Identification of Miranda protein domains regulating asymmetric cortical localization, cargo binding, and cortical release

An important question in cellular and developmental biology is how a cell divides to produce daughter cells with different fates. Drosophila neuroblasts are a model system for studying asymmetric cell division: at each division, neuroblasts retain stem cell-like features, whereas their sibling ganglion mother cell (GMC) has a more restricted fate. Establishing neuroblast/GMC differences involves the asymmetric localization of proteins (Inscuteable, Miranda, Prospero, and Staufen) and RNA (prospero). All of these factors are apically localized during interphase, and all except Inscuteable move to the basal cortex at mitosis prior to being partitioned solely into the GMC. In this study, we show that Miranda is colocalized with Staufen and Prospero in neuroblasts, and is required for the asymmetric cortical localization of both proteins. Analysis of miranda mutants reveals three functional domains within the Miranda protein: (1) an N-terminal domain (1-290 aa) sufficient for association of Miranda with the cell cortex and basal localization in mitotic neuroblasts; (2) a central domain (446-727 aa) necessary for apical localization in interphase neuroblasts as well as for "cargo binding" of Prospero, Staufen, and prospero mRNA; and (3) a C-terminal domain (727-830 aa) necessary for the timely degradation of Miranda and release of its cargo from the cortex of the newborn GMC. In addition, Miranda is asymmetrically localized in epithelial cells that lack Inscuteable and divide symmetrically; thus the mechanism regulating Miranda localization is common to epithelial cells and neuroblasts, and Inscuteable is not an obligate component. Finally, we define a C-terminal domain of Staufen sufficient for Miranda-dependent cortical localization in neuroblasts.