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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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Plaque sizes and developmental phenotypes of torA− and mek1 mutants. (A) Cells were mixed with K. aerogenes and clonally seeded on agar plates. Photographs were taken after 6 d at 22°C. Typical examples of each phenotype are shown (B). Cells were allowed to develop on DB-agar at 1.5 × 106 cells/cm2 and were photographed at the indicated time points. (C) Northern Blot analysis of Mek1 expression during development of wild-type and torA− cells. Cells were developed as in B, and, at the indicated times, cells were washed off the plate, and total RNA was extracted. Bars: (A) 10 mm; (B) 1 mm.
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Figure 1: Plaque sizes and developmental phenotypes of torA− and mek1 mutants. (A) Cells were mixed with K. aerogenes and clonally seeded on agar plates. Photographs were taken after 6 d at 22°C. Typical examples of each phenotype are shown (B). Cells were allowed to develop on DB-agar at 1.5 × 106 cells/cm2 and were photographed at the indicated time points. (C) Northern Blot analysis of Mek1 expression during development of wild-type and torA− cells. Cells were developed as in B, and, at the indicated times, cells were washed off the plate, and total RNA was extracted. Bars: (A) 10 mm; (B) 1 mm.

Mentions: To isolate novel mutants with defects in chemotaxis, we used REMI to generate 30,000 transformants with random insertions in the genome. These were clonally seeded on bacterial lawns, and plaques that expanded slowly under these conditions were selected. 18 small plaque mutants were isolated, and Tortoise (torA−) was chosen for further study. The plaque size and additional phenotypes of torA−, described in detail below, closely resembled those of mek1− (Fig. 1A and Fig. 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)

Plaque sizes and developmental phenotypes of torA− and mek1 mutants. (A) Cells were mixed with K. aerogenes and clonally seeded on agar plates. Photographs were taken after 6 d at 22°C. Typical examples of each phenotype are shown (B). Cells were allowed to develop on DB-agar at 1.5 × 106 cells/cm2 and were photographed at the indicated time points. (C) Northern Blot analysis of Mek1 expression during development of wild-type and torA− cells. Cells were developed as in B, and, at the indicated times, cells were washed off the plate, and total RNA was extracted. Bars: (A) 10 mm; (B) 1 mm.
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Related In: Results  -  Collection

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

Figure 1: Plaque sizes and developmental phenotypes of torA− and mek1 mutants. (A) Cells were mixed with K. aerogenes and clonally seeded on agar plates. Photographs were taken after 6 d at 22°C. Typical examples of each phenotype are shown (B). Cells were allowed to develop on DB-agar at 1.5 × 106 cells/cm2 and were photographed at the indicated time points. (C) Northern Blot analysis of Mek1 expression during development of wild-type and torA− cells. Cells were developed as in B, and, at the indicated times, cells were washed off the plate, and total RNA was extracted. Bars: (A) 10 mm; (B) 1 mm.
Mentions: To isolate novel mutants with defects in chemotaxis, we used REMI to generate 30,000 transformants with random insertions in the genome. These were clonally seeded on bacterial lawns, and plaques that expanded slowly under these conditions were selected. 18 small plaque mutants were isolated, and Tortoise (torA−) was chosen for further study. The plaque size and additional phenotypes of torA−, described in detail below, closely resembled those of mek1− (Fig. 1A and Fig. 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