The Septin AspB in Aspergillus nidulans Forms Bars and Filaments and Plays Roles in Growth Emergence and Conidiation

The ΔaspB mutant shows a hyper-emergence of growth and abnormal conidiophores. (A) The ΔaspB mutant forms multiple germ tubes, whereas the wild-type strain forms one germ tube. (B) The ΔaspB mutant forms multiple abnormal and stunted branches per compartment, whereas the wild-type strain forms one branch per compartment. (C) The ΔaspB mutant forms disorganized conidiophores. An arrow denotes a new aerial hypha arising from a vesicle. The wild-type strain forms many conidia, and the conidiophores are organized with different layers (V, vesicle; P, phialide; M, metulae; Cc, conidial chain). Conditions for growth were as described in Fig. 1. Scale bar, 5 μm.

The ΔaspB mutant shows clumped nuclei. (A) Wild-type nuclei positioned along hyphae. (B) The ΔaspB mutant results in clumped nuclei, particularly near branches. (C) The ΔaspB mutant shows clumped nuclei. The ΔaspB mutant showed more nuclei in 15 μm around the most basal branch than did the wild-type strain. To delineate the area for nuclear counts, the most basal branch was identified; 7.5 μm from the center of the branch to the left and right of the branch was measured (total area = 15 μm; n = 200). Spores were incubated for 14 to 16 h at 30°C, and nuclei were stained with Hoechst 33258. Scale bar, 5 μm.

AspB forms rings, dots, bars, and filaments. The AspB-GFP strain was incubated at 30°C for 4 to 5 h to view isotropic growth, 6 to 7 h to view early germ tube emergence, 9 to 11 h to view hyphal elongation and septation, and 12 to 16 h to view branching. (A) AspB forms bars (*), rings (<), and dots (arrowhead) in dormant and germinating spores. The inset shows an enlarged view of a ring (scale bar, 0.5 μm). (B to D) AspB forms “X's” (#) in addition to bars, rings, and dots as conidia swell. The inset shows an enlarged view of an “X” (scale bar, 0.75 μm). AspB forms caps and collars as the germ tube emerges, bars that localize to conidia and filaments (arrows) that localize to tips. (E to G) AspB forms bars that localize subapically and filaments that localize to tips as the hypha extends. Cytoplasmic GFP fluorescence is excluded from nuclei (N). (H) AspB forms rings at septa. (I to R) AspB forms filaments in branching compartments, caps as the branch emerges, and dots at the tips of branches. Filaments localize to newly formed branches. (S) Filaments and bars localize to longer branches. (T) Top view of a branch cap. Scale bar, 5 μm.

AspB forms bars, filaments, caps, and rings during conidiophore formation. (A) AspB-GFP localization at several stages of early conidiophore development. AspB-GFP forms bars (*) and filaments (arrow) in conidiophore stalks. AspB-GFP forms caps (C) in aerial hyphae. AspB-GFP caps are diffuse in swollen vesicles. (B) AspB-GFP remains localized at the phialide-conidium interface as conidia form. AspB-GFP forms rings (>) at the base of budding layers. (C) Conidial chains attached to conidiophores show AspB-GFP localization as dots and bars. (D) AspB-GFP forms bars on conidia freshly detached from conidiophores. Some bars looked “frayed” (X) at the ends. Spores were incubated in agar between coverslips for 1, 2, and 3 days at 30°C and viewed by confocal microscopy. Scale bar, 5 μm.

Motion of AspB bars and filaments. (A) AspB-GFP bar (*) in a germinating conidium moves away from the cell periphery, oscillates in and out of the plane of focus, and breaks in two. “Frayed” ends (X) can be observed. (B) AspB-GFP filaments (arrow) show slower movement than bars at the hyphal tips. Cytoplasmic GFP fluorescence is excluded from nuclei. Time lapse fluorescence microscopy was used. Numbers represent time lapse frames from Movies S1 and S2 in the supplemental material. Frames are 5 to 10 s apart. Conditions for growth as in Fig. 4. Scale bar, 5 μm.

AspB structures are lost in the absence of AspA and AspC and filaments and bars are lost in hyphae in the absence of AspE. (I) AspB-GFP structures in septin deletion backgrounds. (A) AspB-GFP structures. Rings (>), dots (arrowhead), bars (*) filaments (arrow), and at septa (S) are indicated. (B) All AspB-GFP structures are lost in the ΔaspA ΔaspC mutant. (C) All AspB-GFP structures are found in ΔaspD strains. (D) All AspB-GFP structures are found in early ΔaspE cells but are mostly seen as dots and/or “X's” in hyphae. Conditions for growth were as described in Fig. 4. Scale bar, 5 μm. (II) AspB-GFP structures found in septin deletion backgrounds. Spores were incubated for 10 h at 30°C, and AspB structures were classified as filament, bar, and spot (rings, dots, and X's) and cytoplasmic (n = 100). Scale bar, 5 μm.

AspB bars are lost in conidiophores in the absence of AspA and AspC. (A) AspB-GFP bars (*) in conidiophores. (B) AspB-GFP bars are lost in conidiophores in the ΔaspA ΔaspC mutant. (C) AspB-GFP bars and dots (arrowhead) in the ΔaspD mutant. (D) AspB-GFP bars and dots in the ΔaspE mutant. Conditions for growth were as described as in Fig. 5. Scale bar, 5 μm.