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. 2019 Jun 4;27(10):2921–2933.e5. doi: 10.1016/j.celrep.2019.05.023

Figure 5.

Figure 5

Cka and Mob4 Cooperate to Promote NSC Reactivation and Are Required for PP2A/Hippo Interaction

(A–G) NSC-specific cka or cka and mob4 double overexpression leads to premature enlargement and increased mitotic NSCs. Double overexpression results in stronger effects (see also Figure 3). Brain lobes of control (A and D, insc-gal4 > CD8-GFP), cka (B and E, insc-gal4 > CD8-GFP, cka), and double cka and mob4 (C and F, insc-gal4 > CD8-GFP, cka, mob4) overexpressing brains at 6 and 18 h ALH. NSCs in green (GFP) and red (Dpn), and divisions in blue (pH3). Dashed circles: MbNSCs; yellow arrowheads: quiescent NSCs; white arrowheads: prematurely enlarging (B and C) and dividing NSCs (D–F). Anterior up. Scale bars: 10 μm.

(G) Quantification of NSC diameters (6 h ALH: insc-gal4 > CD8-GFP n = 1,106 NSCs, 15 BLs, 10 brains; insc-gal4 > CD8-GFP, mob4 n = 639 NSCs, 10 BLs, 5 brains; insc-gal4 > CD8-GFP, cka n = 332 NSCs, 5 BLs, 5 brains; insc-gal4 > CD8-GFP, cka, mob4 n = 284 NSCs, 8 BLs, 8 brains; 18 h ALH: insc-gal4 > CD8-GFP n = 1,717 NSCs, 19 BLs, 14 brains; insc-gal4 > CD8-GFP, mob4 n = 1,004 NSCs, 13 BLs, 9 brains; insc-gal4 > CD8-GFP, cka n = 554 NSCs, 8 BLs, 4 brains; insc-gal4 > CD8-GFP, cka, mob4 n = 400 NSCs, 6 BLs, 4 brains) and divisions (6 h ALH: insc-gal4 > CD8-GFP n = 56 BLs, 28 brains; insc-gal4 > CD8-GFP, mob4 n = 22 BLs, 11 brains; insc-gal4 > CD8-GFP, cka n = 18 BLs, 9 brains; insc-gal4 > CD8-GFP, cka, mob4 n = 20 BLs, 10 brains; 18 h ALH: insc-gal4>CD8-GFP n = 70 BLs, 38 brains; insc-gal4 > CD8-GFP, mob4 n = 30 BLs, 15 brains; insc-gal4 > CD8-GFP, cka n = 18 BLs, 9 brains; insc-gal4 > CD8-GFP, cka, mob4 n = 20 BLs, 10 brains). Wilcoxon rank-sum tests, ∗∗∗p < 0.001; p > 0.05: ns. See also Figure S4.

(H and I) Depletion of Mob4 and/or Cka inhibits PP2A/Mts association to Hippo.

(H) CoIP assays using S2R+ cells expressing Myc-Mts and FLAG-Hippo or control FLAG-NTAN, in addition to RNAi against mob4 and/or cka or control DsRed (see also Figure S5). Lysates and FLAG-purified immunoprecipitates analyzed by western blot with indicated antibodies.

(I) Quantification of relative binding of Myc-Mts to FLAG-Hippo shown as a mean of the ratio between Myc-Mts and FLAG-Hippo signal intensities relative to control (DsRed RNAi) levels (n = 3 independent assays; error bars: SEMs; Student’s t tests; ∗p < 0.05 and ∗∗p < 0.01).