Extended Data Fig. 1: Cells generate two mutually exclusive dynamic states in the membrane during migration and ventral wave propagation.

(a) Coordinated localization dynamics of signalling (PIP3) and cytoskeletal components (F-actin) in migrating Dictyostelium cell protrusions. Top panel: Live-cell images (Arrowheads: Protrusions enriched in both F-actin and PIP3). Bottom panel: 360° membrane kymographs showing consistency of coordination. Here and in all other kymographs, numbers on the left denote time in seconds, unless otherwise mentioned. Throughout this study, PIP3 level is marked by PH_crac in Dictyostelium and by PH_AKT in macrophages, whereas, newly polymerizing F-actin is marked by LimE_Δcoil (‘LimE’ hereafter) in Dictyostelium and by Lifeact in macrophages. (b, c) Coordinated propagation of signalling (PIP3) and cytoskeletal (F-actin) components in ventral-surface cortical waves of Dictyostelium (b) and RAW 264.7 macrophages (c). Top two panels show live-cell images and bottom panels show line-scan intensity profile along the solid white lines. Similar convention is followed throughout this article. (d, e) Activated Ras and PIP3 colocalizing in the protrusions (d), whereas PTEN selectively dissociating from it (e), in migrating Dictyostelium cells. Left: Live-cell images, Right: 360° membrane kymographs. Arrowheads: Protrusions/front-states. Throughout this study, Ras-Binding Domain of Raf1 (RBD) was used as a detector of Ras activation. (f, g) In propagating waves of Dictyostelium, activated Ras and PIP3 dynamically co-localized and defined the front-state regions (f), whereas PIP3 and PTEN exhibit consistent complementarity (g). Live-cell images, line-scan intensity profiles, and line kymographs are shown. (h) Complementary distribution of PIP3 and PTEN is independent of cytoskeleton. Here Dictyostelium cells are imaged in presence of Latrunculin A (F-actin polymerization inhibitor). Arrowheads denoting front-states. (i) Schematic showing the front-back complementary patterning in three different scenarios: migrating cell protrusions, cytoskeleton-independent cortical symmetry breaking, and propagating ventral waves. For first two cases, we could study a 1D profile, whereas for ventral waves, we observed a 2D profile at the substrate-attached surface. Several examples of established signalling and cytoskeletal components are listed and categorized. In all situations, when a front-state was created from the back/basal state of the membrane, back markers moved away from that particular domain, maintaining complementarity. All scale bars are 10 μm.