Figure 8.
Figure 8. Loss of HookA or the HookA–dynein–dynactin interaction significantly weakens the interaction between dynein and early endosomes. (A) Western blots showing that the amount of mCherry-RabA–labeled early endosomes pulled down with GFP–dynein HC from the ΔhookA mutant extract is significantly lower than that from the wild type. The mCherry-RabA signals in the extracts used for the pull-down experiments are also shown. (B) A quantitative analysis of the Western results (shown in A). The ratio of mCherry-RabA to GFP–dynein HC (RabA/dynein) was calculated. Values are relative to the wild-type value, which is set at 1. The mean ± SD value for the ΔhookA mutant is 0.19 ± 0.22 (n = 4, P < 0.001). (C) The HookAL150P,E151K-GFP signals were concentrated at the hyphal tip where mCherry-RabA–marked early endosomes accumulate, and the GFP and mCherry signals largely overlap. Bars, 5 µm. (D) The dynein HC and the p150 subunit of dynactin could be pulled down with HookA-GFP, but the amounts of these proteins pulled down with HookAL150P,E151K-GFP were obviously decreased. For the ratio of dynein to HookA, if we set the wild-type mean values to 1, the mean ± SD value for HookAL150P,E151K was 0.05 ± 0.09 (n = 3, P < 0.001). For the ratio of dynactin to HookA, if we set the wild-type mean values to 1, values for HookAL150P,E151K were 0.09 ± 0.16 (n = 3, P < 0.001). In contrast, the amount of mCherry-RabA–labeled early endosomes pulled down was apparently not decreased. The mCherry-RabA signals in the extracts used for the pull-down experiments are also shown. (E) Western blots showing that the amount of mCherry-RabA–labeled early endosomes pulled down with dynein HC-GFP from the HookAL150P,E151K mutant extract is significantly lower than that from the wild type. The mCherry-RabA signals in the extracts used for the pull-down experiments are also shown. (F) A quantitative analysis of the Western results (shown in E). The ratio of mCherry-RabA to GFP–dynein HC (RabA/dynein) was calculated. Values are relative to the wild-type value, which is set at 1. The mean ± SD value for the ΔhookA mutant is 0.25 ± 0.15 (n = 4, P < 0.001). (G) A working model showing that HookA on an early endosome links dynein–dynactin to the cargo for its movement along the microtubule track. Several dimers of HookA are depicted. A possibility not excluded is that HookA also facilitates cargo–track interaction, which is likely to be dynamic rather than static. For simplicity, HookA is depicted as the only protein linking dynein–dynactin to the early endosome, but it is likely that additional proteins are required for bridging the HookA–dynein–dynactin and HookA–early endosome interactions.

Loss of HookA or the HookA–dynein–dynactin interaction significantly weakens the interaction between dynein and early endosomes. (A) Western blots showing that the amount of mCherry-RabA–labeled early endosomes pulled down with GFP–dynein HC from the ΔhookA mutant extract is significantly lower than that from the wild type. The mCherry-RabA signals in the extracts used for the pull-down experiments are also shown. (B) A quantitative analysis of the Western results (shown in A). The ratio of mCherry-RabA to GFP–dynein HC (RabA/dynein) was calculated. Values are relative to the wild-type value, which is set at 1. The mean ± SD value for the ΔhookA mutant is 0.19 ± 0.22 (n = 4, P < 0.001). (C) The HookAL150P,E151K-GFP signals were concentrated at the hyphal tip where mCherry-RabA–marked early endosomes accumulate, and the GFP and mCherry signals largely overlap. Bars, 5 µm. (D) The dynein HC and the p150 subunit of dynactin could be pulled down with HookA-GFP, but the amounts of these proteins pulled down with HookAL150P,E151K-GFP were obviously decreased. For the ratio of dynein to HookA, if we set the wild-type mean values to 1, the mean ± SD value for HookAL150P,E151K was 0.05 ± 0.09 (n = 3, P < 0.001). For the ratio of dynactin to HookA, if we set the wild-type mean values to 1, values for HookAL150P,E151K were 0.09 ± 0.16 (n = 3, P < 0.001). In contrast, the amount of mCherry-RabA–labeled early endosomes pulled down was apparently not decreased. The mCherry-RabA signals in the extracts used for the pull-down experiments are also shown. (E) Western blots showing that the amount of mCherry-RabA–labeled early endosomes pulled down with dynein HC-GFP from the HookAL150P,E151K mutant extract is significantly lower than that from the wild type. The mCherry-RabA signals in the extracts used for the pull-down experiments are also shown. (F) A quantitative analysis of the Western results (shown in E). The ratio of mCherry-RabA to GFP–dynein HC (RabA/dynein) was calculated. Values are relative to the wild-type value, which is set at 1. The mean ± SD value for the ΔhookA mutant is 0.25 ± 0.15 (n = 4, P < 0.001). (G) A working model showing that HookA on an early endosome links dynein–dynactin to the cargo for its movement along the microtubule track. Several dimers of HookA are depicted. A possibility not excluded is that HookA also facilitates cargo–track interaction, which is likely to be dynamic rather than static. For simplicity, HookA is depicted as the only protein linking dynein–dynactin to the early endosome, but it is likely that additional proteins are required for bridging the HookA–dynein–dynactin and HookA–early endosome interactions.

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