Arp11 affects dynein-dynactin interaction and is essential for dynein function in Aspergillus nidulans

Abstract

The dynactin complex contains proteins including p150 that interacts with cytoplasmic dynein and an actin-related protein Arp1 that forms a minifilament. Proteins including Arp11 and p62 locate at the pointed end of the Arp1 filament, but their biochemical functions are unclear (Schroer TA. Dynactin. Annu Rev Cell Dev Biol 2004;20:759-779). In Aspergillus nidulans, loss of Arp11 or p62 causes the same nuclear distribution (nud) defect displayed by dynein mutants, indicating that these pointed-end proteins are essential for dynein function. We constructed a strain with S-tagged p150 of dynactin that allows us to pull down components of the dynactin and dynein complexes. Surprisingly, while the ratio of pulled-down Arp1 to S-p150 in Arp11-depleted cells is clearly lower than that in wild-type cells, the ratio of pulled-down dynein to S-p150 is significantly higher. We further show that the enhanced dynein-dynactin interaction in Arp11-depleted cells is also present in the soluble fraction and therefore is not dependent upon the affinity of these proteins to the membrane. We suggest that loss of the pointed-end proteins alters the Arp1 filament in a way that affects the conformation of p150 required for its proper interaction with the dynein motor.

Figure 1. Phenotype of the ΔArp11 mutant

A) Deletion of the Arp11 gene in Aspergillus nidulans causes the same colony growth defect as that exhibited by the null mutant of nudA that encodes dynein HC (65). Cells were grown on YUU medium at 32°C for 3 days. B) DAPI staining showing that the ΔArp11 mutant exhibits a typical nud phenotype after an overnight incubation at 32°C. Bar, approximately 5 μm.

Figure 2. The alcA-based Arp11, p62, p50 and Arp1 mutants all show a typical nud phenotype when the alcA promoter is shut off by glucose

A) DAPI staining of cells grown on YUU medium at 32°C overnight. Bar, approximately 5 μm. B) Percentage of cells showing the nud phenotype (defined by the presence of a cluster of four or more nuclei). More than 200 cells were counted for each mutant.

Figure 3. Localizations of dynein and dynactin components in various dynactin mutants

A) Downregulation (or depletion) of Arp11, p62, p50 and Arp1 dramatically decreases the microtubule plus-end accumulation of GFP-NUDA (or GFP–HC), which is represented by the comet-like structures at the hyphal tip in wild-type cells (see Movies S2–S6 for images of multiple cells in the same field). In Arp11- or p62-depleted cells (alcA-Arp11 or alcA-p62), GFP–HC accumulates at dots along filament-like structures (Movies S3 and S4). The GFP-nudA (HC) fusion gene is driven by nudA’s endogenous promoter. B) Besides the dot along filament-like structures, GFP–HC also accumulates at or close to septa in alcA-Arp11 mutant cells on glucose (arrow points to a septum). Cells were grown on minimal glucose medium overnight at room temperature and shifted to 32°C for about 5 h. C) The alcA-driven GFP–HC, GFP–p150 and GFP–p50 all accumulate at or close to septa in the ΔArp11 mutant grown on glycerol overnight at 32°C. But the alcA-driven GFP–p62 is not seen to accumulate at any specific place in the ΔArp11 mutant. Bar, approximately 5 μm.

Figure 4. Arp1 depletion but not Arp11 or p62 depletion significantly decreases the protein level of p150

Total protein extracts from cells grown on glucose overnight at 32°C were loaded on the gel and probed with an affinity-purified anti-p150 antibody. PONCEAU S staining of the blot shows similar loading of proteins in each lane.

Figure 5. The S-tagged p150 is functional and is associated with other dynactin components such as Arp11 and Arp1

A) The S-tagged p150 is functional. The strain with S-p150 forms a wild-type-like colony after a 3-day incubation at 32°C (top). It has a normal nuclear distribution pattern (middle) and exhibits a normal dynein localization pattern (the bottom panel shows images of dynein HC localization) after an overnight incubation at 32°C. Bar, approximately 5 μm. B) S-p150 pulls down Arp11, Arp1 and a low level of dynein HC. Because GFP–Arp11 is under the control of the alcA promoter, glycerol medium was used to allow expression of GFP–Arp11 and glucose medium was used to shut off its expression. We adjusted loading to make the levels of S-p150 appear similar in the two lanes. Cells were grown at 32°C overnight.

Figure 6. Biochemical analyses of the alcA-Arp11 or alcA-p62 mutant grown on glucose

A) Western blots showing a typical purification result. In this experiment, similar amounts of total proteins from different strains were used for affinity purification. Note that the yield of purified S-tagged p150 is significantly decreased in the alcA-Arp11 mutant. B and C) Western blots showing typical purification results. Because depletion of Arp11 or p62 decreases the yield of purified S-tagged p150, we adjusted loading so that the intensities of purified S-tagged p150 in all lanes appear similar. It was obvious that S-tagged p150 pulled down more dynein HC or IC when either Arp11 or p62 was depleted. This was not because of increased levels of HC and IC in the total protein extracts isolated from Arp11- or p62-depleted cells (B and D). In contrast, the amount of Arp1 pulled down was obviously decreased in the mutants (C). E) A quantitative analysis on the effects of Arp11 depletion. Values were all relative to the wild-type values, which were all set at 1. The mean and standard error values were calculated from three independent experiments. Note that the intensity ratio of HC to S-tagged p150 is significantly increased (p < 0.05), while that of Arp1 to S-tagged p150 is significantly decreased (p < 0.001) in the mutant. Cells were all grown on YUU medium overnight at 32°C.

Figure 7. The enhanced dynein–dynactin interaction and the decreased Arp1 association with p150 in Arp11-depleted cells could be detected in the soluble fraction

A) Western blots showing typical purification results. Cell extracts in the absence (left) and presence (right) of 0.4% Triton-X-100 as detergent were used as starting materials and supernatants from a 100 000× g spin were collected for the S-tag-based purification. B) A quantitative analysis on the effects of Arp11 depletion. Values were all relative to the wild-type values, which were all set at 1. The mean and standard error values were calculated from four independent experiments. Note that in the absence of detergent, the intensity ratio of HC to S-tagged p150 is significantly increased (p < 0.01), while that of Arp1 to S-tagged p150 is significantly decreased (p < 0.001) in the mutant. Similarly, a significantly increased intensity ratio of HC to S-tagged p150 (p < 0.005) and a significantly decreased intensity ratio of Arp1 to S-tagged p150 (p<0.001) in the mutant were also observed in the presence of the detergent (bottom). Cells were grown on YUU medium overnight at 32°C.

Figure 8. A diagram illustrating a model that a loss of pointed-end proteins such as Arp11 and p62 enhances the interaction between dynein and dynactin but weakens the interaction between p150 and the Arp1 filament

Drawing of the wild-type dynactin complex is mainly based on the dynactin complex depicted in Schroer (1). Our current data support the idea that the extreme C-terminus of p150 is exposed. For simplicity, dynactin subunits such as p24, p25, p27 and capping proteins are not shown (note that these proteins are not included in this study), and only dynein HCs and ICs are shown for the dynein complex. We speculate that in the absence of the pointed-end complex, some Arp1 subunits may get lost from the pointed end and/or the interaction between p150 and the Arp1 filament is weakened. These possibilities are combined in the diagram (note that a shortening of the Arp1 filament may weaken the interaction between Arp1 and p50/p150). Dynein–dynactin interaction is mediated by a direct binding between p150 and dynein IC (1,7,8). This study suggests that the conformation of p150 may be changed upon a loss of the pointed-end complex, which may lead to an increase in the affinity between the two complexes. However, because the exact conformational change of p150 is not revealed by this study, it is simply represented by a gray-to-black color change in the diagram. Similarly, a possible change in the p50 dynamitin multimer associated with p150 is represented by a black-to-gray color change in the diagram.