Investigating the cellular and molecular mechanisms involved in establishing and maintaining centrosome asymmetry in Drosophila neuroblasts

Abstract

Centrosome asymmetry has been implicated in stem cell fate maintenance in both flies and vertebrates. Drosophila neuroblasts, the neural precursors of the fly’s central nervous system, contain molecularly and physically asymmetric centrosomes. For instance, the apical daughter centrosome maintains a stable microtubule organizing center (MTOC) activity and remains tethered to the apical cortex throughout the cell cycle. The basal mother centrosome, however, loses MTOC activity and only regains it during prophase. This centrosome asymmetry is important for centrosome positioning, spindle orientation and centrosome segregation during asymmetric cell division. In a gene candidate approach, we identified Bld10, fly ortholog of Cep135 and the uncharacterized gene CG7337, the fly ortholog of WDR62, as a regulator of centrosome asymmetry during interphase. In bld10 mutant neuroblasts the mother centrosome does not downregulate MTOC activity resulting in two mature active centrosomes. As a consequence of perturbed centrosome asymmetry, we observed spindle misalignment during metaphase and centrosome missegregation. In contrast, we were able to show that in CG7337/wdr62 mutant neuroblasts both centrosomes lose MTOC activity, resulting in interphase neuroblasts containing two untethered centrioles. Moreover, cold treatment of wdr62 mutant neuroblasts displayed microtubule instability while over expression of Wdr62 had hyper stabilized microtubules. We also observed decrease in Polo levels on the apical centrosome in mutant neuroblasts. Wdr62 localizes to the microtubules during the cell cycle. Taken together, we concluded that Wdr62 plays an important role in stabilizing microtubules, which are necessary for recruitment of Polo, and hence, maintains centrosome asymmetry. In addition, wdr62 mutants display cell cycle delay and decrease in brain size. To gain further insight into the neuroblast centrosome cycle and centrosome asymmetry establishment, we used 3D-SIM (Structured Illumination Microscopy). We observed that centriole duplication begins soon after centriole separation and molecular markers involved in establishing centrosome asymmetry have a very precise segregation pattern based on the age of the centrioles. Using 3D-SIM, we are able to define the time points of the occurrence of these events