Interplay between Drosophila importin-alpha2, beta and kelch during oogenesis and early embryogenesis

This work describes the interplay between importin-α2 (imp-α2), kelch and importin-β (imp-β) during two developmental periods of the fruit fly Drosophila namely, oogenesis and early embryogenesis. In particular, we emphasize on the role played by Imp-α2 in localizing Kelch to the ring canals (RC) during oogenesis. Imp-α2 is critically involved in RC assembly. In mutant imp-α2 females, the RCs are occluded and dumping of nurse cell cytoplasm into the oocyte is prevented. In the egg chambers, Kelch is synthesized but unable to bind RCs and mediate their opening. kelch mutations produce similar RC occlusion. In wild-type, Kelch strongly decorates RCs, yet Imp-α2 remains in the cytoplasm. Further analyses reveal no direct interaction between Kelch and Imp-α2, suggesting a mechanism by which Imp-α2 acts upon a factor regulating Kelch function (Gorjánácz et al., 2002). The first part of this study focuses on the interactions that take place between imp-α2 and kelch as well as kelch and imp-β . Using a sensitized background we were able to show that genetic interaction could take place between imp-α2 and kelch. Moreover, the interaction between imp-β and kelch is even stronger because we can detect interaction genetically and physically. Analysis of the distribution of Imp-α2 in wild type and kel∆ ovaries indicated an interdependence of Imp-α2 and Kelch in their cellular localization. Confocal analysis showed that the Kelch protein can be detected in preblastodermic embryos. Kelch was found to decorate the centrosomes and the spindle during mitosis although its pattern of distribution is generally distinct from that of Imp-α2 but overlaps during anaphase. The occurrence of both proteins in the nuclei during mitosis suggests that they may to interact. Hence, we suggest a new role for Kelch in mitosis during early embryogenesis. Further analysis showed that Imp-α2ΔIBB, which is unable to bind Imp-β, blocks oogenesis whereas Imp-α2NLSB-, which is able to bind an NLS bearing cargo protein, allows oogenesis to fully proceed in mutant imp-α2 females but subsequently arrests nuclear division in embryos, indicating that Imp-α2 exerts specific functions in distinct processes, such as RC assembly and mitosis. Genetic interaction between Kelch and Imp-β was detected by using the recessive imp-βRE34 allele whose binding affinity for Imp-α2 is affected. Heterozygous kelΔ/imp-βRE34 females, similar to imp-α2D14/imp-βRE34, produced eggs whose development was arrested during early embryogenesis. Moreover, pull-down assays showed that Kelch and Imp-β can physically interact. Based on our results we propose a model, wherein Imp-β could be the mediator between Imp-α2 and Kelch during oogenesis and that binding of Imp-α2 to Imp-β could release Kelch which will be able to gain access to RCs. In conclusion, this study reveals a possible mechanism through which Imp-α2 controls the localization of Kelch to RCs and a new role played by Kelch during early embryogenesis