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Dictyostelium IQGAP-related protein specifically involved in the completion of cytokinesis.

Adachi H, Takahashi Y, Hasebe T, Shirouzu M, Yokoyama S, Sutoh K - J. Cell Biol. (1997)

Bottom Line: These results indicate that the GAPA protein is specifically involved in the completion of cytokinesis.Recently, it was reported that IQGAPs are putative effectors for Rac and CDC42, members of the Rho family of GTPases, and participate in reorganization of the actin cytoskeleton.Thus, it is possible that Dictyostelium GAPA participates in the severing of the midbody by regulating the actin cytoskeleton through an interaction with a member of small GTPases.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153. f37771@m-unix.cc.u-tokyo.ac.jp

ABSTRACT
The gapA gene encoding a novel RasGTPase-activating protein (RasGAP)-related protein was found to be disrupted in a cytokinesis mutant of Dictyostelium that grows as giant and multinucleate cells in a dish culture. The predicted sequence of the GAPA protein showed considerable homology to those of Gap1/Sar1 from fission yeast and the COOH-terminal half of mammalian IQGAPs, the similarity extending beyond the RasGAP-related domain. In suspension culture, gapA- cells showed normal growth in terms of the increase in cell mass, but cytokinesis inefficiently occurred to produce spherical giant cells. Time-lapse recording of the dynamics of cell division in a dish culture revealed that, in the case of gapA- cells, cytokinesis was very frequently reversed at the step in which the midbody connecting the daughter cells should be severed. Earlier steps of cytokinesis in the gapA- cells seemed to be normal, since myosin II was accumulated at the cleavage furrow. Upon starvation, gapA- cells developed and formed fruiting bodies with viable spores, like the wild-type cells. These results indicate that the GAPA protein is specifically involved in the completion of cytokinesis. Recently, it was reported that IQGAPs are putative effectors for Rac and CDC42, members of the Rho family of GTPases, and participate in reorganization of the actin cytoskeleton. Thus, it is possible that Dictyostelium GAPA participates in the severing of the midbody by regulating the actin cytoskeleton through an interaction with a member of small GTPases.

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Related in: MedlinePlus

Southern blot (A) and reverse transcription–PCR (B)  analyses of wild-type AX2 (W) and mutant D42-2 (M) cells. (A)  2.5 μg of genomic DNA digested with either ClaI or HindIII was  loaded into each lane. The transferred DNA was probed with the  full-length gapA cDNA. (B) DNA amplified with the same amount  of total RNA and specific primers was loaded onto each lane. For  details, see Materials and Methods.
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Figure 3: Southern blot (A) and reverse transcription–PCR (B) analyses of wild-type AX2 (W) and mutant D42-2 (M) cells. (A) 2.5 μg of genomic DNA digested with either ClaI or HindIII was loaded into each lane. The transferred DNA was probed with the full-length gapA cDNA. (B) DNA amplified with the same amount of total RNA and specific primers was loaded onto each lane. For details, see Materials and Methods.

Mentions: Second, genetic complementation of the mutant phenotype with the intact gene was examined. To clone the entire gene, EcoRI and HindIII fragments of the D42-2 DNA including the 5′ and 3′ halves of the disrupted gene (Fig. 1) were rescued. Through ligation of the rescued fragments, the intact HindIII–ClaI fragment (4.1 kb) was reconstructed and inserted into a Dictyostelium shuttle vector, and the resultant plasmid, p43-L, was used to transform the D42-2 strain. All the transformants showed the wild-type phenotype (Fig. 2, D and H), indicating that this 4.1-kb fragment carries the gene involved in cytokinesis. Southern blot analysis showed that the gene exists as a single copy (Fig. 3 A). The absence of a transcript of the gene in vegetative mutant cells was confirmed by the reverse transcription– PCR method, using the myosin II heavy chain transcript as a control (Fig. 3 B).


Dictyostelium IQGAP-related protein specifically involved in the completion of cytokinesis.

Adachi H, Takahashi Y, Hasebe T, Shirouzu M, Yokoyama S, Sutoh K - J. Cell Biol. (1997)

Southern blot (A) and reverse transcription–PCR (B)  analyses of wild-type AX2 (W) and mutant D42-2 (M) cells. (A)  2.5 μg of genomic DNA digested with either ClaI or HindIII was  loaded into each lane. The transferred DNA was probed with the  full-length gapA cDNA. (B) DNA amplified with the same amount  of total RNA and specific primers was loaded onto each lane. For  details, see Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139833&req=5

Figure 3: Southern blot (A) and reverse transcription–PCR (B) analyses of wild-type AX2 (W) and mutant D42-2 (M) cells. (A) 2.5 μg of genomic DNA digested with either ClaI or HindIII was loaded into each lane. The transferred DNA was probed with the full-length gapA cDNA. (B) DNA amplified with the same amount of total RNA and specific primers was loaded onto each lane. For details, see Materials and Methods.
Mentions: Second, genetic complementation of the mutant phenotype with the intact gene was examined. To clone the entire gene, EcoRI and HindIII fragments of the D42-2 DNA including the 5′ and 3′ halves of the disrupted gene (Fig. 1) were rescued. Through ligation of the rescued fragments, the intact HindIII–ClaI fragment (4.1 kb) was reconstructed and inserted into a Dictyostelium shuttle vector, and the resultant plasmid, p43-L, was used to transform the D42-2 strain. All the transformants showed the wild-type phenotype (Fig. 2, D and H), indicating that this 4.1-kb fragment carries the gene involved in cytokinesis. Southern blot analysis showed that the gene exists as a single copy (Fig. 3 A). The absence of a transcript of the gene in vegetative mutant cells was confirmed by the reverse transcription– PCR method, using the myosin II heavy chain transcript as a control (Fig. 3 B).

Bottom Line: These results indicate that the GAPA protein is specifically involved in the completion of cytokinesis.Recently, it was reported that IQGAPs are putative effectors for Rac and CDC42, members of the Rho family of GTPases, and participate in reorganization of the actin cytoskeleton.Thus, it is possible that Dictyostelium GAPA participates in the severing of the midbody by regulating the actin cytoskeleton through an interaction with a member of small GTPases.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153. f37771@m-unix.cc.u-tokyo.ac.jp

ABSTRACT
The gapA gene encoding a novel RasGTPase-activating protein (RasGAP)-related protein was found to be disrupted in a cytokinesis mutant of Dictyostelium that grows as giant and multinucleate cells in a dish culture. The predicted sequence of the GAPA protein showed considerable homology to those of Gap1/Sar1 from fission yeast and the COOH-terminal half of mammalian IQGAPs, the similarity extending beyond the RasGAP-related domain. In suspension culture, gapA- cells showed normal growth in terms of the increase in cell mass, but cytokinesis inefficiently occurred to produce spherical giant cells. Time-lapse recording of the dynamics of cell division in a dish culture revealed that, in the case of gapA- cells, cytokinesis was very frequently reversed at the step in which the midbody connecting the daughter cells should be severed. Earlier steps of cytokinesis in the gapA- cells seemed to be normal, since myosin II was accumulated at the cleavage furrow. Upon starvation, gapA- cells developed and formed fruiting bodies with viable spores, like the wild-type cells. These results indicate that the GAPA protein is specifically involved in the completion of cytokinesis. Recently, it was reported that IQGAPs are putative effectors for Rac and CDC42, members of the Rho family of GTPases, and participate in reorganization of the actin cytoskeleton. Thus, it is possible that Dictyostelium GAPA participates in the severing of the midbody by regulating the actin cytoskeleton through an interaction with a member of small GTPases.

Show MeSH
Related in: MedlinePlus