Fig. 3.

IRSp53 is involved in CG endocytosis. a Schematic depicting the domain organisation of IRSp53. IRSp53 exists in an inactive dimer state, which upon binding to GTP-CDC42 is activated, allowing SH3 domain to bind to its effectors. b Histogram (left) shows 5-min uptake of TfR and fluid phase in IRSp53 WT cells normalised to IRSp53−/− cells, along with representative images (right). Data were pooled from two independent experiments and the number of cells indicated in the figure. c Histogram (top) shows normalised 5-min fluid uptake in IRSp53−/− and IRSp53WT cells when treated with LG186 or vehicle (DMSO) along with representative images (bottom). Data were pooled from two independent experiments and the number of cells indicated in the figure. d Histogram (top) shows an average number of endocytic structures quantified per field from the electron microscope images (bottom). Data pooled from three independent blocks. Untreated WT MEFs (WT, top row), IRSp53 null MEFs (IRSp53−/−, bottom left) or LG186-treated WT MEFs (LG186, bottom right) were incubated for 5 min at 37 °C with 10 mg/ml HRP as a fluid-phase marker before processing for electron microscopy. Endocytic structures close to the plasma membrane (PM) are filled with the electron dense peroxidase precipitate. WT cells show a range of endocytic structures including vesicular structures (double arrowheads) and tubular/ring-shaped putative CLIC/GEECs (large arrowheads) but the IRSp53−/− cells and LG186-treated cells only show predominant labelling of vesicular profiles. p value <0.05 (*), 0.001 (**) Mann–Whitney U test (b–c) and two-sample Student’s t test (d). Error bars, s.d. (b–d). Scale bar, 20 µm (b–c), 1 µm (d), respectively. Schematic (a) was adapted with permission from MBInfo (www.mechanobio.info) Mechanobiology Institute, National University of Singapore