Limits...
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.

Show MeSH
Chemotaxis and motility analysis of torA− and Mek1/torA− cells. (A) Perimeter tracks of control, torA−, and Mek1/torA− cells moving in a spatial gradient of cAMP generated across the bridge of a gradient chamber in which buffer alone was placed in one trough and buffer plus 10−6 M cAMP in the other trough (SOURCE) bordering the bridge. The motion analysis of individual cells was initiated after 5 min, the time necessary to establish a steep gradient of cAMP. (B) Transfilter assay for chemotaxis of torA− and control cells. 1,000 cells were applied to multiwell chemotaxis chambers with 5-μm pores. 10−6 M cAMP or DB was added to the well underneath the filter, and after 3 h cells that had reached the bottom of the well were counted. (C) The behavior of individual control and torA− cells in natural aggregation territories in submerged cultures. Three representative cells of each cell type in close proximity but not touching were videorecorded for 50–70 min, and motion analyzed. In each case, instantaneous velocity was plotted as a function of time. Periodicity, in minutes, was computed from velocity peaks. To the right of each velocity plot is the centroid track of a portion of the analysis. Troughs, which represent the deduced peak (P) and back (B) of each wave are numbered in the velocity plot and respective centroid track for comparison. In the case of control cells, a common aggregation center (agg center) was identifiable. Arrows reflect the general direction of translocation. No common aggregation center was identifiable in the torA− territories.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196008&req=5

Figure 3: Chemotaxis and motility analysis of torA− and Mek1/torA− cells. (A) Perimeter tracks of control, torA−, and Mek1/torA− cells moving in a spatial gradient of cAMP generated across the bridge of a gradient chamber in which buffer alone was placed in one trough and buffer plus 10−6 M cAMP in the other trough (SOURCE) bordering the bridge. The motion analysis of individual cells was initiated after 5 min, the time necessary to establish a steep gradient of cAMP. (B) Transfilter assay for chemotaxis of torA− and control cells. 1,000 cells were applied to multiwell chemotaxis chambers with 5-μm pores. 10−6 M cAMP or DB was added to the well underneath the filter, and after 3 h cells that had reached the bottom of the well were counted. (C) The behavior of individual control and torA− cells in natural aggregation territories in submerged cultures. Three representative cells of each cell type in close proximity but not touching were videorecorded for 50–70 min, and motion analyzed. In each case, instantaneous velocity was plotted as a function of time. Periodicity, in minutes, was computed from velocity peaks. To the right of each velocity plot is the centroid track of a portion of the analysis. Troughs, which represent the deduced peak (P) and back (B) of each wave are numbered in the velocity plot and respective centroid track for comparison. In the case of control cells, a common aggregation center (agg center) was identifiable. Arrows reflect the general direction of translocation. No common aggregation center was identifiable in the torA− territories.

Mentions: The defects in motility and/or chemotaxis suggested from time-lapse movies were further analyzed by computer-assisted methods (Table and Fig. 3) (Soll 1995; Soll and Voss 1998; Wessels et al. 2000). In spatial gradients of cAMP, the morphology parameters (length, area, and roundness) of control, torA− and Mek1/torA−, were similar. Velocity was reduced 30%, and the rate of turning was higher in torA− cells relative to control cells (Table ). The defect in velocity was not reversed in Mek1/torA− cells, whereas that in directional change was. The control and Mek1/torA− cells carried out chemotaxis up the spatial gradient in a highly efficient manner, exhibiting mean chemotactic indices of +0.60 and +0.52, respectively. The torA− cells exhibited a mean chemotactic index of only +0.12, close to that of randomly moving cells.


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)

Chemotaxis and motility analysis of torA− and Mek1/torA− cells. (A) Perimeter tracks of control, torA−, and Mek1/torA− cells moving in a spatial gradient of cAMP generated across the bridge of a gradient chamber in which buffer alone was placed in one trough and buffer plus 10−6 M cAMP in the other trough (SOURCE) bordering the bridge. The motion analysis of individual cells was initiated after 5 min, the time necessary to establish a steep gradient of cAMP. (B) Transfilter assay for chemotaxis of torA− and control cells. 1,000 cells were applied to multiwell chemotaxis chambers with 5-μm pores. 10−6 M cAMP or DB was added to the well underneath the filter, and after 3 h cells that had reached the bottom of the well were counted. (C) The behavior of individual control and torA− cells in natural aggregation territories in submerged cultures. Three representative cells of each cell type in close proximity but not touching were videorecorded for 50–70 min, and motion analyzed. In each case, instantaneous velocity was plotted as a function of time. Periodicity, in minutes, was computed from velocity peaks. To the right of each velocity plot is the centroid track of a portion of the analysis. Troughs, which represent the deduced peak (P) and back (B) of each wave are numbered in the velocity plot and respective centroid track for comparison. In the case of control cells, a common aggregation center (agg center) was identifiable. Arrows reflect the general direction of translocation. No common aggregation center was identifiable in the torA− territories.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Chemotaxis and motility analysis of torA− and Mek1/torA− cells. (A) Perimeter tracks of control, torA−, and Mek1/torA− cells moving in a spatial gradient of cAMP generated across the bridge of a gradient chamber in which buffer alone was placed in one trough and buffer plus 10−6 M cAMP in the other trough (SOURCE) bordering the bridge. The motion analysis of individual cells was initiated after 5 min, the time necessary to establish a steep gradient of cAMP. (B) Transfilter assay for chemotaxis of torA− and control cells. 1,000 cells were applied to multiwell chemotaxis chambers with 5-μm pores. 10−6 M cAMP or DB was added to the well underneath the filter, and after 3 h cells that had reached the bottom of the well were counted. (C) The behavior of individual control and torA− cells in natural aggregation territories in submerged cultures. Three representative cells of each cell type in close proximity but not touching were videorecorded for 50–70 min, and motion analyzed. In each case, instantaneous velocity was plotted as a function of time. Periodicity, in minutes, was computed from velocity peaks. To the right of each velocity plot is the centroid track of a portion of the analysis. Troughs, which represent the deduced peak (P) and back (B) of each wave are numbered in the velocity plot and respective centroid track for comparison. In the case of control cells, a common aggregation center (agg center) was identifiable. Arrows reflect the general direction of translocation. No common aggregation center was identifiable in the torA− territories.
Mentions: The defects in motility and/or chemotaxis suggested from time-lapse movies were further analyzed by computer-assisted methods (Table and Fig. 3) (Soll 1995; Soll and Voss 1998; Wessels et al. 2000). In spatial gradients of cAMP, the morphology parameters (length, area, and roundness) of control, torA− and Mek1/torA−, were similar. Velocity was reduced 30%, and the rate of turning was higher in torA− cells relative to control cells (Table ). The defect in velocity was not reversed in Mek1/torA− cells, whereas that in directional change was. The control and Mek1/torA− cells carried out chemotaxis up the spatial gradient in a highly efficient manner, exhibiting mean chemotactic indices of +0.60 and +0.52, respectively. The torA− cells exhibited a mean chemotactic index of only +0.12, close to that of randomly moving cells.

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