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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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Cytokinesis in  wild-type and rasG− cells. (a)  Video frames showing cytokinesis in wild-type and rasG−  cells. Cells were observed by  time-lapse videomicroscopy  after synchronization by  aphidicolin. Representative  cytokineses from each strain  are shown. (b) Synchronized  wild-type and rasG− cells  were stained with Texas red-conjugated phalloidin and  Hoechst 33342 to visualize  F-actin and nuclei, respectively. RasG− cells show a  characteristic failure to separate after formation of the  cleavage furrow. Bar, 10 μm.
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Figure 8: Cytokinesis in wild-type and rasG− cells. (a) Video frames showing cytokinesis in wild-type and rasG− cells. Cells were observed by time-lapse videomicroscopy after synchronization by aphidicolin. Representative cytokineses from each strain are shown. (b) Synchronized wild-type and rasG− cells were stained with Texas red-conjugated phalloidin and Hoechst 33342 to visualize F-actin and nuclei, respectively. RasG− cells show a characteristic failure to separate after formation of the cleavage furrow. Bar, 10 μm.

Mentions: We wished to discern whether rasG− cells were unable to perform cytokinesis because of a mechanical defect preventing the physical separation of the daughter cells, like that in mhcA− cells, or because of a problem with the initiation of cytokinesis due to the loss of a signal communicating the completion of nuclear division to the cytoplasm. To answer these questions, synchronized cells in mitosis were required, but we found existing methods of synchronizing Dictyostelium cells unsatisfactory. By releasing cells whose cell cycle progression was blocked by aphidicolin (an inhibitor of DNA polymerase; Pedrali-Noy et al., 1980), we observed good synchrony. 50% of cells passed through mitosis ∼4 h after release. When synchronized wild-type cells are observed by time-lapse video microscopy, cytokinesis is observed as a rounding of the cell followed by a rapid pinching movement, taking ∼5 min to complete (Fig. 8 A, top rows). The initial stages of cytokinesis in rasG− cells appear indistinguishable from those in wild-type cells (Fig. 8 A, lower rows). However, the daughter cells appear unable to separate completely and remain attached by a thin bridge of cytoplasm, which is eventually resolved by traction between the daughter cells (Fig. 8 A).


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)

Cytokinesis in  wild-type and rasG− cells. (a)  Video frames showing cytokinesis in wild-type and rasG−  cells. Cells were observed by  time-lapse videomicroscopy  after synchronization by  aphidicolin. Representative  cytokineses from each strain  are shown. (b) Synchronized  wild-type and rasG− cells  were stained with Texas red-conjugated phalloidin and  Hoechst 33342 to visualize  F-actin and nuclei, respectively. RasG− cells show a  characteristic failure to separate after formation of the  cleavage furrow. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2141629&req=5

Figure 8: Cytokinesis in wild-type and rasG− cells. (a) Video frames showing cytokinesis in wild-type and rasG− cells. Cells were observed by time-lapse videomicroscopy after synchronization by aphidicolin. Representative cytokineses from each strain are shown. (b) Synchronized wild-type and rasG− cells were stained with Texas red-conjugated phalloidin and Hoechst 33342 to visualize F-actin and nuclei, respectively. RasG− cells show a characteristic failure to separate after formation of the cleavage furrow. Bar, 10 μm.
Mentions: We wished to discern whether rasG− cells were unable to perform cytokinesis because of a mechanical defect preventing the physical separation of the daughter cells, like that in mhcA− cells, or because of a problem with the initiation of cytokinesis due to the loss of a signal communicating the completion of nuclear division to the cytoplasm. To answer these questions, synchronized cells in mitosis were required, but we found existing methods of synchronizing Dictyostelium cells unsatisfactory. By releasing cells whose cell cycle progression was blocked by aphidicolin (an inhibitor of DNA polymerase; Pedrali-Noy et al., 1980), we observed good synchrony. 50% of cells passed through mitosis ∼4 h after release. When synchronized wild-type cells are observed by time-lapse video microscopy, cytokinesis is observed as a rounding of the cell followed by a rapid pinching movement, taking ∼5 min to complete (Fig. 8 A, top rows). The initial stages of cytokinesis in rasG− cells appear indistinguishable from those in wild-type cells (Fig. 8 A, lower rows). However, the daughter cells appear unable to separate completely and remain attached by a thin bridge of cytoplasm, which is eventually resolved by traction between the daughter cells (Fig. 8 A).

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