Phenotype of the eedE16 (kinA ^K895 *) mutant and position of the kinA ^K895 * mutation in KinA kinesin-1. (A) Microscopic images showing the distributions of GFP-dynein and mCherry-RabA-labeled early endosomes (mCherry-RabA) in wild type and the mutant. Bar, 5 μm. Although bi-directional movements of mCherry-RabA-labeled early endosomes are not completely abolished, most of the eedE16 hyphal tips (∼80%) show an obvious accumulation of mCherry-RabA signals (n = 130). Hyphal tip is indicated by a yellow arrowhead. (B) Line scans of GFP-dynein fluorescence intensity in wild type and the mutant. XY graphs with mean (solid lines) and SEM (shading) were generated by Prism 9. GFP-dynein intensity near the hyphal tip (between 0 and 2.145 μm from hyphal tip) was significantly higher in the mutant than in wild type (P < 0.0001, two-way ANOVA with Bonferroni’s multiple comparisons test, n = 30 hyphae for wild type, n = 30 hyphae for the mutant). (C) Line scans of mCherry-RabA (early endosomes) fluorescence intensity in wild type and the mutant. XY graphs with mean (solid lines) and SEM (shading) were generated by Prism 9. The intensity of mCherry-RabA near the hyphal tip (between 0.585 and 2.730 μm from hyphal tip) was significantly higher in the mutant than in wild type (P < 0.0001, two-way ANOVA with Bonferroni’s multiple comparisons test, n = 30 hyphae for wild type, n = 30 hyphae for the mutant). (D) Domain structures of kinesin-1 proteins in human (KIF5A) and A. nidulans (KinA). Positions of the IAK motif involved in kinesin-1 autoinhibition and the eedE16 mutation kinA^K895* within the IAK motif are indicated. (E) Colony phenotypes of a wild-type strain, the kinA^K895* mutant and the ∆kinA mutant. (F) A quantitative analysis of colony diameter of the wild-type strain (n = 23), the kinA^K895* mutant (n = 24) and the ∆kinA mutant (n = 23; ordinary one-way ANOVA with Tukey’s multiple comparisons test). Scatter plots with mean and SD values were generated by Prism 9. (G) Kymographs showing mCherry-RabA signals (diagonal lines indicating movements of mCherry-RabA-marked early endosomes). Yellow arrows indicate dynein-mediated (or minus-end-directed) movements away from the hyphal tip. (H) A quantitative analysis on the frequency of minus-end-directed early endosome transport in wild type (n = 33 hyphal tips) and the kinA^K895* mutant (n = 31 hyphal tips; unpaired t test, two-tailed, Prism 9). Scatter plots with mean and SD values were generated by Prism 9. (I) A quantitative analysis on the speed of minus-end-directed transport in wild type (n = 92 movements) and the kinA^K895* mutant (n = 34 movements; unpaired t test, two-tailed, Prism 9). (J) A quantitative analysis on the frequency of plus-end-directed early endosome transport in wild type (n = 28 hyphal tips) and the kinA^K895* mutant (n = 38 hyphal tips; unpaired t test, two-tailed, Prism 9). Scatter plots with mean and SD values were generated by Prism 9. (K) A quantitative analysis on the speed of plus-end-directed transport in wild type (n = 51 movements) and the kinA^K895* mutant (n = 32 movements; unpaired t test, two-tailed, Prism 9).