Phi opening partially compensates for NudF/LIS1 deficiency and mildly enhances the formation of the dynein–dynein–ΔC-HookA complex. (A) Images of GFP-dynein and mCherry-RabA in a nudF6 strain, a nudF6, nudA^R1602E strain, and a nudF6, nudA^{R1602E, K1645E} strain. Hyphal tips are indicated by arrowheads and septa by arrows. Note that GFP-dynein with nudA^{R1602E, K1645E} accumulates at a septum with early endosomes (mCherry-RabA) in the nudF6 mutant. (B) A quantitative analysis on GFP-dynein comet intensity in wild-type (n = 35), nudA^R1602E (n = 71), nudA^{R1602E, K1645E} (n = 49), nudF6, nudA^R1602E (n = 62), and nudF6, nudA^{R1602E, K1645E} (n = 33) strains. The average value for wild type is set as 1. (C) A quantitative analysis on septal dynein intensity in wild-type (n = 55), nudA^R1602E (n = 62), nudA^{R1602E, K1645E} (n = 42), nudF6, nudA^R1602E (n = 32), and nudF6, nudA^{R1602E, K1645E} (n = 30) strains. The average value for the nudA^{R1602E, K1645E} strain is set as 1. For both B and C, scatterplots with mean and SD values were generated by Prism 8. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 (Kruskal–Wallis ANOVA test with Dunn’s multiple comparisons test, unpaired). (D) Western blots showing the effects of the nudA^R1602E and nudA^{R1602E, K1645E} mutations on dynein–dynein–ΔC-HookA complex formation. A ΔC-hookA-S strain without GFP-dynein was used as a negative control. (E) A quantitative analysis on the ratios of pulled-down dynactin p150, ΔC-HookA-S, and NudF/LIS1 to GFP-dynein HC, indicated as Dynactin/Dynein, ∆C-HookA/Dynein, and LIS1/Dynein, respectively. The values were generated from western analyses of three independent pull-down experiments (n = 3 for all). The wild-type values are set as 1. Scatterplots with mean and SD values were generated by Prism 8. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (one-way ANOVA, unpaired).