APC/C-dependent degradation of Spd2 regulates centrosome asymmetry in Drosophila neural stem cells (2023)
Figure 5

Figure 5 Spd2 accumulation increases the mobility of Spd2 at the interphase centrosome, preventing accumulation of Polo-dependent phosphorylation

(A) Representative images of interphase NBs in fixed Spd2WT-RES (top panels) and Spd2DK-RES (lower panels) third instar larval brains expressing Polo-GFP from the native promoter and stained for DAPI and Asl. Dotted yellow squares highlight centrosomes and their magnified images are shown in insets. While Spd2 was localised more symmetrically on the two centrosomes in Spd2DK-RES NBs compared to Spd2WT-RES NBs, Polo was still asymmetrically localised, being enriched at the apical centrosome, in Spd2DK-RES NBs. Scale bar: 10 µm.

(B) Asymmetric indexes of RFP-Spd2, Asl and Polo-GFP in Spd2WT-RES and Spd2DK-RES interphase NBs expressing Polo-GFP. The asymmetric distribution of Polo-GFP at interphase centrosomes was unaffected in Spd2DK-RES, unlike Spd2. 40 NBs (n) from at least three brains were analysed in each line. Red bars represent means ± SD.

(C, D) Representative images of interphase NBs in fixed wor>lacZ (control, top panels) and HA-Spd2WT-OE larval brains expressing Polo-GFP from the native promoter and stained for DAPI and Spd2 (C). Asymmetric indexes of Polo-GFP in interphase NBs wor>lacZ (control, top panels) and HA-Spd2WT-OE larval brains (D). 38 and 39 NBs (n) from at least three brains were analysed in each line. The asymmetric distribution of Polo-GFP at interphase centrosomes was unaffected by HA-Spd2WT overexpression. Red bars represent means ± SD.

(E) Representative images of interphase NBs in fixed mCh-Spd2WT-RES (top panels) and mCH-Spd2DK-RES larval brains and stained for DAPI, γTub and Cnn. Both multichannel and single-channel images of mCh-Spd2, γTub and Cnn are shown. Dotted yellow squares highlight centrosomes and their magnified images are shown in insets. Similar to Spd2DK-RES NBs, in mCh-Spd2CONS-RES NBs, Spd2 and γTub were more symmetrically accumulated at the two centrosomes than in mCh-Spd2WT-RES NBs, but Cnn was still asymmetrically distributed. Scale bar: 10 µm.

(F) Asymmetric indexes of mCh-Spd2, γTub and Cnn were analysed in mCh-Spd2WT-RES and mCh-Spd2CONS-RES interphase NBs. n = 34 and 47 from 5 and 9 brains of each line, respectively. Red bars represent means ± SD.

(G, H) FRAP analyses of the centrosomal Spd2 fluorescent signals in Spd2WT-RES and Spd2DK-OE NBs. (G) Representative images from time-lapse movies (Movies EV13, EV14) of Spd2 fluorescent signals in Spd2WT-RES NBs (top panels) and Spd2DK-OE NBs (lower panels) upon photobleaching. (H) The recoveries of the fluorescent intensities of centrosomal Spd2 signals after photobleaching were measured in Spd2WT-RES (n = 7) and Spd2DK-OE NBs (n = 5). Means ± SEM of the normalised fluorescent intensities are shown in a line graph. Centrosomal Spd2 signals recovered more fully after photobleaching in Spd2DK-OE NBs than in Spd2WT-RES NBs.

(I) A schematic diagram of a model for the role of APC/CFzr-dependent Spd2 degradation in the regulation of centrosomes in the Drosophila larval NB. The model is explained in detail in Discussion.

Data information: p-values in all the plots in this figure were calculated using unpaired non-parametric Mann Whitney U-tests.

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