Calcineurin localizes to the hyphal septum in Aspergillus fumigatus: implications for septum formation and conidiophore development

Abstract

A functional calcineurin A fusion to enhanced green fluorescent protein (EGFP), CnaA-EGFP, was expressed in the Aspergillus fumigatus DeltacnaA mutant. CnaA-EGFP localized in actively growing hyphal tips, at the septa, and at junctions between the vesicle and phialides in an actin-dependent manner. This is the first study to implicate calcineurin in septum formation and conidiophore development of a filamentous fungus.

FIG. 1.

(A) Genomic DNA from wild-type (AF293), ΔcnaA mutant, and cnaA-egfp strains digested with HincII and probed with a digoxigenin-labeled 650-bp PCR fragment in the cnaA open reading frame. Lanes 1, 2, 3, and 4 indicate the digoxigenin-labeled marker ladder and DNA from the wild-type, ΔcnaA mutant, and cnaA-egfp strains, respectively. The difference in band size observed between the wild-type strain (1,156 bp; Fig. 1A, lane 2) and the cnaA-egfp strain (1,044 bp; lane 3) is due to the presence of an intron in the wild-type strain. (B) Radial growth on glucose minimal agar media (GMM) inoculated with 10³ conidia and colony diameters measured of the wild-type, ΔcnaA mutant, and the cnaA-egfp strains over 96 h at 37°C. Experiments were performed in triplicate, and a representative picture of the growth assay is shown. (C) Wild-type and the cnaA-egfp strains were grown as mentioned in panel B in the presence of 20 ng of FK506/ml. (D) For microscopic observation, 10³ conidia of the respective strains were inoculated in 100 μl of GMM liquid medium in the absence or presence of FK506 and grown for a period of 24 h. Scale bar, 20 μm.

FIG. 2.

Localization of CnaA-EGFP during growth of A. fumigatus. The cnaA-egfp strain was grown in 100 μl of GMM liquid medium on coverslips for 24 h and observed by fluorescence microscopy. (A) Distribution of CnaA-EGFP in dormant conidia at 0 h of growth. (B) Swollen conidium at 3 h of growth. CnaA-EGFP is localized in small vesicular structures at the region of germ tube formation. (C) Germling at 6 h of growth showing a newly formed septum to which CnaA-EGFP is localized. (D) The CnaA-EGFP is localized to the hyphal tip in dot-like structures. (E) Middle regions of hypha showing mature septa with CnaA-EGFP. (F) CnaA-EGFP fusion protein in dot-like structures seen closely associated with the central region of the septum. (G) Vesicle showing the localization of CnaA-EGFP at its septum. Scale bar, 10 μm.

FIG. 3.

Localization of CnaA-EGFP during conidiophore formation. The cnaA-egfp strain was grown in 100 μl of GMM liquid medium on coverslips for 24 h and observed by fluorescence microscopy. (A) Dot-like cortical patches in a newly forming conidiophore (indicated by arrows). (B) Dot-like structures decorating a newly forming septum (indicated by arrowheads) in a conidiophore. (C) CnaA-EGFP is localized to the septum in the conidiophore. (D) CnaA-EGFP is seen localized to junctions between the vesicle and phialides in a mature conidiophore. Scale bar, 10 μm.

FIG. 4.

Effect of cytochalasin A on the localization of CnaA-EGFP. The cnaA-egfp strain was grown in 100 μl of GMM liquid medium for 16 h and then cytochalasin A (80 μg/ml [panels C and D]) was added. The cultures were grown for an additional 8 h and examined by fluorescence microscopy. (A and B) Control cultures with CnaA-EGFP localized to septa in the absence of the inhibitor; (C and D) cytosolic localization of CnaA-EGFP due to actin inhibition. Scale bar, 10 μm.