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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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Phenotypic analysis of parasites overexpressing MyoB. Detection of residual bodies in intracellular parasites expressing myc–MyoB (clone MyoB/1) by confocal microscopy. (A) The residual bodies detected with anti-myc antibodies did not stain with DAPI. (B) Colocalization of MyoB (polyclonal anti-myc) with actin illustrated their coenrichment in residual bodies. (C) Residual bodies were not significantly stained with the microneme marker anti-TgMIC6. In contrast, they stained with antibodies recognizing the glycosyl phosphatidylinositol–anchored surface antigen SAG1 (D), but were not detected by antibodies recognizing a protein of the IMC (E). The latter marker visualized the high frequency of dividing parasites in the population by detecting the presence of newly formed daughter cell IMCs (E, arrows). (F) Western blot analysis of independent recombinant T. gondii clones expressing MyoB at different levels. N, nucleus; RB, residual body. Bars, 1 μm.
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fig8: Phenotypic analysis of parasites overexpressing MyoB. Detection of residual bodies in intracellular parasites expressing myc–MyoB (clone MyoB/1) by confocal microscopy. (A) The residual bodies detected with anti-myc antibodies did not stain with DAPI. (B) Colocalization of MyoB (polyclonal anti-myc) with actin illustrated their coenrichment in residual bodies. (C) Residual bodies were not significantly stained with the microneme marker anti-TgMIC6. In contrast, they stained with antibodies recognizing the glycosyl phosphatidylinositol–anchored surface antigen SAG1 (D), but were not detected by antibodies recognizing a protein of the IMC (E). The latter marker visualized the high frequency of dividing parasites in the population by detecting the presence of newly formed daughter cell IMCs (E, arrows). (F) Western blot analysis of independent recombinant T. gondii clones expressing MyoB at different levels. N, nucleus; RB, residual body. Bars, 1 μm.

Mentions: The integration of a second copy of MyoB controlled by the TUB1 promoter led to significant overexpression of MyoB compared with the wild-type situation. In contrast, MyoC was expressed at a level comparable to the endogenous protein (Fig. 2 C). We isolated several independent clones exhibiting variable levels of MyoB expression (Fig. 8 F). In addition to full-length MyoB, clone B1 accumulated notable amounts of a degradation product, mimicking MyoB/CΔtail. Indeed, the truncation corresponds to a large deletion in its tail region, as demonstrated by using antibodies recognizing the NH2-terminal epitope tag. Upon morphological examination, this clone showed strong phenotypic abnormalities during cell division. By IFA, most vacuoles were shown to contain one or multiple large residual bodies staining for MyoB, but failed to stain with DAPI (Fig. 8 A). The same phenotype was observed with other clones, though with a variable severity. MyoB and actin accumulated significantly in these structures (Fig. 8 B), but they were only weakly stained for rhoptries (unpublished data) or microneme markers (Fig. 8 C). Such residual bodies were most frequently located at the posterior of the parasites, whereas bleb-like protrusions were found distributed more randomly around the parasites. The presence of the glycosyl phosphatidylinositol–anchored plasma membrane marker, SAG1 (Fig. 8 D), around the residual bodies concomitant with the absence of staining with an IMC marker (Fig. 8 E) indicated that these bodies were surrounded solely by the plasma membrane. Numerous vacuoles contained parasites that had not accurately separated, and the geometrical organization in rosettes was rarely seen in the overexpressers compared with wild-type parasites. DAPI and IMC staining revealed an unusual proportion of parasites undergoing cell division (Fig. 8 E, arrows). Quantification at the EM level revealed that the process of cell division was slower in MyoB overexpressers, since more than 40% of the parasite population was in the middle of endodyogeny (two nuclei per cell) compared with less than 10% in wild-type parasites.


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)

Phenotypic analysis of parasites overexpressing MyoB. Detection of residual bodies in intracellular parasites expressing myc–MyoB (clone MyoB/1) by confocal microscopy. (A) The residual bodies detected with anti-myc antibodies did not stain with DAPI. (B) Colocalization of MyoB (polyclonal anti-myc) with actin illustrated their coenrichment in residual bodies. (C) Residual bodies were not significantly stained with the microneme marker anti-TgMIC6. In contrast, they stained with antibodies recognizing the glycosyl phosphatidylinositol–anchored surface antigen SAG1 (D), but were not detected by antibodies recognizing a protein of the IMC (E). The latter marker visualized the high frequency of dividing parasites in the population by detecting the presence of newly formed daughter cell IMCs (E, arrows). (F) Western blot analysis of independent recombinant T. gondii clones expressing MyoB at different levels. N, nucleus; RB, residual body. Bars, 1 μm.
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Related In: Results  -  Collection

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fig8: Phenotypic analysis of parasites overexpressing MyoB. Detection of residual bodies in intracellular parasites expressing myc–MyoB (clone MyoB/1) by confocal microscopy. (A) The residual bodies detected with anti-myc antibodies did not stain with DAPI. (B) Colocalization of MyoB (polyclonal anti-myc) with actin illustrated their coenrichment in residual bodies. (C) Residual bodies were not significantly stained with the microneme marker anti-TgMIC6. In contrast, they stained with antibodies recognizing the glycosyl phosphatidylinositol–anchored surface antigen SAG1 (D), but were not detected by antibodies recognizing a protein of the IMC (E). The latter marker visualized the high frequency of dividing parasites in the population by detecting the presence of newly formed daughter cell IMCs (E, arrows). (F) Western blot analysis of independent recombinant T. gondii clones expressing MyoB at different levels. N, nucleus; RB, residual body. Bars, 1 μm.
Mentions: The integration of a second copy of MyoB controlled by the TUB1 promoter led to significant overexpression of MyoB compared with the wild-type situation. In contrast, MyoC was expressed at a level comparable to the endogenous protein (Fig. 2 C). We isolated several independent clones exhibiting variable levels of MyoB expression (Fig. 8 F). In addition to full-length MyoB, clone B1 accumulated notable amounts of a degradation product, mimicking MyoB/CΔtail. Indeed, the truncation corresponds to a large deletion in its tail region, as demonstrated by using antibodies recognizing the NH2-terminal epitope tag. Upon morphological examination, this clone showed strong phenotypic abnormalities during cell division. By IFA, most vacuoles were shown to contain one or multiple large residual bodies staining for MyoB, but failed to stain with DAPI (Fig. 8 A). The same phenotype was observed with other clones, though with a variable severity. MyoB and actin accumulated significantly in these structures (Fig. 8 B), but they were only weakly stained for rhoptries (unpublished data) or microneme markers (Fig. 8 C). Such residual bodies were most frequently located at the posterior of the parasites, whereas bleb-like protrusions were found distributed more randomly around the parasites. The presence of the glycosyl phosphatidylinositol–anchored plasma membrane marker, SAG1 (Fig. 8 D), around the residual bodies concomitant with the absence of staining with an IMC marker (Fig. 8 E) indicated that these bodies were surrounded solely by the plasma membrane. Numerous vacuoles contained parasites that had not accurately separated, and the geometrical organization in rosettes was rarely seen in the overexpressers compared with wild-type parasites. DAPI and IMC staining revealed an unusual proportion of parasites undergoing cell division (Fig. 8 E, arrows). Quantification at the EM level revealed that the process of cell division was slower in MyoB overexpressers, since more than 40% of the parasite population was in the middle of endodyogeny (two nuclei per cell) compared with less than 10% in wild-type parasites.

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