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. 2007 Mar;6(3):555-62.
doi: 10.1128/EC.00266-06. Epub 2007 Jan 19.

Aspergillus nidulans Dis1/XMAP215 protein AlpA localizes to spindle pole bodies and microtubule plus ends and contributes to growth directionality

Cathrin Enke  1 , Nadine Zekert, Daniel Veith, Carolin Schaaf, Sven Konzack, Reinhard Fischer
Affiliations

Aspergillus nidulans Dis1/XMAP215 protein AlpA localizes to spindle pole bodies and microtubule plus ends and contributes to growth directionality

Cathrin Enke et al. Eukaryot Cell. 2007 Mar.

Abstract

The dynamics of cytoplasmic microtubules (MTs) is largely controlled by a protein complex at the MT plus end. In Schizosaccharomyces pombe and in filamentous fungi, MT plus end-associated proteins also determine growth directionality. We have characterized the Dis1/XMAP215 family protein AlpA from Aspergillus nidulans and show that it determines MT dynamics as well as hyphal morphology. Green fluorescent protein-tagged AlpA localized to MT-organizing centers (centrosomes) and to MT plus ends. The latter accumulation occurred independently of conventional kinesin or the Kip2-familiy kinesin KipA. alpA deletion strains were viable and only slightly temperature sensitive. Mitosis, nuclear migration, and nuclear positioning were not affected, but hyphae grew in curves rather than straight, which appeared to be an effect of reduced MT growth and dynamics.

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Figures

FIG. 1.
FIG. 1.
AlpA belongs to the third group of the Dis1/XMAP215 family. (A) Two TOG domains, eight HEAT repeats, and a coiled-coil region were identified, which are common to all class three members, including S. pombe Alp14 and Dis1, which is the Dis1/XMAP215 family-founding protein, and S. cerevisiae Stu2. Members of the first group include human ch-TOG, Xenopus XMAP215, Drosophila melanogaster Msps, D. discoideum DdCP224, and Arabidopsis thaliana MOR1. So far, there is only one known group two member, namely ZYG-9 of Caenorhabditis elegans. (B) Phylogenetic analysis of S. pombe Alp14 (Sp) homologues with S. cerevisiae (Sc), A. nidulans (An), A. fumigatus (Af), and A. oryzae (Ao). Accession numbers are indicated.
FIG. 2.
FIG. 2.
AlpA localization during mitosis and in interphase. (A) During mitosis, GFP-AlpA was distributed along short spindles (arrowhead in the first frame). As the spindle elongated GFP-AlpA was redistributed to the spindle poles (arrowheads in the last frame). Frames are shown in 2-min intervals. The strain was SCE05. (B) GFP-AlpA movement can be seen as comet-like structures, indicating the association with the MT plus ends (see Movie S01 in the supplemental material) (C, D) MTs were visualized by decoration with a red-labeled kinesin rigor mutant protein (mRFP1-KipBrigor) during interphase (C) and mitosis (D) (strain SDV96). The arrows point to a GFP-AlpA signal at MT plus ends (C) and to the spindle pole bodies (D). The arrow head in panel D points to the center of the spindle, where the protein could be associated with the kinetochores. Bars, 3 μm (A) and 2 μm (B to D).
FIG. 3.
FIG. 3.
Phenotype of an alpA deletion strain. (A) In comparison to a control strain (top, RMS011), the ΔalpA strain (bottom, SDV83b) grew slower and colonies were more compact. While wild-type hyphae grew straight (B), hyphae of the alpA deletion strain showed a curved growth phenotype (C). (D) In a strain having the only functional copy of alpA under the control of the inducible alcA promoter (SCE05), curved growth was observed under repressing conditions (glucose), but wild-type hyphal morphology was restored when grown under inducing conditions (ethanol) (E).
FIG. 4.
FIG. 4.
Localization of the Spitzenkörper in hyphae and germination pattern. The Spitzenkörper was observed in growing hyphae as described previously (19). (A) Colonies of the wild-type (RMS011) (WT), ΔalpA (SDV83), ΔkipA (SSK44), and ΔalpA ΔkipA (SAD1c) strains on an agar plate after 3 days of growth at 37°C. (B) Representative hyphae with a Spitzenkörper in the center of the cell or noncentral. To indicate the position of the organelle, we introduced a cross into the hypha. (C) Quantification of the location of the Spitzenkörper in the strains listed for panel A. Dark blue columns represent hyphae with the Spitzenkörper in the center and gray columns the ones where the Spitzenkörper was noncentral. Between 50 and 64 hyphae were analyzed for each strain. (D) Quantification of the germination pattern of conidiospores as displayed in the pictures. Wild type (RMS011), n = 200; alpA mutant (SDV83), n = 268.
FIG. 5.
FIG. 5.
alpA affects MTs and MT plus end-localized proteins. (A) Several MTs and bundles thereof stained with GFP are obvious in the wild type (SJW02). (B) The number and dynamics (see Movies S02 and S03 in the supplemental material) of MTs in an alpA deletion strain (SDV86) were reduced compared to the wild type. Arrowheads point to nuclei and arrows to spindle pole bodies. (C) While GFP-KipA localized to MT plus ends and moved as comets in the wild type (SSK92) (see Movie S05 in the supplemental material), (D) GFP-KipA decorated short fragments of MTs in the ΔalpA background (SDV87) (see Movie S06 in the supplemental material). Fragments of MTs were also GFP decorated in ΔalpA strains with GFP-NudA (E) and GFP-NudF (F) fusion proteins (strains SDV100 and SDV101). Arrows in panels D to F point to MT plus ends as determined by the growth at this end. Bars in panel B, 5 μm (A, C, and D), 6 μm (B), and 4 μm (E and F).
FIG. 6.
FIG. 6.
Benomyl sensitivity of the wild-type (upper row of colonies) and alpA deletion (lower row) strains. Benomyl was added in concentrations from 0 to 0.8 μg/ml, and colonies were grown for 2 days at 37°C.
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