14-3-3 proteins genetically interact with Scrib and Mud and function as a molecular bridge between Dlg and Mud. (A) Quantification of mitotic spindle alignments in different genetic backgrounds. Knockdown of 14-3-3ε alone or the 14-3-3εj2B10 heterozygous background alone (14-3-3εj2B10/+) did not affect planar spindle orientation. Reduction of 14-3-3ε levels by 14-3-3ε-RNAi (HMS01229) or 14-3-3εj2B10/+ in mud-RNAi or scrib-RNAi discs significantly increased abnormal spindle orientation, while reduction of pins levels by pins-RNAi (HMS01462) or pinsP62/+ in mud-RNAi did not. Data are shown as box plots (median ± quartiles). Each point represents a cell. *, P < 0.01; **, P < 0.001; ***, P < 0.0001; n.s., not significant (P > 0.05) by Kolmogorov–Smirnov test. The number of analyzed spindles is as follows: nub-Gal4/+, n = 86; 14-3-3ε-RNAi, n = 58; 14-3-3εj2B10/+, n = 83; mud-RNAi, n = 83; mud-RNAi+pins-RNAi, n = 73; mud-RNAi+pinsP62/+, n = 35; mud-RNAi+14-3-3ε-RNAi, n = 109; mud-RNAi+14-3-3εj2B10/+, n = 128; scrib-RNAi, n = 92; scrib-RNAi +14-3-3εj2B10/+, n = 68; and mud-RNAi+scrib-RNAi, n = 114. (B–D) PLA between Scrib and Mud in control (nub-Gal4/+; B), dlg-knockdown (Cnub-Gal4>dlg-RNAi; C), and 14-3-3ζ/14-3-3ε-knockdown (nub-Gal4>14-3-3s-RNAi as 14-3-3ε-RNAi and 14-3-3ζ-RNAi from Ren et al., 2010; D) wing discs. (E) Quantification of the number of PLA (Scrib/Mud) spots for control (n = 15), dlg-RNAi (n = 12), and 14-3-3s-RNAi (n = 16) wing discs. Error bars are SD. ****, P < 0.0001 by Kolmogorov–Smirnov test. Scale bars: 5 µm. (I) Model illustrating how junctional proteins Scrib and Dlg may control planar spindle orientation by interacting with 14-3-3 proteins. 14-3-3 proteins could work as a molecular link between Dlg and Mud, which in turn interact with microtubules (purple) and motor proteins (cyan). Adherens junctions (red), septate junctions (green), spindle poles (yellow), and potential interacting proteins (small circles).
