Cohesin Cleavage by Separase Required for Anaphase and Cytokinesis in Human Cells Silke Hauf, Irene C. Waizenegger, and Jan-Michael Peters

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• Movie 1
  Selected pictures of this movie are shown in Fig. 4A. Cell line WT_1 was stably transfected with a GFP-tagged version of histone H2B to label chromatin. Cells were induced to express SCC1-myc WT for 2 days and then recorded both in Nomarski and in epifluorescence. Time in (h:min) is indicated. The upper part shows the GFP-recording, the lower part shows a merge of Nomarski (red) and GFP (green). The movie starts with a cell in interphase. Chromosomes begin to condense at 0:20 and congress to the metaphase plate. The last chromosome is aligned on the metaphase plate between 1:10 and 1:20 and anaphase is initiated (1:30). Cytokinesis only starts when anaphase has taken place (1:40). DNA decondenses and two daughter cells are formed.

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• Movie 2
  Selected pictures of this movie are shown in Fig. 4B. Cell line mtNC_1 was transfected, induced and recorded as described for Supplemental movie 1. The recording starts when the cell has already reached metaphase. No unaligned chromosomes are visible. Yet the cell stays in metaphase for an additional 45 min, when cytokinesis is initiated without anaphase having taken place (0:47). The cleavage furrow ingresses, constricting the chromosomes in the spindle midzone (1:07). At the same time DNA starts to decondense, the cleavage furrow finally regresses (1:52) and the cell enters an interphase state (2:22), now containing a nucleus increased in size and irregular in shape.

Supplemental method for generation of stable HeLa SCC1-myc cell lines.

HeLa tet-on cells (Clontech) were co-transfected with pTRE2 (Clontech) containing wild-type or mutant forms of SCC1 (Fig. 1E) tagged with 9 myc epitopes at the COOH-terminus and pTK-Hyg (Clontech) in a ratio of 20 : 1 using Lipofectamine (Gibco) according to manufacturer's instructions. Cells were replated after transfection and Hygromycin B (Gibco) was added to 350 g/ml 2 days later. Medium was changed every 4 to 7 days and colonies were selected after 2.5 weeks. Expression was tested by immunoblotting after 2 to 3 days of induction with Doxycycline (Sigma). Several clones were selected and expanded for further analysis. 2 g/ml Doxycycline were used for induction.

Supplemental method for FACS analysis.

For FACS analysis, cells were fixed with 4% paraformaldehyde in PBS for 1 h, washed with PBS, permeabalized with 0.1% Triton X-100 in PBS, washed with PBS-T (PBS, 0.01% Triton X-100), and blocked with 3% BSA in PBS-T for 1 h at room temperature. Cells were labeled with mouse-anti-myc, followed by goat-anti-mouse-Alexa 488. Labeling was in blocking buffer for 1 h, cells were washed three times with PBS-T between incubations. Cells were resuspended in buffer containing propidiumiodide (10 mM Tris [pH 7.5], 5 mM MgCl₂, 10 (g/ml RNAse A, 50 g/ml propidiumiodide), incubated at 37°C for 30 min and analyzed on a FACScan.

Supplemental method for stable transfection with H2B-GFP.

HeLa SCC1-myc cell lines were transfected with a H2B-GFP expression vector also encoding a blasticidin resistance [T. Kanda, K. F. Sullivan, G. M. Wahl, Curr. Biol. 8, 377 (1998)] using Lipofectamine (Gibco) according to manufacturer's instructions. Cells were replated after 2 days and Blasticidin S (Calbiochem) was added to 5 g/ml 3 days after transfection. Medium was changed to 2 g/ml blasticidin 6 days later. After 2 weeks of drug selection, uniformly GFP-expressing colonies were selected and further expanded.

Supplemental method for video microscopy.

Cells were observed with a Zeiss Axiovert 100M microscope equipped with an incubation chamber (Zeiss) to keep cells at 37°C and 5% CO₂. Images were taken with a Cool Snap FX camera (Photometrics) using a 40 objective and processed using MetaMorph Software (Universal Imaging Corp.).

Supplemental method for hypotonic chromosome spreading procedure as in Fig. 4C.

Cells were induced to express SCC1-myc for 3 d. 2 h before harvesting colchicine was added to a final concentration of 5 g/ml. Spreading was done as described [S. Czvitkovich et al., Mech. Dev., in press]. Briefly, cells were harvested by mitotic shake off, spun down, and the pellet was resuspended in 0.6% prewarmed KCl solution and incubated at 37°C for 8 min. Cells were fixed in Carnoy's fixative (75% methanol, 25% acetic acid). Chromosomes were spread by dropping onto glass slides. Slides were stained with Giemsa (Fluka) and mounted with Entellan (Merck).

Supplemental method for chromosome spreading as in Fig. 1, A and B.

Cells were induced to express SCC1-myc for 3 d. 18 h before harvesting nocodazole was added to a final concenctration of 0.1 g/ml. Cells were harvested by mitotic shake off, resuspended in PBS and spun onto slides for 5 min at 1500 rpm using a Cytospin (Shandon). Slides were incubated in 0.1% Triton X-100 in PBS for 2 min, washed in PBS, and fixed in 2% paraformaldehyde in PBS for 15 min at room temperature (RT). Slides were washed again in PBS, incubated in 0.5% NP-40 in PBS for 5 min, washed with PBS and blocked in 3% BSA in PBS for 1 h at RT. Specimens were stained with a polyclonal rabbit-anti-myc antibody (13), goat-anti-rabbit IgG-Alexa 568 (Molecular Probes, Eugene, OR), human CREST serum (kind gift from A. Kromminga, Hamburg, Germany) and goat-anti-human IgG-Alexa 488 (Molecular Probes). DNA was counterstained with DAPI.

Supplemental method for mapping of cleavage sites in human SCC1.

For generating truncated versions of human SCC1 cDNA, polymerase chain reactions (PCRs) were used. For NH₂-terminal deletions different 5'-primers containing a T7 promotor region, a start codon and appropriate SCC1 sequences were used. For COOH-terminal deletions different 3'-primers with appropriate SCC1 sequences and a stop-codon were used (primer sequences are available upon request). The obtained PCR fragments were transcribed and translated in the presence of ³⁵S-labeled amino acids in reticulocyte lysate in vitro (TNT system, Promega). The in vitro translated products were separated by SDS-PAGE and immunoblotted with antibodies specific for the COOH- or NH₂-terminus of SCC1.

Supplemental method for in vitro SCC1 cleavage assay.

Inactive human separase immunoprecipitates were activated in mitotic Xenopus egg extracts as described [I. C. Waizenegger, S. Hauf, A. Meinke, J. M. Peters, Cell 103, 399 (2000)]. Separase bound to beads was used for the SCC1 in vitro cleavage assay: 25 (l beads were mixed with 30 (l of the following SCC1-myc in vitro translation (IVT) mix (15 l SCC1-myc IVT (wild type or mutant forms generated by PCR, primer sequences are available upon request) + 0.3 l 1 M MgCl₂, 0.3 l 100 mM ATP, 0.12 l 250 mM EGTA, 13.78 l XB + 1 mM DTT) and incubated in a thermomixer at 22°C and 1200 rpm. Because addition of active Polo-like kinase stimulates SCC1 cleavage in vitro (I.W., unpublished observation), 0.5 g human GST-Polo-like kinase (kind gift from N. Redemann) was added to each reaction mix. 6 l were taken per time point, the reaction was stopped by addition of SDS loading buffer. Samples were analyzed by immunoblotting with mouse monoclonal antibodies against the myc epitope (9E10).

Supplemental method for sucrose density gradient fractionation.

For density gradient centrifugation, whole cell extracts were centrifuged for 20 min at 4°C and 13,000 rpm and supernatants were loaded on 5-30% sucrose gradients. Gradients were prepared using a Biocom gradient master. Centrifugation was performed for 16 h at 4°C and 36,000 rpm in a SW40 rotor (Beckman Coulter) and 600 l fractions were collected with a density gradient fractionator (ISCO).

Supplemental method for immunofluorescence analysis.

When extracted prior to fixation (Fig. 3, C, D, and E), cells were processed for immunostaining as described (13). For not-extracted cells the first incubation with 0.1% Triton X-100 was omitted.