APC/C-dependent degradation of Spd2 regulates centrosome asymmetry in Drosophila neural stem cells (2023)
Figure 2

Figure 2. Spd2 compromises asymmetric PCM loading and activity of interphase centrosomes in larval NBs

(A) A schematic diagram representing the centrosome behaviour during interphase in wild-type larval NBs and NBs with increased levels of Spd2. The daughter and mother centrosomes are represented by blue and yellow circles, respectively; the centrosomes become inactive and detached from the cortex are represented by red circles. The size of the circles indicate the microtubule nucleation activity of the centrosome. In the wild type NB the daughter centrosome keeps actively nucleating microtubules and attached to the apical cortex during interphase, while in NBs with increased Spd2 levels both centrosomes become inactive and move around in the cytoplasm.

(B) Selected images from time-lapse movies of the control (pUbq-Sas6-GFP, top panels), fzrRNAi (carrying pUbq-Sas6-GFP to visualise centrosomes, middle panels) and Spd2DK-OE larval NBs (See also Movies EV1, EV2, EV4, EV5). All were expressing wor>mCh-αTub to visualise microtubules. mCherry are signals shown in red and GFP signals in green. In the control one of the two centrosome retained microtubule nucleation activity and stayed attached to the apical cortex (blue squares) while the other centrosome became inactive and moved toward the basal side (yellow squares). In fzrRNAi and Spd2DK-OE NBs, both centrosomes (orange squares) frequently became inactivated in interphase and were dissociated from the cortex, and dynamically moved in the cytoplasm. Higher magnification images of the centrosomes are shown in the insets. Scale bars: 5μm.

(C) In time lapse movies, NBs that showed an active centrosome that was attached to the cortex ("Attached") and NBs with both two centrosomes being inactivated and detached from the cortex ("Detached") in interphase were counted in third instar larval brains of the indicated fly lines and their proportions are presented in bar graphs. The numbers of NBs analysed from at least four brains (n) are Control: 29, fzrRNAi: 80, Spd2WT-RES: 45, Spd2DK-RES: 52, Spd2WT-OE: 43, Spd2DK-OE: 40, Spd2WT-OE + fzrRNAi: 72. p-values are fzrRNAi: 0.0069, Spd2WT-RES: 0.2479, Spd2DK-RES: 0.0116, Spd2WT-OE: <0.0001, Spd2DK-OE: <0.0001, Spd2WT-OE + fzrRNAi: 0.0008 (Pearson's chi-squared tests were performed between control and each condition).

(D) Representative images of NBs of immunostained Spd2WT-RES and Spd2DK-RES larval brains. Endogenous Asl, γTub or Cnn were detected by their specific antibodies. AurA was visualised pUbq-AurA-GFP transgene. Scale bars: 10 µm.

(E) The signal intensities of the indicated centriolar and PCM proteins at two centrosomes were quantified in individual interphase NBs in Spd2WT-RES and Spd2DK-RES larval brains, and their asymmetric distributions of their centrosomal signals between the two centrosomes are presented as Asymmetric Indexes (see Materials and Method) in a scatted dot plot. Red bars represent means ± SD. The numbers of NBs analysed from at least 3 different brains of Spd2WT-RES and Spd2DK-RES (n) were 40 and 40 for Asl, 40 and 41 for RFP-Spd2, 42 and 37 for Cnn, 40 and 41 for γTub, and 40 and 40 for AurA. p-values were calculated performing non-parametric unpaired Mann Whitney U tests and were shown above the plots.