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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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Epifluorescence micrographs of  stationary rasG− cells stained with Texas red– phalloidin to visualize F-actin and Hoechst  33342 to visualize the nuclei. The cells adopt  abnormal morphologies and adhere to the  glass through large pads of F-actin. Small, intensely stained masses of F-actin are also  present in some cells. Bar, 5 μm.
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Figure 4: Epifluorescence micrographs of stationary rasG− cells stained with Texas red– phalloidin to visualize F-actin and Hoechst 33342 to visualize the nuclei. The cells adopt abnormal morphologies and adhere to the glass through large pads of F-actin. Small, intensely stained masses of F-actin are also present in some cells. Bar, 5 μm.

Mentions: The distribution of F-actin is also aberrant in axenically grown rasG− cells. Staining with fluorescent phalloidin (Fig. 3 B) reveals two major differences between rasG− and wild-type. Firstly, the lamellipodia seen at the leading edges of polarized wild-type cells are replaced by large numbers of elongated filopodia. While similar filopodia are seen in a proportion of wild-type cells (∼10–15%; data not shown), they are present in nearly all rasG− cells, frequently in large numbers, often reaching considerably greater lengths than in wild-type cells (Fig. 3 B). Again, nearly all rasG− cells show no obvious polarity; the filopodia appear to be spread randomly around the perimeter of most cells. Wild-type cells in the rounded phase frequently show a continuous, broad cortex of F-actin, with no actin-rich protrusions; equivalent rasG− cells exhibit a similar cortex, but usually also possess long filopodia (Fig. 3 C). Various highly unusual morphologies are also common in rasG− cells, in particular crescent and hour glass shapes (Fig. 4). In these cells the cortex, rather than being continuous, is divided into discrete F-actin–rich lobes, as if the different ends of the cell were trying to move in opposite directions. The nuclei are usually found between the lobes of F-actin, and the two halves of the cells pulling in opposite directions can squeeze the nuclei into cylindrical shapes.


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)

Epifluorescence micrographs of  stationary rasG− cells stained with Texas red– phalloidin to visualize F-actin and Hoechst  33342 to visualize the nuclei. The cells adopt  abnormal morphologies and adhere to the  glass through large pads of F-actin. Small, intensely stained masses of F-actin are also  present in some cells. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Epifluorescence micrographs of stationary rasG− cells stained with Texas red– phalloidin to visualize F-actin and Hoechst 33342 to visualize the nuclei. The cells adopt abnormal morphologies and adhere to the glass through large pads of F-actin. Small, intensely stained masses of F-actin are also present in some cells. Bar, 5 μm.
Mentions: The distribution of F-actin is also aberrant in axenically grown rasG− cells. Staining with fluorescent phalloidin (Fig. 3 B) reveals two major differences between rasG− and wild-type. Firstly, the lamellipodia seen at the leading edges of polarized wild-type cells are replaced by large numbers of elongated filopodia. While similar filopodia are seen in a proportion of wild-type cells (∼10–15%; data not shown), they are present in nearly all rasG− cells, frequently in large numbers, often reaching considerably greater lengths than in wild-type cells (Fig. 3 B). Again, nearly all rasG− cells show no obvious polarity; the filopodia appear to be spread randomly around the perimeter of most cells. Wild-type cells in the rounded phase frequently show a continuous, broad cortex of F-actin, with no actin-rich protrusions; equivalent rasG− cells exhibit a similar cortex, but usually also possess long filopodia (Fig. 3 C). Various highly unusual morphologies are also common in rasG− cells, in particular crescent and hour glass shapes (Fig. 4). In these cells the cortex, rather than being continuous, is divided into discrete F-actin–rich lobes, as if the different ends of the cell were trying to move in opposite directions. The nuclei are usually found between the lobes of F-actin, and the two halves of the cells pulling in opposite directions can squeeze the nuclei into cylindrical shapes.

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