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Disassembly activity of actin-depolymerizing factor (ADF) is associated with distinct cellular processes in apicomplexan parasites.

Haase S, Zimmermann D, Olshina MA, Wilkinson M, Fisher F, Tan YH, Stewart RJ, Tonkin CJ, Wong W, Kovar DR, Baum J - Mol. Biol. Cell (2015)

Bottom Line: Strikingly, mutation of a key basic residue (Lys-72), previously implicated in disassembly in PfADF1, had no detectable phenotypic effect on parasite growth, motility, or development.Biochemical analyses of each ADF protein confirmed the reduced ability of lysine mutants to mediate actin depolymerization via filament disassembly although not severing, in contrast to previous reports.These data suggest that actin filament disassembly is essential for apicomplexan parasite development but not for motility, as well as pointing to genus-specific coevolution between ADF proteins and their native actin.

View Article: PubMed Central - PubMed

Affiliation: Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia Department of Medical Biology, University of Melbourne, Parkville, VIC 3052, Australia.

No MeSH data available.


Related in: MedlinePlus

TgADFK68A-cMyc–expressing parasites cannot restore TgADF cKO loss of function. (A–E) Plaque assays of plated human fibroblasts grown with each parasite treated with or without ATc for 8 d. White plaques represent characteristic areas where host cells have been lysed due to parasite propagation. A growth defect is seen in A (bottom right) for the TgADF cKO line, as expected. A similar phenotype is seen in D (bottom for both clones), where no plaques have been formed for TgADFK68A-cMyc–expressing parasites grown in the presence of ATc. No obvious growth defect is detectable in other complementing lines.
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Figure 2: TgADFK68A-cMyc–expressing parasites cannot restore TgADF cKO loss of function. (A–E) Plaque assays of plated human fibroblasts grown with each parasite treated with or without ATc for 8 d. White plaques represent characteristic areas where host cells have been lysed due to parasite propagation. A growth defect is seen in A (bottom right) for the TgADF cKO line, as expected. A similar phenotype is seen in D (bottom for both clones), where no plaques have been formed for TgADFK68A-cMyc–expressing parasites grown in the presence of ATc. No obvious growth defect is detectable in other complementing lines.

Mentions: No plaques were formed for TgADF cKO parasites grown in ATc (Figure 2A), as expected (Mehta and Sibley, 2011). Down-regulation of TgADF-HA in clones of the wild-type TgADF-cMyc and PfADF1-cMyc–expressing parasite lines, however, showed effectively wild-type levels of plaque formation, suggesting functional complementation (Figure 2, B and C). Surprisingly, no obvious differences in size and number of plaques were observed for the PfADF1K72A-cMyc mutant parasites grown in ATc (Figure 2E and Supplemental Table S1). In contrast, a mutant phenotype consistent with the full knockdown phenotype was seen with the TgADFK68A-cMyc–expressing clones (Figures 2D). Thus, by plaque assay alone, it appears that whereas the ectopically expressed wild-type proteins and the PfADF1K72A-cMyc mutant clones are functional with respect to successful parasite propagation, the loss of Lys-68 in TgADF severely affects the parasite at one or several points within the lytic cycle. The ability of the PfADF1K72A-cMyc mutant to complement native TgADF absence in contrast to the TgADFK68A mutant suggests major differences in either the activity of the ADFs or their interactions with native actin in the respective species.


Disassembly activity of actin-depolymerizing factor (ADF) is associated with distinct cellular processes in apicomplexan parasites.

Haase S, Zimmermann D, Olshina MA, Wilkinson M, Fisher F, Tan YH, Stewart RJ, Tonkin CJ, Wong W, Kovar DR, Baum J - Mol. Biol. Cell (2015)

TgADFK68A-cMyc–expressing parasites cannot restore TgADF cKO loss of function. (A–E) Plaque assays of plated human fibroblasts grown with each parasite treated with or without ATc for 8 d. White plaques represent characteristic areas where host cells have been lysed due to parasite propagation. A growth defect is seen in A (bottom right) for the TgADF cKO line, as expected. A similar phenotype is seen in D (bottom for both clones), where no plaques have been formed for TgADFK68A-cMyc–expressing parasites grown in the presence of ATc. No obvious growth defect is detectable in other complementing lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: TgADFK68A-cMyc–expressing parasites cannot restore TgADF cKO loss of function. (A–E) Plaque assays of plated human fibroblasts grown with each parasite treated with or without ATc for 8 d. White plaques represent characteristic areas where host cells have been lysed due to parasite propagation. A growth defect is seen in A (bottom right) for the TgADF cKO line, as expected. A similar phenotype is seen in D (bottom for both clones), where no plaques have been formed for TgADFK68A-cMyc–expressing parasites grown in the presence of ATc. No obvious growth defect is detectable in other complementing lines.
Mentions: No plaques were formed for TgADF cKO parasites grown in ATc (Figure 2A), as expected (Mehta and Sibley, 2011). Down-regulation of TgADF-HA in clones of the wild-type TgADF-cMyc and PfADF1-cMyc–expressing parasite lines, however, showed effectively wild-type levels of plaque formation, suggesting functional complementation (Figure 2, B and C). Surprisingly, no obvious differences in size and number of plaques were observed for the PfADF1K72A-cMyc mutant parasites grown in ATc (Figure 2E and Supplemental Table S1). In contrast, a mutant phenotype consistent with the full knockdown phenotype was seen with the TgADFK68A-cMyc–expressing clones (Figures 2D). Thus, by plaque assay alone, it appears that whereas the ectopically expressed wild-type proteins and the PfADF1K72A-cMyc mutant clones are functional with respect to successful parasite propagation, the loss of Lys-68 in TgADF severely affects the parasite at one or several points within the lytic cycle. The ability of the PfADF1K72A-cMyc mutant to complement native TgADF absence in contrast to the TgADFK68A mutant suggests major differences in either the activity of the ADFs or their interactions with native actin in the respective species.

Bottom Line: Strikingly, mutation of a key basic residue (Lys-72), previously implicated in disassembly in PfADF1, had no detectable phenotypic effect on parasite growth, motility, or development.Biochemical analyses of each ADF protein confirmed the reduced ability of lysine mutants to mediate actin depolymerization via filament disassembly although not severing, in contrast to previous reports.These data suggest that actin filament disassembly is essential for apicomplexan parasite development but not for motility, as well as pointing to genus-specific coevolution between ADF proteins and their native actin.

View Article: PubMed Central - PubMed

Affiliation: Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia Department of Medical Biology, University of Melbourne, Parkville, VIC 3052, Australia.

No MeSH data available.


Related in: MedlinePlus