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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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Determination of the subcellular localization of MyoB and MyoC. Transgenic parasites expressing myc–MyoB (A) or myc–MyoC (B and E) were analyzed by IFA and confocal microscopy after staining with anti-myc (A, B, and E) and either anti-MyoA (A) or anti-MIC6 (B) antibodies. MIC6 is a microneme marker defining the apical pole of the parasite. (C) Western blot analysis of parasites expressing myc–MyoB or myc–MyoC with anti-myc antibodies. (D) Schematic representation of some major morphological features of a tachyzoite (adapted from Nichols and Chiappino, 1987). (E) A parasitophorous vacuole containing a rosette of 32 tachyzoites. Note the homogeneous orientation of the parasites with their apical poles pointing outwards. APR, anterior polar ring; MTs, microtubules; PM, plasma membrane; PPR, posterior polar ring. Bars, 1 μm.
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fig5: Determination of the subcellular localization of MyoB and MyoC. Transgenic parasites expressing myc–MyoB (A) or myc–MyoC (B and E) were analyzed by IFA and confocal microscopy after staining with anti-myc (A, B, and E) and either anti-MyoA (A) or anti-MIC6 (B) antibodies. MIC6 is a microneme marker defining the apical pole of the parasite. (C) Western blot analysis of parasites expressing myc–MyoB or myc–MyoC with anti-myc antibodies. (D) Schematic representation of some major morphological features of a tachyzoite (adapted from Nichols and Chiappino, 1987). (E) A parasitophorous vacuole containing a rosette of 32 tachyzoites. Note the homogeneous orientation of the parasites with their apical poles pointing outwards. APR, anterior polar ring; MTs, microtubules; PM, plasma membrane; PPR, posterior polar ring. Bars, 1 μm.

Mentions: To analyze more closely the localization of the MyoB/C gene products in nondividing parasites, we generated recombinants stably expressing myc–MyoB or myc–MyoC. We confirmed the expression of polypeptides of the anticipated sizes by Western blotting with anti-myc antibodies (Fig. 5 C). The stably transformed parasites were then examined by immunofluorescence assay (IFA), revealing distinct, subcellular steady-state distributions (Fig. 5, A and B). As previously observed in transient transfections, MyoB was spread throughout the cytoplasm associated with a punctate structure, whereas the distribution of MyoC was mostly restricted to the posterior and weakly to the anterior poles of the parasites (Fig. 5 E). Double immunofluorescence staining of MyoB and MyoA showed no major overlap. Additionally, the double staining against MyoC and the apical micronemal protein TgMIC6 confirmed the predominant posterior localization of MyoC. High resolution confocal microscopy distinctly identified the staining of MyoC as a ring structure, likely corresponding to the posterior polar ring and more weakly to the apical polar ring at the terminal regions of the IMC. A schematic representation of a tachyzoite cytoskeleton based on previous EM studies (Nichols and Chiappino, 1987) is depicted in Fig. 5 D. At the apical pole, the conoid is associated with the apical polar ring, the IMC, and the sets of 22 pellicular microtubules. The posterior polar ring delineates the termination of the IMC at the rear end of the parasite. A large parasitophorous vacuole containing 32 parasites arranged in rosette illustrates the focused staining at the posterior pole of the parasites, which corresponds to the center of the rosette. A weaker staining at the periphery corresponds to the apical tip of each parasite (Fig. 5 E). The restricted localization of MyoC is unlikely to depend only on direct interactions with actin filaments since the treatment of recombinant parasites with 10 μM cytochalasin D for 2 h did not alter the ring structures visualized by IFA (unpublished data).


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)

Determination of the subcellular localization of MyoB and MyoC. Transgenic parasites expressing myc–MyoB (A) or myc–MyoC (B and E) were analyzed by IFA and confocal microscopy after staining with anti-myc (A, B, and E) and either anti-MyoA (A) or anti-MIC6 (B) antibodies. MIC6 is a microneme marker defining the apical pole of the parasite. (C) Western blot analysis of parasites expressing myc–MyoB or myc–MyoC with anti-myc antibodies. (D) Schematic representation of some major morphological features of a tachyzoite (adapted from Nichols and Chiappino, 1987). (E) A parasitophorous vacuole containing a rosette of 32 tachyzoites. Note the homogeneous orientation of the parasites with their apical poles pointing outwards. APR, anterior polar ring; MTs, microtubules; PM, plasma membrane; PPR, posterior polar ring. Bars, 1 μm.
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Related In: Results  -  Collection

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fig5: Determination of the subcellular localization of MyoB and MyoC. Transgenic parasites expressing myc–MyoB (A) or myc–MyoC (B and E) were analyzed by IFA and confocal microscopy after staining with anti-myc (A, B, and E) and either anti-MyoA (A) or anti-MIC6 (B) antibodies. MIC6 is a microneme marker defining the apical pole of the parasite. (C) Western blot analysis of parasites expressing myc–MyoB or myc–MyoC with anti-myc antibodies. (D) Schematic representation of some major morphological features of a tachyzoite (adapted from Nichols and Chiappino, 1987). (E) A parasitophorous vacuole containing a rosette of 32 tachyzoites. Note the homogeneous orientation of the parasites with their apical poles pointing outwards. APR, anterior polar ring; MTs, microtubules; PM, plasma membrane; PPR, posterior polar ring. Bars, 1 μm.
Mentions: To analyze more closely the localization of the MyoB/C gene products in nondividing parasites, we generated recombinants stably expressing myc–MyoB or myc–MyoC. We confirmed the expression of polypeptides of the anticipated sizes by Western blotting with anti-myc antibodies (Fig. 5 C). The stably transformed parasites were then examined by immunofluorescence assay (IFA), revealing distinct, subcellular steady-state distributions (Fig. 5, A and B). As previously observed in transient transfections, MyoB was spread throughout the cytoplasm associated with a punctate structure, whereas the distribution of MyoC was mostly restricted to the posterior and weakly to the anterior poles of the parasites (Fig. 5 E). Double immunofluorescence staining of MyoB and MyoA showed no major overlap. Additionally, the double staining against MyoC and the apical micronemal protein TgMIC6 confirmed the predominant posterior localization of MyoC. High resolution confocal microscopy distinctly identified the staining of MyoC as a ring structure, likely corresponding to the posterior polar ring and more weakly to the apical polar ring at the terminal regions of the IMC. A schematic representation of a tachyzoite cytoskeleton based on previous EM studies (Nichols and Chiappino, 1987) is depicted in Fig. 5 D. At the apical pole, the conoid is associated with the apical polar ring, the IMC, and the sets of 22 pellicular microtubules. The posterior polar ring delineates the termination of the IMC at the rear end of the parasite. A large parasitophorous vacuole containing 32 parasites arranged in rosette illustrates the focused staining at the posterior pole of the parasites, which corresponds to the center of the rosette. A weaker staining at the periphery corresponds to the apical tip of each parasite (Fig. 5 E). The restricted localization of MyoC is unlikely to depend only on direct interactions with actin filaments since the treatment of recombinant parasites with 10 μM cytochalasin D for 2 h did not alter the ring structures visualized by IFA (unpublished data).

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