The Place of Noncentrosomal Microtubule Nucleation

<p>The initial stages of noncentrosomal microtubule nucleation revealed by an endogenous GFP–α-tubulin fusion (left) and phase contrast (right). Following the corresponding videos, it is possible to unmistakably tell the chromosomes (arrows) apart form the other phase-dark objects that are present over the nuclear region (asterisks). The cell in (A) is shown as a single timeframe and the cell in (B) as a time-lapse series. In both cells, noncentrosomal microtubule nucleation (arrowheads) takes place close to the remains on the NE and does not overlap with the major chromosomes. Nucleation sites can be clustered (A) or dispersed (B). In the time-lapse series (B), only the chromosomes that are in focus are labelled. Timepoint 0 min in these series corresponds to the first sign of noncentrosomal microtubule nucleation, around 11 min after NEB. A white bar marks the growing end of a microtubule bundle that at timepoint 93 min reaches one of the bivalents.</p

Chromosome Segregation in Anastral Spindles in <i>Drosophila</i> Spermatocytes

<div><p>(Control [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020008#pbio-0020008-s005" target="_blank">Video S5</a>]) At metaphase I (0), the bivalents (revealed by a His2Avd–YFP fusion, shown by double arrowheads) are aligned in the middle of the spindle (revealed by a GFP–α-tubulin fusion), at the metaphase plate. At the onset of anaphase (3 min), the homologue chromosomes start to migrate towards opposite poles (single arrowheads) and to decondense. During anaphase B (4 min and 6 min), the spindle poles move apart from each other and the two sets of decondensed chromosomes become further separated.</p> <p>(<i>asp</i> [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020008#pbio-0020008-s006" target="_blank">Video S6</a>]) At timepoint 0, the bivalents align at the metaphase plate. Homologue chromosomes split apart at the onset of anaphase I (4 min). However, anaphase A migration is highly impaired. By the time the chromosomes start to decondense, they have barely moved towards the spindle poles (8 min and 14 min), and often homologue chromosomes end up included in the same daughter nucleus.</p> <p>(Colcemid [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020008#pbio-0020008-s008" target="_blank">Video S8</a>]) As in <i>asp</i> spermatocytes, the asters (arrows) remain at the plasma membrane at metaphase I in colcemid-treated cells, and the bivalents align in a metaphase plate-like within the acentrosomal spindles (0 min). Homologue chromosomes split apart at the onset of anaphase (upper cell, 6 min) and significantly segregate from one another (upper cell, 8 min; lower cell, 3 min). Further separation of the daughter nuclei during anaphase B is very limited in these cells (8 min), and cytokinesis does not occur.</p></div

Centriole Migration in Primary Spermatocytes

<div><p>(A) Time-lapse series of confocal images from a wild-type primary spermatocyte expressing GFP-PACT (centrioles) and His2AvD–GFP (chromosomes). The centrioles (arrows) can be seen moving away from the plasma membrane (0) towards the nucleus (N) and then migrating diametrically apart as the chromatin condenses. The chromosomes are fully condensed at timepoint 121 min.</p> <p>(B–D) The two centriole pairs (green) projected over the phase-contrast view (grey) can be seen close to the fenestrated NE and away from the plasma membrane (pm) in control cells (B), while they remain plasma membrane-bound in <i>asp</i> (C) and in colcemid-treated wild-type cells (D). In <i>asp</i> spermatocytes (C), the position of the membrane-bound centrioles correlates tightly with the pointed end of phase-dark protrusions (arrows) that are not present in colcemid-treated cells. These reflect the distribution of phase-contrast membranes known to overlap microtubules in these cells.</p> <p>(E–J) XY projections (E–G) and their corresponding optical sections (H–J) of control (E and H), <i>asp</i> (F and I), colcemid-treated spermatocytes (G and J) expressing an endogenous GFP–α-tubulin confirm that the two major MTOCs in control cells are close to the nucleus, but remain near the plasma membrane in the two experimental conditions. MTOC activity in colcemid-treated spermatocytes was assayed following a 1-s pulse of 350 nm light to inactivate the drug, thus allowing microtubule regrowth. The yellow bar in the XY projections (E–G) marks the position of the corresponding XZ optical sections (H–J).</p></div

Noncentrosomal Microtubules and Spindle Assembly

<div><p>(Central column) Spindle assembly in <i>Drosophila</i> spermatocytes with membrane-bound centrosomes. At the time of NEB, the chromatin (pale blue) starts to condense, and the membrane-bound centrosomes (red) organise asters (yellow) at a significant distance from the nuclear region. Around 12 min after NEB, the first noncentrosomal microtubules (green) start to nucleate near the remnants of the NE (grey), as the chromosomes achieve full condensation (dark blue). These microtubules then bundle, associate with the chromosomes, and eventually end up organised into a bipolar anastral array whose shape is reminiscent of the female meiotic spindle.</p> <p>(Left column) Spindle assembly in wild-type <i>Drosophila</i> oocytes (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020008#pbio-0020008-Theurkauf1" target="_blank">Theurkauf and Hawley 1992</a>; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020008#pbio-0020008-Matthies1" target="_blank">Matthies et al. 1996</a>). NEB starts at the beginning of stage 13 of oocyte development. At this stage, the oocyte does not contain centrosomes and the chromosomes (karyosome) are tightly condensed (dark blue). Microtubules (green) appear 11–15 min after NEB within the nuclear region in association with the karyosome. These microtubules form bundles and are sorted around the chromatin into a bipolar spindle. Evidence suggests that ER components may be required for spindle assembly in these cells (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020008#pbio-0020008-Kramer1" target="_blank">Kramer and Hawley 2003</a>). At metaphase I, recombined bivalents are aligned at the spindle equator, while those that have not recombined are found closer to the spindle poles. Meiosis remains arrested at this point (stage 14) until oocyte activation. Despite the obvious morphological similitude, the equivalence between these and the anastral spindles organised in spermatocytes with membrane-bound centrosomes is unclear.</p> <p>(Right column) Hypothesis regarding the contribution of centrosomal and noncentrosomal microtubules to spindle assembly during meiosis I in wild-type <i>Drosophila</i> spermatocytes. Before NEB, the centrosomes are located at opposite positions near the nucleus. Shortly after NEB, astral microtubules enter the nuclear region and make the first contact with the condensing chromatin. No evidence of noncentrosomal microtubule polymerisation near the nuclear region at this stage has been found yet. Once chromosomes are fully condensed, microtubule bundles of centrosomal origin (yellow) connecting centrosomes to chromosomes already exist. At this stage, noncentrosomal microtubules (green) start to polymerise in association with the remnants of the NE. These microtubules form bundles that interact with the chromosomes and intermingle with the microtubules of centrosomal origin. The fully mature spindle in these cells would therefore contain a spindle-shaped structure made of microtubules of noncentrosomal origin (green) embedded in another spindle-shape array made of two overlapping asters (yellow). We propose that each of these subsets may perform to a certain extent some of the functions carried out by normal spindles, but neither of them can on its own mediate robust cell division.</p></div

Table S2. Results from the high-content screen. from An <i>in vivo</i> genetic screen in <i>Drosophila</i> identifies the orthologue of human cancer/testis gene <i>SPO11</i> among a network of targets to inhibit <i>lethal(3)malignant brain tumour</i> growth

The table contains all the information regarding the high-content screen including the list of all the RNAi lines screened, the corresponding VDRC UAS-RNAi line ID, FlyBase ID, CG name, Symbol, and the behaviour in the screen assay. Lines that performed as mbt tumor suppressors only in the first in both first and second rounds of screen are labeled orange and green, respectively. Only the latter (green) were tagged as confirmed mbt-SPRs. Lines that lead to larval lethality or larval brains smaller than wild type are labeled brown and purple, respectively

Supplemental Figures and figure legends 1 to 6 from An <i>in vivo</i> genetic screen in <i>Drosophila</i> identifies the orthologue of human cancer/testis gene <i>SPO11</i> among a network of targets to inhibit <i>lethal(3)malignant brain tumour</i> growth

Using transgenic RNAi technology, we have screened over 4.000 genes to identify targets to inhibit malignant growth caused by the loss of function of <i>lethal(3)malignant brain tumour</i> (mbt) in <i>Drosophila in vivo</i>. We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in mbt tumours hence showing that, rather counterintuitively, tumour-linked overexpression is not a good predictor of functional requirement. Moreover, we have found that most of the genes upregulated in mbt tumours remain overexpressed in tumour-suppressed double-mutant conditions, hence revealing that most of the tumour transcriptome signature is not necessarily correlated with malignant growth. One of the identified target genes is <i>meiotic W68</i> (<i>mei-W68</i>), the <i>Drosophila</i> orthologue of the human Cancer Testes gene <i>Sporulation-specific protein 11</i> (<i>SPO11</i>), the enzyme that catalyses the formation of meiotic double-strand breaks. We show that <i>Drosophila mei-W68/SPO11</i> drives oncogenesis by causing DNA damage in a somatic tissue, hence providing the first instance in which a <i>SPO11</i> orthologue is unequivocally shown to have a pro-tumoural role. Altogether, the results from this screen point to the possibility of investigating the function of human cancer relevant genes in a tractable experimental model organism like <i>Drosophila.</i