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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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TorA is Triton X-100/sodium carbonate insoluble. (A) Anti-GFP immunoblots of TorA–GFP-expressing cells (lanes 1–4) and Ax3 (lane 5). Cells were lysed through 5 μM nucleopore filters (Corning) and separated in low speed supernatants (lane 1), and pellets (lanes 2 and 5). Insoluble fraction after extraction of whole cells with 0.5% Triton X-100 according to Spudich 1987 (lane 3). Insoluble fraction after extraction of pellets with a mixture of 0.5% Triton X-100, 0.2 M sodium carbonate (lane 4). (B) GFP fluorescence of insoluble fractions after extraction of whole cells with 0.5% Triton X-100. (C) Silver-stained gel of insoluble 0.5% Triton X-100/sodium carbonate–insoluble fractions. Lane 1, TorA–GFP; lane 2, TorA (1–676)–GFP; lane 3, Ax3. Bar, 10 μm.
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Figure 9: TorA is Triton X-100/sodium carbonate insoluble. (A) Anti-GFP immunoblots of TorA–GFP-expressing cells (lanes 1–4) and Ax3 (lane 5). Cells were lysed through 5 μM nucleopore filters (Corning) and separated in low speed supernatants (lane 1), and pellets (lanes 2 and 5). Insoluble fraction after extraction of whole cells with 0.5% Triton X-100 according to Spudich 1987 (lane 3). Insoluble fraction after extraction of pellets with a mixture of 0.5% Triton X-100, 0.2 M sodium carbonate (lane 4). (B) GFP fluorescence of insoluble fractions after extraction of whole cells with 0.5% Triton X-100. (C) Silver-stained gel of insoluble 0.5% Triton X-100/sodium carbonate–insoluble fractions. Lane 1, TorA–GFP; lane 2, TorA (1–676)–GFP; lane 3, Ax3. Bar, 10 μm.

Mentions: Investigation of the subcellular localization of TorA–GFP and its colocalization with mitochondria revealed that TorA displayed some unusual properties for an integral component of mitochondria. Using immunoblots (Fig. 9 A), we showed that nearly all TorA–GFP was localized to the particulate fraction. After extraction of the particulate fraction with Triton X-100, TorA–GFP localized to the Triton X-100–insoluble fraction. Under these conditions, ∼70% of the mitochondrial marker TopA (Komori et al. 1997) was solubilized (data not shown). As shown by fluorescence microscopy, Triton X-100 extraction does not disrupt the TorA–GFP clusters (Fig. 9 B). Surprisingly, TorA–GFP remained insoluble, even after treatment of the particulate fraction with Triton X-100 in 200 mM sodium carbonate (Fig. 9A and Fig. C). These fractions do not contain mitochondria or cytoskeleton, suggesting that TorA–GFP may be part of a novel structure. TorA–GFP was the predominant protein in the final fraction, which contained only six or seven major protein bands (Fig. 9 C). The coiled coil region in TorA is not required for these properties, since a truncated version, TorA (1–676), lacking the coiled coil region, still targeted GFP to the Triton/sodium carbonate–insoluble fraction (Fig. 9 C).


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)

TorA is Triton X-100/sodium carbonate insoluble. (A) Anti-GFP immunoblots of TorA–GFP-expressing cells (lanes 1–4) and Ax3 (lane 5). Cells were lysed through 5 μM nucleopore filters (Corning) and separated in low speed supernatants (lane 1), and pellets (lanes 2 and 5). Insoluble fraction after extraction of whole cells with 0.5% Triton X-100 according to Spudich 1987 (lane 3). Insoluble fraction after extraction of pellets with a mixture of 0.5% Triton X-100, 0.2 M sodium carbonate (lane 4). (B) GFP fluorescence of insoluble fractions after extraction of whole cells with 0.5% Triton X-100. (C) Silver-stained gel of insoluble 0.5% Triton X-100/sodium carbonate–insoluble fractions. Lane 1, TorA–GFP; lane 2, TorA (1–676)–GFP; lane 3, Ax3. Bar, 10 μm.
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Figure 9: TorA is Triton X-100/sodium carbonate insoluble. (A) Anti-GFP immunoblots of TorA–GFP-expressing cells (lanes 1–4) and Ax3 (lane 5). Cells were lysed through 5 μM nucleopore filters (Corning) and separated in low speed supernatants (lane 1), and pellets (lanes 2 and 5). Insoluble fraction after extraction of whole cells with 0.5% Triton X-100 according to Spudich 1987 (lane 3). Insoluble fraction after extraction of pellets with a mixture of 0.5% Triton X-100, 0.2 M sodium carbonate (lane 4). (B) GFP fluorescence of insoluble fractions after extraction of whole cells with 0.5% Triton X-100. (C) Silver-stained gel of insoluble 0.5% Triton X-100/sodium carbonate–insoluble fractions. Lane 1, TorA–GFP; lane 2, TorA (1–676)–GFP; lane 3, Ax3. Bar, 10 μm.
Mentions: Investigation of the subcellular localization of TorA–GFP and its colocalization with mitochondria revealed that TorA displayed some unusual properties for an integral component of mitochondria. Using immunoblots (Fig. 9 A), we showed that nearly all TorA–GFP was localized to the particulate fraction. After extraction of the particulate fraction with Triton X-100, TorA–GFP localized to the Triton X-100–insoluble fraction. Under these conditions, ∼70% of the mitochondrial marker TopA (Komori et al. 1997) was solubilized (data not shown). As shown by fluorescence microscopy, Triton X-100 extraction does not disrupt the TorA–GFP clusters (Fig. 9 B). Surprisingly, TorA–GFP remained insoluble, even after treatment of the particulate fraction with Triton X-100 in 200 mM sodium carbonate (Fig. 9A and Fig. C). These fractions do not contain mitochondria or cytoskeleton, suggesting that TorA–GFP may be part of a novel structure. TorA–GFP was the predominant protein in the final fraction, which contained only six or seven major protein bands (Fig. 9 C). The coiled coil region in TorA is not required for these properties, since a truncated version, TorA (1–676), lacking the coiled coil region, still targeted GFP to the Triton/sodium carbonate–insoluble fraction (Fig. 9 C).

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