Figure 5

S102D expression perturbs cytokinesis. Panel A HeLa cells on glass coverslips were transiently transfected with GFP-FIP3, or the indicated mutants as described. After 48 h, cells were fixed and microtubules (anti-tubulin) and DNA (DAPI) stained as described. The fraction of cells expressing each FIP3 species that were binucleate was then determined and is plotted graphically. The data shown are from a representative experiment: in this case over 200 GFP-positive cells were counted per condition. *indicates a significant increase over control (GFP-FIP3) cells p = 0.02, and **p < 0.05. Similar results were obtained in two other experiments of this type, but the fraction of binucleate cells varied between experiments (e.g. the% of binucleate cells expressing GFP-FIP3-I737E varied between 16 and 33%). Panel B shows an immunoblot from the same experiment in which equal amounts of lysate were immunoblotted with anti-GFP to show that similar levels of each mutant were expressed. Anti-GAPDH is used as a loading control. Panel C shows the cytosol/membrane ratio of the indicated mutants in cells synchronised in metaphase. Data from a representative experiment is shown. Panel D HeLa cells on glass coverslips were transiently transfected with GFP-FIP3 (pseudo-coloured green), or the indicated mutants as described. After 24 h, cells were fixed and immunostained with anti-tubulin (pseudo-coloured red) and the distribution of cells in telophase examined. Shown are cells in early or late telophase. Data from a representative experiment, repeated 5 times are shown. Note that the categorisation of these images into 'early' or 'late' telophase is dictated by the appearance of FIP3 (see [11] for details) which is highly dynamic in telophase, moving from a characteristic perientrosomal localisation into the intercellular bridge region as cells move from early to late telophase. Panel E HeLa cells expressing HA-FIP3 or HA-FIP3-4A were analysed by immunofluoresence microscopy using monoclonal anti-HA in telophase to reveal the localisation of epitope-tagged FIP3. Shown are typical images of cells in early or late telophase, pseudo-coloured green. Data from a representative experiment, repeated 3 times are shown.