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A requirement for the Abnormal Spindle protein to organise microtubules of the central spindle for cytokinesis in Drosophila

¹ University of Siena, Department of Evolutionary Biology, Via Mattioli 4, I-53100 Siena, Italy
² University of Cambridge, Department of Genetics, Downing Street, Cambridge CB2 3EH, UK

View larger version (125K): [in a new window]   Fig. 1. Localisation of Asp protein in mitosis and meiosis during spermatogenesis. The merged image (left hand panels) shows DNA (blue), microtubules (green) and Asp (red). The channel showing Asp protein alone is shown in the central panels and microtubules alone in the right hand panels. (A) A four-cell cyst at metaphase and (B) an eight-cell cysts during late telophase; note the Asp ring between daughter nuclei (arrow). Progression through the first meiotic division: (C) Prometaphase meiotic spindles with Asp at the poles. (D) Metaphase meiotic cells with Asp staining strongly at the spindle poles and weakly in the midpoint of the spindles (arrowheads). (E) Telophase cells of meiosis I in which the Asp at the spindle poles falls into two clusters around the centrioles that are beginning to separate. In addition, these cells show prominent Asp staining along the putative minus ends of the central spindle microtubules, which are shown at higher magnification in (F) (arrowhead). The arrow in (E) and (F) point to a very central band of Asp staining that can also be seen in the central spindle. Scale bar is 10 µm.

View larger version (62K): [in a new window]   Fig. 2. Cysts of aspdd4 spermatocytes. The cells are stained to reveal DNA (blue), microtubules (green) and either Centrin (red; A) or -tubulin (red; B). The equivalent monochrome images for the centrosomal antigens and microtubules are shown in the respective central and right hand panels. Although the meiotic spindles look abnormal, their poles appear to be organised in discrete structures. Note the wide range of meiotic stages throughout the same cyst.

View larger version (139K): [in a new window]   Fig. 3. aspdd4 meiocytes show well defined spindle poles and a range of central spindle defects. (A,B) Centrin (red in merged image and monochrome in central panels) is seen in a typical V-shaped pattern corresponding to adjacent centrioles at the spindle poles (A). The telophase tetrapolar cell in (B) shows no organisation of a central spindle. It has failed to undergo cytokinesis in meiosis I and appears set to fail the second meiotic division. (C) A cyst stained to reveal - tubulin (red in merged image, and monochrome in central panel). This illustrates the variation in the extent of formation of central spindle structures with this mutant allele. Central spindle structures are missing in several cells of the cyst. In this panel, three cells with central spindle structures are shown — in one it is poorly formed (small arrow), in another it is displaced (mid-sized arrow) and in the third it is comparable to wild type (large arrow). In all cases the merged image shows DNA in blue and microtubules in green. The corresponding monochrome images of microtubules are also shown in the bottom panels. (D) A cyst stained to reveal centrosomin (red in the merged image, and monochrome in the central panel). The arrowhead points to a cell lacking central spindle structure.

View larger version (150K): [in a new window]   Fig. 4. Mislocalisation of mid-zone antigens in telophase cells of aspdd4 mutants. Localisation of actin (A), Pav-KLP (B) and Peanut (C) are shown in red in the merged image and in monochrome in the central panels. The DNA is blue and the microtubules are green. The corresponding images of microtubules are also shown in monochrome in the bottom panels. In each panel the inset shows the localisation of the corresponding proteins at an equivalent stage of wild-type meiosis. (A) Actin is not organised into a contractile ring as in the wild type but is dispersed in the central part of the cell. One exceptional cell in which the actin is sufficiently well organised to cause constriction of the cell is seen in the very bottom left of this field. Pav-KLP is present in ring-like structures in some cells (large arrow), but in the majority, it has a more diffuse distribution in the central part of the cell (the small arrow indicates one such example). It does stain distinct ring canals (arrowheads), indicating that cytokinesis has occurred in earlier division cycles. (C) In contrast to the wild-type telophase cell, Peanut does not accumulate in a contracted ring-like structure but remains in a wide band around the circumference of the mutant telophase cell. In some cells (arrow), the central spindle is better organised than others (arrowhead).

View larger version (103K): [in a new window]   Fig. 5. Localisation of Asp protein (red) during meiosis in wild-type females. In all panels, microtubules are counter-stained green and DNA is blue. (A) Metaphase of meiosis I. Asp is present at the spindle poles. (B) Early telophase of meiosis I. (C) Late telophase of meiosis I. Note the accumulation of Asp in a ring-like structure in the central spindle around the central pole body. (D) Anaphase of meiosis II. Asp remains in the more diffuse structure connecting the two spindles and is in well defined regions at the spindle poles. (E) Asp at the centre of the sperm aster. (F) Asp at the poles of the gonomeric spindle of the first mitosis of the zygote.

View larger version (177K): [in a new window]   Fig. 6. Female meiosis in aspdd1. DNA is stained red and microtubules are green. (A) This field shows the telophase spindle of meiosis I (arrowhead) and the male pronucleus with associated sperm aster (arrow). (B) Telophase of meiosis II (arrowhead). Unlike wild type, the sperm aster remains diminutive (arrow). (C) Dissociation of duplicated centrosomes from the male pronucleus (arrow). (D) This field shows polar bodies with condensed chromosomes (large arrow), and three haploid anastral mitotic spindles carrying DNA derived from the male pronucleus (arrows). There are also four independent asters of microtubules (arrowheads) derived from the sperm aster that has dissociated from the spindle.

View larger version (10K): [in a new window]   Fig. 7. Model of Asp function during mitosis/meiosis in males (A) and meiosis in females (B). Asp is depicted in orange and is localised at the minus ends of microtubules (green). DNA is shown in blue. (A) Asp is associated with the free microtubule minus ends at the metaphase spindle poles (left). At early telophase (centre), the central part of the spindle is reorganised, and a subpopulation of Asp molecules participate in nucleating central spindle microtubules. Following the completion of cytokinesis, Asp protein remains associated with the mid-body (somatic mitoses) or the ring canals (mitosis and meiosis in spermatogenesis). (B) Asp is at the acentriolar poles of the female meiotic spindles at metaphase I. The structure of the central MTOC starts to form at early telophase I by a reversal of the polarity of the central spindle microtubules. Asp is recruited to the central region at this time and is shown participating in the nucleation of the minus ends of these central spindle microtubules.