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Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division.

Delbac F, Sänger A, Neuhaus EM, Stratmann R, Ajioka JW, Toursel C, Herm-Götz A, Tomavo S, Soldati T, Soldati D - J. Cell Biol. (2001)

Bottom Line: MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis.When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation.Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Biologie, Universität Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACT
In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

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Overexpression of MyoB delays cell division and reduces virulence in mice. (A) Parasites expressing GFP or MyoB were allowed to invade host cells at 37°C for 10 min; the cells were then further incubated for 12 and 24 h. For each condition, 100 vacuoles were analyzed, and the number of parasites per vacuole is presented in a histogram. (B) Overexpression of MyoB allowed mice to survive acute infection with T. gondii RH. Summary of two independent experiments: a group of five mice was inoculated with the two parasite lines in the first experiment. Groups of 10 mice (RHGFP) and 20 mice (RHMyoB overexpresser) were inoculated per parasite line in the second experiment. (Ba) The RHMyoB strain was compared with RH expressing GFP. (Bb) Numerators correspond to the number of surviving mice 4 wk after infection; denominators indicate the total number of mice inoculated in two independent experiments. (Bc) Numerators represent numbers of surviving mice that were positive for T. gondii serology; denominators represent the total number of mice surviving in the two separate experiments. (Bd) The percentage was corrected according to the real infection rate; the seronegative mice were not taken into account. (C) Isolation of cysts in brains of infected mice 2 mo after intraperitoneal inoculation with mutant parasites overexpressing MyoB. The cysts were stained specifically with the Dolichos biflorus FITC–labeled lectin, which recognizes the cell wall of T. gondii cysts. Bar, 10 μm.
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fig10: Overexpression of MyoB delays cell division and reduces virulence in mice. (A) Parasites expressing GFP or MyoB were allowed to invade host cells at 37°C for 10 min; the cells were then further incubated for 12 and 24 h. For each condition, 100 vacuoles were analyzed, and the number of parasites per vacuole is presented in a histogram. (B) Overexpression of MyoB allowed mice to survive acute infection with T. gondii RH. Summary of two independent experiments: a group of five mice was inoculated with the two parasite lines in the first experiment. Groups of 10 mice (RHGFP) and 20 mice (RHMyoB overexpresser) were inoculated per parasite line in the second experiment. (Ba) The RHMyoB strain was compared with RH expressing GFP. (Bb) Numerators correspond to the number of surviving mice 4 wk after infection; denominators indicate the total number of mice inoculated in two independent experiments. (Bc) Numerators represent numbers of surviving mice that were positive for T. gondii serology; denominators represent the total number of mice surviving in the two separate experiments. (Bd) The percentage was corrected according to the real infection rate; the seronegative mice were not taken into account. (C) Isolation of cysts in brains of infected mice 2 mo after intraperitoneal inoculation with mutant parasites overexpressing MyoB. The cysts were stained specifically with the Dolichos biflorus FITC–labeled lectin, which recognizes the cell wall of T. gondii cysts. Bar, 10 μm.

Mentions: To assess the influence of stable MyoB overexpression on growth rate, we counted parasites per vacuole at 12 and 24 h after infection. Parasites expressing MyoB exhibited a significant growth delay compared with parasites expressing GFP (Fig. 10 A) and GFP–MyoBtail (unpublished data), suggesting that the motor is necessary for the phenotype. To determine if this mutant exhibits an alteration of virulence in vivo, two series of experiments were conducted with groups of mice infected in the intraperitoneal cavity with 20 parasites of either RH or RHMyoB (Fig. 10 B). As reported previously, the inoculation of wild-type RH is always lethal in mice (Mercier et al., 1998), and all infected (seropositive) mice died within 7 d. In contrast, a large proportion of mice infected with RHMyoB survived the challenge. 2 wk later, the mice were tested for seropositivity to confirm infection. To examine if the attenuated parasites were able to establish a chronic infection in the animal, we searched for the presence of cysts using specific staining with the Dolichos biflorus lectin (Boothroyd et al., 1997). The presence of a few cysts per brain was indicative of a chronic infection in all mice analyzed (Fig. 10 C).


Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division.

Delbac F, Sänger A, Neuhaus EM, Stratmann R, Ajioka JW, Toursel C, Herm-Götz A, Tomavo S, Soldati T, Soldati D - J. Cell Biol. (2001)

Overexpression of MyoB delays cell division and reduces virulence in mice. (A) Parasites expressing GFP or MyoB were allowed to invade host cells at 37°C for 10 min; the cells were then further incubated for 12 and 24 h. For each condition, 100 vacuoles were analyzed, and the number of parasites per vacuole is presented in a histogram. (B) Overexpression of MyoB allowed mice to survive acute infection with T. gondii RH. Summary of two independent experiments: a group of five mice was inoculated with the two parasite lines in the first experiment. Groups of 10 mice (RHGFP) and 20 mice (RHMyoB overexpresser) were inoculated per parasite line in the second experiment. (Ba) The RHMyoB strain was compared with RH expressing GFP. (Bb) Numerators correspond to the number of surviving mice 4 wk after infection; denominators indicate the total number of mice inoculated in two independent experiments. (Bc) Numerators represent numbers of surviving mice that were positive for T. gondii serology; denominators represent the total number of mice surviving in the two separate experiments. (Bd) The percentage was corrected according to the real infection rate; the seronegative mice were not taken into account. (C) Isolation of cysts in brains of infected mice 2 mo after intraperitoneal inoculation with mutant parasites overexpressing MyoB. The cysts were stained specifically with the Dolichos biflorus FITC–labeled lectin, which recognizes the cell wall of T. gondii cysts. Bar, 10 μm.
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Related In: Results  -  Collection

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fig10: Overexpression of MyoB delays cell division and reduces virulence in mice. (A) Parasites expressing GFP or MyoB were allowed to invade host cells at 37°C for 10 min; the cells were then further incubated for 12 and 24 h. For each condition, 100 vacuoles were analyzed, and the number of parasites per vacuole is presented in a histogram. (B) Overexpression of MyoB allowed mice to survive acute infection with T. gondii RH. Summary of two independent experiments: a group of five mice was inoculated with the two parasite lines in the first experiment. Groups of 10 mice (RHGFP) and 20 mice (RHMyoB overexpresser) were inoculated per parasite line in the second experiment. (Ba) The RHMyoB strain was compared with RH expressing GFP. (Bb) Numerators correspond to the number of surviving mice 4 wk after infection; denominators indicate the total number of mice inoculated in two independent experiments. (Bc) Numerators represent numbers of surviving mice that were positive for T. gondii serology; denominators represent the total number of mice surviving in the two separate experiments. (Bd) The percentage was corrected according to the real infection rate; the seronegative mice were not taken into account. (C) Isolation of cysts in brains of infected mice 2 mo after intraperitoneal inoculation with mutant parasites overexpressing MyoB. The cysts were stained specifically with the Dolichos biflorus FITC–labeled lectin, which recognizes the cell wall of T. gondii cysts. Bar, 10 μm.
Mentions: To assess the influence of stable MyoB overexpression on growth rate, we counted parasites per vacuole at 12 and 24 h after infection. Parasites expressing MyoB exhibited a significant growth delay compared with parasites expressing GFP (Fig. 10 A) and GFP–MyoBtail (unpublished data), suggesting that the motor is necessary for the phenotype. To determine if this mutant exhibits an alteration of virulence in vivo, two series of experiments were conducted with groups of mice infected in the intraperitoneal cavity with 20 parasites of either RH or RHMyoB (Fig. 10 B). As reported previously, the inoculation of wild-type RH is always lethal in mice (Mercier et al., 1998), and all infected (seropositive) mice died within 7 d. In contrast, a large proportion of mice infected with RHMyoB survived the challenge. 2 wk later, the mice were tested for seropositivity to confirm infection. To examine if the attenuated parasites were able to establish a chronic infection in the animal, we searched for the presence of cysts using specific staining with the Dolichos biflorus lectin (Boothroyd et al., 1997). The presence of a few cysts per brain was indicative of a chronic infection in all mice analyzed (Fig. 10 C).

Bottom Line: MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis.When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation.Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Biologie, Universität Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACT
In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

Show MeSH
Related in: MedlinePlus