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. 2003 Sep 15;162(6):1017-29.
doi: 10.1083/jcb.200302129. Epub 2003 Sep 8.

NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization

Tim Raemaekers  1 , Katharina Ribbeck, Joel Beaudouin, Wim Annaert, Mark Van Camp, Ingrid Stockmans, Nico Smets, Roger Bouillon, Jan Ellenberg, Geert Carmeliet
Affiliations

NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization

Tim Raemaekers et al. J Cell Biol. .

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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Figures

Figure 1.
Figure 1.
Identification of NuSAP. (A and B) Deduced amino acid sequence of mouse and human NuSAP and its alignment with predicted proteins from other species, and with the SAP motif consensus sequence. (A) Identical and similar residues are shaded in black. Homologous residues were taken as follows: positively charged (R and K), negatively charged (E and D), and hydrophobic (L,V,I,F, and M). Gaps, indicated by dashes or numbers between parentheses, were introduced for optimal alignment. Boxed at the NH2 terminus is the potential SAP motif, and at the COOH terminus (in dashed lines) is a conserved stretch of highly charged residues, with a predicted helical structure, which we have named the ChHD domain. The potential PEST sequence is shaded in gray, and the putative KEN boxes are double underlined. The potential NLS identified in the mouse sequence is underlined. (B) Residues within the SAP motif consensus sequence have been defined by Aravind and Koonin (2000): h (hydrophobic), p (polar), l (aliphatic), and b (bulky). Also shown is the sequence of Acinus (GenBank/EMBL/ DDBJ accession no. AAF89661), a SAP module–containing protein. Shaded in black are residues that agree with the consensus sequence, and in gray are residues that conform to the similarity as described in A. Sequences besides those of mouse and human were deduced from ESTs. The GenBank/EMBL/DDBJ accession nos. are as follows: Hs, Homo sapiens (AAG25874); Bt, Bos taurus (BE480183); Mm, Mus musculus (AAG31285); Rn, Rattus norvegicus (AA923940); Gg, Gallus gallus (AJ392813); Xl, Xenopus laevis (AW642384); and Dr, Danio rerio (AI545826, AI958745). (C) SDS-PAGE of radiolabeled, in vitro transcribed and translated NuSAP. The transcription and translation reaction (TNT) was followed by treatment of the sample with calf intestine alkaline phosphatase buffer in the absence (buffer) or presence of (phosphatase) enzyme. The bandshift indicates that in vitro–produced NuSAP is a phosphoprotein. Luciferase DNA was used as a positive control, whereas no DNA template was used in the negative control. (D) Western blot of total cell lysates prepared from MC3T3E1 cells and transfected COS1 cells. For transfections, an empty control or NuSAP-Myc vector was used. The blot was probed for NuSAP expression using both anti-NuSAP and anti-Myc antibodies. The polyclonal anti-NuSAP antibodies include an anti-peptide (Anti-NuSAPp) and an anti-recombinant protein (Anti-NuSAPr) antibody. (E) Western blot analysis for NuSAP expression in different cell lines. The blot, which was prepared from total cell lysates, was also probed for β-actin expression. Arrowhead indicates the 51-kD marker (C–E).
Figure 2.
Figure 2.
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.
Figure 3.
Figure 3.
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.
Figure 4.
Figure 4.
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.
Figure 5.
Figure 5.
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.
Figure 6.
Figure 6.
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.
Figure 7.
Figure 7.
Suppression of NuSAP results in defective anaphase and cytokinesis. (A–E) HeLa cells were analyzed at specific time points after transfection with siRNA duplexes. (A) Anaphase and cytokinesis appearance in synchronized cells 30 h after transfection. Cells were fixed in methanol and double stained for α-tubulin and nucleic acids. (B) Quantification of the observed abnormal anaphase spindles. The percentage of normal and abnormal anaphase spindles is depicted with the total anaphase fraction set as 100% (three independent experiments). (C). Relative number of binucleated cells 10 and 40 h after transfection; 300 interphase cells from each of three independent experiments were scored for normal or binucleate phenotype. (D) Cells were fixed in methanol and stained for γ-tubulin, centrin, and nucleic acids 48 h after transfection. Some centrin foci are not clearly visible, as the image was taken at a specific z-axis section. Insets show a higher magnigfication of γ-tubulin and centrin foci. (E) Quantification of the multipolar spindle phenotype in synchronized cells at the indicated time points corresponding to successive mitosis; 100 mitotic cells from each of three independent experiments were scored for the presence of multipolar spindles. Bars: (A, D, and E) 10 μm.
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