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First published online 29 April 2008
doi: 10.1242/jcs.019174

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EML3 is a nuclear microtubule-binding protein required for the correct alignment of chromosomes in metaphase

¹ Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
² MitoCheck Project Group, European Molecular Biology Laboratory, Meyerhofstr.1, 69117 Heidelberg, Germany
³ Max-Planck-Institut für Molekulare Genetik, Ihnestr. 73, 14195 Berlin, Germany

View larger version (51K): [in this window] [in a new window]   Fig. 1. Purification of microtubule-binding proteins from nuclear extracts. Purification scheme (A) and analysis of nuclear extracts (nu) or cytosolic extracts (cyt) by immunoblot (B) using specific antibodies to the human nuclear MAP TPX2 (hTPX2), the cytosolic chaperone Hsc70, -tubulin, the chromatin protein RCC1, β-actin or the cytoplasmic MAP OP18/Stathmin. (C) Analysis of MAP purification by sedimentation in the presence of taxol or nocodazole. Nuclear extract (T), supernatant (1) or pellet (2) after first microtubule spindown and supernatant (3) or pellet (4 and 5, 10x amount) after second microtubule spindown were analysed by silver staining of total protein (lower panel) and immunoblot using antibodies specific to RCC1 (middle panel) or and human TPX2 (hTPX2, upper panel). Expected running behaviour on SDS gels (B and C, right) and respective molecular weights (B and C, left) are indicated. Note that probably owing to posttranslational modification (see gel retardation in the SDS gel), TPX2 in the microtubule pellet fraction (Fig. 1C, lane 2) was consistently more difficult to detect by immunoblotting than in total (T) or the supernatant (1) and only visible after long exposure times. (D) Protein sequence of human TPX2 (upper panel) and representative ion spectrum (lower panel). Identified peptides are shown in red and green, respectively, which resulted in a sequence coverage of 30%. The fragment ion spectrum of the peptide coloured in green is shown in the lower panel. C-terminal fragment ions of the peptide are labelled as y fragments, N-terminal fragments as b. The numbers of their respective N-terminal amino acid (y) or C-terminal amino acid (b) are indicated. Doubly charged fragments are labelled (++), all other fragments were singly charged.

View larger version (50K): [in this window] [in a new window]   Fig. 2. Characterisation of EML3 localisation by indirect immunofluorescence. Antibodies against human EML3 peptide sequences were used to detect the protein by immunoblot or immunofluorescence. (A) Immunoblots of total lysates of HeLa cells. P, preimmune serum; I, EML3 immune serum. (B) Localisation of EML3 in the different mitotic phases and in interphase of HeLa cells as indicated. Cells were fixed with methanol at –20°C and stained with serum against EML3 (middle left panels and red colour in merge), -tubulin (middle right panels and green colour in merge) and with DAPI to visualise DNA (blue colour in merge). (C) EML3 was detected by indirect immunofluorescence after fixation with formaldehyde in interphase HeLa cells before and after addition of leptomycin B (LMB) to inhibit CRM1-dependent nuclear export. Scale bars: 10 µm.

View larger version (76K): [in this window] [in a new window]   Fig. 3. Characterisation of YFP-EML3 localisation. Images show the localisation of the EYFP-EML3 fusion protein in interphase and in the different mitotic phases as indicated. Cells were fixed with methanol at –20°C and stained with antibodies against GFP (middle left panels and red colour in merge), -tubulin (middle right panels and green colour in merge) and with DAPI to visualise DNA (blue colour in merge). Scale bars: 10 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 4. Domain analysis of Eml3. Schematic representation is shown at the top of the constructs used to prepare images below. From the full-length (FL) human EML3 (see also Fig. 2), two basic amino acids were replaced to corrupt the nuclear localisation signal (NLS). An N-terminal fragment (amino acids 1-168, N) contained the HELP domain, whereas the N-terminal 168 amino acids including the HELP domain were missing in the N construct. The EYFP-Eml3 fusion constructs (EYFP-Eml3) were visualised in interphase or metaphase, respectively. Cells were fixed with methanol at –20°C and stained with antibodies against GFP (middle left panels and red colour in merge), -tubulin (middle right panels and green colour in merge) and with DAPI to visualise DNA (blue colour in merge). Scale bars: 10 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 5. Phenotypic analysis of EML3 knockdown using time-lapse imaging. (A) siRNA oligos specific for Eml3 (80 nM) were used to knockdown the respective mRNAs in HeLa Kyoto cells; the same concentration of scrambled, nontargeting siRNAs served as a control. Left panel, qRT-PCR quantification of siRNA-mediated gene knockdown 24 hours post transfection (see experimental procedures). The relative remaining levels of target mRNA expression normalised by GAPDH mRNA was measured compared with cells transfected with scrambled siRNA oligos. Error bars represent the maximum and minimum expression levels of three individual experiments. Right panel, total cell lysate was prepared from HeLa cells before and after knockdown of EML3 using two different siRNA oligonucleotides (1 and 2) and EML3 was detected by immunoblot using specific antibodies. Tubulin served as a loading control. (B) siRNA oligos were used to knockdown EML3 in HeLa Kyoto cells stably expressing Histone 2B-EGFP. 29 hours after transfection, the EGFP signal was recorded at intervals of 30 minutes for another 48 hours. Automatically analysed phenotypes were classified and indices of mitosis, apoptosis, shape and the overall proliferation rate plotted over time. Results of representative experiments are plotted in blue (continuous line, fitted curve; dotted line, measured data points) and the control confidence band (mean ± s.d.) in grey. (C) The EGFP signal was recorded for 14 hours at intervals of 5 minutes, 60 hours after siRNA oligo transfection. Sections from selected frames were used for still images, times after seeding (i.e. transfection of) the cells (in brackets) and time intervals from the respective starting points are indicated.

View larger version (33K): [in this window] [in a new window]   Fig. 6. Analysis of EML3 function in spindle formation. Knockdown of EML3 was performed as described in Fig. 5 using two different siRNA oligonucleotides specific to EML3 (1 and 2) and cells fixed with paraformaldehyde 80 hours post transfection. (A) Mitotic indices after gene knockdown were determined from 480 cells and means and s.d. from three independent experiments were plotted (left panel). The sums of cells at the different mitotic stages after gene knockdown were determined in the mitotic cells (right panel). (B) Representative immunofluorescence images of aberrant metaphase-like structures with unaligned chromosomes. Merge: DNA (blue) and Tubulin (green). Scale bars: 10 µm. Quantification of phenotypes resulting from EML3 knockdown is shown on the right. Means ± s.d. from three independent experiments (n=100) were plotted. (C) The maximum extension of metaphase chromosomes in a virtual pole-to-pole axis of the mitotic spindle was determined in 20 images after treatment with the respective siRNA oligos as indicated. Single values (left) and average values and s.d. (right) were plotted.

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