Figure 3

Characteristics of the two territories separated by actin waves on the substrate-attached cell surface. The actin waves labeled with mRFP-LimEΔ are shown in red, actin regulatory proteins or phosphoinositides in green. The upper panels depict TIRF images of double-labeled cells, the lower panels show line scans that cross the border between inner territory and external area. Sizes and positions of the scans are shown in the upper panels. A, the presence of PIP3 distinguishes the inner territory from the external one. B, activated Ras, a stimulator of actin polymerization, is bound to the membrane within the inner territory. This cell is also shown in Additional file 1 to ensure that the external area depleted of activated Ras is not out of focus. C, the Arp2/3 complex, responsible for the nucleation of branched actin assemblies, is enriched in the inner territory and in the actin waves surrounding this area. D, PI (4,5)-bis-phosphate (PIP2) is increased in the external area relative to the inner territory. E, filaments of myosin-II are localized to the external area. F, in this area also cortexillin is enriched, a bundling protein with a preference for the anti-parallel arrangement of actin filaments [16]. To prevent binding to PIP2, we probed for the actin structure using a truncated fragment (aa 352-435), which comprises the actin bundling domain without its C-terminal PIP2-binding motif [19]. Dotted lines in the images of (A to C) and arrowheads in the corresponding line scans indicate the cell border where otherwise hard to recognize. Scale bar for all images, 10 μ m.