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HookA is a novel dynein–early endosome linker critical for cargo movement in vivo

HookA is a novel linker protein that binds to endosomes and to dynein–dynactin and promotes dynein–early endosome interaction in Aspergillus.

. A sequence alignment of A. nidulans HookA with Drosophila Hook, and the three human Hook proteins. The alignment was performed using CLUSTALW. A. nidulans HookA (AnHookA): CBF80909, 638 residues; Drosophila Hook (DmHook): Q24185, 679 residues; human Hook1 (HsHook1): Q9UJC3, 728 residues; HsHook2: Q96ED9, 719 residues; HsHook3: Q86VS8, 718 residues. Residues that are identical (asterisks), strongly similar (double dots), or weakly similar (single dots) are shown as red, green, and blue characters, respectively. The amino acid residues within the putative microtubulebinding domain, the coiled-coil domains, and the cargo-binding domain are marked with red, blue, and brown solid lines, respectively, on top of the AnHookA sequence. The amino acid residues deleted in the N, N1, C, and C1 mutants are also marked on top of the sequence using dashed lines with arrows at the ends. The HookA L150P,E151K mutations in the eedA1 allele are marked on top of the AnHookA sequence with two blue stars below the letters P and K. Figure S2. Kymographs showing colocalization of HookA-GFP signals with mCherry-RabA-labeled early endosomes in a hyphal segment. Note that HookA-GFP signals colocalize with both motile and static early endosomes (two examples are indicated by arrows). Out of 34 motile early endosomes, 21 show clear colocalization with HookA-GFP (three examples are indicated by arrowheads). However, the corresponding HookA-GFP signals are not easily detected on 13 mCherry-RabA-labeled endosomes. This suggests that not all mCherry-RabA-labeled endosomes have associated HookA-GFP proteins. However, we could not exclude the possibility that the GFP signals on these structures may be too weak to be detected. Figure S3. The C terminus of HookA physically interacts with early endosomes. (A) Western blots showing that mCherry-RabA-labeled early endosomes are pulled down with either C-HookA-GFP or HookA-GFP. The parental strain (RQ54) used for transformation of the C-HookA-GFP or HookA-GFP construct was used as a negative control for this experiment, and thus, the negative control strain contains the same mCherry-RabA fusion protein as the two experimental strains. (B) A quantitative analysis of the Western results (shown in A). The intensity ratio of the pulled down mCherry-RabA to C-HookA-GFP or HookA-GFP was calculated. The values of RabA/HookA are set as 1, and the values of RabA/C-HookA (1.77 ± 0.2, mean ± SD) are relative to the RabA/ HookA values (P < 0.005, n = 3). Figure S4. The amount of dynein pulled down with the C-HookA-GFP is significantly lower than that pulled down with p150-GFP. (A) Western blots showing the positions of p150-GFP, C-HookA-GFP, and the pulled down dynein HC. (B) A quantitative analysis of the pull-down results. For this analysis, both the p150-GFP fusion and the C-HookA-GFP fusion were probed by the same solution containing the anti-GFP antibody. The ratios of pulled down dynein to p150-GFP (dynein/p150) and that of pulled down dynein to C-HookA-GFP (dynein/C-HookA) were calculated. The values of dynein/p150 are set as 1, the values of dynein/C-HookA-GFP are relative to the dynein/p150 values, and the mean ± SD value is 0.31 ± 0.095 (P < 0.001, n = 3). Figure S5. HookA-microtubule interaction is not readily detectable. (A) HookA-microtubule interaction is not readily detectable by a microtubule (MT)-pelleting assay. Total protein extract from a strain containing p150-GFP was used as a positive control for the microtubule-pelleting assay (Yao et al., 2012). The strain containing C1-HookA-GFP in the p25 background was used for the experiment to ensure that HookA is not associated with early endosomes and that any detected microtubule pelleting is a result of MT binding by HookA rather than that mediated by HookA-dynein-dynactin interaction. The strain containing N-C1-HookA-GFP that lacks the microtubule-binding domain was used as a negative control. All lysates were centrifuged at 100,000 g at 4°C for 40 min, and the supernatant was used for the microtubule-pelleting assay. Samples with no microtubules added (MT) were used as negative controls for the assay. P, pellet; S, supernatant. (B) A Western blot showing the N-HookA-GFP fusion containing the N-terminal 134 aa of the putative microtubule-binding domain of HookA. The blot was probed by an anti-GFP antibody, and the sample containing the C-HookA-GFP fusion was included in the same blot for comparison. (C) N-HookA-GFP does not obviously decorate microtubules in live cells. Because the wild-type hookA allele is replaced by the N-HookA-GFP allele, early endosomes are abnormally accumulated at the hyphal tip (80% of cells show this accumulation, n > 40). The N-HookA-GFP fusion does not appear to decorate any specific cellular structures (n > 40). Bars, 5 µm. Video 1. Movements of early endosomes in a wild-type strain of A. nidulans. Early endosomes are labeled by the mCherry-RabA fusion protein that was initially introduced to A. nidulans by transformation of the DNA encoding the fusion. The mCherry-RabA signals undergo bidirectional movements in wild-type hyphae. An inverted fluorescence microscope (IX70; Olympus) was used for capturing images. 30 frames were taken with a 0.1-s exposure time and a 0.3-s interval between frames. Binning is 1 × 1. The video speed has been increased fivefold.
Video 2. Early endosomes in the hookA strain. Early endosomes labeled by mCherry-RabA are largely accumulated at the hyphal tip. Directed movements of early endosomes away from the hyphal tip are not observed. The hookA construct was transformed into a wild-type strain carrying the mCherry-RabA marker. An inverted fluorescence microscope (IX70; Olympus) was used for capturing images. 30 frames were taken with a 0.1-s exposure time and a 0.3-s interval between frames. Binning is 1 × 1. The video speed has been increased fivefold.
Video 3. Early endosomes in the C-HookA strain. Early endosomes labeled by mCherry-RabA are largely accumulated at the hyphal tip. Directed movements of early endosomes away from the hyphal tip are not observed. The construct containing the C-HookA mutation was transformed into a wild-type strain carrying the mCherry-RabA marker. An inverted fluorescence microscope (IX70; Olympus) was used for capturing images. 30 frames were taken with a 0.1-s exposure time and a 0.3-s interval between frames. Binning is 1 × 1. The video speed has been increased fivefold.
Video 4. HookA-GFP signals in a wild-type strain. Bidirectional movements of the signals can be observed, and many of these signals are associated with early endosomes (Fig. S2). The DNA encoding the HookA-GFP fusion protein was transformed into a wild-type strain carrying the mCherry-RabA marker although only GFP signals are shown in this video. An inverted fluorescence microscope (IX70; Olympus) was used for capturing images. 12 frames were taken with a 0.1-s exposure time and a 1-s interval between frames. Binning is 2 × 2. The video speed has been increased fivefold.
Video 5. C-HookA-GFP signals. The signals are largely diffuse, but some puncta can be observed to move along microtubule-like structures. The DNA encoding the C-HookA-GFP fusion protein was transformed into a wild-type strain carrying the mCherry-RabA marker although only GFP signals are shown in this video. An inverted fluorescence microscope (IX70; Olympus) was used for capturing images. 12 frames were taken with a 0.1-s exposure time and a 1-s interval between frames. Binning is 2×2. The video speed has been increased fivefold.
Video 6. Early endosomes in the N-HookA strain. Early endosomes labeled by mCherry-RabA are largely accumulated at the hyphal tip, but a few can be seen to move away from the hyphal tip. The construct containing the N-HookA mutation was transformed into a wild-type strain carrying the mCherry-RabA marker. An inverted fluorescence microscope (IX70; Olympus) was used for capturing images. 30 frames were taken with a 0.1-s exposure time and a 0.3-s interval between frames. Binning is 1 × 1. The video speed has been increased fivefold.