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Dictyostelium RasG is required for normal motility and cytokinesis, but not growth.

Tuxworth RI, Cheetham JL, Machesky LM, Spiegelmann GB, Weeks G, Insall RH - J. Cell Biol. (1997)

Bottom Line: Unexpectedly, RasG- cells are able to grow at nearly wild-type rates.Despite their lack of polarity and abnormal cytoskeleton, mutant cells perform normal chemotaxis.Taken together, these data suggest a principal role for RasG in coordination of cell movement and control of the cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom.

ABSTRACT
RasG is the most abundant Ras protein in growing Dictyostelium cells and the closest relative of mammalian Ras proteins. We have generated mutants in which expression of RasG is completely abolished. Unexpectedly, RasG- cells are able to grow at nearly wild-type rates. However, they exhibit defective cell movement and a wide range of defects in the control of the actin cytoskeleton, including a loss of cell polarity, absence of normal lamellipodia, formation of unusual small, punctate polymerized actin structures, and a large number of abnormally long filopodia. Despite their lack of polarity and abnormal cytoskeleton, mutant cells perform normal chemotaxis. However, rasG- cells are unable to perform normal cytokinesis, becoming multinucleate when grown in suspension culture. Taken together, these data suggest a principal role for RasG in coordination of cell movement and control of the cytoskeleton.

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Normal myosin function in rasG− cells. Cells adhering  to glass coverslips were perfused with buffer containing 0.01%  azide. Cells lacking myosin II (mhcA−, ▴) remain flattened and  adherent, while both wild-type cells (▪) and rasG− cells (•)  round up and detach from the glass.
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Figure 7: Normal myosin function in rasG− cells. Cells adhering to glass coverslips were perfused with buffer containing 0.01% azide. Cells lacking myosin II (mhcA−, ▴) remain flattened and adherent, while both wild-type cells (▪) and rasG− cells (•) round up and detach from the glass.

Mentions: Nearly all the cytokinesis mutants that have been previously described in Dictyostelium have defects in the structural proteins of the cytoskeleton. The most comprehensively studied mutants lack myosin II function (De Lozanne and Spudich, 1987; Knecht and Loomis, 1987; Chen et al., 1995). These cells are unable to complete cytokinesis because of an inability to generate the contractile ring which pinches the daughter cells apart. The phenotype of rasG disruptants is not caused by a lack of myosin II activity. Treatment of cells with sodium azide causes a rapid depletion of cellular ATP levels, which causes myosin II to bind irreversibly to actin (Patterson and Spudich, 1995). When wild-type cells in a perfusion chamber are treated with 0.01% sodium azide, they round up and are washed off the substratum. mhcA− cells, which lack the heavy chain of myosin II, remain flattened and stuck down. Azide treatment caused nearly normal rounding and loss of rasG− cells (Fig. 7). The small number of mutant cells that did not wash away had rounded up in the same way as wild-type cells but were still adhering to the glass coverslip. This seems to be caused by the aberrant adhesion of rasG− cells rather than any lack of myosin II function.


Dictyostelium RasG is required for normal motility and cytokinesis, but not growth.

Tuxworth RI, Cheetham JL, Machesky LM, Spiegelmann GB, Weeks G, Insall RH - J. Cell Biol. (1997)

Normal myosin function in rasG− cells. Cells adhering  to glass coverslips were perfused with buffer containing 0.01%  azide. Cells lacking myosin II (mhcA−, ▴) remain flattened and  adherent, while both wild-type cells (▪) and rasG− cells (•)  round up and detach from the glass.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141629&req=5

Figure 7: Normal myosin function in rasG− cells. Cells adhering to glass coverslips were perfused with buffer containing 0.01% azide. Cells lacking myosin II (mhcA−, ▴) remain flattened and adherent, while both wild-type cells (▪) and rasG− cells (•) round up and detach from the glass.
Mentions: Nearly all the cytokinesis mutants that have been previously described in Dictyostelium have defects in the structural proteins of the cytoskeleton. The most comprehensively studied mutants lack myosin II function (De Lozanne and Spudich, 1987; Knecht and Loomis, 1987; Chen et al., 1995). These cells are unable to complete cytokinesis because of an inability to generate the contractile ring which pinches the daughter cells apart. The phenotype of rasG disruptants is not caused by a lack of myosin II activity. Treatment of cells with sodium azide causes a rapid depletion of cellular ATP levels, which causes myosin II to bind irreversibly to actin (Patterson and Spudich, 1995). When wild-type cells in a perfusion chamber are treated with 0.01% sodium azide, they round up and are washed off the substratum. mhcA− cells, which lack the heavy chain of myosin II, remain flattened and stuck down. Azide treatment caused nearly normal rounding and loss of rasG− cells (Fig. 7). The small number of mutant cells that did not wash away had rounded up in the same way as wild-type cells but were still adhering to the glass coverslip. This seems to be caused by the aberrant adhesion of rasG− cells rather than any lack of myosin II function.

Bottom Line: Unexpectedly, RasG- cells are able to grow at nearly wild-type rates.Despite their lack of polarity and abnormal cytoskeleton, mutant cells perform normal chemotaxis.Taken together, these data suggest a principal role for RasG in coordination of cell movement and control of the cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom.

ABSTRACT
RasG is the most abundant Ras protein in growing Dictyostelium cells and the closest relative of mammalian Ras proteins. We have generated mutants in which expression of RasG is completely abolished. Unexpectedly, RasG- cells are able to grow at nearly wild-type rates. However, they exhibit defective cell movement and a wide range of defects in the control of the actin cytoskeleton, including a loss of cell polarity, absence of normal lamellipodia, formation of unusual small, punctate polymerized actin structures, and a large number of abnormally long filopodia. Despite their lack of polarity and abnormal cytoskeleton, mutant cells perform normal chemotaxis. However, rasG- cells are unable to perform normal cytokinesis, becoming multinucleate when grown in suspension culture. Taken together, these data suggest a principal role for RasG in coordination of cell movement and control of the cytoskeleton.

Show MeSH
Related in: MedlinePlus