Figure 5.
The kinAK895* mutation affects cargo-adapter-mediated dynein activation in vivo. (A) Images of HookA-GFP in the nudF6 single mutant, the nudF6, phi double mutant, the kinAk895* single mutant and the kinAk895*, phi double mutant at 32°C (note that phi stands for nudAR1602,K1645E). Since we did the experiments at both 32°C (a semi-restrictive temperature for nudF6) and 37°C (a nearly restrictive temperature for nudF6), HookA-GFP was used as an early endosome marker since the HookA-GFP signals are clearer than those of mCherry-RabA at 37°C. Note that although the function of NudF/LIS1 is not completely lost at 32°C, the hyphal-tip accumulation of HookA-GFP signals is very obvious in the nudF6 single mutant. Hyphal tip is indicated by a yellow arrowhead. Bar, 5 μm. (B) A quantitative analysis on hyphal-tip accumulated HookA-GFP signals (Kruskal–Wallis with Dunn’s multiple comparisons test, unpaired). The average value for the nudF6 strain is set as 1. Scatter plots with mean and SD values were generated by Prism 9. (C) Line scans of HookA-GFP fluorescence intensity in the nudF6 single mutant, the nudF6, phi double mutant, the kinAk895* single mutant, and the kinAk895*, phi double mutant grown at 37°C. XY graphs with mean (solid lines) and SEM (shading) were generated by Prism 9. The intensity of HookA-GFP near the hyphal tip (between 0.39 and 1.82 μm from hyphal tip) was significantly different between the nudF6 single mutant and the nudF6, phi double mutant (P < 0.0001) but not significantly different between the kinAk895* single mutant and the kinAk895*, phi double mutant (P values are in between 0.192 and >0.999; two-way ANOVA with Tukey’s multiple comparisons test). (D) Dynein localization upon overexpression of the cargo adapter ∆C-HookA in a strain with wild-type kinA (gpdA-∆C-hookA-S) and in a strain with the kinAK895* mutation (kinAK895*, gpdA-∆C-hookA-S). Bright-field images are shown below to indicate hyphal shape and position of septum. Hyphal tip is indicated by a yellow arrowhead and septum by a brown arrow. Bar, 5 μm. (E) A quantitative analysis of dynein signals at septa (unpaired t test, two-tailed, Prism 9). All values are relative to the average value for the gpdA-∆C-hookA-S strain, which is set as 1. Scatter plots with mean and SD values were generated by Prism 9. (F) Line scans of GFP-dynein fluorescence intensity in the gpdA-∆C-hookA-S and the kinAK895*, gpdA-∆C-hookA-S strains. XY graphs with mean (solid lines) and SEM (shading) were generated by Prism 9. GFP-dynein intensity near the hyphal tip (between 0.26 and 1.69 μm from hyphal tip) was significantly higher in the kinAK895*, gpdA-∆C-hookA-S strain than in the gpdA-∆C-hookA-S strain (P < 0.0001, two-way ANOVA with Bonferroni’s multiple comparisons test, n = 30 hyphae for each strain).

The kinA K895 * mutation affects cargo-adapter-mediated dynein activation in vivo. (A) Images of HookA-GFP in the nudF6 single mutant, the nudF6, phi double mutant, the kinAk895* single mutant and the kinAk895*, phi double mutant at 32°C (note that phi stands for nudAR1602,K1645E). Since we did the experiments at both 32°C (a semi-restrictive temperature for nudF6) and 37°C (a nearly restrictive temperature for nudF6), HookA-GFP was used as an early endosome marker since the HookA-GFP signals are clearer than those of mCherry-RabA at 37°C. Note that although the function of NudF/LIS1 is not completely lost at 32°C, the hyphal-tip accumulation of HookA-GFP signals is very obvious in the nudF6 single mutant. Hyphal tip is indicated by a yellow arrowhead. Bar, 5 μm. (B) A quantitative analysis on hyphal-tip accumulated HookA-GFP signals (Kruskal–Wallis with Dunn’s multiple comparisons test, unpaired). The average value for the nudF6 strain is set as 1. Scatter plots with mean and SD values were generated by Prism 9. (C) Line scans of HookA-GFP fluorescence intensity in the nudF6 single mutant, the nudF6, phi double mutant, the kinAk895* single mutant, and the kinAk895*, phi double mutant grown at 37°C. XY graphs with mean (solid lines) and SEM (shading) were generated by Prism 9. The intensity of HookA-GFP near the hyphal tip (between 0.39 and 1.82 μm from hyphal tip) was significantly different between the nudF6 single mutant and the nudF6, phi double mutant (P < 0.0001) but not significantly different between the kinAk895* single mutant and the kinAk895*, phi double mutant (P values are in between 0.192 and >0.999; two-way ANOVA with Tukey’s multiple comparisons test). (D) Dynein localization upon overexpression of the cargo adapter ∆C-HookA in a strain with wild-type kinA (gpdA-∆C-hookA-S) and in a strain with the kinAK895* mutation (kinAK895*, gpdA-∆C-hookA-S). Bright-field images are shown below to indicate hyphal shape and position of septum. Hyphal tip is indicated by a yellow arrowhead and septum by a brown arrow. Bar, 5 μm. (E) A quantitative analysis of dynein signals at septa (unpaired t test, two-tailed, Prism 9). All values are relative to the average value for the gpdA-∆C-hookA-S strain, which is set as 1. Scatter plots with mean and SD values were generated by Prism 9. (F) Line scans of GFP-dynein fluorescence intensity in the gpdA-∆C-hookA-S and the kinAK895*, gpdA-∆C-hookA-S strains. XY graphs with mean (solid lines) and SEM (shading) were generated by Prism 9. GFP-dynein intensity near the hyphal tip (between 0.26 and 1.69 μm from hyphal tip) was significantly higher in the kinAK895*, gpdA-∆C-hookA-S strain than in the gpdA-∆C-hookA-S strain (P < 0.0001, two-way ANOVA with Bonferroni’s multiple comparisons test, n = 30 hyphae for each strain).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal