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First published online 23 January 2003
doi: 10.1242/jcs.00295

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Drosophila dd4 mutants reveal that TuRC is required to maintain juxtaposed half spindles in spermatocytes

Vitor Barbosa^1,*, Melanie Gatt¹, Elena Rebollo², Cayetano Gonzalez² and David M. Glover^1,

¹ University of Cambridge, Department of Genetics, Downing Street, Cambridge CB2 3EH, UK
² European Molecular Biology Laboratory, Cell Biology and Biophysics Programme, Meyerhofstrasse 1, 69117 Heidelberg, Germany
^* Present address: NYU School of Medicine, Skirball Institute of Biomolecular Medicine, Developmental Genetics Program, 450 First Avenue, New York, NY 10016, USA

View larger version (123K): [in a new window]   Fig. 1. Monopolar and conical spindles in dd4 testes. (A-F) Living cells at different stages of meiosis in wild-type (A-C) and dd4S (D-F) testes viewed by phase-contrast light microscopy. (A) Cyst of primary spermatocytes at the onset of meiosis I with parafusorial membranes becoming visible (arrow). (B) This parafusorial material is seen along the meiotic spindle and concentrates in the equatorial region in late anaphase I (arrow). (C) A cyst at the onion stage showing spermatids, each containing a nucleus (light sphere) adjacent to a Nebenkern (phase dense sphere). (D) Cyst of primary spermatocytes from dd4S testis with an abnormal number of cells. One of the cells is larger than the others (arrow), whereas the morphology of both germline and somatic cells (cyst precursor cell, arrowhead) seems normal. (E) Example of the asymmetric distribution of the phase-dense material in dd4S meiocytes. Both conical (arrows) and biconical (arrowhead) figures are visible. (F) A cyst of early dd4S spermatids with very disorganized Nebenkerns (dark inclusions). The nuclei associated with each Nebenkern are variable in size and number (arrow). Note also what seem to be bundles of sperm tails on the right of this cyst. (G-L) Localization of -tubulin (red) with respect to spindle microtubules (green) in spermatocytes from fixed wild-type (G-I) and dd4S (J-L) testes. DNA is stained blue. (G) A wild-type 16-cell cyst in prometaphase with duplicated and separated centrosomes. (H) Cells in anaphase before formation of the central spindle. (I) Late anaphase/telophase with fully formed central spindle marked by two bands of microtubules (paired arrowheads) separating the central spindle mid-zone (single arrowhead). The -tubulin-containing MTOCs have separated before meiosis II. (J) A dd4S 16-cell cyst in which condensing chromatin is surrounded by masses of microtubules (arrows) in early meiosis and -tubulin staining is undetectable. (K) Field of dd4S cells in meiosis showing one cone that has a central spindle-like structure (arrow) separating two masses of chromatin and three hemi-spindles. The DNA in the hemi-spindles is present both at the centre of the asters and around the periphery (e.g. arrowhead). (L) The left-most arrow points to a rare example of a bipolar spindle. The cones in this panel (remaining arrows) show a pronounced constriction at their apexes compared with the cone in panel K. Measurement of the frequency of the different types of defective meiotic figure in a sample of 162 cells indicated that 38% were hemi-spindles, 7% sharp cones and 33% biconical. 22% of this group of cells had a morphology that suggested either apoptosis or necrosis. Bars, (A-F), 20 µm; (G-L), 50 µm.

View larger version (140K): [in a new window]   Fig. 5. Pav-KLP becomes focused at the apexes of cones. Pav-KLP (red), -tubulin (green), and DNA (blue) in wild-type (A and B) and dd4S (C and D) meiocytes. (A) Wild-type cyst in prometaphase/metaphase revealing ring canals derived from premeiotic divisions containing Pav-KLP (arrow). (B) Meiocytes in telophase II showing contracting rings with Pav-KLP staining around the central-spindle mid-zone (arrow). (C) A dd4S cyst with Pav-KLP-containing ring canals (large arrow). This cysts also contains two hemi-spindles with Pav-KLP staining at the putative plus ends of microtubules at the periphery of the asters (arrowheads). A cone is also indicated by the small arrow in with Pav-KLP accumulated at its apex. (D) dd4S meiocytes with Pav-KLP at the constriction point of cones (arrowheads), and at the apex of two biconical structures (arrows). Bar, 50 µm.

View larger version (96K): [in a new window]   Fig. 2. CNN is associated with centrosomal MTOCs in dd4S spermatocytes. (A-C) Wild-type meiosis showing CNN (red) localized at the spindle poles during wild-type meiosis. Microtubules are stained green and DNA is stained blue. Cysts are shown at prometaphase (A), anaphase (B) and telophase/cytokinesis (C). (E-H) CNN localization in dd4S meiocytes. (D) Spermatocytes in early meiosis in which discrete bodies containing CNN do not form two discrete foci but are dispersed. In this field the three uppermost cells have at least three such bodies accumulated at the centre of the asters (arrow), although some CNN-containing bodies can be dispersed (arrowhead). (E) Hemi-spindles with variable numbers of CNN-containing bodies at the focus of the asters. (F) Two cones that show constrictions around the mid-zone (arrowheads). Punctate CNN staining is found at the astral poles of both cones and hemi-spindles (arrows). Bar, 50 µm.

View larger version (98K): [in a new window]   Fig. 3. Asp associates with the poles but not the central spindle of dd4S spermatocytes. Asp localization (red) in relation to microtubules (green) and DNA (blue) in meiotic wild-type (A-C) or dd4S (D-F) cells. Wild-type cysts are shown at prometaphase (A) and anaphase/telophase (B). Asp is localized at the spindle poles (arrowhead in B) and in association with the tips of microtubules in the central spindle during anaphase/telophase (arrows in B). (C) Later telophase stage in which cells show pronounced constriction around the central spindle mid-zone. Asp interacts with the putative minus ends of central spindle microtubules, more distant from the poles at this stage (arrows and arrowheads in the inset). The inset shows an enlargement of the spindle marked with small arrowhead. (D) Cyst of dd4S cells apparently at early meiosis with Asp staining at the centre of the hemi-spindle asters (arrowheads). The arrows indicate cells attempting to organize a bipolar spindle with defects in Asp distribution at the poles. The inset shows a higher magnification of the distribution of Asp in a hemi-spindle. (E) Cyst of dd4S cells in which the arrow points to a sharp cone with Asp staining only at the astral pole. The arrowheads highlight microtubule tips at the periphery of three hemi-spindles. Inset shows a more detailed view of a cone with fibrous Asp-containing material emanating from the astral pole and in this case also localizing near a central-spindle-like bundle of microtubules. (F) dd4S cyst displaying a anaphase cell lacking central spindle microtubules and with fragmented Asp staining at the poles (arrow). The large arrowhead points to a fibrous distribution of Asp emanating from the centre of a hemi-spindle aster. The inset shows a higher magnification of the spindle indicated by the small arrowhead showing a cone with some Asp localizing in the central-spindle-like region. Bars, 50 µm. Bar in insets of D and E, 10 µm.

View larger version (163K): [in a new window]   Fig. 4. Time-lapse series of meiosis I in wild-type and dd4 spermatocytes. (A-E) Wild-type division. (A) Prophase with opposed asters (asterisks). (B) Prometaphase with elongated nuclear region within which bivalents (arrows) acquire biorientation. (C) Rapid movement of homologues (arrows) towards opposite poles as the phase-dense material (pdm) starts accumulating along the spindle. (D) Anaphase B where the parafusorial membranes are fully visible. (E) Telophase in which the cleavage furrow (arrowheads) will separate the daughter nuclei (nu). (F-J) Late spindle collapse in a dd4S spermatocyte. (F) Asters initially segregate. (G) pdm accumulates along the equatorial region of bipolar spindles as apparently bioriented homologues move between the poles (arrows). (H) The spindle poles approach each other before any visible segregation of homologues takes place. The pdm accumulates at the centre of the spindle, which becomes the apical regions of two nascent conical structures. (I) These cones elongate further and their apexes become darker. (J) The cell becomes disorganized as individualization of nuclear-like (nu) vesicles takes place. (K-L) Defective MTOC segregation or early spindle collapse in a dd4S spermatocyte. (K) In this cell, a bona fide biastral spindle never forms at the onset of meiosis and the hemi-spindle structure seen in this panel persists until later stages. (L) Individualized bivalents tend to undergo rapid movements towards the pole containing the aster (arrow) as the pdm accumulate distally. (M) In later stages all visible chromosomes localize in the vicinity of the astral pole without evident segregation of the homologues (the curved arrow indicates the direction of a rotation of the cell). (N) The `hemi-spindle' then becomes conical as the pdm accumulates at the apex. (O) The pdm tends to fray and disorganize and several nuclear-like vesicles (nu) form in the region previously occupied by the asters. Bar, 10 µm.

View larger version (94K): [in a new window]   Fig. 6. Peanut localizes to the apexes of some cones. Merged images on the top row show the distribution of DNA (blue), Peanut (red), and -tubulin (green) in dd4S (main panels) and wild-type (inset) cells. The individual channels for Peanut and -tubulin are shown in the centre and bottom panels, respectively. (A) dd4S cells showing localization of Peanut in rings around the midzone-like region of cones (arrows). (B) Cone with sharp apexes and no detectable Peanut (arrow). The arrowhead points to a hemi-spindle devoid of Peanut staining. Bar, 50 µm.

View larger version (32K): [in a new window]   Fig. 7. Model to explain the abnormal microtubule structures during dd4S meiosis. Mutations in the TuRC component Dgrip91 may result in either failure of separation of the MTOCs or the collapse of a bipolar spindle shortly after its formation in prophase I (EC, early collapse). Alternatively, a bipolar spindle may be assembled, only to undergo a late collapse (LC) before anaphase I. In the first case monopolar structures, that we term hemi-spindles, form and are able to attach bivalents (blue ellipses), which, because of the lack of biorientation do not congress at metaphase. Some of these chromatin masses, located distal to the astral pole, may stabilize microtubules (green lines) with an opposite polarity relative to those nucleated by the main spindle pole (+ and - represent the putative polarity of microtubules). A structure resembling a central spindle could then form. Only these cases (cones) seem to be able to assemble a contractile ring containing Peanut and F-actin (orange hoop) and undergo cytokinesis. LCs generally result in biconical figures that are probably unable to assemble a central spindle due to a drastic loss of orientation of bivalents relative to a single pole. Consistently, Pav-KLP (yellow squares), which localizes close to the putative plus ends of the microtubules (+) in hemi-spindles, is only seen at the apex of the cones and at the vertices of biconical figures.

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