GFP-Forked, a genetic reporter for studying Drosophila oocyte polarity.

The polarized organization of the Drosophila oocyte can be visualized by examining the asymmetric localization of mRNAs, which is supported by networks of polarized microtubules (MTs). In this study, we used the gene forked, the putative Drosophila homologue of espin, to develop a unique genetic reporter for asymmetric oocyte organization. We generated a null allele of the forked gene using the CRISPR-Cas9 system and found that forked is not required for determining the axes of the Drosophila embryo. However, ectopic expression of a truncated form of GFP-Forked generated a distinct network of asymmetric Forked, which first accumulated at the oocyte posterior and was then restricted to the anterolateral region of the oocyte cortex in mid-oogenesis. This localization pattern resembled that reported for the polarized MTs network. Indeed, pharmacological and genetic manipulation of the polarized organization of the oocyte showed that the filamentous Forked network diffused throughout the entire cortical surface of the oocyte, as would be expected upon perturbation of oocyte polarization. Finally, we demonstrated that Forked associated with Short-stop and Patronin foci, which assemble non-centrosomal MT-organizing centers. Our results thus show that clear visualization of asymmetric GFP-Forked network localization can be used as a novel tool for studying oocyte polarity.

Drosophila oocyte polarity and cytoskeleton organization require regulation of Ik2 activity by Spn-F and Javelin-like.

The Drosophila melanogaster Spn-F, Ik2, and Javelin-like (Jvl) proteins interact to regulate oocyte mRNA localization and cytoskeleton organization. However, the mechanism by which these proteins interact remains unclear. Using antibodies to activated Ik2, we showed that this protein is found at the region of oocyte and follicle cell where microtubule minus ends are enriched. We demonstrate that germ line Ik2 activation is diminished both in jvl and in spn-F mutant ovaries. Structure-function analysis of Spn-F revealed that the C-terminal end is critical for protein function, since it alone was able to rescue spn-F sterility. On the other hand, germ line expression of Spn-F lacking its conserved C-terminal region (Spn-FΔC) phenocopied ik2, leading to production of ventralized eggshell and bicaudal embryos. In Spn-FΔC-expressing oocytes, Gurken protein is mislocalized and oskar mRNA and protein localization is disrupted. Expression of Ik2 rescued Spn-FΔC ovarian phenotypes. We found that whereas Spn-F physically interacts with Ik2 and Jvl, Spn-FΔC physically interacts with Ik2 but not with Jvl. Thus, expression of Spn-FΔC, which lacks the Jvl-interacting domain, probably interferes with interaction of Ik2 and Jvl. In summary, our results demonstrate that Spn-F mediates the interaction between Ik2 and Jvl to control Ik2 activity.