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Differential Support of Aspergillus fumigatus Morphogenesis by Yeast and Human Actins.

LeClaire LL, Fortwendel JR - PLoS ONE (2015)

Bottom Line: Although highly conserved, there are key differences among actins of fungal species as well as between mammalian and fungal actins.For example, the F-actin stabilizing molecules, phalloidin and jasplakinolide, bind to actin structures in yeast and human cells, whereas phalloidin does not bind actin structures of Aspergillus.We also show that human β-actin cannot support Aspergillus viability, even though the amino acid sequences of human and Aspergillus actins are 89.3% identical.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, Alabama, United States of America.

ABSTRACT
The actin cytoskeleton is highly conserved among eukaryotes and is essential for cellular processes regulating growth and differentiation. In fungi, filamentous actin (F-actin) orchestrates hyphal tip structure and extension via organization of exocytic and endocytic processes at the hyphal tip. Although highly conserved, there are key differences among actins of fungal species as well as between mammalian and fungal actins. For example, the F-actin stabilizing molecules, phalloidin and jasplakinolide, bind to actin structures in yeast and human cells, whereas phalloidin does not bind actin structures of Aspergillus. These discrepancies suggest structural differences between Aspergillus actin filaments and those of human and yeast cells. Additionally, fungal actin kinetics are much faster than those of humans, displaying 5-fold faster nucleation and 40-fold faster nucleotide exchange rates. Limited published studies suggest that these faster actin kinetics are required for normal growth and morphogenesis of yeast cells. In the current work, we show that replacement of Aspergillus actin with yeast actin generates a morphologically normal strain, suggesting that Aspergillus actin kinetics are similar to those of yeast. In contrast to wild type A. fumigatus, F-actin in this strain binds phalloidin, and pharmacological stabilization of these actin structures with jasplakinolide inhibits germination and alters morphogenesis in a dose-dependent manner. We also show that human β-actin cannot support Aspergillus viability, even though the amino acid sequences of human and Aspergillus actins are 89.3% identical. Our findings show that minor differences in actin protein sequence account for loss of phalloidin and jasplakinolide sensitivity in Aspergillus species.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0142535.g005: 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.

Mentions: To confirm that treatment is associated with changes in F-actin dynamics, the wild type and Scact1 strains were germinated on cover slips and subsequently exposed to jasplakinolide for two hours. After treatment, each strain was immunolabeled with an anti-actin antibody to reveal actin structures. The jasplakinolide-treated wild type strain displayed typical actin dynamics, including tip-concentrated actin coupled with sub-apical cortical actin patches (Fig 5A). In contrast, upon exposure to jasplakinolide, the Scact1 mutant displayed large, intensely stained aggregates of actin located to the hyphal tip and regularly spaced along the length of each hypha (Fig 5D). The regular spacing of the actin aggregates somewhat resembled the distribution of nuclei along Aspergillus spp. hyphae [36, 37]. Because, perinuclear F-actin structures have recently been identified in cultures of Cryptococcus neoformans treated with microtubule inhibitors [38], we co-stained jasplakinolide-treated strains for actin and nuclei to examine possible co-localization of these structures in the Scact1 mutant. Hoechst staining revealed normally spaced nuclei in the wild type strain upon F-actin stabilization (Fig 5B). Spacing of nuclei in the Scact1 mutant treated with jasplakinolide also appeared normal and nuclei never co-localized with actin aggregates. Instead, nuclei were interspersed with the aggregated actin structures (Fig 5E). Calcofluor white staining further revealed altered cell wall structure in the Scact1 mutant after F-actin stabilization. In contrast to the homogenous cell wall staining revealed in the wild type strain (Fig 5C), the Scact1 mutant displayed patches of intense cell wall staining at hyphal tips and in sub-apical compartments after jasplakinolide treatment (Fig 5F).


Differential Support of Aspergillus fumigatus Morphogenesis by Yeast and Human Actins.

LeClaire LL, Fortwendel JR - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4640809&req=5

pone.0142535.g005: 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.
Mentions: To confirm that treatment is associated with changes in F-actin dynamics, the wild type and Scact1 strains were germinated on cover slips and subsequently exposed to jasplakinolide for two hours. After treatment, each strain was immunolabeled with an anti-actin antibody to reveal actin structures. The jasplakinolide-treated wild type strain displayed typical actin dynamics, including tip-concentrated actin coupled with sub-apical cortical actin patches (Fig 5A). In contrast, upon exposure to jasplakinolide, the Scact1 mutant displayed large, intensely stained aggregates of actin located to the hyphal tip and regularly spaced along the length of each hypha (Fig 5D). The regular spacing of the actin aggregates somewhat resembled the distribution of nuclei along Aspergillus spp. hyphae [36, 37]. Because, perinuclear F-actin structures have recently been identified in cultures of Cryptococcus neoformans treated with microtubule inhibitors [38], we co-stained jasplakinolide-treated strains for actin and nuclei to examine possible co-localization of these structures in the Scact1 mutant. Hoechst staining revealed normally spaced nuclei in the wild type strain upon F-actin stabilization (Fig 5B). Spacing of nuclei in the Scact1 mutant treated with jasplakinolide also appeared normal and nuclei never co-localized with actin aggregates. Instead, nuclei were interspersed with the aggregated actin structures (Fig 5E). Calcofluor white staining further revealed altered cell wall structure in the Scact1 mutant after F-actin stabilization. In contrast to the homogenous cell wall staining revealed in the wild type strain (Fig 5C), the Scact1 mutant displayed patches of intense cell wall staining at hyphal tips and in sub-apical compartments after jasplakinolide treatment (Fig 5F).

Bottom Line: Although highly conserved, there are key differences among actins of fungal species as well as between mammalian and fungal actins.For example, the F-actin stabilizing molecules, phalloidin and jasplakinolide, bind to actin structures in yeast and human cells, whereas phalloidin does not bind actin structures of Aspergillus.We also show that human β-actin cannot support Aspergillus viability, even though the amino acid sequences of human and Aspergillus actins are 89.3% identical.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, Alabama, United States of America.

ABSTRACT
The actin cytoskeleton is highly conserved among eukaryotes and is essential for cellular processes regulating growth and differentiation. In fungi, filamentous actin (F-actin) orchestrates hyphal tip structure and extension via organization of exocytic and endocytic processes at the hyphal tip. Although highly conserved, there are key differences among actins of fungal species as well as between mammalian and fungal actins. For example, the F-actin stabilizing molecules, phalloidin and jasplakinolide, bind to actin structures in yeast and human cells, whereas phalloidin does not bind actin structures of Aspergillus. These discrepancies suggest structural differences between Aspergillus actin filaments and those of human and yeast cells. Additionally, fungal actin kinetics are much faster than those of humans, displaying 5-fold faster nucleation and 40-fold faster nucleotide exchange rates. Limited published studies suggest that these faster actin kinetics are required for normal growth and morphogenesis of yeast cells. In the current work, we show that replacement of Aspergillus actin with yeast actin generates a morphologically normal strain, suggesting that Aspergillus actin kinetics are similar to those of yeast. In contrast to wild type A. fumigatus, F-actin in this strain binds phalloidin, and pharmacological stabilization of these actin structures with jasplakinolide inhibits germination and alters morphogenesis in a dose-dependent manner. We also show that human β-actin cannot support Aspergillus viability, even though the amino acid sequences of human and Aspergillus actins are 89.3% identical. Our findings show that minor differences in actin protein sequence account for loss of phalloidin and jasplakinolide sensitivity in Aspergillus species.

No MeSH data available.


Related in: MedlinePlus