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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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Biochemical responses towards cAMP in torA−. Cells were developed for 5 h in a shaken suspension with the addition of 100-nM pulses of cAMP every 6 min or on DB-agar plates (in the case of cGMP accumulation) (A) Signal transduction responses: 5 × 10−5 M 2′deoxy-cAMP (cAMP accumulation), 10−7 M cAMP (F-actin), 10-6 M cAMP (cGMP accumulation). •, torA−; ▪, Ax3; ▴, mek1−. (B) Translocation of Crac PH-domains to the leading edge of torA− and wild-type cells in a cAMP gradient. The white dot indicates the position of the micropipette filled with 10−6 M cAMP. Bar, 10 μm.
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Figure 4: Biochemical responses towards cAMP in torA−. Cells were developed for 5 h in a shaken suspension with the addition of 100-nM pulses of cAMP every 6 min or on DB-agar plates (in the case of cGMP accumulation) (A) Signal transduction responses: 5 × 10−5 M 2′deoxy-cAMP (cAMP accumulation), 10−7 M cAMP (F-actin), 10-6 M cAMP (cGMP accumulation). •, torA−; ▪, Ax3; ▴, mek1−. (B) Translocation of Crac PH-domains to the leading edge of torA− and wild-type cells in a cAMP gradient. The white dot indicates the position of the micropipette filled with 10−6 M cAMP. Bar, 10 μm.

Mentions: The following results further show that torA− is specifically defective in chemotaxis. First, the appearance of coordinated cell movements caused by the propagation of cAMP waves after 3–4 h of development indicates that torA− differentiates properly and expresses a cAMP signaling system. Consistent with this, wild-type and torA− cells accumulate equal amounts of cAMP in response to cAMP stimulation, indicating that torA− cells have normal chemoattractant-induced activation of adenylyl cyclase (Fig. 4 A). Previous studies suggest that appropriate activation of adenylyl cyclase requires wild-type levels of surface receptors, G protein subunits, adenylyl cyclase, and several cytosolic regulators. Second, since it has previously been reported that mek1− cells fail to accumulate cGMP in response to chemoattractant stimulation, we tested whether torA− cells had a defect in cGMP metabolism. Under our conditions, both the torA− and the mek1− cells displayed a wild-type cGMP response. Third, chemoattractant-induced polymerization of actin was normal in both mutants (data shown for torA− only). Since the chemotaxis defect was caused by failure to detect the gradient or a failure to respond properly, we assessed whether the cells can sense the direction of a gradient. We transformed torA− and wild-type cells with a GFP fusion of the PH domain of cytosolic regulator of adenylyl cyclase (Crac). When wild-type cells are placed in a cAMP gradient this fusion protein translocates to the side of the highest concentration (Parent et al. 1998). As shown in Fig. 4 B, this translocation also occurred in torA− cells, showing that these cells are able to detect external gradients. These results suggest that torA− cells are unable to respond to a detected gradient with proper pseudopod extension.


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)

Biochemical responses towards cAMP in torA−. Cells were developed for 5 h in a shaken suspension with the addition of 100-nM pulses of cAMP every 6 min or on DB-agar plates (in the case of cGMP accumulation) (A) Signal transduction responses: 5 × 10−5 M 2′deoxy-cAMP (cAMP accumulation), 10−7 M cAMP (F-actin), 10-6 M cAMP (cGMP accumulation). •, torA−; ▪, Ax3; ▴, mek1−. (B) Translocation of Crac PH-domains to the leading edge of torA− and wild-type cells in a cAMP gradient. The white dot indicates the position of the micropipette filled with 10−6 M cAMP. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196008&req=5

Figure 4: Biochemical responses towards cAMP in torA−. Cells were developed for 5 h in a shaken suspension with the addition of 100-nM pulses of cAMP every 6 min or on DB-agar plates (in the case of cGMP accumulation) (A) Signal transduction responses: 5 × 10−5 M 2′deoxy-cAMP (cAMP accumulation), 10−7 M cAMP (F-actin), 10-6 M cAMP (cGMP accumulation). •, torA−; ▪, Ax3; ▴, mek1−. (B) Translocation of Crac PH-domains to the leading edge of torA− and wild-type cells in a cAMP gradient. The white dot indicates the position of the micropipette filled with 10−6 M cAMP. Bar, 10 μm.
Mentions: The following results further show that torA− is specifically defective in chemotaxis. First, the appearance of coordinated cell movements caused by the propagation of cAMP waves after 3–4 h of development indicates that torA− differentiates properly and expresses a cAMP signaling system. Consistent with this, wild-type and torA− cells accumulate equal amounts of cAMP in response to cAMP stimulation, indicating that torA− cells have normal chemoattractant-induced activation of adenylyl cyclase (Fig. 4 A). Previous studies suggest that appropriate activation of adenylyl cyclase requires wild-type levels of surface receptors, G protein subunits, adenylyl cyclase, and several cytosolic regulators. Second, since it has previously been reported that mek1− cells fail to accumulate cGMP in response to chemoattractant stimulation, we tested whether torA− cells had a defect in cGMP metabolism. Under our conditions, both the torA− and the mek1− cells displayed a wild-type cGMP response. Third, chemoattractant-induced polymerization of actin was normal in both mutants (data shown for torA− only). Since the chemotaxis defect was caused by failure to detect the gradient or a failure to respond properly, we assessed whether the cells can sense the direction of a gradient. We transformed torA− and wild-type cells with a GFP fusion of the PH domain of cytosolic regulator of adenylyl cyclase (Crac). When wild-type cells are placed in a cAMP gradient this fusion protein translocates to the side of the highest concentration (Parent et al. 1998). As shown in Fig. 4 B, this translocation also occurred in torA− cells, showing that these cells are able to detect external gradients. These results suggest that torA− cells are unable to respond to a detected gradient with proper pseudopod extension.

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