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First published online 28 August 2008
doi: 10.1242/dev.022624

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Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis

¹ Wellcome Trust Centre for Cell Biology, The University of Edinburgh, Edinburgh EH9 3JR, UK.
² Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, NJ 08854-8020, USA.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Ectopic poles in the equatorial region of meiotic metaphase spindle in the Drosophila incenp mutant. Metaphase I-arrested oocytes from wild type (A) and the incenpQA26 mutant (B,C) were immunostained for DNA, tubulin and the pole protein D-TACC. Ectopic poles, which often accumulate D-TACC, were formed in the incenp mutant. (D) Frequencies of abnormal morphology of meiotic spindles in wild type and the incenp mutant. More than 30 spindles were examined. The difference is significant (P<0.001). Scale bar: 10 µm.

View larger version (131K): [in this window] [in a new window]   Fig. 2. Instability of the metaphase I spindle equatorial region in the incenp mutant. A time lapse sequence of a metaphase I-arrested spindle in wild type (A) and the incenpQA26 mutant (B). Oocytes expressing GFP--tubulin were dissected and observed under a confocal microscope. The numbers represent minutes:seconds after the arbitrary point. Scale bar: 10 µm.

View larger version (92K): [in this window] [in a new window]   Fig. 3. Transitory spindle poles during spindle formation in the incenp mutant. A time lapse sequence of spindle formation after nuclear envelope breakdown to metaphase I in wild type (A) and the incenpQA26 mutant (B). Time zero indicated the first appearance of spindle microtubules (arrowheads) around the chromosomes. At 10:12 in the mutant, an ectopic pole (arrow) was formed around the spindle equatorial region and eventually merged with one of the poles. Scale bar: 10 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 4. The spindle equatorial region is partially defective in the incenp mutant. (A,B) Immunolocalisation of Incenp and Aurora B localises to the equatorial region of meiotic metaphase I spindle in wild type and the incenpQA26 mutant. (C) Immunolocalisation of Cyclin B to the equatorial region of meiotic metaphase I spindle in wild type and the incenpQA26 mutant. (D,E) Examples of GFP signal intensity plots along the spindles from wild-type and incenp oocytes expressing GFP-tubulin, as marked with the red line in E. (F) The relative intensities of the spindle equatorial region (maximum intensity within the central 4 µm) over pole regions (average maximum intensity of 2 µm from each pole) are shown as the mean values (bars; 1.4 in wild type and 1.1 in incenp) and standard deviations (lines) for multiple wild-type and incenp mutant spindles. The difference is significant (P<0.015; n=15). Scale bars: 10 µm.

View larger version (19K): [in this window] [in a new window]   Fig. 5. The incenp mutation delays spindle microtubule assembly in female meiosis. (A) Time taken from nuclear envelope breakdown to the first appearance of spindle microtubules around the chromosomes in wild-type and incenpQA26 mutant oocytes expressing GFP--tubulin. The mean values (bars) are shown with standard deviations (lines). The difference is significant (P<0.001; n8). (B) The length of metaphase I-arrested spindles in wild type and the incenpQA26 mutant. (C) The relative amounts of -and -tubulins in ovaries from wild type and the incenpQA26 mutant were examined by immunoblots. Blotted membranes are stained with MemCode for loading and transfer controls.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Subito is required for stability of the spindle equatorial region but not for spindle microtubule assembly in female meiosis. (A) Subito localises to equatorial regions in wild type and the incenpQA26 mutant. (B) Instability of the spindle equatorial region in the subito1 mutant. An example of metaphase I-arrested subito oocytes expressing GFP--tubulin. An ectopic pole (arrowhead) was formed at the spindle equatorial region and merged with one of the main poles. (C) Time taken from nuclear envelope breakdown to the first appearance of spindle microtubules. The subito mutant did not show a significant difference from wild type (P=0.45; n=6). Scale bars: 10 µm.

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