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Tortoise, a novel mitochondrial protein, is required for directional responses of Dictyostelium in chemotactic gradients.

van Es S, Wessels D, Soll DR, Borleis J, Devreotes PN - J. Cell Biol. (2001)

Bottom Line: Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype.TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker.The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
We have identified a novel gene, Tortoise (TorA), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA-green fluorescent protein (GFP) is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.

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Effects of constitutive expression of TorA and TorA–GFP on plaque size of torA− and growth in suspension. (A) Plaques of torA− and cells expressing TorA or TorA–GFP, grown for 6 d on K. aerogenes lawns. (B) Growth curves of Ax3 cells and Ax3 cells expressing TorA. Cells were grown to confluency in petri dishes and then shaken in flasks at 3 × 105 cells/ml and counted at the indicated time points. •, Ax3; ▪, Ax3 cells overexpressing TorA.
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Figure 5: Effects of constitutive expression of TorA and TorA–GFP on plaque size of torA− and growth in suspension. (A) Plaques of torA− and cells expressing TorA or TorA–GFP, grown for 6 d on K. aerogenes lawns. (B) Growth curves of Ax3 cells and Ax3 cells expressing TorA. Cells were grown to confluency in petri dishes and then shaken in flasks at 3 × 105 cells/ml and counted at the indicated time points. •, Ax3; ▪, Ax3 cells overexpressing TorA.

Mentions: In performing control experiments to confirm that the Tortoise phenotype was caused by a deletion in the isolated gene, we discovered a fascinating new phenotype caused by overexpression of the TorA gene. Cells with high levels of expression lost their ability to grow in shaken suspension and were entirely dependent on the surface of a petri dish for growth. We expressed the full-length gene under a constitutive Actin 15 promoter in torA− cells. The resulting TorA/torA− cell line formed larger plaques and fruiting bodies; however, the fruiting bodies were slightly smaller than those of wild type (Fig. 5 A). On growth plates, detached TorA-overexpressing cells did not proliferate but became smaller and rounder, whereas the attached cells continued to grow. Typically, wild-type cells grow at a similar rate whether attached or in suspension. We next designed an experiment to directly show that the mutant grows normally when attached to the surface of a petri dish but cannot grow in suspension. Wild-type cells and cells overexpressing TorA were grown to confluency in petri dishes, harvested, and shaken in flasks. Under these conditions, cells overexpressing TorA stopped growing, whereas wild-type cells displayed normal growth rates. Thus, TorA overexpression leads to surface-dependent growth (Fig. 5 B).


Tortoise, a novel mitochondrial protein, is required for directional responses of Dictyostelium in chemotactic gradients.

van Es S, Wessels D, Soll DR, Borleis J, Devreotes PN - J. Cell Biol. (2001)

Effects of constitutive expression of TorA and TorA–GFP on plaque size of torA− and growth in suspension. (A) Plaques of torA− and cells expressing TorA or TorA–GFP, grown for 6 d on K. aerogenes lawns. (B) Growth curves of Ax3 cells and Ax3 cells expressing TorA. Cells were grown to confluency in petri dishes and then shaken in flasks at 3 × 105 cells/ml and counted at the indicated time points. •, Ax3; ▪, Ax3 cells overexpressing TorA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196008&req=5

Figure 5: Effects of constitutive expression of TorA and TorA–GFP on plaque size of torA− and growth in suspension. (A) Plaques of torA− and cells expressing TorA or TorA–GFP, grown for 6 d on K. aerogenes lawns. (B) Growth curves of Ax3 cells and Ax3 cells expressing TorA. Cells were grown to confluency in petri dishes and then shaken in flasks at 3 × 105 cells/ml and counted at the indicated time points. •, Ax3; ▪, Ax3 cells overexpressing TorA.
Mentions: In performing control experiments to confirm that the Tortoise phenotype was caused by a deletion in the isolated gene, we discovered a fascinating new phenotype caused by overexpression of the TorA gene. Cells with high levels of expression lost their ability to grow in shaken suspension and were entirely dependent on the surface of a petri dish for growth. We expressed the full-length gene under a constitutive Actin 15 promoter in torA− cells. The resulting TorA/torA− cell line formed larger plaques and fruiting bodies; however, the fruiting bodies were slightly smaller than those of wild type (Fig. 5 A). On growth plates, detached TorA-overexpressing cells did not proliferate but became smaller and rounder, whereas the attached cells continued to grow. Typically, wild-type cells grow at a similar rate whether attached or in suspension. We next designed an experiment to directly show that the mutant grows normally when attached to the surface of a petri dish but cannot grow in suspension. Wild-type cells and cells overexpressing TorA were grown to confluency in petri dishes, harvested, and shaken in flasks. Under these conditions, cells overexpressing TorA stopped growing, whereas wild-type cells displayed normal growth rates. Thus, TorA overexpression leads to surface-dependent growth (Fig. 5 B).

Bottom Line: Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype.TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker.The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
We have identified a novel gene, Tortoise (TorA), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA-green fluorescent protein (GFP) is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.

Show MeSH
Related in: MedlinePlus