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First published online 1 April 2003
doi: 10.1242/jcs.00412

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Role for NudC, a dynein-associated nuclear movement protein, in mitosis and cytokinesis

¹ Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA
² Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama 35487, USA
³ Department of Molecular Pathology, MD Anderson Cancer Center, Houston, Texas 77030, USA
⁴ Program in Cell and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA
⁵ Department of Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA
⁶ Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA

View larger version (84K): [in a new window]   Fig. 1. NudC localization during M phase. Randomly growing HeLa cells were treated briefly with detergent, as described in Materials and Methods, to remove the soluble portion of NudC, and stained with anti-NudC C peptide antibodies (red) and anti-ß-tubulin antibodies (green). The DAPI staining pattern of DNA is used to stage the cells during mitosis (M phase). The first two columns are shown in black and white for contrast. The last column is shown in color to indicate NudC localization to microtubule-containing mitotic structures (overlay, yellow). (A-C) Prophase. (D-F) Prometaphase. (G-I) Metaphase. (J-L) Anaphase/Telophase. (M-O) Cytokinesis. Bar, 10 µm for all panels.

View larger version (37K): [in a new window]   Fig. 2. NudC downregulation by siRNA-mediated gene silencing. HeLa cells were transfected with Luc siRNA or NudC siRNA for 24-96 hours. (A) NudC protein levels were determined by immunoblotting with anti-NudC C peptide antibodies (top). Equal protein loading was determined by immunoblotting with anti-ß-actin antibodies (bottom). A representative of four independent experiments is shown. (B) The proliferation of HeLa cells transfected with either Luc siRNA or NudC siRNA, or left untransfected was determined over 96 hours. The total cell number x104 in 1 ml of growth medium was determined. Identical results were obtained counting triplicate samples in two independent experiments.

View larger version (69K): [in a new window]   Fig. 3. NudC downregulation induces multinucleation. HeLa cells were transfected with either Luc siRNA or NudC siRNA for 72 hours and stained with anti-NudC C peptide antibodies (red) and anti-ß-tubulin antibodies (green), and counterstained with DAPI (blue). (A) Luc-siRNA-transfected cells. Mitotic cells show more NudC staining (arrow). The MTOC (asterisk) is prominent in interphase cells. Cells undergoing cytokinesis are indicated (arrowhead). (B) NudC-siRNA-transfected cells. An increase in the number of large, flat cells containing multiple nuclei (arrows) and cells connected by persistent intercellular bridges (arrowheads) is seen. Bar, 10 µm. (C) Proportion of multinucleate cells after transfection with NudC siRNA or Luc siRNA, or left untransfected. Results are essentially identical in two independent experiments.

View larger version (72K): [in a new window]   Fig. 4. NudC downregulation induces mitotic and cytokinetic phenotypes. HeLa cells prepared as in Fig. 3 were analysed for mitotic phenotypes. (A-D) Control siRNA-treated cells in different M phases. (A) Cell in prometaphase. (B) Cell in metaphase. (C) Cleavage-furrow (arrowhead) and midzone microtubules forming during anaphase/telophase. (D) Cell undergoing cytokinesis. (E-K) NudC-siRNA-treated cells. (E) Mitotic spindles appear to be less focused during prometaphase in NudC-siRNA-treated cells. (F) Cells exhibit misaligned chromosome (arrow; see inset) in metaphase. (G) Midzone microtubules are missing (arrowhead) in NudC-siRNA-treated cells in anaphase/telophase. (H,I) Midzone microtubules are missing or disorganized (arrowhead) in cytokinesis. (J,K) Midbody structure is unusually thick or very elongated (arrowhead) between divided cells, suggesting problems in cell separation in NudC-siRNA-treated cells. A much reduced though still detectable level of NudC was observed in NudC-siRNA-treated cells undergoing mitosis. NudC, red; ß-tubulin, green; DNA, blue. Bar, 10 µm in all panels. Scale is the same in A-I.

View larger version (63K): [in a new window]   Fig. 5. Plk1 is mislocalized in NudC downregulated cells. HeLa cells were transfected with Luc siRNA or NudC siRNA for 72 hours. (A) Total cell lysates (10 µg) were immunoblotted with anti-NudC C peptide (top), anti-Plk1 (middle) or anti-ß-actin (bottom) antibodies. (B) Subcellular localization of Plk1. Cells were stained with anti-Plk1 antibodies and counterstained with DAPI as indicated. In Luc-siRNA-treated control cells, Plk1 staining was found on the centrosomes during prometaphase (a) and metaphase (c), and at the midbody during cytokinesis (e). By contrast, in NudC-siRNA-treated cells, Plk1 staining was diffuse and not localized to the centrosomes (g,i) or midbody (k). Notice the lagging DNA in the midzone during telophase in NudC-siRNA-treated cells (l, arrow). Stages in M phase were determined by DAPI stains of DNA (b,d,f,h,j,l). Bar, 10 µm for all panels.

View larger version (36K): [in a new window]   Fig. 6. NudC overexpression by adenovirus-mediated gene transfer. HeLa cells were transduced with Ad-Luc or Ad-NudC and analyzed from 24 hours to 96 hours. (A) Total cell lysates (5 µg) were immunoblotted with anti-MBP-NudC fusion protein antibodies (top) or anti-ß-actin antibodies (bottom). In the NudC blot, lanes 1-4 were exposed five times longer than lanes 5-8 in order to detect endogenous NudC expression in the Ad-Luc control cells. The faster-migrating band below NudC in lanes 6-8 might be due to degradation products that were only observed in the NudC-overexpressing cells. (B) The proliferation of HeLa cells transduced with either Ad-Luc or Ad-NudC was determined over 96 hours. Total cell number x 104 in 1 ml of growth medium was determined. Identical results were obtained counting triplicate samples in two independent experiments.

View larger version (88K): [in a new window]   Fig. 7. NudC overexpression induces multinucleation. HeLa cells transduced with either Ad-Luc or Ad-NudC were examined by immunofluorescence microscopy. Cells were stained with anti-NudC C peptide antibodies (red) and counterstained with DAPI (blue), and the same fluorescence intensity setting was used to capture NudC staining for control and Ad-NudC transduced cells. (A-C) Control Ad-Luc cells showing normal increases in cell numbers from 24 hours to 72 hours. At the selected fluorescence setting, no endogenous NudC staining was detectable in control cells. (D-H) Ad-NudC transduced cells showed high levels of NudC staining from 24 hours to 96 hours of infection. Cells became multinucleate as early as 24 hours after Ad-NudC transduction (D, arrows), large and flat by 72 hours (F), and over 90% of the cells contained numerous nuclei (G) or a few enlarged nuclei (H) by 96 hours. Some of these multinucleate cells also contained micronuclei (F, arrow and inset, G,H, arrows). See also Fig. 8D,E for multinucleate cells in the Ad-NudC population. Bar, 20 µm.

View larger version (180K): [in a new window]   Fig. 8. NudC overexpression induces cytokinesis failure and multiple centrosomes. HeLa cells transduced with Ad-NudC for 96 hours were stained with anti-ß-tubulin and counterstained with DAPI. (A,B) Abnormal midbody structures and unusual microtubule matrix are found in NudC-overexpressing cells. (A, inset) Lagging chromosomes are observed in the abnormal midzone/midbody structure. (C) Interphase cells are connected by intercellular bridge with disorganized, curving microtubules (arrows). (D) Large multinucleate cells are connected by an unusually dense midbody-like structure (inset, multiple nuclei and micronuclei in both cells). (E) Many MTOCs are detected in large multinucleate cells. (F) Multipolar spindles are observed (inset, DNA with unattached chromosomes). (G) Elongated, apolar, fragmented and curving microtubules are detected at the cell cortex (arrow). Bar, 10 µm for all panels.

View larger version (97K): [in a new window]   Fig. 9. Plk1 is mislocalized in NudC-overexpressing cells. HeLa cells were transduced with Ad-Luc or Ad-NudC for 96 hours and cells were stained with anti-Plk1 antibodies and counterstained with DAPI as indicated. In Ad-Luc-transduced control cells, Plk1 staining is found on the centrosomes during metaphase (A) and at the midbody during cytokinesis (C). In Ad-NudC-transduced cells, Plk1 staining is punctate and irregular throughout the cell, as observed in cells that are connected by an intercellular bridge (E) and in multinucleate cells (G). (H) Multiple nuclei and micronuclei are observed by DAPI staining of DNA. Bar, 10 µm for all panels.

View larger version (101K): [in a new window]   Fig. 10. nud-1 RNAi in C. elegans: cleavage-furrow regression. Selected Nomarski images from digital time-lapse video recordings of live C. elegans embryos progressing through the two-cell stage in a wild-type (A-D) and a nud-1 RNAi embryo (E-L). Each image displays the time elapsed (minute:second) since initiating sequence capture, approximately 20 minutes after fertilization. Embryos are aligned with the anterior to the left, as determined by the presence of the polar body and denoted by asterisks (some polar bodies are out of the focal plane). (A,E) In wild-type and nud-1 RNAi embryos, the pronuclei have fused and the spindle is beginning to elongate. (B,F) Spindle elongation is not affected in nud-1 RNAi embryos. (C,G) The first cleavage furrow has been initiated. (D,H) Telophase is completed in the wild-type embryo. However, the cleavage furrow is incomplete in the nud-1 RNAi embryo (arrow). An `x' is used to label each nucleus. (I-L) The furrow in the nud-1 RNAi embryo quickly regresses and the nuclei aggregate in the center of the embryo. Bar, 5 µm in all panels. Digital time-lapse videos of these embryos are available online (http://jcs.biologists.org/supplemental).

View larger version (92K): [in a new window]   Fig. 11. nud-1 RNAi in C. elegans: absence of midzone microtubules and abortive cytokinesis. Fluorescent images from embryos displaying anaphase-stage microtubules in a one-cell-stage wild-type embryo (A-C) and nud-1 RNAi embryos (D-L). The embryos were stained with anti-tubulin antibodies (green) (A,D,G,J-L) and counter-stained with DAPI (blue) (B,E,H,J-L). Merged images are shown in C,F,I-L. Embryos are aligned as described in Fig. 9. (A-C) Anaphase-stage wild-type embryo with midzone microtubules. (D-I) nud-1 RNAi one-cell-stage embryos during anaphase. Midzone microtubules are absent (D,F) or weak (G,I). Weak midzone microtubules usually correspond with the presence of chromatin bridges during anaphase (H). NUD-1-depleted embryos continue the cell cycle without cytokinesis (J-L). Bar, 5 µm in all panels.