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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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Comparison of the expression of T. gondii myosin transcripts and proteins between tachyzoites and bradyzoites. (A) RT-PCR amplification of T. gondii myosin transcripts from total tachyzoite RNA with two distinct sets of primers specific to MyoA and one set of primers specific for MyoB, MyoC, and MyoE. (B) Semiquantitative RT-PCR analysis on total RNA prepared from tachyzoites cultivated in vitro and bradyzoites encysted in vivo. After first strand synthesis, the cDNAs of MyoA, MyoB, MyoC, MyoD, and MyoE, as well as the tubulin mRNA, TUB1 (used as a control), were detected using appropriate primer pairs. Serial dilutions of the cDNAs were 1:10 and 1:100. The size of the PCR products were as expected from the respective cDNA sequence. (C) Western blot analysis of lysates prepared from extracellular RH, the persistent strain Prugniaud, RHMyoC, and RHMyoB. In the upper half, the membrane was incubated with the antiserum raised against peptides B/C1 and B/C2. The monoclonal anti-myc was used in the lower half.
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fig2: Comparison of the expression of T. gondii myosin transcripts and proteins between tachyzoites and bradyzoites. (A) RT-PCR amplification of T. gondii myosin transcripts from total tachyzoite RNA with two distinct sets of primers specific to MyoA and one set of primers specific for MyoB, MyoC, and MyoE. (B) Semiquantitative RT-PCR analysis on total RNA prepared from tachyzoites cultivated in vitro and bradyzoites encysted in vivo. After first strand synthesis, the cDNAs of MyoA, MyoB, MyoC, MyoD, and MyoE, as well as the tubulin mRNA, TUB1 (used as a control), were detected using appropriate primer pairs. Serial dilutions of the cDNAs were 1:10 and 1:100. The size of the PCR products were as expected from the respective cDNA sequence. (C) Western blot analysis of lysates prepared from extracellular RH, the persistent strain Prugniaud, RHMyoC, and RHMyoB. In the upper half, the membrane was incubated with the antiserum raised against peptides B/C1 and B/C2. The monoclonal anti-myc was used in the lower half.

Mentions: Toward the identification of a myosin contributing to cell division, we analyzed the pattern of expression of the five known myosins in the two invasive life stage forms of T. gondii present in intermediate hosts. Specific transcripts coding for myosins A, B, C, and D (not shown), but not E, were amplified by RT-PCR using total RNAs isolated from the rapidly replicating tachyzoites (Fig. 2 A). Furthermore, a comparison of the amount of specific transcripts between tachyzoites and the dormant, encysted bradyzoites was assessed by semiquantitative RT-PCR (Fig. 2 B), as previously described (Yahiaoui et al., 1999). Rather unexpectedly and with the exception of MyoA, the myosin mRNAs were more abundant in the persistent stage. This analysis did not distinguish between the increased rate of transcription and the increased stability of transcripts in bradyzoites compared with tachyzoites. Expressed sequence tag (EST) clones corresponding to myosins are extremely underrepresented in the 10,000 ESTs found in the T. gondii database (Ajioka et al., 1998). Nevertheless, the same tendency toward a predominant representation in bradyzoite compared with tachyzoite cDNAs was observed (http://www.cbil.upenn.edu/ParaDBs/). Indeed, 11 EST clones specific for MyoC were present in the in vivo bradyzoite cDNA library compared with two ESTs in the much larger pool of T. gondii RH strain tachyzoite ESTs. This imbalance reflects and strengthens the results obtained by RT-PCR. The two transcripts encoding MyoB and MyoC were previously described and presumed to derive from alternative RNA splicing (Heintzelman and Schwartzman, 1997).


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)

Comparison of the expression of T. gondii myosin transcripts and proteins between tachyzoites and bradyzoites. (A) RT-PCR amplification of T. gondii myosin transcripts from total tachyzoite RNA with two distinct sets of primers specific to MyoA and one set of primers specific for MyoB, MyoC, and MyoE. (B) Semiquantitative RT-PCR analysis on total RNA prepared from tachyzoites cultivated in vitro and bradyzoites encysted in vivo. After first strand synthesis, the cDNAs of MyoA, MyoB, MyoC, MyoD, and MyoE, as well as the tubulin mRNA, TUB1 (used as a control), were detected using appropriate primer pairs. Serial dilutions of the cDNAs were 1:10 and 1:100. The size of the PCR products were as expected from the respective cDNA sequence. (C) Western blot analysis of lysates prepared from extracellular RH, the persistent strain Prugniaud, RHMyoC, and RHMyoB. In the upper half, the membrane was incubated with the antiserum raised against peptides B/C1 and B/C2. The monoclonal anti-myc was used in the lower half.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198869&req=5

fig2: Comparison of the expression of T. gondii myosin transcripts and proteins between tachyzoites and bradyzoites. (A) RT-PCR amplification of T. gondii myosin transcripts from total tachyzoite RNA with two distinct sets of primers specific to MyoA and one set of primers specific for MyoB, MyoC, and MyoE. (B) Semiquantitative RT-PCR analysis on total RNA prepared from tachyzoites cultivated in vitro and bradyzoites encysted in vivo. After first strand synthesis, the cDNAs of MyoA, MyoB, MyoC, MyoD, and MyoE, as well as the tubulin mRNA, TUB1 (used as a control), were detected using appropriate primer pairs. Serial dilutions of the cDNAs were 1:10 and 1:100. The size of the PCR products were as expected from the respective cDNA sequence. (C) Western blot analysis of lysates prepared from extracellular RH, the persistent strain Prugniaud, RHMyoC, and RHMyoB. In the upper half, the membrane was incubated with the antiserum raised against peptides B/C1 and B/C2. The monoclonal anti-myc was used in the lower half.
Mentions: Toward the identification of a myosin contributing to cell division, we analyzed the pattern of expression of the five known myosins in the two invasive life stage forms of T. gondii present in intermediate hosts. Specific transcripts coding for myosins A, B, C, and D (not shown), but not E, were amplified by RT-PCR using total RNAs isolated from the rapidly replicating tachyzoites (Fig. 2 A). Furthermore, a comparison of the amount of specific transcripts between tachyzoites and the dormant, encysted bradyzoites was assessed by semiquantitative RT-PCR (Fig. 2 B), as previously described (Yahiaoui et al., 1999). Rather unexpectedly and with the exception of MyoA, the myosin mRNAs were more abundant in the persistent stage. This analysis did not distinguish between the increased rate of transcription and the increased stability of transcripts in bradyzoites compared with tachyzoites. Expressed sequence tag (EST) clones corresponding to myosins are extremely underrepresented in the 10,000 ESTs found in the T. gondii database (Ajioka et al., 1998). Nevertheless, the same tendency toward a predominant representation in bradyzoite compared with tachyzoite cDNAs was observed (http://www.cbil.upenn.edu/ParaDBs/). Indeed, 11 EST clones specific for MyoC were present in the in vivo bradyzoite cDNA library compared with two ESTs in the much larger pool of T. gondii RH strain tachyzoite ESTs. This imbalance reflects and strengthens the results obtained by RT-PCR. The two transcripts encoding MyoB and MyoC were previously described and presumed to derive from alternative RNA splicing (Heintzelman and Schwartzman, 1997).

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