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First published online October 12, 2007
doi: 10.1242/10.1242/dev.010900

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Asymmetric localisation of Miranda and its cargo proteins during neuroblast division requires the anaphase-promoting complex/cyclosome

Cathy Slack^1,*,, Paul M. Overton¹, Richard I. Tuxworth¹ and William Chia^1,2,

¹ MRC Centre for Developmental Neurobiology, New Hunt's House, King's College London, Guy's Campus, London SE1 1UL, UK.
² Temasek Lifesciences Laboratory and Department of Biological Sciences, 1 Research Link, National University of Singapore, 117604, Singapore.

View larger version (56K): [in this window] [in a new window]   Fig. 1. APC/C function is required for Miranda localisation to the basal cortex. (A-D,H,I) In idaPL17 mutant neuroblasts (NBs), Miranda is exclusively cytoplasmic during prophase (B) and accumulates pericentrosomally during metaphase (D) and anaphase (I), rather than forming a cortical crescent as in wild-type larval NBs (A,C,H). (E) Expression of a GFP::Ida fusion protein fully rescues the Miranda localisation defects observed in idaPL17 mutant NBs. (F,G) Loss-of-function mutants for APC/C subunits cdc27 (F) and morula (mr, APC2; G) also show Miranda pericentrosomal accumulation. (J-M) Miranda mislocalisation occurs independently of both centrosome function and microtubules. cnn; ida double mutants still accumulate pericentrosomal Miranda (L) and Miranda mislocalisation in ida mutant NBs is insensitive to colchicine treatment, which depolymerises microtubules (M). Broken circle in M indicates the outline of the NB. Miranda, red (A-M); PH3, green (A,B); GFP, green (E); Cnn, green (J-M); DNA, blue (A-M). (N) Quantification of Miranda pericentrosomal mislocalisation in allelic combinations of ida mutants. Scale bar: 10 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 2. Ida functions specifically to control the cortical localisation of Miranda and its associated proteins, but not to control PON/Numb. (A-G) Wild-type localisation of Inscuteable (Insc, green; A,B), Prospero (Pros, green; C), Staufen (Stauf, green; D), Brat (green; E), Numb (green; F), PON (green; G) and Miranda (Mira, red; A-G) in mitotic larval neuroblasts (NBs). (H,I) Inscuteable (green) localisation is unaffected in ida mutant NBs and a cortical Inscuteable crescent is always seen. Where Miranda (red in H',I') crescents form, Inscuteable and Miranda crescents occupy opposite sides of the cell cortex (I,I'). (J-L) Prospero (green, J), Staufen (green, K) and Brat (green, L) accumulate pericentrosomally with Miranda (J'-L') in ida mutant NBs. (M,N) By contrast, Numb (green, M) and PON (green, N) remain associated with the NB cortex even in ida mutant NBs that show strong Miranda mislocalisation defects (M',N'). Miranda localisation is shown in red (H'-N'); DNA in blue. Scale bar: 10 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 3. The C-terminal domain of Miranda is required for its ubiquitylation. (A) Constructs encoding FLAG-Miranda or FLAG-Miranda in which the C-terminal 103 amino acids were deleted (FLAG-MirandaCterm) under the control of an actin promoter were cotransfected into Drosophila S2 cells with DNA encoding HA-ubiquitin under the control of a heat-shock promoter. Extracts were prepared from cells both without (-) and with (+) heat shock. FLAG-Miranda but not FLAG-MirandaCterm was detected in the immune complex after the cells were heat shocked. (B) FLAG-Miranda and FLAG-MirandaCterm were expressed in S2 cells, and cell extracts were subjected to immunoprecipitation (IP) with either anti-FLAG or anti-ßgal antibodies followed by detection of ubiquitylated protein by western blotting using anti-ubiquitin antibody. Ubiquitylated Miranda was detected in the immune complex after immunoprecipitation of FLAG-Miranda but not FLAG-MirandaCterm. (C) FLAG-tagged Miranda was expressed in larval neuroblasts (NBs), and larval brain extracts were immunoprecipitated with either anti-FLAG or anti-ßgal antibodies; ubiquitylated protein was detected using anti-ubiquitin antibody.

View larger version (75K): [in this window] [in a new window]   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). mirandaRR127 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 mirandaRR127 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 mirandaRR127 mutants with ubiquitin restores normal protein localisation. GFP::MiraCterm-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 mirandaRR127 stage 9 NBs. Miranda is mislocalised pericentrosomally in a high proportion of mirandaRR127 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.