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Fig. 4. The C-terminal region of Miranda is required for its correct cortical localisation. (A-F') Larval neuroblast (NB) clones generated using the MARCM system. NB clones are labelled with CD8::GFP (green, A-F), Miranda (red, A'-E') and Prospero (red, F'); DNA (blue). miranda^RR127 mutant NBs do not properly localise Miranda to the cell cortex during prophase (B,B') and accumulate both Miranda (D,D') and Prospero (F,F') pericentrosomally at the expense of cortical protein during metaphase. Miranda mislocalisation in miranda^RR127 mutant NBs occurs independently of microtubule function and is still observed after colchicine treatment to depolymerise microtubules (E,E'). (G,G') Replacement of the C-terminal domain of Miranda in miranda^RR127 mutants with ubiquitin restores normal protein localisation. GFP::Mira^Cterm-ubi, green; Miranda, red. (H,H') NB clones mutant for the proteasome regulatory subunit Tbp-1 show pericentrosomal accumulation of Miranda. (I,I') However, after treatment with colchicine to depolymerise microtubules, Miranda disperses throughout the cytoplasm (compare to E,E'). (J,J') Prospero is uniformly cytoplasmic in Tbp-1 mutant NBs (compare to Fig. 2I). (K-M) Miranda localisation in miranda^RR127 stage 9 NBs. Miranda is mislocalised pericentrosomally in a high proportion of miranda^RR127 zygotic (90%, n=105; L) and germline clone (GLC) mutant NBs (90%, n=94; M) at the expense of basally localised protein. Miranda, red; -tubulin, green. Scale bar: 10 µm.

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