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. 2018 Jul 30;8(1):11419.
doi: 10.1038/s41598-018-29818-4.

The actin capping protein in Aspergillus nidulans enhances dynein function without significantly affecting Arp1 filament assembly

Jun Zhang  1 , Rongde Qiu  1 , Xin Xiang  2
Affiliations

The actin capping protein in Aspergillus nidulans enhances dynein function without significantly affecting Arp1 filament assembly

Jun Zhang et al. Sci Rep. .

Abstract

The minus-end-directed microtubule motor cytoplasmic dynein requires the dynactin complex for in vivo functions. The backbone of the vertebrate dynactin complex is the Arp1 (actin-related protein 1) mini-filament whose barbed end binds to the heterodimeric actin capping protein. However, it is unclear whether the capping protein is a dynactin component in lower eukaryotic organisms, especially because it does not appear to be a component of the budding yeast dynactin complex. Here our biochemical data show that the capping protein is a component of the dynactin complex in the filamentous fungus Aspergillus nidulans. Moreover, deletion of the gene encoding capping protein alpha (capA) results in a defect in both nuclear distribution and early-endosome transport, two dynein-mediated processes. However, the defect in either process is less severe than that exhibited by a dynein heavy chain mutant or the ∆p25 mutant of dynactin. In addition, loss of capping protein does not significantly affect the assembly of the dynactin Arp1 filament or the formation of the dynein-dynactin-∆C-HookA (Hook in A. nidulans) complex. These results suggest that fungal capping protein is not important for Arp1 filament assembly but its presence is required for enhancing dynein function in vivo.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Components of the dynactin complex are pulled down with CapA-GFP. (A) A schematic representation of the dynactin complex. Conventional actin was not depicted as we do not have evidence from our pull-down experiments that conventional actin is a component of the A. nidulans dynactin complex. (B) Western blots showing that dynactin p150, Arp1 and the dynein HC were pulled down with CapA-GFP. A strain without any GFP tag was used as a negative control. Cropped pieces with black outlines indicate blots probed by different antibodies against the indicated proteins (see Supplemental Fig. 5 for the original blots). The antibody against GFP (from Clontech) has been used previously. The affinity-purified antibodies against dynein HC, dynactin p150 and Arp1 have been described and used previously,.
Figure 2
Figure 2
Colony and nuclear-distribution phenotypes of the ∆capA mutant. (A) Colony phenotypes of the ∆capA mutant and control strains including wild type, the nudA1 mutant and ∆p25 mutant. The plate was incubated at 37 °C for 2 days. (B) Images showing the nuclear distribution phenotype of the ∆capA mutant in comparison with that of wild type or the nudA1 mutant. Cells were grown at 37 °C for ~8 hours in MM + glucose medium. Bar, 5 μm. (C) A quantitative analysis on the percentage of germ tubes containing 0, 1, 2, 3 or ≥4 nuclei in the spore head. In the wild-type control strain, 60.3% of the germ tubes contain one nucleus, 32.4% contain no nucleus and 7.3% contain two nuclei in the spore head (n = 68). In the ∆capA mutant, 10.7% contain one nucleus, 38.1% contain two nuclei, 31% contain three nuclei, 17.8% contain four nuclei and 2.4% contain five nuclei in the spore head (n = 84). The mean ranks of these two sets of data are significantly different at p = 0.05 (the actual p-value is smaller than 0.000000000000001, two-tailed) based on a nonparametric test that assumes no information about the distribution (unpaired, Mann-Whitney test, Prism 7 for Mac OS X, version 7.0c, 2017). In the nudA1 mutant, 100% of the germ tubes (n = 50) show a cluster of 4–8 nuclei in the spore head, and we express the number as “4 or >4” because the exact number of nuclei in the cluster is hard be determined accurately.
Figure 3
Figure 3
The nuclear-distribution phenotype of the ∆capA mutant in the bimC4 background. (A) Images showing the position of the single nucleus in the bimC4 mutant, the nudA1, bimC4 double mutant and the ∆capA, bimC4 double mutant. Cells were grown at 42 °C or ~8 hours in MM + glucose medium. Bar, 5 μm. (B) A quantitative analysis on the percentage of germ tubes with the single nucleus in the spore head. The percentage values are 16.9% for the bimC4 single mutant (n = 201), 50.2% for the ∆capA, bimC4 double mutant (n = 263) and 100% for the nudA1, bimC4 double mutant (n = 60). Error bars represent the 95% confidence interval values generated by Prism 7.
Figure 4
Figure 4
Early endosome-distribution phenotype of the ∆capA mutant. (A) Images showing large early endosomes labeled by overexpressed GFP-RabA in wild type and the ∆capA mutant. Cells were grown at 37 °C for ~8 hours in MM + glucose medium. The ∆p25 mutant defective in the dynein-early endosome interaction was used as a control. Bar, 5 μm. (B) A quantitative analysis on the percentage of germ tubes with a large early endosome located at the hyphal tip. The percentage values are 5.1% for wild type (n = 234), 59.4% for the ∆capA mutant (n = 271), and 96% for the ∆p25 mutant (n = 100). Error bars represent the 95% confidence interval values generated by Prism 7.
Figure 5
Figure 5
Western analyses of the dynactin complex in the ∆capA mutant. (A) Western blots showing that normal amounts of dynein HC and Arp1 are pulled down with p150-GFP in the ∆capA mutant extract. Cells were cultured for overnight at 37 °C in MM + glucose medium. A strain without any GFP tag was used as a negative control. Another control is the strain containing GFP-dynein HC in the alcA-Arp1 background, in which the expression of Arp1 was repressed by glucose and the stability of p150 is decreased. Cropped pieces with black outlines indicate blots probed by different antibodies against the indicated proteins (see Supplemental Fig. 10 for the original blots). (B) Western blots showing that dynein HC, dynactin p150 and Arp1 are pulled down with ∆C-HookA-GFP in the ∆capA mutant extract. The antibody against GFP (from Clontech) has been used previously. The affinity-purified antibodies against dynein HC, dynactin p150 and Arp1 have been described and used previously,. Cropped pieces with black outlines indicate blots probed by different antibodies against the indicated proteins (see Supplemental Fig. 11 for the original blots). (C) A quantitative analysis on the ratio of pulled-down p150, Arp1 or dynein HC to ∆C-HookA-GFP as well as the ratio of pulled-down dynein HC or Arp1 to p150. The values were generated from western analyses (shown in B) of three independent pull-down experiments. The wild-type values are set as 1. Scatter plots with mean values were generated by using Prism 7. For all the ratios, there is no significant difference between wild-type and ∆capA at the 95% confidence level based on nonparametric tests without assuming any information on the distribution (p = 0.1 for Arp1/∆C-HookA, p = 0.7 for p150/∆C-HookA, p = 0.7 for dynein HC/∆C-HookA, p = 0.1 for dynein HC/p150 and p = 0.7 for Arp1/p150, two-tailed) (unpaired, Mann-Whitney test, Prism 7).
Figure 6
Figure 6
A quantitative analysis of dynein-mediated early endosome movement upon loss of CapA. (A) Images showing TagGFP2-RabA-labeled early endosomes in wild type, the ∆capA mutant and the ∆p25 mutant. Cells were cultured for overnight at 37 °C in MM + glycerol medium. Bar, 5 μm. (B) Kymographs showing early endosome movements. Two kymographs are shown for wild type (WT-1 and WT-2) and three are shown for the ∆capA mutant (∆capA-1, ∆capA-2, and ∆capA-3). A ∆p25 kymograph is shown as a control as there is no diagonal lines that indicate movements. For each kymograph, position of the hyphal tip is on the right side and indicated by a short arrow (the word “hyphal tip” is on the last kymograph for each strain). (C) Percent of hyphal-tip cells (called “tip cells” for simplicity) showing different numbers of dynein-mediated early endosome movement events within 16 seconds (n = 34 for wild type and n = 41 for the ∆capA mutant). The mean ranks of the wild type and ∆capA numbers of events are significantly different at p = 0.05 (p = 0.0022, two-tailed). (D) Percent of hyphal-tip cells showing different numbers of kinesin-3-mediated early endosome movements within 16 seconds (n = 34 for wild type and n = 41 for the ∆capA mutant). The mean ranks of the wild type and ∆capA numbers of events are not significantly different at p = 0.05 (p = 0.962, two-tailed). (E) Velocity of dynein-mediated early endosome movement in wild type and the ∆capA mutant (n = 34 for wild type and n = 32 for the ∆capA mutant). The mean ranks of the two sets of values are significantly different at p = 0.05 (p = 0.000000008666721, two-tailed). (F) Velocity of kinesin-3-mediated early endosome movement in wild type and the ∆capA mutant (n = 25 for wild type and n = 33 for the ∆capA mutant). The mean ranks of the two sets of values are significantly different at p = 0.05 (p = 0.013, two-tailed). Scatter plots with mean and SD values were generated by Prism 7. All the statistical analyses were done using nonparametric tests assuming no information about the distribution (unpaired, Mann-Whitney test, Prism 7).
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