) Interphase MC3T3E1 cells were briefly permeabilized before PFA fixation, and double stained for α-tubulin and endogenous NuSAP or NuMA. NuSAP (but not NuMA) colocalizes to perinuclear microtubules. (E and F) Full-length and various GFP-tagged NuSAP fragments were analyzed in transfected COS1 cells for subcellular localization and microtubule-binding potential. In the latter assay, cells were permeabilized (perm.) before glutaraldehyde fixation. The microtubule-binding domain of NuSAP lies toward the COOH terminus, as fragments 243–427 and 129–367, like full-length NuSAP, associates with microtubules. For subcellular localization studies, nonpermeabilized cells (nonperm.) were fixed in PFA. Bars: (D and F) 10 μm.

Mentions: NuSAP's specific localization to central spindle microtubules in mitotic cells led us to study whether it interacts with pure prepolymerized microtubules in a sedimentation assay. First, we used in vitro translated NuSAP in the assay, and as shown in Fig. 4 A, NuSAP was recovered in the microtubule pellet when microtubules were present, but remained soluble in the absence of microtubules (Fig. 4 A). This interaction was specific, as a well-characterized MAP (MAP2) also bound to microtubules, whereas BSA did not (unpublished data). To estimate the affinity of NuSAP for microtubules, NuSAP was mixed with different concentrations of microtubules in the same assay. Plotting of bound NuSAP versus the microtubule concentration and direct hyperbolic curve-fitting yielded an equilibrium dissociation constant of ∼1 μM (the tubulin concentration required to bind 50% of NuSAP; Fig. 4 B), indicating microtubule binding with moderately high affinity. To determine whether NuSAP interacts directly with microtubules, the sedimentation assay was repeated in a more defined system using purified recombinant NuSAP protein and pure prepolymerized microtubules. Recombinant NuSAP co-pelleted to large parts with microtubules, and only little remained in the microtubule-unbound supernatant fraction (Fig. 4 C). These results indicate that NuSAP not only colocalizes, but also directly binds to microtubules, fulfilling the criteria of a bona fide MAP.


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 binds to microtubules in vitro and in vivo. (A and B) Microtubule sedimentation assay of in vitro produced NuSAP with pure prepolymerized microtubules. (A) The assay was performed using crude in vitro translation product in the presence (+) or absence (−) of microtubules. Recovery of NuSAP in the microtubule pellet (P) fraction, as opposed to the soluble supernatant (SN) fraction, was determined by immunoblotting with anti-NuSAP and anti-α-tubulin antibodies. As a negative control, the assay was performed using reaction product with no template DNA. Included on the blot is an in vitro transcription and translation product of NuSAP (TNT) and total cell lysate prepared from MC3T3E1 cells (L). (B) Affinity of NuSAP for microtubules was determined by plotting bound NuSAP versus the tubulin concentration yielding a dissociation constant (Kd) of ∼1 μM. (C) Microtubule sedimentation assay with purified recombinant NuSAP (detected by Western blot) and pure prepolymerized microtubules (Coomassie). (D) Interphase MC3T3E1 cells were briefly permeabilized before PFA fixation, and double stained for α-tubulin and endogenous NuSAP or NuMA. NuSAP (but not NuMA) colocalizes to perinuclear microtubules. (E and F) Full-length and various GFP-tagged NuSAP fragments were analyzed in transfected COS1 cells for subcellular localization and microtubule-binding potential. In the latter assay, cells were permeabilized (perm.) before glutaraldehyde fixation. The microtubule-binding domain of NuSAP lies toward the COOH terminus, as fragments 243–427 and 129–367, like full-length NuSAP, associates with microtubules. For subcellular localization studies, nonpermeabilized cells (nonperm.) were fixed in PFA. Bars: (D and F) 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172854&req=5

fig4: NuSAP binds to microtubules in vitro and in vivo. (A and B) Microtubule sedimentation assay of in vitro produced NuSAP with pure prepolymerized microtubules. (A) The assay was performed using crude in vitro translation product in the presence (+) or absence (−) of microtubules. Recovery of NuSAP in the microtubule pellet (P) fraction, as opposed to the soluble supernatant (SN) fraction, was determined by immunoblotting with anti-NuSAP and anti-α-tubulin antibodies. As a negative control, the assay was performed using reaction product with no template DNA. Included on the blot is an in vitro transcription and translation product of NuSAP (TNT) and total cell lysate prepared from MC3T3E1 cells (L). (B) Affinity of NuSAP for microtubules was determined by plotting bound NuSAP versus the tubulin concentration yielding a dissociation constant (Kd) of ∼1 μM. (C) Microtubule sedimentation assay with purified recombinant NuSAP (detected by Western blot) and pure prepolymerized microtubules (Coomassie). (D) Interphase MC3T3E1 cells were briefly permeabilized before PFA fixation, and double stained for α-tubulin and endogenous NuSAP or NuMA. NuSAP (but not NuMA) colocalizes to perinuclear microtubules. (E and F) Full-length and various GFP-tagged NuSAP fragments were analyzed in transfected COS1 cells for subcellular localization and microtubule-binding potential. In the latter assay, cells were permeabilized (perm.) before glutaraldehyde fixation. The microtubule-binding domain of NuSAP lies toward the COOH terminus, as fragments 243–427 and 129–367, like full-length NuSAP, associates with microtubules. For subcellular localization studies, nonpermeabilized cells (nonperm.) were fixed in PFA. Bars: (D and F) 10 μm.
Mentions: NuSAP's specific localization to central spindle microtubules in mitotic cells led us to study whether it interacts with pure prepolymerized microtubules in a sedimentation assay. First, we used in vitro translated NuSAP in the assay, and as shown in Fig. 4 A, NuSAP was recovered in the microtubule pellet when microtubules were present, but remained soluble in the absence of microtubules (Fig. 4 A). This interaction was specific, as a well-characterized MAP (MAP2) also bound to microtubules, whereas BSA did not (unpublished data). To estimate the affinity of NuSAP for microtubules, NuSAP was mixed with different concentrations of microtubules in the same assay. Plotting of bound NuSAP versus the microtubule concentration and direct hyperbolic curve-fitting yielded an equilibrium dissociation constant of ∼1 μM (the tubulin concentration required to bind 50% of NuSAP; Fig. 4 B), indicating microtubule binding with moderately high affinity. To determine whether NuSAP interacts directly with microtubules, the sedimentation assay was repeated in a more defined system using purified recombinant NuSAP protein and pure prepolymerized microtubules. Recombinant NuSAP co-pelleted to large parts with microtubules, and only little remained in the microtubule-unbound supernatant fraction (Fig. 4 C). These results indicate that NuSAP not only colocalizes, but also directly binds to microtubules, fulfilling the criteria of a bona fide MAP.

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