Fig 1.
Injection of Chromator mAb 6H11 arrests cell cycle progression prior to NE breakdown.
(A) Diagram of the dynamics of Chromator (in green) localization during mitosis in unperturbed embryos based on the results of Yao et al. (Fig 1 in Yao et al. [5]). Chromosomes, the nuclear envelope, centrosomes, and the midbody are in grey and color intensity is proportional to relative protein levels in this and subsequent diagrams. (B) Confocal time-lapse sequence of a mAb 6H11 injected syncytial embryo expressing Chromator-GFP (in green) and H2Av-RFP (in red). The antibody was injected at interphase and the image sequence starts at metaphase of the first cell cycle which progressed normally. However, although the second cell cycle is initiated as indicated by the condensed chromosomes, the normal redistribution of Chromator-GFP away from the chromosomes as well as NEB did not occur (last panel). (C) Confocal time-lapse sequence of a mAb 6H11 injected syncytial embryo expressing Chromator-GFP (in green) and Tubulin-mCherry (in red). This embryo was injected at interphase with a smaller amount of mAb 6H11 where the blocking effect is confined to the immediate vicinity of the injection site (region indicated by the white bracket) due to limited diffusion. The image sequence starts prior to initiation of the first cell cycle. The first mitosis is as in uninjected embryos; however, while the second mitosis is initiated and proceeds normally in the surrounding areas; at the injection site the nuclei arrest prior to NEB with Chromator still present on the condensed chromosomes. Time is indicated in minutes and seconds.
Fig 2.
Cell cycle arrest in mAb 6H11 and colchicine arrested embryos.
(A) Diagram of proposed model for mAb 6H11-mediated cell cycle arrest. At the time of injection at interphase the antibody does not have access to the Chromator epitope because it is excluded from the chromosomes by the intact nuclear envelope. At the time of NEB of the first cell cycle the antibody is now free to bind to Chromator and this association is maintained as the daughter nuclei become enclosed by the reforming nuclear envelope. That the first cell cycle after NEB proceeds normally even in the presence of the antibody suggests that the presence of mAb 6H11 antibody does not interfere with cell cycle protein function at any point after NEB, with any checkpoint proteins, with cytokinesis, or with formation of the daughter nuclei. However, at the entry of the second cell cycle as indicated by chromosome condensation, the antibody now is bound to Chromator, preventing Chromator dissociation from the chromosomes and spindle matrix formation leading to cell cycle arrest. (B) Comparison of Chromator dynamics in colchicine- and mAb 6H11-arrested embryos. In wild-type, Chromator is localized to the chromosomes at interphase and relocalizes to the spindle matrix and centrosomes at prometaphase and metaphase. In colchicine-arrested embryos Chromator dynamics are as in wild-type embryos (Fig 2 in Yao et al. [5]); however, in mAb 6H11-arrested embryos Chromator remains on the condensed chromosomes, the spindle matrix does not form, and NEB does not occur.
Fig 3.
Confocal time-lapse sequence of an α-GST mAb injected control embryo expressing Megator-YFP (in green) and Tubulin-mCherry (in red).
The antibody was injected at interphase and the image sequence starts at metaphase of the first cell cycle after the injection. The embryo completed two complete mitotic cycles and initiated a third without any observable defects as compared to wild-type embryos. Time is indicated in minutes and seconds.
Fig 4.
mAb 6H11 injection prevents Megator redistribution from the nuclear envelope during mitosis.
(A) Diagram of the dynamics of Megator localization (in green) during mitosis in unperturbed embryos based on the results of Yao et al. (Fig 6 in Yao et al. [5]). (B) Confocal time-lapse sequence of a mAb 6H11 injected syncytial embryo expressing Megator-YFP (in green) and Tubulin-mCherry (in red). The antibody was injected at interphase and the image sequence starts at metaphase of the first cell cycle, which progressed normally, while the second cycle is arrested prior to NEB. The last image was obtained approximately 35 min after the first cell cycle was completed. The chromosomes have condensed (dark regions indicated by white arrows) and Megator-YFP is still present on the NE at a time when it normally would have relocalized to the spindle matrix. Time is indicated in minutes and seconds. (C) Comparison of Megator dynamics in colchicine- and mAb 6H11-arrested embryos. In wild-type, Megator is localized to the nuclear interior and the NE at interphase and relocalizes to the spindle matrix at prophase. In colchicine-arrested embryos Megator dynamics is as in wild-type embryos (Fig 5E in Yao et al. [5]); however, in mAb 6H11-arrested embryos Megator remains at the nuclear rim, the spindle matrix does not form, and NEB does not occur.
Fig 5.
Cyclin B dynamics in un-, colchicine-, and mAb 6H11-injected embryos.
(A) Confocal image sequence of the relative dynamics of Cyclin-B-GFP (in green) and H2Av-RFP (in red) during a mitotic cycle in an untreated embryo. Arrowheads point to enhanced Cyclin-B-GFP localization at the nuclear rim at the time of NEB. Time is indicated in minutes and seconds. (B) Diagram of the dynamics of Cyclin B (in green) localization during mitosis in unperturbed embryos. (C) Confocal image sequence of Cyclin-B-GFP (in green) and H2Av-RFP (in red) dynamics during a mitotic cycle in a colchicine-injected embryo. Arrowheads indicate Cyclin B localization to the nuclear rim and kinetochores similar to that observed in untreated embryos. (D) Confocal image sequence of a Cyclin-B-GFP (in green) and H2Av-RFP (in red) expressing embryo injected with Chromator mAb 6H11. The antibody was injected at interphase and the image sequence starts approximately 2 min after the first cell cycle was completed. Although Cyclin-B-GFP levels appear to be continuously increasing within the nucleus there is no indication of its normal enrichment at the nuclear rim, kinetochores of the condensed chromosomes, or centrosomes. (E) Comparison of Cyclin B dynamics in colchicine- and mAb 6H11-arrested embryos. In wild-type Cyclin B is present at low levels in the nuclear interior, accumulates within the spindle matrix at prometaphase and transiently relocates to the nuclear rim, the kinetochores, and the centrosomes. In colchicine-arrested embryos Cyclin B dynamics are as in wild-type embryos; however, in mAb 6H11-arrested embryos Cyclin B-levels increase within the nucleus without localization to the nuclear rim, kinetochores, or centrosomes. The asterisk indicates the transient Cyclin B localization to the nuclear rim just prior to NEB in colchicine-treated embryos. (F) Plot of average pixel density within the nucleus as a function of time for Cyclin B-GFP. Average pixel density for an area outside the nuclei was subtracted at each time point. Increased levels of Cyclin B-GFP accumulate in the nucleus as chromosome condensation commences (arrow).
Fig 6.
Ran dynamics in un-, colchicine-, and mAb 6H11-injected embryos.
(A) Confocal image sequence of the relative dynamics of Ran-Venus (in green) and Tubulin-mCherry (in red) during a mitotic cycle in an untreated embryo. Time is indicated in minutes and seconds. (B) Diagram of the dynamics of Ran (in green) localization during mitosis in unperturbed embryos. (C) Confocal image sequence of Ran-Venus (in green) and Tubulin-mCherry (in red) dynamics during a mitotic cycle in a colchicine-injected embryo. Ran-Venus is maintained within the nuclear domain even after NEB. Note also that after NEB free tubulin accumulates within the spindle matrix at much higher levels than in the surrounding syncytial cytoplasm. (D) Confocal image sequence of a Ran-Venus (in green) and Tubulin-mCherry (in red) expressing embryo injected with Chromator mAb 6H11. The antibody was injected at interphase and the image sequence starts approximately 4 min after the first cell cycle was completed. No changes to the localization of Ran-Venus was observed and it maintained its nuclear rim localization, as there is no NEB. (E) Comparison of Ran dynamics in colchicine- and mAb 6H11-arrested embryos. In wild-type Ran is present at the nuclear interior and at the nuclear rim at interphase as well as at prometaphase prior to NEB. In colchicine-arrested embryos Ran dynamics is as in wild-type embryos; however, in mAb 6H11-arrested embryos there are no changes to the localization of Ran and it maintains its nuclear rim localization, as there is no NEB. The asterisk indicates the transient Ran localization to the nuclear rim just prior to NEB in colchicine-treated embryos.
Fig 7.
Polo dynamics in un-, colchicine-, and mAb 6H11-injected embryos.
(A) Confocal image sequence of Polo-GFP during the cell cycle in an untreated embryo. Time is indicated in minutes and seconds. (B) Diagram of Polo (in green) dynamics during mitosis in unperturbed embryos. (C) Comparison of Polo localization after cell cycle arrest with colchicine- and mAb 6H11-injections, respectively. In wild-type, Polo is present at centrosomes but not in the nuclear interior at interphase; however, it accumulates during pro- and prometaphase and transiently relocalizes to the nuclear rim and the kinetochores. After colchicine-arrest Polo localizes to the spindle matrix, the nuclear rim, and kinetochores as in wild-type preparations. However, in mAb 6H11 arrested embryos Polo does not accumulate in the nucleus and is present only at centrosomes. (D) Confocal image of polo-GFP from an image sequence from a colchicine-arrested embryo showing Polo-GFP enrichment at the nuclear rim and at the kinetochores. (E) Confocal image of Polo-GFP from the end of an image sequence of a mAb 6H11-arrested embryo where Polo-GFP is only present at the centrosomes.
Fig 8.
The Greatwall kinase is not a spindle matrix protein.
(A) Confocal image sequence of the relative dynamics of Greatwall-GFP and 70 kDa Dextran-TRITC during a mitotic cycle in an unperturbed embryo. Time is indicated in minutes and seconds. (B) Diagram of Greatwall (in green) dynamics during mitosis. (C) Plots of average pixel density within the nucleus as a function of time for Greatwall-GFP and 70 kDa Dextran-TRITC. Greatwall-GFP (Gwl-GFP) is being cleared from the nucleus during prophase prior to NEB (arrow) and coincident with spindle matrix formation.
Fig 9.
Endoplasmic reticulum and other membranes are excluded from the spindle matrix even when permeable to microtubules.
(A) Confocal images at metaphase from a mitotic image sequence from an embryo expressing Rtnl-GFP (in green) and Megator-mCherry (in red). Arrowheads indicate membranes accumulated in the gap between the spindle matrix as represented by Megator-mCherry and the centrosome (black area surrounded by the membranes). (B) Confocal images at metaphase from a mitotic image sequence from an embryo expressing Pdi-GFP (in green) and Tubulin-mCherry (in red). Arrowheads indicate membranes accumulated in the gap between the spindle matrix and the centrosome through which microtubules extend as indicated by the yellow color. (C) Confocal images from a Pdi-GFP expressing embryo injected with 70 kDa Dextran-TRITC as well as with colchicine. After NEB Dextran-TRITC invades the nuclear space; however, Pdi-GFP-labeled membranes are still excluded from the nuclear interior.