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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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Disruption of the  rasG gene. (a) Schematic  representation of the cloning  strategy employed to disrupt  the rasG gene. A 1.7-kb fragment encoding the cDNA for  the blasticidin resistance  gene (bsr) driven by the constitutive actin15 promoter  was inserted by homologous  recombination into the rasG  promoter between the promoter and the ATG start  codon. A probe from the  rasG coding sequence  (shaded bar) was used to detect correct disruptants by  Southern blotting of genomic  DNA. The expected bands in  the parental strain and disruptants are indicated by  dotted lines. (b) Southern  blot of rasG− and wild-type  parental genomic DNA. Nuclear DNA from strains IR15  (rasG−) and AX2 (wt) was  digested with EcoRI and  HindIII, separated on an  0.8% agarose gel, blotted  onto nylon, and probed with  the rasG coding sequence  (see above). The 1.9-kb parental band and 3.2-kb rasG−  disrupted band are marked. (C) Western blot of rasG− and AX2 wild-type cells. Whole cell lysates were separated by PAGE using a  15% acrylamide gel, blotted onto PVDF, and probed with the general Ras antibody Y13-259 (left) and a RasG specific antibody (right).  Y13-259 recognizes several different Dictyostelium Ras proteins with varying efficiency.
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Figure 1: Disruption of the rasG gene. (a) Schematic representation of the cloning strategy employed to disrupt the rasG gene. A 1.7-kb fragment encoding the cDNA for the blasticidin resistance gene (bsr) driven by the constitutive actin15 promoter was inserted by homologous recombination into the rasG promoter between the promoter and the ATG start codon. A probe from the rasG coding sequence (shaded bar) was used to detect correct disruptants by Southern blotting of genomic DNA. The expected bands in the parental strain and disruptants are indicated by dotted lines. (b) Southern blot of rasG− and wild-type parental genomic DNA. Nuclear DNA from strains IR15 (rasG−) and AX2 (wt) was digested with EcoRI and HindIII, separated on an 0.8% agarose gel, blotted onto nylon, and probed with the rasG coding sequence (see above). The 1.9-kb parental band and 3.2-kb rasG− disrupted band are marked. (C) Western blot of rasG− and AX2 wild-type cells. Whole cell lysates were separated by PAGE using a 15% acrylamide gel, blotted onto PVDF, and probed with the general Ras antibody Y13-259 (left) and a RasG specific antibody (right). Y13-259 recognizes several different Dictyostelium Ras proteins with varying efficiency.

Mentions: Cells containing a disrupted rasG gene were generated by homologous recombination by the strategy shown in Fig. 1 A. A construct was made containing 1.9 kb of rasG genomic DNA (Robbins et al., 1992), with a bsr marker (Sutoh, 1993) inserted between the promoter and coding sequence of the gene, in the same orientation so the strong act8 terminator blocked any read-through from the rasG or bsr promoters. The construct was transfected into AX2 cells, and transformants were cloned after 7 d of blasticidin selection. 2 independent clones, out of 74 examined, were found to contain a disruption in rasG (Fig. 1 B), with the rest apparently containing nonhomologous integration of the vector. Both independent lines were found to behave similarly, so one (IR15) was used for all the work described here. When Western blots are probed with the broad-spectrum Ras antibody Y13-259 (Furth et al., 1982), disrupted cells show approximately half the wild-type level of Ras proteins (Fig. 1 C, left). Y13-259 detects the diverged RasB and RasC proteins, albeit less well than RasG or RasD. The Ras protein seen in IR15 therefore presumably derives from the rasB and rasC genes (which are expressed at lower levels than rasG in wild-type cells) or rasD (which is barely expressed in growing wild-type cells). No RasG protein is found in disruptants when a specific RasG antibody is used (Fig. 1 C, right).


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)

Disruption of the  rasG gene. (a) Schematic  representation of the cloning  strategy employed to disrupt  the rasG gene. A 1.7-kb fragment encoding the cDNA for  the blasticidin resistance  gene (bsr) driven by the constitutive actin15 promoter  was inserted by homologous  recombination into the rasG  promoter between the promoter and the ATG start  codon. A probe from the  rasG coding sequence  (shaded bar) was used to detect correct disruptants by  Southern blotting of genomic  DNA. The expected bands in  the parental strain and disruptants are indicated by  dotted lines. (b) Southern  blot of rasG− and wild-type  parental genomic DNA. Nuclear DNA from strains IR15  (rasG−) and AX2 (wt) was  digested with EcoRI and  HindIII, separated on an  0.8% agarose gel, blotted  onto nylon, and probed with  the rasG coding sequence  (see above). The 1.9-kb parental band and 3.2-kb rasG−  disrupted band are marked. (C) Western blot of rasG− and AX2 wild-type cells. Whole cell lysates were separated by PAGE using a  15% acrylamide gel, blotted onto PVDF, and probed with the general Ras antibody Y13-259 (left) and a RasG specific antibody (right).  Y13-259 recognizes several different Dictyostelium Ras proteins with varying efficiency.
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Related In: Results  -  Collection

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Figure 1: Disruption of the rasG gene. (a) Schematic representation of the cloning strategy employed to disrupt the rasG gene. A 1.7-kb fragment encoding the cDNA for the blasticidin resistance gene (bsr) driven by the constitutive actin15 promoter was inserted by homologous recombination into the rasG promoter between the promoter and the ATG start codon. A probe from the rasG coding sequence (shaded bar) was used to detect correct disruptants by Southern blotting of genomic DNA. The expected bands in the parental strain and disruptants are indicated by dotted lines. (b) Southern blot of rasG− and wild-type parental genomic DNA. Nuclear DNA from strains IR15 (rasG−) and AX2 (wt) was digested with EcoRI and HindIII, separated on an 0.8% agarose gel, blotted onto nylon, and probed with the rasG coding sequence (see above). The 1.9-kb parental band and 3.2-kb rasG− disrupted band are marked. (C) Western blot of rasG− and AX2 wild-type cells. Whole cell lysates were separated by PAGE using a 15% acrylamide gel, blotted onto PVDF, and probed with the general Ras antibody Y13-259 (left) and a RasG specific antibody (right). Y13-259 recognizes several different Dictyostelium Ras proteins with varying efficiency.
Mentions: Cells containing a disrupted rasG gene were generated by homologous recombination by the strategy shown in Fig. 1 A. A construct was made containing 1.9 kb of rasG genomic DNA (Robbins et al., 1992), with a bsr marker (Sutoh, 1993) inserted between the promoter and coding sequence of the gene, in the same orientation so the strong act8 terminator blocked any read-through from the rasG or bsr promoters. The construct was transfected into AX2 cells, and transformants were cloned after 7 d of blasticidin selection. 2 independent clones, out of 74 examined, were found to contain a disruption in rasG (Fig. 1 B), with the rest apparently containing nonhomologous integration of the vector. Both independent lines were found to behave similarly, so one (IR15) was used for all the work described here. When Western blots are probed with the broad-spectrum Ras antibody Y13-259 (Furth et al., 1982), disrupted cells show approximately half the wild-type level of Ras proteins (Fig. 1 C, left). Y13-259 detects the diverged RasB and RasC proteins, albeit less well than RasG or RasD. The Ras protein seen in IR15 therefore presumably derives from the rasB and rasC genes (which are expressed at lower levels than rasG in wild-type cells) or rasD (which is barely expressed in growing wild-type cells). No RasG protein is found in disruptants when a specific RasG antibody is used (Fig. 1 C, right).

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