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First published online 25 August 2004
doi: 10.1242/jcs.01347

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Novel small GTPase subfamily capable of associating with tubulin is required for chromosome segregation

Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

View larger version (54K): [in a new window]   Fig. 1. Identification of Gie as a novel subfamily of the small GTPases. (A) Comparison of amino-acid sequences of human Gie1, Gie2 and other small GTPase members. The amino-acid sequences were aligned using the ClustalW program. Amino-acid residues conserved among all the proteins are shaded in black, and those with 66-99% identity are in light gray. G1-G5 indicate well-conserved regions important for binding to phosphate/Mg2+ and the guanine base. The region considered to be a putative effecter domain is overlined. The C-terminal sequence for prenylation (CAAX box: A, aliphatic; X, any amino acid) and the N-terminal sequence for myristoylation (MG box) are boxed. The human sequence data are available from GenBank/EMBL/DDBJ under the accession numbers AK001564 (Gie1) and BC015408 (Gie2). (B) An unrooted tree constructed using ClustalW with standard parameters and viewed using TreeView. (C) Deduced amino-acid sequences from the indicated genes were aligned using the ClustalW. Amino-acid residues conserved among all the proteins are shaded in black, and those with 75-99% identity are in light gray. The accession numbers of Drosophila melanogaster CG7891 and Caenorhabditis elegans Y57G11C.13 are NP649769 and CAB16514, respectively, in the NCBI Entrez Protein database.

View larger version (46K): [in a new window]   Fig. 2. Expression of Gie in various human tissues and cell lines. (A) Expression of hGie1 and hGie2 mRNAs in human tissues. Northern blots of poly(A)+ RNAs from various human tissues (Clontech) were hybridized with 32P-labeled Gie probes. (B) Extracts from PC12 cells were separated by SDS-PAGE and immunoblotted with pre-immune serum (left), the anti-Gie antibody (middle) or the same antibody that had been incubated with the antigen peptide (right). (C) Extracts from the indicated cell lines were also immunoblotted with the anti-Gie (top) and anti-GAPDH (bottom) antibodies.

View larger version (34K): [in a new window]   Fig. 3. Overexpression of dominant-negative Gie mutants induces abnormal morphology in the chromosomes of HeLa cells. (A) Identification of nucleotide-bound forms of Gie1 and Gie2. HeLa cells were transfected with expression vectors encoding the FLAG-proteins listed at the top and metabolically radiolabeled with 32P. The expressed proteins were immunoprecipitated with an anti-FLAG monoclonal antibody, and nucleotides associating with the proteins were separated by thin-layer chromatography. The radioactivity of GTP and GDP was quantified, and the proportions of GTP-bound form in total Gie proteins are shown at the bottom. (B) HeLa cells were grown on poly-L-lysine-coated coverglasses and transfected with the various mutants of Gie1 or with pCMV5 vector alone. DNA (green) and Gie1 (red) were detected with PicoGreen and the anti-Gie antibody, respectively. Phase-contrast photographs images (right) are also shown. Most of the cells expressing Gie1/T34N and Gie1/N130I exhibited phenotypes characterized as micronuclei (white arrows). Scale bar, 5 µm. (C) The appearance of abnormal nuclei was measured, and the data are represented as percentages of means±s.e.m. from at least three independent experiments (each of 200-400 cells).

View larger version (51K): [in a new window]   Fig. 4. Drosophila Gie is required for chromosome segregation. (A) Reduction of dGie mRNA levels by RNAi. Drosophila S2 cells were cultured with the control dsRNA (left) or dGie dsRNA (right) for 4 days, and the level of dGie transcript was measured by semiquantitative reverse-transcription PCR with decreasing amounts of the templates. Aurora-B mRNA was used as a loading control. (B) Flow cytometry analyses of the control (left) and dGie-dsRNA-treated (right) S2 cells. (C) The control (top) and dsRNA-treated (middle and bottom) S2 cells were stained with PicoGreen and the anti--tubulin antibody to reveal DNA (green) and microtubules (red), respectively. Gie-depleted S2 cells displayed no separation of their chromatids before cytokinesis and remained with a thin chromatin string (arrowhead). They also had lagging chromosomes (arrows). Scale bar, 5 µm. (D, left) Representative immuno-stains of control (top) and dGie-dsRNA-treated S2 cells (bottom). The appearance of cells that had a chromatin string (arrowhead) or lagging chromosomes (arrows) at anaphase stage was measured. (D, right) The data are represented as percentages of means±s.e.m. from at least three independent experiments (each of 100 cells).

View larger version (68K): [in a new window]   Fig. 5. Cell-cycle-dependent dynamics of Gie localization. PC12 cells were fixed and stained for endogenous Gie with the affinity-purified anti-Gie antibody (green). Microtubules (red) and DNA (blue) were also visualized with anti--tubulin antibody and DAPI, respectively. Gie displayed cytoplasmic distribution in interphase (white arrows) but moved prominently to the spindle mid-zone (yellow arrows) in anaphase and to the mid-body in late-telophase (yellow arrowhead). Scale bar, 5 µm.

View larger version (33K): [in a new window]   Fig. 6. Gie associates with tubulin independent of its guanine-nucleotide-bound forms or the absence of its effector domains. (A) Extracts from HeLa cells expressing FLAG-tagged wild-type Gie1 and Ha-Ras were subjected to immunoprecipitation (IP) with the anti-FLAG antibody. The precipitants were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (left) or immunoblotting (IB) with an anti-ß-tubulin antibody (right). The asterisk denotes the position of interacting protein. (B) Microtubule co-sedimentation assay. Gie stayed mainly soluble (sup) when microtubule polymerization was inhibited by keeping on ice (lanes 2,4), and was specifically recovered in the pellet (pell) in the presence of Taxol-stabilized microtubules (lanes 1,3). The behavior of the Rab5 is shown as a negative control. (C,D) HeLa cells were transfected with expression vectors encoding the proteins listed at the bottom and the cell extracts were immunoprecipitated with the anti-FLAG antibody. The precipitants were subjected to immunoblotting using anti-FLAG and anti-ß-tubulin antibodies.

View larger version (52K): [in a new window]   Fig. 7. Overexpression of Gie1 mutants lacking the switch regions impairs chromosome segregation in HeLa cells. (A) HeLa cells that had been transfected with the indicated Gie1 and cultured for 24-30 hours were analysed by immunofluorescence microscopy. DNA (green) and Gie1 (red) were detected with PicoGreen and the anti-Gie antibody, respectively. Impaired chromosome segregation was observed in cells expressing Gie1/31-60 and Gie1/61-90 (white arrows). Scale bar, 5 µm. (B) The appearance of abnormal nuclei was measured and the data are represented as percentages of means±s.e.m. from at least three independent experiments (each of 200-400 cells).