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  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.
Collins et al. BMC Cell Biology 2012 13:4 doi:10.1186/1471-2121-13-4