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. 1998 Feb 17;95(4):1595-600.
doi: 10.1073/pnas.95.4.1595.

Cell cycle-dependent subcellular localization of the TSG101 protein and mitotic and nuclear abnormalities associated with TSG101 deficiency

W Xie  1 , L Li, S N Cohen
Affiliations

Cell cycle-dependent subcellular localization of the TSG101 protein and mitotic and nuclear abnormalities associated with TSG101 deficiency

W Xie et al. Proc Natl Acad Sci U S A. .

Abstract

TSG101 is a recently discovered tumor susceptibility gene whose functional inactivation in mouse fibroblasts results in cell transformation and the ability to form metastatic tumors in nude mice. Although restoration of TSG101 activity reverses tumorigenesis, neoplasia is irreversible in some cells, suggesting that permanent genetic alteration can occur during TSG101 inactivation. Here we describe studies that support this notion. We find that localization of TSG101 is cell cycle-dependent, occurring in the nucleus and Golgi complex during interphase, and in mitotic spindles and centrosomes during mitosis; cells made neoplastic by a deficiency in TSG101 expression show a series of mitosis-related abnormalities, including multiple microtubule organizing centers, aberrant mitotic spindles, abnormal distribution of metaphase chromatin, aneuploidy, and nuclear anomalies. Our findings suggest that TSG101 deficiency may lead to genome instability in addition to previously reported reversible neoplastic transformation.

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Figures

Figure 1
Figure 1
Generation and analysis of anti-TSG101 antibody. (a) Map of TSG101 protein-coding region showing the location of the N-terminal (amino acids 2–17), coiled-coil domain (amino acids 282–300), and C-terminal peptides (amino acids 364–380), which are identical or highly conserved in human and mouse and were used as antigens. Peptide locations are designated according to the TSG101 sequence shown in ref. . (b) Polyclonal antibody detecting a band of 43 kDa in mammalian extract (lane 2); analysis using prebleed serum from the same rabbit (lane 1). Lane 3 shows affinity-purified antibody against the TSG101 coiled-coil domain detecting a single band, migrating at the same location as the mammalian signal, in an extract of Sf9 cells overexpressing TSG101. Purified antibodies generated against the N-terminal or C-terminal domains of TSG101 detected the same band.
Figure 2
Figure 2
Cell cycle-dependent localization of TSG101. Cells were synchronized at G0 (a), G1 (b), G1/S (i), and S (j) stages of the cell cycle and stained with affinity-purified anti-TSG101 antibody. GFP-TSG101 fusion protein localization in cycling cells: c Lower, nuclear staining; c Upper, diffuse cytoplasmic staining; c Right, asymmetric cytoplasmic staining. Cells stained with the Golgi-specific marker β-COP (d) and TSG101 (e). Cells costained with anti-TSG101 antibody (red) and Bodipy FL C5-ceramide (green) showing colocalization (yellow) (f) Brefeldin A-treated cells stained for β-COP (g) and TSG101 (h) showing cytoplasmic dispersion. Mitotic cells (k) showing staining of TSG101 (green) in mitotic spindle and propidium iodide-stained metaphase chromatin (red). (l Upper × 3) Localization of tubulin (red), TSG101 (green), and both (yellow) in centrosomes. (l Lower × 3) The staining of a mitotic spindle, as above. (m) Staining of TSG101 in midbody (green); condensed chromatin is stained in red by propidium iodide. Cells stained with propidium iodide (red) and purified anti-TSG101 antibody (green) (n), and with the same antibody preincubated with the antigen peptide (o) or two irrelevant TSG101 peptides (q and r), are compared with cells stained with prebleed from the same rabbit purified in the same fashion as the immune sera (p). All photographs except for GFP-fusion study were made by using confocal microscopy.
Figure 3
Figure 3
TSG101-specific RNA (Upper) at different stages of the cell cycle, with β-actin RNA control (Lower). Lanes 1–8, RNA extracted from cells synchronized in G0, early G1, mid-G1, late G1, G1/S, S, G2/M, and M stages of the cell cycle. Lane 9, RNA extracted from nonsynchronized cycling cells.
Figure 4
Figure 4
Mitotic spindles, microtubule organizing centers, and nuclear morphology of SL6 cells. (a) NIH 3T3 (Upper) and SL6 (Lower) cells stained by anti-TSG101 antibody. (b) Cell stained by anti-tubulin antibody (green) and propidium iodide to detect DNA (red). (c) Tubulin (Left), TSG101 (Center), or both (Right) staining in an SL6 cell containing five centrosomes; other metaphase cells in this preparation showed similar abnormalities. (d) “H”-shaped metaphase DNA (red) and tubulin-stained mitotic spindle (green) in SL6 cell. (e–g) Nuclear abnormalities in SL6 cells, stained for tubulin (green) and DNA (red). All photographs were made by using confocal microscopy.
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