Figure 2. Macropinocytic cups contain a central domain of PIP3 surrounded by a ring of SCAR.
(A, B, C) Evidence that fluorescently tagged SCAR complex faithfully marks regions of active actin polymerisation: (A) An aggregation-competent cell moving under an agarose overlay (optimal conditions for visualising pseudopods) showing HSPC300-GFP recruited to the two pseudopods; (B) Kymograph of expansion of the pseudopod arrowed in (A) showing that the SCAR reporter is present during periods of expansion, but absent in the plateaus when the pseudopod is not expanding; (C) Membrane speed and SCAR complex accumulation are positively correlated. The HSPC300-GFP signal and local membrane speed was measured at 100 points along the membrane of a motile cell. Data of eight independent cells was combined and plotted as a 2D histogram. Green arrow indicates data points with high levels of SCAR and positive displacement. Red arrow indicates data points due to blebs, which are actin-free and expand much faster than pseudopods (Zatulovskiy et al., 2014). (D) SCAR is recruited as a ring to the lip of macropinocytic cups. The upper panels show top and side views of a surface rendering of a cell with three macropinocytic cups and the lower shows the same cell with a SCAR reporter. (E) SCAR is recruited to pseudopods in distinct blocks, not as a ring. Pitted appearance of the 3D surface is a rendering artefact caused by small vesicles that reside just underneath the cell membrane. (F) SCAR is recruited to the edge of an intense PIP3 patch in the macropinocytic cup. The white dotted line in the left panel corresponds to the position of the vertical plane in the right panel. (G) 3D reconstruction of the cell in the previous panel. (H) F-actin is nearly uniformly distributed in the macropinocytic cup and does not predict the localization of SCAR. Ax2 cells were used in all panels. HSPC300 was used as a marker for the SCAR complex, PH-CRAC as a reporter for PI(3,4,5)P3 and Lifeact as a reporter for F-actin. 3D images were reconstructed from Z-stacks taken on a spinning disk microscope.