JCS022525 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Fab fragments against the C-terminal domain of gp210 do not prevent extracts becoming mitotic after addition of cyclin B Δ90. Extracts were incubated with control or inhibitory Fab fragments for 30 minutes. Recombinant cyclin B Δ90 was added and aliquots of the extracts taken at the indicated time points and assayed for histone H1 kinase activity.

• Supplemental Figure S2 -

  Fig. S2. Nuclei inhibited for NEBD do not lose nucleoporins. Nuclei were assembled in the presence of either control Fab fragments or inhibitory Fab fragments. After two hours, a twofold volume of mitotic extract was added and incubated for another 2 hours. Samples were analyzed using the indicated antibodies (green and red in overlays). Chromatin was stained with DAPI (blue in overlays). Bars, 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Nuclei inhibited for NEBD do not become mitotic. Nuclei were assembled in the presence of either control Fab fragments or inhibitory Fab fragments. After two hours, a twofold volume of mitotic extract was added and incubated for another 2 hours. Samples were analyzed using antibodies against phospho-histone H1 (green) and the monoclonal antibody mAb414 (red). Chromatin was stained with DAPI (blue in upper row). Bars, 10 µm.

• Supplemental Figure S4 -

  Fig. S4. Live recording of NEBD in vitro. Nuclei were assembled in the presence of either control or inhibitory Fab fragments. After 2 hours, cyclin B Δ90 was added and incubated for another 30 minutes. Chromatin was stained with DAPI, membranes with DiIC18, and a fluorescently labeled 70-kDa dextran (Galy et al., 2003) was added to the reaction. The nuclear suspension was then observed by confocal time-lapse microscopy. Note the entry of the 70-kDa dextran into nuclei assembled in the presence of control but not inhibitory Fab fragments; instead, the NE remained intact and the nuclei grew further, as indicated by the scale bar.

• Supplemental Figure S5 -

  Fig. S5. Fab fragments do not change the localization of gp210 on the NE and NPC. Nuclei were assembled in the presence of no, control or inhibitory Fab fragments for 90 minutes. Samples were fixed, incubated with no antibody, antibody against gp210 or monoclonal antibody mAb414. After incubation with colloidal gold-labeled protein-A, samples were processed for EM. On EM slices, gold particles were counted and categorized according to the NPC localization or NE localization. Unclear NE or NPC localizations were counted as undecided. Examples of NPC and NE localization are shown. Bar, 100 nm.

• Supplemental Figure S6 -

  Fig. S6. RNAi-mediated depletion of cyclin B does not change the localization of LEM-2. Confocal image and corresponding DIC image from a time-lapse recording showing a cyb-1-siRNA-treated two-cell-stage embryo expressing GFP-LEM2. The duplicated nuclei (arrows) induced upon cyb-1 RNAi-mediated depletion are surrounded by a normal NE-like LEM-2 signal. Bar, 10 µm.

• Movie 1 -

  Movie 1. Time-lapse confocal recordings of control-siRNA-treated (top) and gp210-siRNA-treated (bottom) embryos expressing YFP-lamin. Recordings were synchronized using the onset of anaphase as being t=0 seconds. Playback speed is increased by a factor of 120.

• Movie 2 -

  Movie 2. Time-lapse confocal recording of gp210-siRNA-treated embryos expressing GFP-histone H2B. Playback speed is increased by a factor of 120.

• Movie 3 -

  Movie 3. Time-lapse confocal recordings of control-siRNA-treated (top) and gp210-siRNA-treated (bottom) embryos expressing GFP-emerin. Recordings were synchronized using the onset of anaphase as being t=0 seconds. Playback speed is increased by a factor of 120.