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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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Localization of TorA–GFP. (A) TorA–GFP localization in living cells. (B) Deconvoluted images for GFP (green) and DAPI (blue) in TorA–GFP-expressing cells that were fixed and stained with 5 μM DAPI. Bars, 10 μM.
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Figure 6: Localization of TorA–GFP. (A) TorA–GFP localization in living cells. (B) Deconvoluted images for GFP (green) and DAPI (blue) in TorA–GFP-expressing cells that were fixed and stained with 5 μM DAPI. Bars, 10 μM.

Mentions: To localize TorA in living cells, we fused GFP to its COOH terminus and expressed the construct under the Actin 15 promoter. The phenotype of the TorA–GFP/torA− cell line was identical to that of TorA/torA− (Fig. 5 A), including the acquisition of surface-dependent growth, indicating that the GFP fusion did not significantly affect TorA function (data not shown). The observed TorA–GFP fluorescence was localized to punctate regions throughout the cell (Fig. 6 A; see Video 4, available at http://www.jcb.org/cgi/content/full/152/3/621/DC1).


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)

Localization of TorA–GFP. (A) TorA–GFP localization in living cells. (B) Deconvoluted images for GFP (green) and DAPI (blue) in TorA–GFP-expressing cells that were fixed and stained with 5 μM DAPI. Bars, 10 μM.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Localization of TorA–GFP. (A) TorA–GFP localization in living cells. (B) Deconvoluted images for GFP (green) and DAPI (blue) in TorA–GFP-expressing cells that were fixed and stained with 5 μM DAPI. Bars, 10 μM.
Mentions: To localize TorA in living cells, we fused GFP to its COOH terminus and expressed the construct under the Actin 15 promoter. The phenotype of the TorA–GFP/torA− cell line was identical to that of TorA/torA− (Fig. 5 A), including the acquisition of surface-dependent growth, indicating that the GFP fusion did not significantly affect TorA function (data not shown). The observed TorA–GFP fluorescence was localized to punctate regions throughout the cell (Fig. 6 A; see Video 4, available at http://www.jcb.org/cgi/content/full/152/3/621/DC1).

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