Fig. 2. Miranda localization is a dynamic, multistep process in NBs and NE cells. Dynamic localization of Miranda detected by live imaging corresponds to its localization in fixed tissue, as detected by immunohistochemistry. Single confocal sections are shown. Embryos expressing Miranda-GFP under control of neuralized-Gal4 (neura-Gal4) were examined by time-lapse confocal microscopy for neuroblasts (NB) or neuroepithelial (NE) cells undergoing mitosis. In the majority of NBs (A) and in all NE cells (B), Miranda-GFP localizes uniformly to the cytoplasm and the cortex, but not to an apical crescent at prophase. At pro/metaphase cytoplasmic Miranda-GFP is more intense (white arrowhead), and includes nuclear and cortical areas. At metaphase, the basal cortical crescent is formed and Miranda-GFP gradually disappears from the remaining areas of the cell. Miranda-GFP is inherited by the GMC at telophase in NBs (A; white circle), and by both NE cells in a symmetric division (B). Miranda-GFP shows a very similar cytoplasm-to-basal cortex localization pattern when expressed under the control of V32-Gal4 (C) and scabrous-Gal4 (sca-Gal4) (D). Cytoplasmic Miranda accumulation is indicated by white arrows. (E) Miranda-GFP recapitulates the localization pattern of total Miranda protein. In fixed embryos, the location of Miranda-GFP is indistinguishable from that of total Miranda, in the cytoplasm at prophase and at the basal crescent at metaphase. Miranda, red; GFP, green; DNA, blue. (F) In embryos expressing an unphosphorylatable form of Lgl, UAS-Lgl^3A, Miranda-GFP is found uniformly around the cortex and cytoplasmic localization is abolished. (G,H) Miranda-GFP localizes to a tight metaphase crescent overlapping with total Miranda in two additional transgenic lines (line 2, G; line 3, H). (I) Immunoblotting using a Miranda antibody (top panel) and a GFP antibody (middle panel) reveals that ectopically expressed Miranda-GFP represented by the 130 kDa band is specifically expressed in UAS-Miranda-GFP/scabrous-Gal4 embryos, but not in UAS-Miranda-GFP or scabrous-Gal4 embryos (controls). Miranda-GFP levels are low compared with total Miranda protein, which runs at 75-100 kDa. Tubulin was detected as a control for equal loading (bottom panel).