No abstract

Mass movements of cytoplasm, known as cytoplasmic streaming, occur in some large eukaryotic cells. In Drosophila oocytes there are two forms of microtubule-based streaming. Slow, poorly ordered streaming occurs during stages 8-10A, while pattern formation determinants such as oskar mRNA are being localized and anchored at specific sites on the cortex. Then fast well-ordered streaming begins during stage 10B, just before nurse cell cytoplasm is dumped into the oocyte. We report that the plus-end-directed microtubule motor kinesin-1 is required for all streaming and is constitutively capable of driving fast streaming. Khc mutations that reduce the velocity of kinesin-1 transport in vitro blocked streaming yet still supported posterior localization of oskar mRNA, suggesting that streaming is not essential for the oskar localization mechanism. Inhibitory antibodies indicated that the minus-end-directed motor dynein is required to prevent premature fast streaming, suggesting that slow streaming is the product of a novel dynein-kinesin competition. As F-actin and some associated proteins are also required to prevent premature fast streaming, our observations support a model in which the actin cytoskeleton triggers the shift from slow to fast streaming by inhibiting dynein. This allows a cooperative self-amplifying loop of plus-end-directed organelle motion and parallel microtubule orientation that drives vigorous streaming currents and thorough mixing of oocyte and nurse-cell cytoplasm.

Drosophila bicoid mRNA is synthesized in the nurse cells and transported to the oocyte where microtubules and Exuperantia protein mediate localization to the anterior pole. Fluorescent bicoid mRNA injected into the oocyte displays nonpolar microtubule-dependent transport to the closest cortical surface, and the oocyte microtubule cytoskeleton lacks clear axial asymmetry. Nonetheless, bicoid mRNA injected into the nurse cell cytoplasm, withdrawn, and injected into a second oocyte shows microtubule-dependent transport to the anterior cortex. Nurse cells require microtubules and Exuperantia to support anterior transport of bicoid mRNA, and microtubules are required for bicoid mRNA-Exuperantia particle coassembly. We propose that microtubule-dependent Exuperantia-bicoid mRNA complex formation in the nurse cell cytoplasm allows anterior-specific transport on a grossly nonpolar oocyte microtubule network.

Microtubules and the plus-end-directed microtubule motor Kinesin I are required for the selective accumulation of oskar mRNA at the posterior cortex of the Drosophila melanogaster oocyte, which is essential to posterior patterning and pole plasm assembly. We present evidence that microtubule minus ends associate with the entire cortex, and that Kinesin and microtubules are not required for oskar mRNA association with the posterior pole, but prevent ectopic localization of this transcript and the pole plasm proteins Oskar and Vasa to other cortical regions. Cortical binding of oskar mRNA seems to be dependent on the actin cytoskeleton. We conclude that most of the actin-rich oocyte cortex can support pole plasm assembly, and propose that Kinesin restricts pole plasm formation to the posterior by moving oskar mRNA away from microtubule-rich lateral and anterior cortical regions.