Marrari, Y., Rouvière, C. and Houliston, E (2004)
Dev. Biol. in press

In this article we showed that dynein is required for the cortical rotation by injecting the dynactin subunit dynamitin, which uncouples cytoplasmic dynein from its cargos, below the vegetal surface of the egg.

Movie 1 (256KB) : Z-series of confocal images acquired at successive 2 µm steps beneath the vegetal surface of an egg injected with dynamitin before the formation of the array (0.48 NT), fixed 15 minutes later and processed for anti-tubulin immunofluorescence. A severe reduction in the number of vegetal subcortical microtubules was observed in a zone of about 150 µm of diameter. An accumulation of microtubules is detectable at a 16 µm depth beneath the affected zone. In contrast, in unaffected regions, microtubules are well aligned and accumulated immediately beneath the cortex.

Movie 2 (1.0MB): Sequence of images acquired every 20 seconds of DiOC6(3)-labelled mitochondrial islands embedded in the vegetal cytoplasmic mass, moving with respect to the immobilised cortex. A local inhibition of cortical rotation in a zone about 100 µm diameter was provoked following dynamitin injection at about 0.48 NT.

Movie 3 (1.2MB): The cortical rotation in an egg injected with BSA analysed by DiOC6(3)-labelling as in movie 4 is not perturbed.

Movies 4-6: Sequences of 13 images of cytoplasmic mitochondrial island translocation during the cortical rotation in the presence of dynamitin injected after the formation of the array at various times show different degrees of cortical rotation inhibition.
Movie 4 (384KB): Egg injected with dynamitin at 0.6 NT. The translocation was completely inhibited around the site of injection (index of inhibition = 3).
Movie 5 (384KB): Egg injected at 0.64 NT. The translocation slowed down locally (index of inhibition = 2).

Movie 6 (384KB): Egg injected at 0.74 NT. The translocation was not disrupted (index of inhibition = 1).

We also showed that microtubules are present within the vegetal array during cortical rotation that move with the cortex relative to the cytoplasm, and others that move with the cytoplasm relative to the cortex .

Movie 7 (768 KB) : This sequence of images acquired every 5 seconds following photo-bleaching shows that most microtubule bundles labelled by incorporation of fluorescein-tubulin translocate relative to the vegetal egg cortex, immobilised against the coverslip. The image before bleaching is shown in Figure 1 A of the manuscript. Cytoplasmic yolk platelets displace in the same direction as the microtubule bundles.

Movie 8 (768 KB) : This movie shows the same sequence as movie 1, realigned with respect to cytoplasmic yolk platelets. The realignment of the images simulates the “natural“ situation in which gravity maintains the position of the vegetal yolk mass and the cortex displaces. Microtubules are observed to displace within bundles relative to the cytoplasm as revealed by their entry into the bleached zone.