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Fig. 1. Expression of the Syne-1 fragment GSSF1 blocks cytokinesis in animal cells. (A) A map of Syne-1 protein showing the positions of the two fragments used in these studies, the kinesin binding fragment GSSF1 and the control fragment GSSF5 that does not bind kinesin. Also shown are the two Golgi binding sites identified previously (light blue), the proposed nuclear envelope binding site (yellow) and the kinesin II binding site identified in this study (dark blue). (B) When COS-7 cells were transfected with GFP-tagged GSSF1 (a) and nuclei were stained with DAPI (b), we found that a large proportion of transfected cells (a and b, arrow) were binucleate, whereas untransfected cells remained mononucleate (a and b, arrowheads). (C) COS-7 cell transfected with GSSF1 (a), stained with DAPI (b), examined by phase contrast microscopy (c) appears as a single binucleate cell (rather than two adjacent mononucleate cells). (D) Fluorescence activated cell sorting (FACS) assay for binucleate cells. HeLa cells were transfected with GFP-GSSF1 (black bar), GSSF5 (striped bar) or mock transfected (white bar). GFP positive cells were isolated by fluorescence activated cell sorting and the proportion of total GFP-positive cell population possessing twice the normal amount of DNA was quantified by propidium iodide fluorescence. Because mock transfected cells do not express GFP, the proportion of the total cell population with twice the normal amount of DNA is given in percent. Error bars indicate standard deviation of triplicate FACS experiments (* indicates the statistically significant difference compared to the control obtained by Student's t-test). (E) DAPI-stained cells transfected with dominant-negative fragment GSSF1 (a-c), the control fragment GSSF5 (d-f).
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