Limits...
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.

Show MeSH

Related in: MedlinePlus

Restriction map around the Dictyostelium gapA gene  and the tag integration site of the REMI mutant, D42-2. Genomic  DNA from the D42-2 strain was digested with the restriction enzymes indicated, and then analyzed by Southern blotting using  linearized pUC118, a portion of the tag, as a probe. Information  obtained on restriction analysis of the rescued plasmids is also included. The four exons of the gapA gene are indicated by thick  lines. bsr, blasticidin S-resistance marker.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139833&req=5

Figure 1: Restriction map around the Dictyostelium gapA gene and the tag integration site of the REMI mutant, D42-2. Genomic DNA from the D42-2 strain was digested with the restriction enzymes indicated, and then analyzed by Southern blotting using linearized pUC118, a portion of the tag, as a probe. Information obtained on restriction analysis of the rescued plasmids is also included. The four exons of the gapA gene are indicated by thick lines. bsr, blasticidin S-resistance marker.

Mentions: The general methods for recombinant DNA were described previously (Sambrook et al., 1989). Fragments of the gapA gene were cloned from the genomic DNA of the D42-2 strain by the plasmid rescue method as previously described (Adachi et al., 1994), except that Escherichia coli STBL2 (GIBCO BRL) was used as the host for the plasmids to prevent deletion of the Dictyostelium DNA in E. coli cells. First, EcoRI (12.6-kb) and HindIII (7.5-kb) fragments containing E. coli plasmid pUC118 (Vieira and Messing, 1987) with the 5′ or 3′ half of the gapA gene were rescued (see Fig. 1) and termed p42-2Eco and p42-2Hind, respectively. The DNA sequences of these fragments were determined with an SQ5500 DNA sequencer (Hitachi Co., Tokyo, Japan) and a Thermo Sequenase core sequencing kit with 7-deaza-dGTP (Amersham Corp.). The intact gapA gene was reconstructed as a 4.1-kb HindIII–ClaI fragment as follows. The gene fragment containing the insertion point of the D42-2 mutant was amplified by PCR from AX2 genomic DNA using an LA-PCR kit (Takara, Tokyo, Japan). The amplified DNA was cloned into pUC118 (Vieira and Messing, 1987), and the sequence from the BamHI site to the BsmI site of the insert was confirmed to be identical to the corresponding regions of the plasmid-rescued fragments. By ligation of this BamHI–BsmI fragment with the rescued HindIII–BamHI (p42-1Eco) and BsmI–ClaI (p42-2Hind) fragments, and cloning into pUC119 (Vieira and Messing, 1987), plasmid p42-2CPX carrying the intact gapA gene was obtained. The reconstructed HindIII–ClaI fragment was inserted into the Dictyostelium shuttle vector, pATANB43 (Dynes and Firtel, 1989), using the linker sequences to construct p43-L, which was used for the genetic complementation experiments.


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)

Restriction map around the Dictyostelium gapA gene  and the tag integration site of the REMI mutant, D42-2. Genomic  DNA from the D42-2 strain was digested with the restriction enzymes indicated, and then analyzed by Southern blotting using  linearized pUC118, a portion of the tag, as a probe. Information  obtained on restriction analysis of the rescued plasmids is also included. The four exons of the gapA gene are indicated by thick  lines. bsr, blasticidin S-resistance marker.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Restriction map around the Dictyostelium gapA gene and the tag integration site of the REMI mutant, D42-2. Genomic DNA from the D42-2 strain was digested with the restriction enzymes indicated, and then analyzed by Southern blotting using linearized pUC118, a portion of the tag, as a probe. Information obtained on restriction analysis of the rescued plasmids is also included. The four exons of the gapA gene are indicated by thick lines. bsr, blasticidin S-resistance marker.
Mentions: The general methods for recombinant DNA were described previously (Sambrook et al., 1989). Fragments of the gapA gene were cloned from the genomic DNA of the D42-2 strain by the plasmid rescue method as previously described (Adachi et al., 1994), except that Escherichia coli STBL2 (GIBCO BRL) was used as the host for the plasmids to prevent deletion of the Dictyostelium DNA in E. coli cells. First, EcoRI (12.6-kb) and HindIII (7.5-kb) fragments containing E. coli plasmid pUC118 (Vieira and Messing, 1987) with the 5′ or 3′ half of the gapA gene were rescued (see Fig. 1) and termed p42-2Eco and p42-2Hind, respectively. The DNA sequences of these fragments were determined with an SQ5500 DNA sequencer (Hitachi Co., Tokyo, Japan) and a Thermo Sequenase core sequencing kit with 7-deaza-dGTP (Amersham Corp.). The intact gapA gene was reconstructed as a 4.1-kb HindIII–ClaI fragment as follows. The gene fragment containing the insertion point of the D42-2 mutant was amplified by PCR from AX2 genomic DNA using an LA-PCR kit (Takara, Tokyo, Japan). The amplified DNA was cloned into pUC118 (Vieira and Messing, 1987), and the sequence from the BamHI site to the BsmI site of the insert was confirmed to be identical to the corresponding regions of the plasmid-rescued fragments. By ligation of this BamHI–BsmI fragment with the rescued HindIII–BamHI (p42-1Eco) and BsmI–ClaI (p42-2Hind) fragments, and cloning into pUC119 (Vieira and Messing, 1987), plasmid p42-2CPX carrying the intact gapA gene was obtained. The reconstructed HindIII–ClaI fragment was inserted into the Dictyostelium shuttle vector, pATANB43 (Dynes and Firtel, 1989), using the linker sequences to construct p43-L, which was used for the genetic complementation experiments.

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