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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 GFP fusions. (A) Transgenic parasites expressing the different GFP fusion constructs with the tails of MyoB (B), MyoC (C and D), MyoB/C (A), or the COOH-terminal 118 residues specific to MyoC (E) were analyzed by confocal immunofluorescence microscopy after staining with anti-myc (A–E) and anti-MyoA (A–C) antibodies. (D) A parasite in the process of cell division showed two ring structures in the middle of the dividing mother cell. (F) Western blot analysis of wild-type parasites (RH) and transgenic parasites expressing GFP alone, GFP–Btail, GFP–Ctail, GFP–B/Ctail, or GFP–C118. PPR, posterior polar ring. Bars, 1 μm.
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fig6: Determination of the subcellular localization of GFP fusions. (A) Transgenic parasites expressing the different GFP fusion constructs with the tails of MyoB (B), MyoC (C and D), MyoB/C (A), or the COOH-terminal 118 residues specific to MyoC (E) were analyzed by confocal immunofluorescence microscopy after staining with anti-myc (A–E) and anti-MyoA (A–C) antibodies. (D) A parasite in the process of cell division showed two ring structures in the middle of the dividing mother cell. (F) Western blot analysis of wild-type parasites (RH) and transgenic parasites expressing GFP alone, GFP–Btail, GFP–Ctail, GFP–B/Ctail, or GFP–C118. PPR, posterior polar ring. Bars, 1 μm.

Mentions: Three distinct domains define the modular structure of most myosins. The catalytic motor domain binds to actin, the regulatory neck region carries the light chains and structurally acts as a lever arm, and the tail domain is responsible for the functional diversity of each molecule. The tail domains appear to contribute predominantly as determiners of subcellular localization; they bring the motor to its site of action. MyoB and MyoC share a common core region in their tail domain and diverge only in their last 21 and 118 amino acids, respectively. To assess the importance of these domains, we fused GFP to fragments encoding the tails of MyoB or MyoC. Additionally, to examine the efficiency of alternative splicing in tachyzoites, we fused GFP to a genomic fragment of the MyoB/C gene that encompasses both tail exons. Stable clones expressing GFP–Btail, GFP–Ctail, or the GFP–B/Ctail chimera were isolated and characterized. The myc–GFP–Btail and myc–GFP–Ctail fusions showed the expected mobility shift compared with GFP on a Western blot (Fig. 6 F). Interestingly, parasites expressing GFP fused to the genomic fragment of MyoB/C predominantly produced GFP–Ctail. Only a trace of a product comigrating with GFP–Btail was visible. This result correlates with the predominance of MyoC versus MyoB in wild-type tachyzoites (Fig. 2 C) and suggests that a fully processed transcript is the major spliced form (Fig. 3 C). The subcellular localization of GFP–tail fusions was determined by IFA and confocal microscopy. Consistent with the localization of full-length MyoB and MyoC, GFP–Btail was distributed in a punctate pattern throughout the cytoplasm, whereas GFP–Ctail accumulated in ring structures at the extremities of the parasites (Fig. 6, B and C). In parasites undergoing cell division and thus containing two nuclei, two rings are visible and likely correspond to the posterior polar rings of the forming daughter cells (Fig. 6 D, and also Fig. 1, b–d). In perfect agreement with the results of Western blotting, the GFP–B/Ctail fusion was mostly found to localize at the polar rings similar to GFP–Ctail (Fig. 6 A). GFP fused to the last COOH-terminal 118 amino acids of MyoC did not localize to the polar rings but was abundant and diffuse in the cytosol (Fig. 6 E). This result indicates that the determinants of MyoC localization are not restricted to the region specific to MyoC, and additional information in the common core of the two tails is important for the selective targeting.


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 GFP fusions. (A) Transgenic parasites expressing the different GFP fusion constructs with the tails of MyoB (B), MyoC (C and D), MyoB/C (A), or the COOH-terminal 118 residues specific to MyoC (E) were analyzed by confocal immunofluorescence microscopy after staining with anti-myc (A–E) and anti-MyoA (A–C) antibodies. (D) A parasite in the process of cell division showed two ring structures in the middle of the dividing mother cell. (F) Western blot analysis of wild-type parasites (RH) and transgenic parasites expressing GFP alone, GFP–Btail, GFP–Ctail, GFP–B/Ctail, or GFP–C118. PPR, posterior polar ring. Bars, 1 μm.
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Related In: Results  -  Collection

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fig6: Determination of the subcellular localization of GFP fusions. (A) Transgenic parasites expressing the different GFP fusion constructs with the tails of MyoB (B), MyoC (C and D), MyoB/C (A), or the COOH-terminal 118 residues specific to MyoC (E) were analyzed by confocal immunofluorescence microscopy after staining with anti-myc (A–E) and anti-MyoA (A–C) antibodies. (D) A parasite in the process of cell division showed two ring structures in the middle of the dividing mother cell. (F) Western blot analysis of wild-type parasites (RH) and transgenic parasites expressing GFP alone, GFP–Btail, GFP–Ctail, GFP–B/Ctail, or GFP–C118. PPR, posterior polar ring. Bars, 1 μm.
Mentions: Three distinct domains define the modular structure of most myosins. The catalytic motor domain binds to actin, the regulatory neck region carries the light chains and structurally acts as a lever arm, and the tail domain is responsible for the functional diversity of each molecule. The tail domains appear to contribute predominantly as determiners of subcellular localization; they bring the motor to its site of action. MyoB and MyoC share a common core region in their tail domain and diverge only in their last 21 and 118 amino acids, respectively. To assess the importance of these domains, we fused GFP to fragments encoding the tails of MyoB or MyoC. Additionally, to examine the efficiency of alternative splicing in tachyzoites, we fused GFP to a genomic fragment of the MyoB/C gene that encompasses both tail exons. Stable clones expressing GFP–Btail, GFP–Ctail, or the GFP–B/Ctail chimera were isolated and characterized. The myc–GFP–Btail and myc–GFP–Ctail fusions showed the expected mobility shift compared with GFP on a Western blot (Fig. 6 F). Interestingly, parasites expressing GFP fused to the genomic fragment of MyoB/C predominantly produced GFP–Ctail. Only a trace of a product comigrating with GFP–Btail was visible. This result correlates with the predominance of MyoC versus MyoB in wild-type tachyzoites (Fig. 2 C) and suggests that a fully processed transcript is the major spliced form (Fig. 3 C). The subcellular localization of GFP–tail fusions was determined by IFA and confocal microscopy. Consistent with the localization of full-length MyoB and MyoC, GFP–Btail was distributed in a punctate pattern throughout the cytoplasm, whereas GFP–Ctail accumulated in ring structures at the extremities of the parasites (Fig. 6, B and C). In parasites undergoing cell division and thus containing two nuclei, two rings are visible and likely correspond to the posterior polar rings of the forming daughter cells (Fig. 6 D, and also Fig. 1, b–d). In perfect agreement with the results of Western blotting, the GFP–B/Ctail fusion was mostly found to localize at the polar rings similar to GFP–Ctail (Fig. 6 A). GFP fused to the last COOH-terminal 118 amino acids of MyoC did not localize to the polar rings but was abundant and diffuse in the cytosol (Fig. 6 E). This result indicates that the determinants of MyoC localization are not restricted to the region specific to MyoC, and additional information in the common core of the two tails is important for the selective targeting.

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