The cell end marker protein TeaC is involved in growth directionality and septation in Aspergillus nidulans

Abstract

Polarized growth in filamentous fungi depends on the correct spatial organization of the microtubule (MT) and actin cytoskeleton. In Schizosaccharomyces pombe it was shown that the MT cytoskeleton is required for the delivery of so-called cell end marker proteins, e.g., Tea1 and Tea4, to the cell poles. Subsequently, these markers recruit several proteins required for polarized growth, e.g., a formin, which catalyzes actin cable formation. The latest results suggest that this machinery is conserved from fission yeast to Aspergillus nidulans. Here, we have characterized TeaC, a putative homologue of Tea4. Sequence identity between TeaC and Tea4 is only 12.5%, but they both share an SH3 domain in the N-terminal region. Deletion of teaC affected polarized growth and hyphal directionality. Whereas wild-type hyphae grow straight, hyphae of the mutant grow in a zig-zag way, similar to the hyphae of teaA deletion (tea1) strains. Some small, anucleate compartments were observed. Overexpression of teaC repressed septation and caused abnormal swelling of germinating conidia. In agreement with the two roles in polarized growth and in septation, TeaC localized to hyphal tips and to septa. TeaC interacted with the cell end marker protein TeaA at hyphal tips and with the formin SepA at hyphal tips and at septa.

FIG. 1.

Comparison of TeaC with related proteins from other fungi. (A) Scheme of A. nidulans TeaC, S. pombe Tea4, and S. cerevisiae Bud14, indicating the location of the SH3 domain. The alignment of the three SH3 domains is displayed below the scheme. (B) Alignment of TeaC homologues from four Aspergillus species as indicated. The SH3 domain is highlighted with the red line below the sequences. The alignments were done with CLUSTAL W with standard parameters. Identical amino acids are shaded in black, and chemically conserved amino acids are shaded in gray using the program box shade. aa, amino acids.

FIG. 2.

Phenotypic comparison of wild-type with ΔteaC and ΔteaA strains. (A) Colonies of wild-type (TN02A3), ΔteaA (SSK91), and ΔteaC (SYH21) strains. Strains were grown on MM glucose agar plates for 2 days. (B) Hyphae of wild-type, ΔteaA, and ΔteaC strains were grown for 1 day on microscope slides coated with MM with 2% glucose and 0.8% agarose. Images were taken with differential interference contrast. Hyphae are 3 to 4 μm in diameter. (C) Z-stack image of wild-type (TN02A3) and ΔteaC (SYH21) strains grown on MM with 2% glucose for 1 day and stained with calcofluor white. (D) Z-stack image of wild-type (TN02A3) and ΔteaC (SYH21) strains grown on glucose MM and stained with calcofluor white and Hoechst 33343. The arrow points to a short, anucleate hyphal compartment. Hyphae are 3 to 4 μm in diameter. (E) Quantification of the effect of different gene deletions on second germ tube formation. Conidia of each strain were germinated in MM with glucose and analyzed for the emergence of the second germ tube. For each strain, 100 germlings were counted. WT, wild type.

FIG. 3.

Effect of overexpression of teaC. (A) Comparison of colonies of SYH03 [alcA(p)-GFP-teaC] and wild-type strains on glycerol- or threonine-containing medium after 4 days of growth at 37°C. (B) Western blot of hyphae of SYH03 grown on MM with glycerol or threonine as a carbon source. Portions (10 μg) of total protein extract were loaded onto the gel and processed for the Western blot as described in Materials and Methods. (C) Fluorescence microscopic picture of a hypha of SYH03 after induction with threonine for 1 day. (D) Germinated conidia of SYH03 grown on MM with threonine as a carbon source for 48 h observed using differential interference contrast. The hyphal tip had lysed. (E) Quantification of the effect of teaC overexpression on the emergence of the second germ tube. For each strain, 100 germlings were counted. (F) Germinated conidia of SYH03 grown on MM with threonine as carbon source for 48 h observed using differential interference contrast, stained with calcofluor white (CW), or observed in the GFP channel. WT, wild type; DIC, differential interference contrast.

FIG. 4.

Effect of overexpression of teaC on septation. (A) Strains SYH17 [teaC(p)-mRFP1-teaC] (WT) and SYH03 [alcA(p)-GFP-teaC] grown on threonine MM and stained with calcofluor white. The red arrows point to the stained septa. Hyphae are 3 to 4 μm in diameter. (B) Strain SYH26 [alcA(p)-GFP-sepA and alcA(p)-mRFP1-teaC] grown on MM with threonine as carbon source for 48 h and stained with calcofluor white. The ringlike structure is a small branch observed from the top. (C) Quantification of the number of septa in strains SYH17 (considered as the wild type), SYH03, SNT28 [alcA(p)-GFP-sepA], and SYH26 grown in threonine MM. A total of 50 germlings were counted for each strain. WT, wild type; CW, calcofluor white.

FIG. 5.

TeaC localization. (A) Hyphae of strain SYH03 [alcA(p)-GFP-teaC] grown on MM with glycerol as a carbon source. The upper row shows differential interference contrast (DIC) pictures; the lower row shows same hyphae in the GFP channel. GFP-TeaC localized to one point in the hyphal apex (enlarged in the inset), at the branching site, and at the septa. (B) Localization of TeaC (GFP) and the Spitzenkörper (visualized with FM4-64). (C) mRFP1-TeaC expressed under the control of the native promoter (SYH17). Fluorescence signals were observed at the hyphal tip and new branches and at the septa. Hyphae are 3 to 4 μm in diameter.

FIG. 6.

Relationship between TeaC and MTs. (A) Observation of GFP-labeled MTs and mRFP1-labeled TeaC (SYH22). The arrows point to the MT plus ends. The same hyphae was observed at time zero and after 2.5 s, as indicated in the images. (B) Effect of benomyl on the localization of TeaC in strain SYH22 [teaC(p)-mRFP1-teaC]. A. nidulans was grown in MM with glycerol for 1 day and then shifted to medium containing 2.5 μg of benomyl/ml for 30 min. (C) In the ΔkipA mutant SYH25 TeaC moved away from the center of the apex to the side of the tip (arrow). (D) Effect of cytochalasin A on TeaC localization. SYH17 was grown in MM with glycerol for 1 day and shifted to medium containing 2 μg of cytochalasin A/ml for 2 min. Hyphae are 3 to 4 μm in diameter.

FIG. 7.

Interaction of TeaC with TeaA and SepA. (A) Colocalization of TeaC and TeaA in strain SYH18; (B) visualization of TeaC and SepA in strain SYH13 at the hyphal tip; (C) partially colcalization of TeaC and SepA in strain SYH13 at the septa; (D) localization of SepA in strain SNT28; (E) localization of TeaC in strain SYH03; (F) interaction of TeaC with SepA (strain SYH05) shown in the bimolecular fluorescence complementation assay; (G) interaction of TeaC with TeaA (strain SYH06) shown in the bimolecular fluorescence complementation assay. Hyphae are 3 to 4 μm in diameter.

FIG. 8.

Yeast two-hybrid assay to confirm the interactions described in Fig. 7. (A) Colonies of the yeast strains. The proteins indicated on the left side were fused to the binding domain (BD), and the proteins indicated above the picture were fused to the activation domain (AD). The mated yeasts were selected on SD−Leu−Trp plates and grown on SD medium (SD−Leu−Trp−His) supplemented with 1 mM or 10 mM 3AT (3-amino-1,2,4-triazole). (B) The β-galactosidase activity was analyzed from liquid cultures using ONPG as substrate and is expressed as Miller units. The data are expressed as the means of three independent experiments. The standard deviation is indicated.

FIG. 9.

Localization dependency of TeaC and TeaA. (A) TeaC localization in the ΔteaA mutant (SYH19); (B) TeaA localization in the ΔteaC mutant (SYH24). Hyphae are 3 to 4 μm in diameter.