NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization.
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.
Affiliation: Laboratory for Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium.
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.
- Cell Nucleolus/genetics/metabolism*
- Microtubule-Associated Proteins/antagonists & inhibitors/genetics/metabolism*
- Nuclear Proteins/antagonists & inhibitors/genetics/metabolism*
- Spindle Apparatus/genetics/metabolism*
- Amino Acid Sequence/genetics
- Base Sequence/genetics
- Cell Line
- Cell Nucleus/genetics/pathology
- Chromosome Segregation/genetics
- DNA, Complementary/analysis
- Eukaryotic Cells/metabolism
- G2 Phase/genetics
- Molecular Sequence Data
- Protein Binding/genetics
- Protein Structure, Tertiary/genetics
- RNA Interference
- RNA, Messenger/metabolism
- Up-Regulation/drug effects/physiology
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fig1: 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).
We compared gene expression patterns of proliferating and differentiating mouse MC3T3E1 osteoblasts by differential display, and identified a cDNA fragment that showed enhanced levels during proliferation. The full-length mouse sequence of this transcript contained a single ORF of a 1,281-bp coding region, which is identical to the sequence of an uncharacterized lymphocyte protein (GenBank/EMBL/DDBJ accession no. AAG31285). The protein was designated NuSAP (see following paragraphs). Cloning of the human NuSAP cDNA and comparison with EST databases showed that NuSAP is highly conserved in vertebrates, but no clear homologues could be identified in invertebrates (Fig. 1 A). Mouse NuSAP cDNA is predicted to encode a protein of 427 aa with a calculated molecular mass of 48.6 kD and an isoelectric point of 9.9. The apparent molecular mass of NuSAP was slightly higher, being ∼55 kD (Fig. 1 C), and this difference can be partially accounted for by phosphorylation as shown by treatment with alkaline phosphatase (Fig. 1 C), but appears to be primarily the result of the high basicity of the protein.