Identification of the augmin complex in the filamentous fungus Aspergillus nidulans

Abstract

Augmin is a protein complex that binds to spindle microtubules (MTs), recruits the potent MT nucleator, γ-tubulin, and thereby promotes the centrosome-independent MT generation within mitotic and meiotic spindles. Augmin is essential for acentrosomal spindle assembly, which is commonly observed during mitosis in plants and meiosis in female animals. In many animal somatic cells that possess centrosomes, the centrosome- and augmin-dependent mechanisms work cooperatively for efficient spindle assembly and cytokinesis. Yeasts have lost the augmin genes during evolution. It is hypothesized that their robust MT nucleation from the spindle pole body (SPB), the centrosome-equivalent structure in fungi, compensates for the lack of augmin. Intriguingly, however, a gene homologous to an augmin subunit (Aug6/AUGF) has been found in the genome of filamentous fungi, which has the SPB as a robust MT nucleation centre. Here, we aimed to clarify if the augmin complex is present in filamentous fungi and to identify its role in mitosis. By analysing the Aug6-like gene in the filamentous fungus Aspergillus nidulans, we found that it forms a large complex with several other proteins that share weak but significant homology to known augmin subunits. In A. nidulans, augmin was enriched at the SPB and also associated with spindle MTs during mitosis. However, the augmin gene disruptants did not exhibit growth defects under normal, checkpoint-deficient, or MT-destabilised conditions. Moreover, we obtained no evidence that A. nidulans augmin plays a role in γ-tubulin recruitment or in mitotic cell division. Our study uncovered the conservation of the augmin complex in the fungal species, and further suggests that augmin has several functions, besides mitotic spindle MT nucleation, that are yet to be identified.

Figure 1. Aug6 is localised at the SPB and the spindle.

(A) Sequence alignment of Aug6 proteins from A. nidulans (An), the red bread mould Neurospora crassa (Nc), Homo sapiens (Hs; also called hDgt6/FAM29A/HAUS6), Drosophila melanogaster (Dm; also called Dgt6), and the moss Physcomitrella patens (Pp). Identical amino acids are boxed, and similar ones are hatched. (B) Detection of Aug6-GFP by immunoblotting. A unique band with the expected molecular weight (arrow) was detected by immunoblotting with the anti-GFP antibody in 2 independent GFP-integrated strains (#1 and #2). Asterisks indicate cross-reactions of the antibody with other proteins. The #2 strain was used throughout this study. (C) Time-lapse imaging of Aug6-GFP during mitosis. Images were acquired every 15 s in a single focal plane. Strong signals were detected at the pole of the spindle (yellow), whereas weak punctate signals were observed along the spindle MT (blue). (D) Time-lapse imaging of Aug6-GFP and γ-tubulin-mCherry. They were co-localised at the SPB during mitosis. See also Movie S1. Bars, 2 µm.

Figure 2. Aug6 forms a large complex and associates with MTs.

(A) Gel filtration chromatography followed by immunoblotting of Aug6-GFP. Stokes radiuses estimated by the size markers are shown at the bottom. (B) MT co-sedimentation assay using Aug6-GFP cell extracts and taxol-stabilised MTs. Results of anti-GFP immunoblotting (Aug6) and Coomassie staining (tubulin [arrowhead] and the whole A. nidulans proteins) are shown. WCE; whole cell extracts, sup; supernatant after centrifugation, pellet; precipitant after centrifugation.

Figure 3. Identification of other augmin subunits that co-precipitate with Aug6.

(A) List of putative augmin subunit proteins co-precipitated with Aug6-GFP. The numbers of peptides identified in the Aug6-GFP immunoprecipitants are shown. No homology to known augmin subunits was found for AN0286. (B) Co-precipitation of Aug6-GFP, but not γ-tubulin, with Aug3-HA. Asynchronous and metaphase-accumulated cell cultures were used. Aug3-HA was immunoprecipitated by the anti-HA antibody, followed by immunoblotting of Aug6-GFP or γ-tubulin. Asterisk indicates cross-reaction of the antibody with the IgG proteins. Sup; proteins unbound to the anti-HA beads, Beads; immunoprecipitants. Equal amounts of Input and Sup were loaded, whereas the immunoprecipitants were 19- (Aug6-GFP, Aug3-HA) or 150 (γ-tubulin)-fold concentrated. (C) Amino acid sequence alignment of Aug1, Aug2, Aug3, and Aug4. Sequences from the red bread mould Neurospora crassa (Nc), Drosophila melanogaster (Dm), Homo sapiens (Hs), and the moss Physcomitrella patens (Pp) are aligned. (D) Co-localisation of Aug1–4 and AN0286 with Aug6 in metaphase. mCherry-tagged Aug1, Aug2, Aug3, Aug4, or AN0286 (purple) was co-imaged with Aug6-GFP (green) using a spinning-disc confocal microscope. See also Movie S2. Bar, 2 µm.

Figure 4. Normal γ-tubulin localisation and mitotic progression in the absence of augmin.

(A) Scheme showing the generation of the aug6 disruptant by one-step gene replacement. (B) Southern blot hybridisation confirmed the aug6 gene deletion via homologous recombination (3 independently selected strains were analysed). The probe is described in (A). We used the #4 strain in this study. (C, D) γ-tubulin localisation to the SPB was not attenuated in the absence of aug6. γ-tubulin signal intensities were quantified in the spindle of WT and aug6Δ in 2 independent experiments, and the result from one experiment is displayed (n = 9 hyphae, total 77 and 73 spindles, respectively). Error bars indicate SD. When multiple spindles were analysed in a hypha, the mean value was used as the γ-tubulin signal intensity of the hypha. Maximum projection images of 7 z-sections are displayed. (E) Normal spindle formation and mitotic progression in aug6Δ, as monitored by time-lapse imaging of mCherry-tubulin. 25 spindles of 10 WT cells and 22 spindles of 12 aug6Δ cells were analysed. Maximum projection images of 5 z-sections are displayed. See also Movie S3. Bar, 2 µm.

Figure 5. Normal cell proliferation in the absence of augmin.

1×10⁴ conidia were spotted on the medium plate containing 0–0.3 µg/mL benomyl, a MT destabilising drug, and cultured for 2 or 2.5 days at 37°C. Deletion of 1 or 2 augmin subunits did not affect cell proliferation in the WT, mad2Δ, or apsBΔ background.