Figure 6.
Figure 6. The N-terminal domain of HookA is critical for the HookA–dynein–dynactin interaction. (A) A diagram showing the wild-type HookA protein and the N-terminal deletion mutant protein, ΔN-HookA, in which the putative microtubule-binding domain is deleted. (B) The ΔN-HookA mutant exhibits the same colony phenotype as that exhibited by ΔhookA. (C) The ΔN-HookA mutant showed an obvious accumulation of mCherry-RabA–labeled early endosomes at the hyphal tip (∼71% of the hyphal tips show this accumulation, n = 112. Also see Video 6). (D) Kymographs showing an obvious accumulation of mCherry-RabA–labeled early endosomes at the hyphal tip in the ΔN-HookA and ΔhookA mutants. An arrowhead indicates one early endosome that moved away from the hyphal tip in the ΔN-HookA mutant. (E) The ΔN-HookA–GFP signals were concentrated at the hyphal tip where mCherry-RabA–marked early endosomes accumulate, and the GFP and mCherry signals largely overlap (100% hyphal tips that show the concentrated GFP signals show the mCherry-RabA accumulation; n = 50). (F) The dynein HC, the p150 subunit of dynactin, and NudF/LIS1 can be pulled down with HookA-GFP, ΔC-HookA–GFP, and ΔC1-HookA–GFP, but the amounts of these proteins pulled down with ΔN-HookA–GFP were obviously decreased. (G) A quantitative analysis of the Western results shown in F. The ratio of pulled down dynein HC, dynactin p150, or NudF/LIS1 to HookA-GFP was calculated. Values of all the mutants are relative to the wild-type values, which are set at 1. Mean and SD values were calculated from multiple independent pull-down experiments, and the number of experiments is indicated as n. For the ratio of dynein to HookA (dynein/HookA), the mean ± SD value for ΔN is 0.08 ± 0.11 (n = 4, P < 0.001), and the values for ΔC and ΔC1 are 1.77 ± 0.96 (n = 4) and 1.26 ± 0.71 (n = 3), respectively. Note that a p-value is provided only when the values are statistically different from the wild-type value, and the values of ΔC and ΔC1 are not different from the wild-type value at P = 0.05. For the ratio of dynactin to HookA (dynactin/HookA), the mean ± SD value for ΔN is 0.22 ± 0.18 (n = 4, P < 0.001), and the values for ΔC and ΔC1 are 1.5 ± 0.28 (n = 4, P < 0.05) and 1.6 ± 0.87 (n = 3), respectively. For the ratio of NudF/LIS1 to HookA (LIS1/HookA), the mean ± SD value for ΔN is 0.15 ± 0.1 (n = 4, P < 0.001), and the values for ΔC and ΔC1 are 1.37 ± 0.21 (n = 4, P < 0.05) and 2.0 ± 1.1 (n = 3), respectively. (H) A diagram showing the wild-type, ΔC-HookA, ΔN-ΔC-HookA, and ΔN1-ΔC-HookA mutant proteins. (I) Dynein HC and dynactin p150 could be pulled down with ΔC-HookA–GFP, but the amounts of these proteins were obviously diminished when the pull-down was performed with ΔN-ΔC-HookA–GFP and were nearly undetectable when the pull-down was performed with ΔN1-ΔC-HookA–GFP. (J) A quantitative analysis of the Western results shown in I. Values of ΔN-ΔC-HookA–GFP and ΔN1-ΔC-HookA–GFP are relative to the ΔC-HookA–GFP values, which are set at 1. Values of ΔN-ΔC-HookA–GFP and ΔN1-ΔC-HookA–GFP are significantly lower than that of ΔC-HookA–GFP. For the ratio of dynein to HookA (dynein/HookA), the mean ± SD values for ΔN-ΔC and ΔN1-ΔC are 0.2 ± 0.3 (n = 3, P < 0.05) and 0 ± 0 (n = 3, P < 0.001), respectively. For the ratio of dynactin to HookA (dynactin/HookA), the mean ± SD values for ΔN-ΔC and ΔN1-ΔC are 0.16 ± 0.2 (n = 3, P < 0.005) and 0.02 ± 0.03 (n = 3, P < 0.001), respectively. However, the values for ΔN-ΔC and ΔN1-ΔC are not significantly different from each other at P = 0.05. Bars, 5 µm.

The N-terminal domain of HookA is critical for the HookA–dynein–dynactin interaction. (A) A diagram showing the wild-type HookA protein and the N-terminal deletion mutant protein, ΔN-HookA, in which the putative microtubule-binding domain is deleted. (B) The ΔN-HookA mutant exhibits the same colony phenotype as that exhibited by ΔhookA. (C) The ΔN-HookA mutant showed an obvious accumulation of mCherry-RabA–labeled early endosomes at the hyphal tip (∼71% of the hyphal tips show this accumulation, n = 112. Also see Video 6). (D) Kymographs showing an obvious accumulation of mCherry-RabA–labeled early endosomes at the hyphal tip in the ΔN-HookA and ΔhookA mutants. An arrowhead indicates one early endosome that moved away from the hyphal tip in the ΔN-HookA mutant. (E) The ΔN-HookA–GFP signals were concentrated at the hyphal tip where mCherry-RabA–marked early endosomes accumulate, and the GFP and mCherry signals largely overlap (100% hyphal tips that show the concentrated GFP signals show the mCherry-RabA accumulation; n = 50). (F) The dynein HC, the p150 subunit of dynactin, and NudF/LIS1 can be pulled down with HookA-GFP, ΔC-HookA–GFP, and ΔC1-HookA–GFP, but the amounts of these proteins pulled down with ΔN-HookA–GFP were obviously decreased. (G) A quantitative analysis of the Western results shown in F. The ratio of pulled down dynein HC, dynactin p150, or NudF/LIS1 to HookA-GFP was calculated. Values of all the mutants are relative to the wild-type values, which are set at 1. Mean and SD values were calculated from multiple independent pull-down experiments, and the number of experiments is indicated as n. For the ratio of dynein to HookA (dynein/HookA), the mean ± SD value for ΔN is 0.08 ± 0.11 (n = 4, P < 0.001), and the values for ΔC and ΔC1 are 1.77 ± 0.96 (n = 4) and 1.26 ± 0.71 (n = 3), respectively. Note that a p-value is provided only when the values are statistically different from the wild-type value, and the values of ΔC and ΔC1 are not different from the wild-type value at P = 0.05. For the ratio of dynactin to HookA (dynactin/HookA), the mean ± SD value for ΔN is 0.22 ± 0.18 (n = 4, P < 0.001), and the values for ΔC and ΔC1 are 1.5 ± 0.28 (n = 4, P < 0.05) and 1.6 ± 0.87 (n = 3), respectively. For the ratio of NudF/LIS1 to HookA (LIS1/HookA), the mean ± SD value for ΔN is 0.15 ± 0.1 (n = 4, P < 0.001), and the values for ΔC and ΔC1 are 1.37 ± 0.21 (n = 4, P < 0.05) and 2.0 ± 1.1 (n = 3), respectively. (H) A diagram showing the wild-type, ΔC-HookA, ΔN-ΔC-HookA, and ΔN1-ΔC-HookA mutant proteins. (I) Dynein HC and dynactin p150 could be pulled down with ΔC-HookA–GFP, but the amounts of these proteins were obviously diminished when the pull-down was performed with ΔN-ΔC-HookA–GFP and were nearly undetectable when the pull-down was performed with ΔN1-ΔC-HookA–GFP. (J) A quantitative analysis of the Western results shown in I. Values of ΔN-ΔC-HookA–GFP and ΔN1-ΔC-HookA–GFP are relative to the ΔC-HookA–GFP values, which are set at 1. Values of ΔN-ΔC-HookA–GFP and ΔN1-ΔC-HookA–GFP are significantly lower than that of ΔC-HookA–GFP. For the ratio of dynein to HookA (dynein/HookA), the mean ± SD values for ΔN-ΔC and ΔN1-ΔC are 0.2 ± 0.3 (n = 3, P < 0.05) and 0 ± 0 (n = 3, P < 0.001), respectively. For the ratio of dynactin to HookA (dynactin/HookA), the mean ± SD values for ΔN-ΔC and ΔN1-ΔC are 0.16 ± 0.2 (n = 3, P < 0.005) and 0.02 ± 0.03 (n = 3, P < 0.001), respectively. However, the values for ΔN-ΔC and ΔN1-ΔC are not significantly different from each other at P = 0.05. Bars, 5 µm.

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