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First published online 25 January 2005
doi: 10.1242/jcs.01663

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Epithelial re-organization and dynamics of progression through mitosis in Drosophila separase complex mutants

BZMB, Department of Genetics, University of Bayreuth, 95440 Bayreuth, Germany

View larger version (91K): [in a new window]   Fig. 1. Exit from mitosis 15 after failure of sister chromatid separation in pim mutant embryos. Time-lapse in vivo imaging was used for the analysis of progression through the fifteenth round of mitosis during Drosophila embryogenesis in pim1 mutant (pim1) and pim+ (pim+) sibling embryos expressing red fluorescent histone H2AvD-mRFP and a green fluorescent microtubule-binding protein. Selected frames showing microtubule distribution (top) or merged color images (bottom) are shown. Numbers below the frames indicate the time in minutes relative to the last metaphase frame, which was set to zero. The appearance of prominent interpolar microtubules characteristic of anaphase is indicated by arrowheads. Central spindle and midbody during telophase are indicated by arrows. Bar in upper right frame, 5 µm.

View larger version (90K): [in a new window]   Fig. 2. Metaphase delay during mitosis 16 in pim mutant embryos. Time-lapse in vivo imaging was used for the analysis of progression through the sixteenth round of mitosis during Drosophila embryogenesis in pim1 mutant (pim1) and pim+ (pim+) sibling embryos expressing red fluorescent histone H2AvD-mRFP and a green fluorescent microtubule-binding protein. Selected frames of merged images are shown. Numbers above and below the frames indicate the time in minutes relative to the last metaphase frame which was set to zero. Bar in upper right frame, 5 µm.

View larger version (71K): [in a new window]   Fig. 3. Metaphase delay during mitosis 16 in pim mutants is caused by diplochromosomes. (A-E) Embryos were labeled with a DNA stain (red) and anti-phospho-histone H3 (PH3, green) at the stage of mitosis 16. (A-D) The number of PH3-positive mitotic cells in a defined epidermal region was counted in 10 embryos homozygous for pim1 (B), dupa1 (C), double mutant for both pim1 and dupa1 (D), as well as in pim+ embryos (A). The resulting average number of mitotic cells (E) in the mutant and the corresponding sibling embryos are indicated by white and black bars, respectively. dup encodes an initiation factor for DNA replication; dupa1 homozygosity inhibits S phase 16 (Whittaker et al., 2000), as well as the accumulation of mitotic figures in pim1 mutants during the subsequent mitosis (see text for further explanations). In metaphase cells with maximal PH3 labeling, chromosomes were arranged in metaphase plates in pim1 dupa1 double mutants (inset in D). In contrast, the single chromatid chromosomes present in dupa1 mutants failed to congress into a plate (inset in C), as previously described (Parry et al., 2003). Bar, 25 µm (A-D). (F) Labeling with anti-cyclin B (green) and a DNA stain (red) demonstrates that progression through mitosis 16 is delayed before cyclin B degradation in pim1 mutants. Bar, 10 µm.

View larger version (35K): [in a new window]   Fig. 4. Centrosome numbers in pim mutants. Embryos were labeled at the stage of mitosis 16 (A,B,D) or mitosis 15 (C) with a DNA stain (blue), with anti-phospho-histone H3 (PH3, green) to identify mitotic cells, with anti-Bazooka (Baz, green) to define cell boundaries, and with anti--tubulin (red, Tub) to reveal centrosomes. Maximal projections of representative cells from a pim1 mutant (B, pim1), a pbl mutant (C, pbl), a pim1pbl double mutant (D, pim1 pbl) and a sibling pim+ embryo (A, pim+) are shown, indicating that centrosomes are duplicated in pim1 mutants during cycle 16 when cytokinesis during mitosis 15 is inhibited by homozygosity for pbl (see text for further explanations). Bar, 3 µm.

View larger version (46K): [in a new window]   Fig. 5. Cytokinesis during mitosis 15 in pim mutants. Time-lapse in vivo imaging was used for the analysis of cytokinesis during the fifteenth round of mitosis during Drosophila embryogenesis in pim1 mutant (pim1) and pim+ (pim+) sibling embryos expressing red fluorescent histone H2AvD-mRFP and a green fluorescent fusion protein marking the cell cortex. Selected frames of merged images are shown. Numbers above the frames indicate the time in minutes relative to the last metaphase frame which was set to zero. Bar in upper right frame, 5 µm.

View larger version (79K): [in a new window]   Fig. 6. Apoptosis in pim mutants. Embryos before (A,B) or after (C,D) progression through mitosis 16 in the dorsolateral epidermis were labeled with a DNA stain (red) and analyzed by TUNEL (green) for the presence of apoptotic cells. pim1 mutant embryos (B,D) do not have more apoptotic cells than the pim+ sibling embryos (A,C), at least before mitosis 16. Bar, 50 µm.

View larger version (108K): [in a new window]   Fig. 7. Epithelial organization in pim mutants. Embryos before mitosis 15 (A,B) or before mitosis 16 (C,D) were labeled with a DNA stain (red) and anti--spectrin (green). Single confocal sections through the nuclear layer of the epidermal epithelium do not reveal differences between pim1 mutant (B,D) and pim+ sibling embryos (A,C) before mitosis 15 (compare A and B). In contrast, a relatively disorganized epidermal epithelium is observed in pim1 mutants before mitosis 16 (compare C and D). Bar, 10 µm.

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