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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).

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