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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 to specific mitochondrial structures. (A–D) Mitotracker staining of wild-type and TorA–GFP-expressing cells. Living wild-type cells (A) and TorA–GFP-expressing cells (B) were stained with Mitotracker red. After staining, some cells were fixed in formaldehyde (C and D). Images show GFP (C) and Mitotracker red (D) fluorescence of one cell. (E–G) Transmission electron microscopy images of TorA–GFP-expressing cells. Examples of three mitochondria from two different TorA–GFP-expressing cells are shown. Arrows indicate submitochondrial bodies apparent in TorA overexpressing cells.
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Figure 8: Localization of TorA to specific mitochondrial structures. (A–D) Mitotracker staining of wild-type and TorA–GFP-expressing cells. Living wild-type cells (A) and TorA–GFP-expressing cells (B) were stained with Mitotracker red. After staining, some cells were fixed in formaldehyde (C and D). Images show GFP (C) and Mitotracker red (D) fluorescence of one cell. (E–G) Transmission electron microscopy images of TorA–GFP-expressing cells. Examples of three mitochondria from two different TorA–GFP-expressing cells are shown. Arrows indicate submitochondrial bodies apparent in TorA overexpressing cells.

Mentions: The clusters appeared to be an intriguing novel mitochondrial structure that stained with the mitochondrial dye Mitotracker (Fig. 8). Expression of wild-type TorA or TorA–GFP resulted in the formation of several structures in the mitochondria that showed intense staining with this dye (Fig. 8 B). The Mitotracker and GFP signals display identical patterns, indicating that the structures observed after Mitotracker staining indeed contain TorA–GFP (Fig. 8C and Fig. D). Importantly, wild-type cells also contained these structures. About 20–30% of wild-type cells showed punctate staining with Mitotracker, but in only a few mitochondria per cell (Fig. 8 A). Thus, the number or size of these structures is enhanced by overexpression of TorA. Mitotracker staining depends on the mitochondrial membrane potential to be specifically imported in mitochondria, suggesting that the novel structure may be a region with high electron potential. Mitotracker does not accumulate at any site outside the mitochondrion and at only one site in each mitochondrion, so the staining of this structure is specific. We also detected some of these structures in torA− mitochondria (data not shown), indicating that the clusters contain other proteins besides TorA. We could not determine whether the had fewer structures. Using electron microscopy, we visualized the clusters as a round electron-dense mass in the mitochondrion (Fig. 8, E–G). The Mitotracker results suggest that these structures also exist in wild-type cells, but they may be harder to find in electron microscopy.


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 to specific mitochondrial structures. (A–D) Mitotracker staining of wild-type and TorA–GFP-expressing cells. Living wild-type cells (A) and TorA–GFP-expressing cells (B) were stained with Mitotracker red. After staining, some cells were fixed in formaldehyde (C and D). Images show GFP (C) and Mitotracker red (D) fluorescence of one cell. (E–G) Transmission electron microscopy images of TorA–GFP-expressing cells. Examples of three mitochondria from two different TorA–GFP-expressing cells are shown. Arrows indicate submitochondrial bodies apparent in TorA overexpressing cells.
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Figure 8: Localization of TorA to specific mitochondrial structures. (A–D) Mitotracker staining of wild-type and TorA–GFP-expressing cells. Living wild-type cells (A) and TorA–GFP-expressing cells (B) were stained with Mitotracker red. After staining, some cells were fixed in formaldehyde (C and D). Images show GFP (C) and Mitotracker red (D) fluorescence of one cell. (E–G) Transmission electron microscopy images of TorA–GFP-expressing cells. Examples of three mitochondria from two different TorA–GFP-expressing cells are shown. Arrows indicate submitochondrial bodies apparent in TorA overexpressing cells.
Mentions: The clusters appeared to be an intriguing novel mitochondrial structure that stained with the mitochondrial dye Mitotracker (Fig. 8). Expression of wild-type TorA or TorA–GFP resulted in the formation of several structures in the mitochondria that showed intense staining with this dye (Fig. 8 B). The Mitotracker and GFP signals display identical patterns, indicating that the structures observed after Mitotracker staining indeed contain TorA–GFP (Fig. 8C and Fig. D). Importantly, wild-type cells also contained these structures. About 20–30% of wild-type cells showed punctate staining with Mitotracker, but in only a few mitochondria per cell (Fig. 8 A). Thus, the number or size of these structures is enhanced by overexpression of TorA. Mitotracker staining depends on the mitochondrial membrane potential to be specifically imported in mitochondria, suggesting that the novel structure may be a region with high electron potential. Mitotracker does not accumulate at any site outside the mitochondrion and at only one site in each mitochondrion, so the staining of this structure is specific. We also detected some of these structures in torA− mitochondria (data not shown), indicating that the clusters contain other proteins besides TorA. We could not determine whether the had fewer structures. Using electron microscopy, we visualized the clusters as a round electron-dense mass in the mitochondrion (Fig. 8, E–G). The Mitotracker results suggest that these structures also exist in wild-type cells, but they may be harder to find in electron microscopy.

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