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First published online 19 March 2009
doi: 10.1242/jcs.043034

Journal of Cell Science 122, 1111-1118 (2009)
Published by The Company of Biologists 2009
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Nucleocytoplasmic shuttling of soluble tubulin in mammalian cells

Tonia Akoumianaki1, Dimitris Kardassis1,2, Hara Polioudaki1, Spyros D. Georgatos3,4 and Panayiotis A. Theodoropoulos1,*

1 Department of Biochemistry, University of Crete, School of Medicine, 71 003 Heraklion, Greece
2 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, 71 110 Heraklion, Greece
3 Stem Cell and Chromatin Group, The Laboratory of Biology, University of Ioannina School of Medicine, 45 110 Ioannina, Greece
4 The Biomedical Institute of Ioannina, IBE/ITE, 45 110 Ioannina, Greece


Figure 1
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  		Fig. 1. Reversible accumulation of endogenous tubulin in the nuclei of HeLa cells is inhibited by LMB. (A) Distribution of endogenous {alpha}/β-tubulin in HeLa cells under microtubule depolymerization conditions (nocodazole+cold) and after a temperature shift to normal temperature (temp. shift). (B) Effect of leptomycin B (LMB) on nuclear export of endogenous tubulin (for details see Materials and Methods). Cells were fixed and stained with anti-tubulin antibodies immediately after treatment as described in Materials and Methods. DNA was stained with Topro. Scale bars: 20 µm.

 

Figure 2
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  		Fig. 2. Distribution of GFP-tagged β-tubulin during mitosis. HeLa cells transiently transfected with GFP-tagged βII-tubulin, cultured for 24 hours and immunostained for lamin B. Representative confocal images from metaphase to telophase are depicted. Scale bar: 20 µm.

 

Figure 3
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  		Fig. 3. Distribution of GFP-tagged β-tubulin in interphase HeLa cells. Cells transiently transfected with GFP-tagged β-tubulin were fixed and surveyed by confocal microscopy. (A) Distribution of βII-tubulin in untreated (upper panel) and Triton-X extracted (lower panel) cells. The linescans on the right depict distance versus fluorescence intensity profiles calculated using the LCS Lite Leica software. Asterisks denote the presence of nucleoli. Note the almost uniform distribution of fluorescence in both the cytoplasm and the nucleus, with the exception of the nucleolus in untreated cells and the microtubule network revealed when cells are treated with Triton X-100. The inset shows a higher magnification of the cell nucleus. (B) Relative distribution of GFP-βII-tubulin in quantitative terms. The fluorescence intensity in similarly sized ROIs (red circles) was measured in the nucleus and the cytoplasm of the transfected cells. Numbers represent the calculated nuclear/cytoplasmic ratios in the cells shown. (C) Changes in nuclear/cytoplasmic ratios in the course of GFP-tubulin expression. Transiently transfected cells were cultured for 3-72 hours and the nuclear/cytoplasmic fluorescence intensity was determined for each time point in 90-200 specimens. Scale bars: 10 µm.

 

Figure 4
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  		Fig. 4. Nuclear export of GFF β-tubulin is mediated by NES motifs. (A) Schematic representation of putative nuclear export sequences (NES) in wild-type (WT) human β-tubulin and two β-tubulin mutants with disrupted NES (disNES-1 and disNES-2). Two Leu residues in each NES sequences were replaced by Ala, as indicated. Hydrophobic amino acids in each NES are shown in red. (B) Schematic diagram of β-tubulin structure using 3D molecule viewer of the Vector NTI Suite software. The segments accommodating the putative NES are shown in red. (C) Histograms showing the ratio of nuclear/cytoplasmic fluorescence intensity in cells expressing wild-type and NES mutated GFP-tagged βII-tubulin. Between 110 and 200 cells were scored per treatment. (D) Cell cycle analysis of cells expressing GFP, wild-type (GFPβII-tb), NES-1 (GFPdisNES-1) and NES-2 (GFPdisNES-2) mutated β-tubulin. Percentages of viable cells in the G0-G1, S and G2-M phase are provided. Values are mean ± s.e.m. of measurements in three separate experiments.

 

Figure 5
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  		Fig. 5. Tubulin binding to histone H3. (A) Blot overlay assay using as a substrate a total cell extract (lanes 1), histone fractions isolated from turkey erythrocytes (lanes 3), and a mixture of histones and BSA and GST used as negative controls (lanes 2, 4 and 5). An SDS-PAGE profile after Coomassie Blue staining (CB) and a blot (Blot) probed with brain tubulin and developed by anti-β-tubulin antibodies are shown. (B) A similar assay with recombinant H3 (rH3) and histones from non treated cells (HeLa); cells treated with taxol (HeLa Tx) and cells treated with TSA (HeLa TSA). An SDS-PAGE profile after Coommasie Blue staining and a blot probed with brain tubulin and developed by anti-β-tubulin antibodies are shown. (C) Blot overlay assays using a fixed amount (input) of recombinant H3 and H4 as substrates and increasing quantities of brain tubulin as a probe. Bound tubulin was quantified by PC-based image analysis. (D) Co-precipitation of tubulin with histone H3. Immunoprecipitation (IP) of tubulin from HeLa extracts (Ext.) in the presence (+) or absence (–) of anti {alpha}-tubulin antibodies (a-tb). The samples were run on SDS-polyacrylamide gels and probed with anti {alpha}-tubulin (tb) and anti H3 (H3) antibodies (WB). For details on assays, see Materials and Methods.

 

Figure 6
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  		Fig. 6. Effect of tubulin on LBR-histone-HP1 complex formation. All panels show SDS-PAGE Coomassie Blue profiles from pull-down assays. (A) Recombinant H3 (rH3) precipitated by HP1β-GST (HP1β) in the presence of increasing amounts (triangle) of purified tubulin (tb). (B) Tubulin precipitated by HP1β-GST or a GST control (GST). (C) Precipitation of H3 after incubation of NtLBR-GST beads with a fixed amount of recombinant H3 and increasing amounts (triangle) of purified tubulin (tb). `d.p.' shows the characteristic proteolytic products of recombinant NtLBR-GST that are produced after expression in bacteria. (D) NtLBR-GST was immobilized on glutathione beads and reacted sequentially with standard amounts of histones H3/H4 (NtLBR-H3/H4) and His-HP1β (NtLBR-H3/H4+HisHP1β), or histones H3/H4 (NtLBR-H3/H4), tubulin and His-HP1β (NtLBR-H3/H4+HisHP1β+tb). For details on assays, see Materials and Methods.

 

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