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Files in this Data Supplement:
Fig. S1. Mars protein levels are controlled by the cell cycle. A wild-type embryo at gastrulation (stage 7) was stained for DAPI (turquoise), phosphohistone H3 (green) and Mars (red). Mars protein levels are strongly elevated in cells undergoing mitosis, which are labeled by phosphohistone H3. Anterior is to the left. Scale bar: 100 µm.
Fig. S2. Subcellular localization of Mars in S2r cells. (A-E) Untransfected S2r cells were stained with antibodies against β-tubulin (green) and Mars (red). DNA was stained with DAPI (turquoise). (A) At interphase, weak Mars staining is detectable in the nucleus. (B) At prometaphase, Mars co-localizes with β-tubulin at microtubule asters, but does not co-localize with cytoplasmic microtubules. At metaphase (C) and (D) anaphase, Mars localizes to the minus ends of spindle microtubules. (E) At telophase, Mars relocalizes to the newly formed nuclei and is absent from the central spindle. (F) In S2r cells treated with double-stranded RNA corresponding to the mars mRNA (RNAi mars), Mars protein is not detectable. Scale bars: 10 µm.
Fig. S3. The centrosomal antigens Centrosomin, γ-Tubulin, Aurora A and DTACC are still associated with centrosomes in mars91 mutant embryos. (A) Wild-type and (B) mars91 homozygous mutant embryos showed centrosomal localization of centrosomin (red). (C) Wild-type and (D) mars91 homozygous mutant embryos showed centrosomal localization of γ-Tubulin (red). (E) Wild-type and (F) mars91 homozygous mutant embryos showed centrosomal localization of Aurora A (red). (G) Wild-type and (H) mars91 homozygous mutant embryos showed centrosomal localization of DTACC (red). Scale bars: 5 µm.
Movie 1. Subcellular localization of GFP-Mars during mitosis. The movie shows the dynamics of GFP-Mars localization in a region of a Drosophila embryo at the syncytial blastoderm (nuclear cycle 11). This movie corresponds to Fig. 2.
Movie 2. Detachment of centrosomes from the nucleus in a mars91 mutant embryo. Microtubules were labeled with α-tubulin−GFP. At the beginning of the movie, both centrosomes (arrows) are attached to the nucleus, before detaching and moving away. This movie corresponds to Fig. 9A.
Movies 3A-C. Detachment of centrosomes from the mitotic spindle in a mars91 mutant embryo. Microtubules were labeled with α-tubulin-GFP. The movie is divided into three parts because the stage had to be moved due to the movements of the nuclei in the living embryo. Movie 3A starts at prophase and shows the formation of the spindle. Movie 3B shows the detachment of one centrosome from the spindle at metaphase. Movie 3C shows the duplication of the two centrosomes. Note that the free centrosome (left) duplicates although it has detached from the spindle. The spindle-associated centrosome (right) becomes attached to the newly formed nucleus after duplication. This movie corresponds to Fig. 9B.
Movie 4. Splitting of spindle-associated centrosome without reattachment to the newly formed nucleus in a mars91 mutant embryo. Microtubules were labeled with α-tubulin-GFP (green); chromatin was labeled with histone-3B-RFP (red). Both centrosomes duplicate before the completion of mitosis and one daughter centrosome of each spindle pole moves away from the spindle.
Movie 5. Synchronous nuclear divisions in a wild-type embryo at the syncytial blastoderm stage. Microtubules were labeled with α-tubulin-GFP (green); chromatin was labeled with histone-3B-RFP (red). In this wild-type embryo, nuclei are evenly spaced and divide synchronously.
Movie 6. Nuclei are arranged in an irregular pattern and frequently show mitotic defects in mars91 mutant embryos at the syncytial blastoderm stage. Microtubules were labeled with α-tubulin-GFP (green); chromatin was labeled with histone-3B-RFP (red). Note the heterogeneous shape of nuclei and spindles and the frequent occurrence of free centrosomes. One mitotic figure leading to an abnormally shaped nucleus is marked with an arrow at the beginning of the movie.
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