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. 2020 Jun 1;31(12):1289-1301.
doi: 10.1091/mbc.E20-03-0166. Epub 2020 Apr 8.

The splicing-factor Prp40 affects dynein-dynactin function in Aspergillus nidulans

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

The splicing-factor Prp40 affects dynein-dynactin function in Aspergillus nidulans

Rongde Qiu et al. Mol Biol Cell. .

Abstract

The multi-component cytoplasmic dynein transports cellular cargoes with the help of another multi-component complex dynactin, but we do not know enough about factors that may affect the assembly and functions of these proteins. From a genetic screen for mutations affecting early-endosome distribution in Aspergillus nidulans, we identified the prp40AL438* mutation in Prp40A, a homologue of Prp40, an essential RNA-splicing factor in the budding yeast. Prp40A is not essential for splicing, although it associates with the nuclear splicing machinery. The prp40AL438* mutant is much healthier than the ∆prp40A mutant, but both mutants exhibit similar defects in dynein-mediated early-endosome transport and nuclear distribution. In the prp40AL438* mutant, the frequency but not the speed of dynein-mediated early-endosome transport is decreased, which correlates with a decrease in the microtubule plus-end accumulations of dynein and dynactin. Within the dynactin complex, the actin-related protein Arp1 forms a mini-filament. In a pull-down assay, the amount of Arp1 pulled down with its pointed-end protein Arp11 is lowered in the prp40AL438* mutant. In addition, we found from published interactome data that a mammalian Prp40 homologue PRPF40A interacts with Arp1. Thus, Prp40 homologues may regulate the assembly or function of dynein-dynactin and their mechanisms deserve to be further studied.

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Figures

FIGURE 1:
FIGURE 1:
Phenotype of the eedD5 mutant. (A) Colony phenotypes of the eedD5 mutant and a wild-type strain. (B) Microscopic images showing the distributions of mCherry–RabA-labeled early endosomes (mCherry-RabA) in wild type and the eedD5 mutant. Bar, 5 μm. Although bidirectional movements of mCherry–RabA-labeled early endosomes are not completely abolished, most of the eedD5 hyphal tips (∼80%) show an abnormal accumulation of mCherry-RabA signals (n = 50). Hyphal tip is indicated by a yellow arrow. (C) Images of nuclei labeled with GFP-tagged Histone H1 in wild type and the eedD5 mutant. Spore head is indicated by a yellow arrowhead.
FIGURE 2:
FIGURE 2:
Phenotypic analysis of the strain containing the Prp40A(1-437)-GFP fusion gene. (A) Domain structures of Prp40 proteins in different organisms including PRPF40A (human), PRPF40B (human), Prp40 (budding yeast), and Prp40A (A. nidulans). Position of the eedD5 mutation, prp40AL438*, is indicated by a black arrow. (B) Colony phenotypes of the strains containing different Prp40A alleles. (C) Localization of Prp40A-GFP or Prp40A(1-437)-GFP and mCherry–RabA-labeled early endosomes in strains containing one of the GFP fusions. Hyphal tip is indicated by a yellow arrow. Bar, 5 μm. (D) A quantitative analysis on the ratio of GFP signal intensity in the cytoplasm to that in the nucleus (Prp40A-GFP: n = 30; Prp40A(1-437)-GFP: n = 30). Scatter plots with mean and SD values were generated by Prism 8. The Mann–Whitney test (unpaired, two-tailed) was used for analyzing the two data sets without assuming normal distribution of the data. ****p < 0.0001. (E) A Western blot detecting the Prp40A(1-437)-GFP fusion, suggesting that the fusion protein is stable. The polyclonal anti-GFP antibody (Clontech) was used to probe the blot.
FIGURE 3:
FIGURE 3:
Phenotype of the ∆prp40A mutant. (A) Colony phenotypes of a wild-type strain, the prp40AL438* mutant and the ∆prp40A mutant. (B) Microscopic images showing the distributions of mCherry–RabA-labeled early endosomes (mCherry-RabA) in wild type and the ∆prp40A mutant. Hyphal tip is indicated by a yellow arrow. Bar, 5 μm. (C) Images of nuclei labeled with GFP-tagged Histone H1 in wild type and the ∆prp40A mutant. Spore head is indicated by a yellow arrowhead. (D) A quantitative analysis on the percentage of germ tubes containing 0, 1, 2, or 3 nuclei in the spore head (wild type: n = 59; prp40AL438*: n = 43; ∆prp40A: n = 57). The number of nuclei in the spore head of the prp40AL438* mutant or the ∆prp40A mutant is higher than that in wild type (p < 0.0001 in both cases, Kruskal–Wallis ANOVA test with Dunn’s multiple comparisons test, unpaired). However, the number of nuclei in the spore head of the prp40AL438* mutant is not significantly different from that in the ∆prp40A mutant (p > 0.05, Kruskal–Wallis ANOVA test with Dunn’s multiple comparisons test, unpaired).
FIGURE 4:
FIGURE 4:
Decreased amounts of Arp1 pulled down with Arp11-GFP and decreased microtubule plus-end accumulation of dynein–dynactin in the prp40AL438* mutant. (A) Western blots showing that the amount of Arp1 pulled down with Arp11-GFP is lower in the prp40AL438* mutant than in the wild-type control. (B) A quantitative analysis on the ratio of pulled-down Arp1 to Arp11-GFP (Arp1/Arp11-GFP). The values were generated from Western analyses of four independent pull-down experiments (n = 4 for all). The wild-type values are set as 1. Scatter plots with mean and SD values were generated by Prism 8. ***p < 0.001 (Student’s t test, two-tailed, unpaired, normal distribution was assumed). (C) Images of GFP-dynein in wild type and the prp40AL438* mutant. Hyphal tip is indicated by a light brown arrow. Bar, 5 μm. (D) Images of dynactin p150-GFP in wild type and the prp40AL438* mutant. Hyphal tip is indicated by a light brown arrow. (E) A quantitative analysis on GFP-dynein comet intensity in wild type (n = 42) and the prp40AL438* mutant (n = 35). (F) A quantitative analysis on p150-GFP comet intensity in wild type (n = 36) and the prp40AL438* mutant (n = 41). For both E and F, all values are relative to the average value for wild type, which is set as 1. Scatter plots with mean and SD values were generated by Prism 8. The Mann–Whitney test (unpaired, two-tailed) was used for analyzing the two data sets without assuming normal distribution of the data. ****p < 0.0001.
FIGURE 5:
FIGURE 5:
A quantitative analysis on dynein-mediated early-endosome transport in the prp40AL438* mutant showing a decrease in the frequency but not the speed of the transport. (A) Kymographs showing the movements of early endosomes in wild type and the prp40AL438* mutant. Green arrows indicate dynein-mediated movements away from the hyphal tip. (B) A quantitative analysis on the frequency of dynein-mediated transport in wild type (n = 37 hyphal tips) and the prp40AL438* mutant (n = 39 hyphal tips). Scatter plots with mean and SD values were generated by Prism 8. ****p < 0.0001 (unpaired, Mann–Whitney test, Prism 8). (C) A quantitative analysis on the speed of dynein-mediated early-endosome movement in wild type (n = 88 movements) and the prp40AL438* mutant (n = 44 movements). The difference between wild type and the mutant is insignificant at p = 0.05 (unpaired, Mann–Whitney test, Prism 8).
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