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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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Structure of the TorA gene. (A) TorA. Solid lines and open bars represent genomic sequences and coding regions (2,427 bp), respectively. The gray box represents the coiled coil domain. Insertion 1 (Ins1) was in the Bcl1 site in the original REMI mutant. Insertion 2 (Ins2) was made in the Spe1 site. (B) Amino acid sequence of TorA. The sequence of TorA is available from Genbank/EMBL/DDBJ under accession number AF303221.
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Figure 2: Structure of the TorA gene. (A) TorA. Solid lines and open bars represent genomic sequences and coding regions (2,427 bp), respectively. The gray box represents the coiled coil domain. Insertion 1 (Ins1) was in the Bcl1 site in the original REMI mutant. Insertion 2 (Ins2) was made in the Spe1 site. (B) Amino acid sequence of TorA. The sequence of TorA is available from Genbank/EMBL/DDBJ under accession number AF303221.

Mentions: Next, we isolated and identified the gene mutated in torA− (Fig. 2). We cloned the regions flanking the insertions and determined the sequence as described in Materials and Methods. To verify whether the observed phenotypes were caused by the REMI insertions, we linearized the recovered plasmids, transformed them into wild-type Ax3 cells, and recreated the genotype through homologous recombination. Fig. 1 A shows that disruption of TorA in fresh wild-type cells regenerates the small plaque and tiny structure phenotype on bacterial lawns. This mutant was used for all further analyses.


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)

Structure of the TorA gene. (A) TorA. Solid lines and open bars represent genomic sequences and coding regions (2,427 bp), respectively. The gray box represents the coiled coil domain. Insertion 1 (Ins1) was in the Bcl1 site in the original REMI mutant. Insertion 2 (Ins2) was made in the Spe1 site. (B) Amino acid sequence of TorA. The sequence of TorA is available from Genbank/EMBL/DDBJ under accession number AF303221.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Structure of the TorA gene. (A) TorA. Solid lines and open bars represent genomic sequences and coding regions (2,427 bp), respectively. The gray box represents the coiled coil domain. Insertion 1 (Ins1) was in the Bcl1 site in the original REMI mutant. Insertion 2 (Ins2) was made in the Spe1 site. (B) Amino acid sequence of TorA. The sequence of TorA is available from Genbank/EMBL/DDBJ under accession number AF303221.
Mentions: Next, we isolated and identified the gene mutated in torA− (Fig. 2). We cloned the regions flanking the insertions and determined the sequence as described in Materials and Methods. To verify whether the observed phenotypes were caused by the REMI insertions, we linearized the recovered plasmids, transformed them into wild-type Ax3 cells, and recreated the genotype through homologous recombination. Fig. 1 A shows that disruption of TorA in fresh wild-type cells regenerates the small plaque and tiny structure phenotype on bacterial lawns. This mutant was used for all further analyses.

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