Fig 1.
Yeast actin supports growth and viability of A. fumigatus.
(A) Western blot analysis of actin protein expression levels in the wild type (WT) and S. cerevisiae ACT1 expressing strains (Scact1). Total protein lysates from 24 hr submerged cultures were separated by SDS-PAGE and detected with an anti-actin antibody (~42 kDa band). Coomassie stained total protein is shown as a loading control. (B) Representative cultures comparing growth and colony morphology of the WT and Scact1 strains. Conidia were point inoculated onto the center of each GMM agar plate and incubated for up to 5 days at 37°C. (C) Quantification of radial outgrowth (colony diameter) over 120 hrs post inoculation growth. Data represents the average of three experiments ± standard deviation.
Fig 2.
Yeast actin supports normal polarity establishment and asexual development of A. fumigatus.
(A) Germination rates of the WT and Scact1 strains are identical. Conidia from each strain were inoculated over cover slips submerged in GMM. Beginning at 4 hrs post-inoculation, cover slips were removed and the number of germ tube producing conidia were scored microscopically. Data represents the average of three experiments ± standard deviation. (B) The WT and Scact1 strains display similar morphologies throughout development. Representative 8 hr germlings (top panels) and 24 hr conidiophores (bottom panels) are shown for the WT and Scact1 strains. Scale bar = 10 μm.
Fig 3.
Actin structures of Scact1 stain with rhodamine-phalloidin.
Staining of Scact1 hyphae with rhodamine-phalloidin detected actin patches and rings. Actin patches were arranged as a sub-apical actin collar (A), as previously identified in filamentous fungi, and also found positioned along the hyphal cortex (B). Actin rings (C) were detected at sites of newly forming septa. Staining of the WT strain with rhodamine-phalloidin produced no detectable fluorescent signal (data not shown). Scale bar = 50 μm.
Fig 4.
The Scact1 strain is sensitive to the F-actin stabilizing agent, jasplakinolide.
(A) Jasplakinolide treatment inhibits Scact1 germination in a dose dependent manner. Conidia from the WT and Scact1 strains were cultured in the presence of increasing doses of jasplakinolide and scored for germ tube formation after 12 hrs incubation at 37°C. Data are presented as the average of three experiments ± standard deviation. (B) Jasplakinolide inhibits growth and morphogenesis of the Scact1 but not WT strain. Conidia were inoculated into a multi-well plate containing liquid GMM and ascending concentrations of jasplakinolide and incubated for 24 hrs at 37°C. Effects on WT and Scact1 growth at 50 μg/ml jasplakinolide are shown. Jasplakinolide treatment was associated with decreased growth, increased branching and swollen hyphal tips (white arrowhead, enlarged panel to right).
Fig 5.
F-actin stabilization alters actin dynamics and cell wall construction in A. fumigatus.
Conidia of the WT and Scact1 strains were inoculated onto cover slips submerged in GMM and incubated for 24 hr at 37°C. Adherent hyphae were subsequently treated with jasplakinolide (50 μg/ml) for 2 hours at 37°C. Cultures were fixed and immunostained with an anti-actin antibody (red) either alone (A and D) or in combination with Hoechst (blue) to detect nuclear position (B and E). White, block arrows indicate areas of actin structure accumulation. Note normal polarization of the cytoskeleton to the hyphal tip in the WT strain in the presence of jasplakinolide (A and B) and the disorganization of aggregated actin into clumps in the jasplakinolide-treated Scact1 strain (D and E). Small white arrowheads denote nuclei (B and E). To detect changes in cell wall deposition, calcofluor white staining was performed on unfixed samples treated with 50 μg/ml jasplakinolide (C and F). White arrowheads denote areas of aberrant cell wall deposition in the jasplakinolide-treated Scact1 strain (C and F). Scale bar = 50 μm.
Fig 6.
Human β-actin does not support A. fumigatus viability.
(A) Hyphal plugs from the wild type and HsactB heterokaryon strains grown for 48 hrs at 37°C on GMM. Cores of agar containing hyphae taken from the colony periphery of previous cultures where transferred to new GMM agar plates for culture. (B) Hyphae of the HsactB heterokaryon growing on GMM agar plates after 48 hrs at 37°C. Blunted hyphal tips (black arrowheads) that regularly lysed (white arrowheads) were noted. Scale bar = 50 μm (C) Conidia harvested from each cultures represented in Panel A were inoculated onto hygromycin selective agar and cultured for 48 hrs at 37°C. Note the lack of germination of the HsactB heterokaryon strain under selection, indicating inability of human actin to support A. fumigatus viability.