Fig. 5.
The PtdIns(3,4,5)P3 spike precedes cup closure. (A) Strategy for identifying the point of cup closure using CFP-MEM-labeled membranes. The top row shows the arrangements of plasma membrane on the top of the cell during macropinosome formation. The bottom row shows the corresponding fluorescence intensity of CFP-MEM during ruffling and macropinosome formation. Fluorescence intensities should increase as the membrane doubles over, reaching maximal values at the point of cup closure. (B) Macropinosome formation in macrophages expressing YFP-BtkPH and CFP-MEM. Arrowheads indicate a forming macropinosome. The top row shows phase-contrast images of the stages of macropinosome formation. The middle row shows the relative density of membrane, as indicated by the intensity of the CFP-MEM images. CFP-MEM fluorescence in the macropinosome increased during the circular stage, indicative of membrane doubling and cup closure. The bottom row shows the YFP-BtkPH:CFP-MEM ratio, which indicates the spike of PtdIns(3,4,5)P3 concentration in membranes just after ruffle closure (red arrowhead). Scale bar: 3 μm. Color bars indicate the signal intensity of CFP-MEM or the molar ratio of YFP-BtkPH:CFP-MEM in each image. (C) Timing of the peak values for YFP-BtkPH:CFP-MEM ratios and CFP-MEM fluorescence, relative to ruffle closure (t=0 seconds), indicated that the maximal fluorescence of CFP-MEM followed the PtdIns(3,4,5)P3 spike (n=ten macropinosomes). The difference between two maxima was significant (*P<0.005). (D) Strategy for identifying cup closure using low concentrations of FM4-64. The top row shows the relative fluorescence of plasma membrane and macropinosome membranes under observation conditions. Plasma membranes will remain fluorescent owing to exchange of FM4-64 with dye in the buffer. Macropinosome closure will limit the reservoir of free FM4-64 to that which is enclosed in the vacuole; hence, the macropinosome fluorescence will diminish faster than the plasma-membrane fluorescence. Cup closure can be identified as the point at which macropinosome fluorescence photobleaches faster than plasma-membrane fluorescence. (E) Photobleaching of FM4-64 in macropinosomes revealed the timing of cup closure relative to the PtdIns(3,4,5)P3 spike. Macrophages expressing YFP-BtkPH were imaged in the presence of M-CSF and 1 μg/ml FM4-64. The top row shows phase-contrast images of the stages of macropinosome formation. The middle row shows the intensity of YFP-BtkPH. YFP-BtkPH fluorescence in the macropinosome increased at t=+20 seconds (relative to ruffle closure), indicative of the PtdIns(3,4,5)P3 spike. The bottom row shows the intensity of FM4-64, which indicates the relative membrane densities in each pixel. FM4-64 fluorescence in the plasma membrane decreased continuously owing to photobleaching and depletion of dye from the buffer. FM4-64 fluorescence in the macropinosome increased during the curved stage then decreased gradually during the circular stage. Scale bar: 3 μm. Color bars indicate the signal intensities of YFP-BtkPH or FM4-64 in each image. (F) Plots of YFP-BtkPH signal intensity in the macropinosome region (black circles) and the entire cell (white circles) of C. Time 0 marks ruffle closure. (G) Intensity of macropinosome (MP):intensity of whole cell area (Cell) for YFP-BtkPH fluorescence (white circles) and FM4-64 fluorescence (black circles) of A. Relative FM4-64 fluorescence in the macropinosome increased during ruffle and cup closure, then decreased afterwards. The point at which the ratio began to decrease indicated cup closure. Comparing the two curves indicates that the PtdIns(3,4,5)P3 spike occurred between ruffle closure (t=0 seconds) and cup closure (t=+100 seconds). (H) Intensity (MP):intensity (Cell) for FM4-64 fluorescence, relative to ruffle closure (t=0 seconds), the PtdIns(3,4,5)P3 spike (spike), the maximum ratio (peak) and the end of the circular stage (end), indicated that cup closure followed the spike of PtdIns(3,4,5)P3 (n=nine macropinosomes). *P<0.01, **P<0.005, ***P<0.001.