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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

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).
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pone.0142535.g004: 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).

Mentions: To test this hypothesis, we cultured conidia of the wild type and Scact1 strains in the presence of ascending doses of the F-actin stabilizing compound. As predicted, germination and hyphal growth of the A. fumigatus wild type strain was not inhibited, even at the highest concentration of jasplakinolide (Fig 4A and 4B). In contrast, germ tube formation and hyphal growth of the Scact1 strain were inhibited by jasplakinolide treatment in a concentration dependent manner. After 12 hours of culture at 37°C, germination of the Scact1 strain was inhibited by 40% at 25 μg/ml of jasplakinolide (Fig 4A). Strikingly, Scact1 germination was inhibited by almost 90%, compared to the non-treated control, when cultured in the presence of 50 μg/ml jasplakinolide (Fig 4A). After 24 hours of culture at 37°C, morphological aberrations in the Scact1 mutant treated with jasplakinolide included the formation of stunted hyphae with increased branching and the production of swollen hyphal tips (Fig 4B). These findings suggested treatment with jasplakinolide produced a loss of polarity and altered cell wall integrity.


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

LeClaire LL, Fortwendel JR - PLoS ONE (2015)

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).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142535.g004: 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).
Mentions: To test this hypothesis, we cultured conidia of the wild type and Scact1 strains in the presence of ascending doses of the F-actin stabilizing compound. As predicted, germination and hyphal growth of the A. fumigatus wild type strain was not inhibited, even at the highest concentration of jasplakinolide (Fig 4A and 4B). In contrast, germ tube formation and hyphal growth of the Scact1 strain were inhibited by jasplakinolide treatment in a concentration dependent manner. After 12 hours of culture at 37°C, germination of the Scact1 strain was inhibited by 40% at 25 μg/ml of jasplakinolide (Fig 4A). Strikingly, Scact1 germination was inhibited by almost 90%, compared to the non-treated control, when cultured in the presence of 50 μg/ml jasplakinolide (Fig 4A). After 24 hours of culture at 37°C, morphological aberrations in the Scact1 mutant treated with jasplakinolide included the formation of stunted hyphae with increased branching and the production of swollen hyphal tips (Fig 4B). These findings suggested treatment with jasplakinolide produced a loss of polarity and altered cell wall integrity.

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