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. 2012 May 25;336(6084):999-1003.
doi: 10.1126/science.1219147. Epub 2012 Apr 12.

Growing microtubules push the oocyte nucleus to polarize the Drosophila dorsal-ventral axis

Tongtong Zhao  1 , Owen S Graham, Alexandre Raposo, Daniel St Johnston
Affiliations

Growing microtubules push the oocyte nucleus to polarize the Drosophila dorsal-ventral axis

Tongtong Zhao et al. Science. .

Abstract

The Drosophila dorsal-ventral (DV) axis is polarized when the oocyte nucleus migrates from the posterior to the anterior margin of the oocyte. Prior work suggested that dynein pulls the nucleus to the anterior side along a polarized microtubule cytoskeleton, but this mechanism has not been tested. By imaging live oocytes, we find that the nucleus migrates with a posterior indentation that correlates with its direction of movement. Furthermore, both nuclear movement and the indentation depend on microtubule polymerization from centrosomes behind the nucleus. Thus, the nucleus is not pulled to the anterior but is pushed by the force exerted by growing microtubules. Nuclear migration and DV axis formation therefore depend on centrosome positioning early in oogenesis and are independent of anterior-posterior axis formation.

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Figures

Fig. 1
Fig. 1. Nuclear migration is driven by a posterior microtubule pushing force
(A) Time course of a migrating nucleus. (B) Analysis of directions of nuclear indentations during migration (left), direction of overall migration (right), and the correlation between them, expressed as the angle between the directional vectors (bottom) (mean±s.d.). Red and cyan dots show the outline of the nucleus at the start and end of migration, respectively; blue Xs show the centroid of the nucleus during migration. (C) Mean angle between migration and indentation directions from four migrating nuclei. (D) Temporal merges of an EB1-GFP movie of a colcemid-treated egg chamber. Each image is a maximum projection of 5 time frames (equal to 10 s). Arrow, MTOC. (A, D) a, anterior; p, posterior; Scale bars, 10 μm.
Fig. 2
Fig. 2. Active centrosomes are localised behind the nuclear indentation
(A) Temporal merges (20 time frames, equal to 10 s) of an EB1-GFP movie reveal active MTOCs (arrows) between the nuclear indentation and the posterior oocyte cortex. (B) The centriolar markers PACT-GFP and Sas-4-GFP and the PCM marker Cnn-GFP are localised behind the nucleus at the onset of migration (top). During migration, MTOCs can be clustered together (yellow arrows), associated with the nucleus (white arrows), or dispersed in the cytoplasm (blue arrows) (bottom). (A-B) a, anterior; p, posterior; Scale bars, 10 μm.
Fig. 3
Fig. 3. Laser ablation of the centrosomes abolishes the nuclear indentation
(A, D) Clusters of centrosomes were bleached for 5 s. 1-4 min after ablation, the nuclear indentation facing the ablated centrosomes disappears. (B, C) When the nuclear membrane or the anterior of the nucleus is bleached, the indentation is maintained. (A-D) a, anterior; p, posterior; Circles, area of bleaching; Arrows, non-ablated, active centrosomes; Scale bar, 10 μm.
Fig. 4
Fig. 4. Mispositioned centrosomes induce ectopic nuclear indentations
(A) Sas4-GFP (top) and a temporal merge of EB1-GFP (20 frames, equals 10 s) (bottom) reveal active, anterior centrosomes in par-16323/par-1W3 mutants, which induce an anterior indentation in the nucleus. (B) Nuclear migration in par-16323/par-1W3 mutants. At the onset of migration, the anterior centrosomes induce an ectopic anterior nuclear indentation. The anterior centrosomes eventually move around the nuclear membrane to cluster with the posterior centrosomes, inducing a broad nuclear indentation and rapid nuclear movement. (A-B) a, anterior; p, posterior; d, dorsal; Arrows, centrosomes; Scale bars, 10 μm.
Fig. 5
Fig. 5. The force of microtubule polymerisation is sufficient to move the nucleus
Quantification of the number of microtubules hitting the posterior of the nucleus. (A) Temporal merge of 20 frames (equal to 10 s) of an EB1-GFP movie. Red arrows indicate tracked microtubules that hit the nuclear indentation. a, anterior; p, posterior; Scale bar, 10 μm. (B) Kymograph of a microtubule that pushes against the nuclear indentation for 3 s; arrows indicate the position of the EB1-GFP comet (plus end of a microtubule). Scale bar, 1 μm. (C) Quantification of the number of microtubules hitting the nuclear indentation, the time that each microtubule pushes, and the resulting average number of microtubules that are pushing against the indentation at any given time (error bars, ± s.e.m.).
Fig. 6
Fig. 6. The nucleus is anchored to the posterior prior to migration
(A) The nuclei often fail to migrate in grk2B6/grk2E12 mutants, but still have prominent posterior indentations (arrows), indicating that they are tethered at the posterior. a, anterior; p, posterior; d, dorsal; Scale bars, 10 μm. (B) A microtubule pushing model for nuclear migration. Before migration, the nucleus is tethered at the posterior with active centrosomes behind it (left panel). The posterior follicle cell signal induces the release of the nucleus from the tether, and growing microtubules then push the nucleus anteriorly (middle panels). This movement is essentially random and continues until the oocyte becomes wedged in an anterior corner (right panel).
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