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First published online 27 June 2006
doi: 10.1242/jcs.03032

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Influence of human Ect2 depletion and overexpression on cleavage furrow formation and abscission

Max Planck Institute for Biochemistry, Department of Cell Biology, Am Klopferspitz 18, 82152 Martinsried, Germany

View larger version (57K): [in a new window]   Fig. 1. Ect2 localizes to the central spindle and the cortex of mitotic cells. (A) HeLa S3 cells grown on coverslips were fixed with paraformaldehyde and permeabilized with Triton X-100. Cells were then stained with anti-Ect2 antibodies. Different cell-cycle phases and a control cell treated for 24 hours with a siRNA targeting Ect2 are shown. Bar, 10 µm. (B) HeLa S3 cells were treated with GL2 and Ect2 siRNAs for 24 and 48 hours. Subsequently, cell lysates were prepared and proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. These were probed with anti-Ect2 antibodies (upper panel) and, as a loading control, with anti--tubulin antibodies (lower panel).

View larger version (32K): [in a new window]   Fig. 2. The BRCT domain targets Ect2 to the central spindle, whereas the PH domain is required for cell cortex localization. (A) Schematic representation of different Ect2 domains and the ability of various N- and C-terminal fragments to localize to the central spindle and the cell cortex. + indicates presence and – absence of localization to the central spindle and cortex. (B) HeLa S3 cells were grown on coverslips and transiently transfected for 36 hours with the indicated myc-Ect2 fragments before fixation with paraformaldehyde and permeabilization with Triton X-100. Cells were stained with anti-myc 9E10 antibodies (left) and FITC-phalloidin (middle). Merged images are shown on the right with 9E10 in red, actin in green and DNA stained with DAPI in blue. (C) HeLa S3 cells were transfected for 36 hours with the indicated myc-Ect2 constructs, fixed and permeabilized as described in B and stained with anti-myc 9E10 antibodies (left) and anti -tubulin antibodies (middle). Merged images are shown on the right, with 9E10 in red, -tubulin in green and DNA stained with DAPI in blue. Bars, 10 µm.

View larger version (66K): [in a new window]   Fig. 3. The MKlp1-MgcRacGAP and MKlp1–Aurora-B complexes are both required for localizing Ect2 to the central spindle. (A) HeLa S3 cells were treated with control (GL2), MKlp1, MgcRacGAP, Aurora-B and MKlp2 siRNAs for 36 hours before fixation and permeabilization with paraformaldehyde and Triton X-100, respectively. Subsequently, cells were stained with anti-Ect2 (left) and anti--tubulin (middle) antibodies. Merged images are shown on the right with Ect2 in red, -tubulin in green and DNA stained with DAPI in blue. Bars, 10 µm. In all cells, the focal plane was adjusted to visualize central spindle rather than cortex staining. (B) Cell lysates from anaphase/telophase synchronized HeLa S3 cells were used for immunoprecipitations (IP's) with anti-Ect2 antibodies (anti-Ect2) or pre-immune antibodies (IgG). Immunoprecipitates and input lysates (Extract) were then probed by western blotting with antibodies against the indicated proteins. (C) Immunoprecipitation using anti-MKlp1 antibodies. (D) Immunoprecipitation using anti-MgcRacGAP antibodies.

View larger version (41K): [in a new window]   Fig. 4. Ect2 displacement from the central spindle does not interfere with cytokinesis. (A) HeLa S3 cells were grown on coverslips and transiently transfected for 36 hours with the indicated myc-Ect2 constructs before fixation and permeabilization with formaldehyde and Triton X-100. Cells were then stained with anti-myc 9E10 antibodies (middle) and antibodies recognizing a C-terminally located epitope on endogenous Ect2 (left). Merged images show transfected myc-Ect2 (9E10) in green, endogenous Ect2 in red and DNA stained with DAPI in blue. A control, non-transfected, cell is shown in the bottom row. Note that this antibody required a simultaneous fixation-permeabilization protocol, so that cortex localization of Ect2 was difficult to see. Bar, 10 µm. (B) Transiently overexpressed myc-Ect2 fragments were immunoprecipitated from HeLa S3 cells synchronized at anaphase/telophase, using the anti-myc 9E10 antibody (IP's). For control, an unrelated myc-tagged hWW45 construct was transfected and analyzed in parallel. Samples were then probed by western blotting with antibodies against the indicated proteins.

View larger version (79K): [in a new window]   Fig. 5. In Ect2-depleted cells cleavage furrow formation is impaired, whereas in cells overexpressing the Ect21-333 fragment cell abscission fails. (A) Western blots with lysates from a stable tetracycline-inducible myc-Ect21-333 cell line, with or without induction with tetracycline for 24 hours. Blots were probed with anti-myc 9E10 antibodies to illustrate induction of the Ect2 fragment and with anti--tubulin antibodies to show equal loading. (B) HeLa S3 cells were treated for 24 hours with either GL2 (control) or Ect2 siRNAs and, in parallel, the inducible myc-Ect21-333 cell line was treated for 24 hours with tetracycline. After fixation and permeabilization with paraformaldehyde and Triton X-100, respectively, the myc-Ect21-333 expressing cells were stained with anti-myc 9E10 antibodies (red), anti -tubulin antibodies (green) and DAPI (blue) and the siRNA-treated cells were stained with anti-Ect2 antibodies (red), anti--tubulin antibodies (green) and DAPI (blue). Images represent merged deconvolved series of Z stacks through each cell. (C) Live-cell imaging of tetracycline (tet)-induced myc-Ect21-333 HeLa S3 cells (upper panel), Ect2-depleted HeLa S3 cells (middle two panels) and HeLa S3 cells treated with GL2 control oligonucleotides (lower panel). Images were taken every 2 minutes and only representative frames are shown. In cells expressing myc-Ect21-333, time `0' was attributed to the last frame in which the cell on the right side still showed an interphase appearance. Similarly, in Ect2-depleted and GL2-treated control cells, time `0' was attributed to the last frame in which the relevant cells still showed an interphase appearance. Bars, 10 µm.

View larger version (58K): [in a new window]   Fig. 6. Ect2 targets RhoA and Citron kinase to the cleavage furrow. (A) HeLa S3 cells were treated for 36 hours with control (GL2) or Ect2 siRNAs and the myc-Ect21-333-expressing cell line was induced for 36 hours with tetracycline. After fixation and permeabilization with 10% TCA and Triton X-100, siRNA-treated cells (upper and middle panels) were stained with anti-Ect2 (left, red) and anti-RhoA (middle, green) antibodies. The stable cell line (lower panel) was stained with anti-myc 9E10 (left, green) and anti-RhoA (middle, red) antibodies. Merged images are shown on the right with DNA in blue. (B) Cells were fixed with paraformaldehyde containing Triton X-100 and stained with anti-Citron kinase antibodies. Merged pictures are shown on the right with Ect2 and myc, respectively, in green, Citron kinase in red and DNA in blue. Bars, 10 µm.

View larger version (57K): [in a new window]   Fig. 7. Influence of nuclear localization on ability of Ect2 N-terminal fragments to interfere with cytokinesis. (A) The myc-tagged Ect2 constructs, Ect21-420, Ect21-388, Ect21-370, Ect21-360, Ect21-333, Ect21-333 containing three SV40 NLS motifs (NLS 1-333) and Ect21-388 with a mutated NLS (NLS mut 1-388) were transfected for 48 hours into HeLa S3 cells before fixation with paraformaldehyde and permeabilization with Triton X-100. Cells were then stained with anti-myc 9E10 antibodies and DAPI and analyzed for the presence of either a single nucleus or binucleation (and occasionally multi-nucleation). Histogram shows the results of three independent experiments (300 cells each) and bars indicate s.d. The localizations of the various constructs in interphase cells are indicated as N (nuclear), N/C (nuclear and cytoplasmic) and C (cytoplasmic). (B) Equal amounts of cell lysates, prepared from mitotic cells transfected with the constructs described in A, were separated by SDS-PAGE and probed by western blotting with anti-myc 9E10 antibody. Anti--tubulin detection is shown as a loading control. (C) HeLa S3 cells were transfected for 36 hours with the indicated myc-Ect2 constructs. Cells were then fixed with paraformaldehyde and permeabilized with Triton X-100, before they were stained with anti-myc 9E10 antibodies (red, left), anti--tubulin antibodies (green). Merged images including DNA stained with DAPI (blue) are shown on the right. Bar, 10 µm.

View larger version (69K): [in a new window]   Fig. 8. Myc-Ect21-333 mislocalizes to a ring-like structure at the cell cortex near the midbody. (A) The indicated myc-Ect2 constructs were overexpressed for 48 hours in HeLa S3 cells before fixation with paraformaldehyde and permeabilization with Triton X-100. Cells were then stained with anti-myc 9E10 in red (left) and anti--tubulin antibodies in green (middle). Merged images including DNA stained with DAPI (blue) are shown on the right. (B) Higher resolution, deconvolved images of the Ect21-333 expressing cells. The three times enlargements on the right show only the myc-Ect2 staining. (C) The myc-Ect21-333 stable cell line was induced for 36 hours and stained with anti-MKlp1 and anti-MgcRacGAP antibodies (middle) in red, anti--tubulin antibodies (left) in green and DNA was labeled with DAPI in blue. Merged images are shown on the right. (D) Quantitative analysis of the number of cells showing displaced myc-Ect2 fragments around the midbody (as shown in A and B). Histogram shows the results of three independent experiments (120 cells each) and bars indicate s.d. Bars, 10 µm.

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