The p25 subunit of the dynactin complex is required for dynein-early endosome interaction

Abstract

Cytoplasmic dynein transports various cellular cargoes including early endosomes, but how dynein is linked to early endosomes is unclear. We find that the Aspergillus nidulans orthologue of the p25 subunit of dynactin is critical for dynein-mediated early endosome movement but not for dynein-mediated nuclear distribution. In the absence of NUDF/LIS1, p25 deletion abolished the localization of dynein-dynactin to the hyphal tip where early endosomes abnormally accumulate but did not prevent dynein-dynactin localization to microtubule plus ends. Within the dynactin complex, p25 locates at the pointed end of the Arp1 filament with Arp11 and p62, and our data suggest that Arp11 but not p62 is important for p25-dynactin association. Loss of either Arp1 or p25 significantly weakened the physical interaction between dynein and early endosomes, although loss of p25 did not apparently affect the integrity of the Arp1 filament. These results indicate that p25, in conjunction with the rest of the dynactin complex, is important for dynein-early endosome interaction.

Figure 1.

The Δp25 mutant in A. nidulans does not exhibit a nud phenotype. (A) Protein sequence alignment of p25 proteins from A. nidulans (A.n.p25), N. crassa (N.c.p25; Lee et al., 2001), and mouse (Eckley et al., 1999). Identical amino acids are boxed in black. Blue lines indicate Hexapeptide repeats in A. nidulans p25 identified using the Simple Modular Architecture Research Tool (SMART) program. (B) The Δp25 mutant grows slightly more slowly than the wild type on plates but is much healthier than a typical nud mutant such as ΔnudA (dynein heavy chain or HC) or ΔArp11. (C) Unlike the ΔnudA and ΔArp11 mutants, the Δp25 mutant exhibits normal nuclear distribution. The strains were grown in liquid Y + UU medium for 7.5 h at 37°C before being fixed and stained with DAPI for visualizing the nuclei. Bars, 5 µm. (D) A quantitative analysis of nuclear distribution in the Δ25 mutant in comparison to that in a wild-type control strain. More than 200 germ tubes were analyzed for each strain. Means and standard deviations (error bars) were calculated from three experiments. No significant differences are revealed at the P-value of 0.05.

Figure 2.

Dynein-mediated retrograde movement of early endosomes is significantly impaired in the Δp25 mutant, although dynein is still able to concentrate at microtubule plus ends. (A) mCherry-RabA–labeled early endosomes move and distribute along the wild-type hyphae, whereas they accumulate as a cloud at the hyphal tip in the Δp25 mutant and in the ΔnudA mutant. (B) A quantitative analysis of the frequency of movements toward or away from the hyphal tip. Because microtubules have mixed polarity in the multi-nucleate hyphae, we only focused on the small region within 10 µm behind the hyphal tip, where most of the plus ends of microtubules should face the hyphal tip. This experiment was done once with data combined from a total of 30 time-lapse sequences from the wild-type cells and a total of 30 time-lapse sequences from the Δp25 cells. For each time-lapse sequence, 30 frames were taken with a 0.1-s exposure time and a 0.3-s interval between frames. Frequency of movements in a total of 360 s is shown. (C) In both the wild type and the Δp25 mutant, GFP-labeled dynein heavy chains (HC) form comet-like structures representing their microtubule plus-end accumulation. Bars, 5 µm. (D) Mean and standard deviation values (error bars) of maximal signal intensity (arbitrary units) of the plus-end GFP-HC comets in Δp25 cells and wild-type cells are shown (n = 14, P < 0.01).

Figure 3.

In NUDF-depleted cells, p25 is required for the localization of dynein (GFP-HC) and dynactin (p150-GFP) to the hyphal tip region, where early endosomes are enriched. (A) Images showing that in the same ΔnudF cells, both GFP-HC and mCherry-RabA localize to the hyphal tip region. (B–D) In NUDF-depleted cells (alcA-nudF grown on glucose), p25 is required for the localization of dynein and dynactin to the hyphal tip region where early endosomes are found. Images of GFP-HC (B), p150-GFP (C), and GFP-RabA (D) are shown. In the absence of NUDF (alcA-nudF), a portion of GFP-HC and p150-GFP proteins localize to the hyphal-tip region, where early endosomes also accumulate (B–D, middle). Introducing the Δp25 mutant allele into this background (alcA-nudF/Δp25) abolishes this hyphal tip accumulation but maintains the plus-end comets of GFP-HC and p150-GFP (B and C, right). Bars, 5 µm.

Figure 4.

p25 and its associated dynactin complex are important for the physical interaction between dynein and early endosomes. (A) Although S-tagged dynein IC was able to pull down GFP-RabA in wild-type extracts in the absence of detergent, the amount of pulled-down GFP-RabA in Arp1-depleted extracts was significantly decreased. (B) A quantitative analysis of the Western results. All the values were relative to wild-type values, which were set as 1. Mean and standard deviation values were based on results from three independent pull-down experiments (P < 0.001). (C) The protein levels of GFP-RabA in the wild type and alcA-Arp1 extracts were similar before the pull-down assay. (D) Although S-tagged dynein IC was able to pull down GFP-RabA in wild-type extracts in the absence of detergent, the amount of pulled-down GFP-RabA in Δp25 extracts was significantly decreased. (E) A quantitative analysis of the Western results. All the values were relative to wild-type values, which were set as 1. Mean and standard deviation values (error bars) were based on results from three independent pull-down experiments (P < 0.001). (F) The protein levels of GFP-RabA in the wild-type and Δp25 extracts were similar before the pull-down assay.

Figure 5.

Although loss of p25 has no apparent effect on the Arp1 filament, the A. nidulans p25 is indeed a component of the dynactin complex. (A) Western blots showing Arp1 proteins pulled down by the S-tagged p150 in wild-type and Δp25 extracts. (B) A quantitative analysis of the Western results on p150–Arp1 interaction in the Δp25 mutant. All the values were relative to wild-type values, which were set as 1. Mean and standard deviation values (error bars) were based on results from three independent experiments. At P = 0.05, there is no significant difference between wild-type and Δp25 values. (C) p25-GFP forms benomyl-sensitive comet-like structures, consistent with being a component of the dynactin complex. For treatment with benomyl to depolymerize microtubules, 2.4 µg/ml of benomyl was used to treat the cells for 20 min. Bars, 5 µm. (D) A Western blot showing that the p25-GFP protein level was dramatically decreased in Arp11-depleted cells but not in p62-depleted cells. (E) S-tagged p150 was able to pull down p25-GFP from both the wild type and the p62-depleted extracts.